USE OF ANP32 PROTEIN IN MAINTAINING THE POLYMERASE ACTIVITY OF INFLUENZA VIRUS IN HOSTS

Abstract

The present invention provides a recombinant sequence information of a key host factor ANP32A/B which is necessary for the replication of influenza virus in a host. More specifically, the present invention relates to a 129-130 motif and a 149 site of the host factor ANP32A/B protein, which are key active sites for exerting its ability to promote the replication of influenza virus, and are also potential targeting sites of anti-influenza drugs.

Claims

1. A mutated ANP32 protein having one or more mutations selected from: the amino acid at position 129 substituted with isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, the amino acid at position 130 substituted with asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, the amino acid at position 149 substituted with alanine A, the amino acid at position 151 substituted with alanine A, or the amino acids at positions 60 and 63, positions 87, 90, 93 and 95, positions 112, 115 and 118 are substituted with alanine, wherein: when the ANP32 protein is chicken ANP32B protein, duck ANP32B protein or turkey ANP32B protein, the amino acid at position 129 is not isoleucine I and the amino acid at position 130 is not asparagine N, when the ANP32 protein is murine ANP32A, the amino acid at position 130 is not alanine A, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of a chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, and wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.

2-27. (canceled)

28. A mutated ANP32 protein, wherein one or more of the the following amino acid segments are deleted or substituted with alanine: amino acids at positions 61-70, amino acids at positions 71-80, amino acids at positions 81-90, amino acids at positions 91-100, amino acids at positions 101-110, amino acids at positions 111-120, amino acids at positions 121-130, amino acids at positions 131-140, amino acids at positions 141-150, or amino acids at positions 151-160, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.

29. A mutated ANP32 protein, wherein when the ANP32 protein is human ANP32B protein, one or more of the following amino acid segments are deleted or substituted with alanine: amino acids at positions 21-30, amino acids at positions 41-50, amino acids at positions 51-60 or amino acids at positions 161-170, or the amino acid positions, which correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1.

30. A mutated ANP32 protein, wherein when the ANP32 protein is chicken ANP32B protein, one or more of the following amino acid segments are deleted or substituted with alanine: amino acids at positions 161-170, amino acids at positions 171-180 or amino acids at positions 191-200, or the amino acid positions, which correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3.

31. A method of reducing the polymerase activity of an influenza virus, comprising mutating the ANP32 protein to have one or more of the following mutations: the amino acid at position 129 is substituted with isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, the amino acid at position 130 is substituted with asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, the amino acid at position 149 is substituted with alanine A, the amino acid at position 151 is substituted with alanine A, or the amino acids at positions 60 and 63, positions 87, 90, 93 and 95, positions 112, 115 and 118 are substituted with alanine, wherein: when the ANP32 protein is a chicken ANP32B protein, duck ANP32B protein or turkey ANP32B protein, the amino acid at position 129 is not isoleucine and the amino acid at position 130 is not asparagine N, when the ANP32 protein is murine ANP32A, the amino acid at position 130 is not alanine A, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, and wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.

32. The method of claim 31, wherein the polymerase activity of influenza virus is lost, wherein the ANP32 protein is subjected to one or more mutations selected from the following: the amino acid at position 129 is substituted with isoleucine I, lysine K or aspartic acid D, the amino acid at position 130 is substituted with asparagine N, phenylalanine F or lysine K, or the amino acids at positions 87, 90, 93 and 95, positions 112, 115 and 118 are substituted with alanine.

33. A method of reducing the polymerase activity of influenza virus, comprising deleting or substituting one or more of the amino acid segments of ANP32 protein with alanine, wherein the one or more of the amino acid segments are selected from the following: amino acids at positions 61-70, amino acids at positions 71-80, amino acids at positions 81-90, amino acids at positions 91-100, amino acids at positions 101-110, amino acids at positions 111-120, amino acids at positions 121-130, amino acids at positions 131-140, amino acids at positions 141-150, or amino acids at positions 151-160, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, and wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.

34. The method of claim 33, wherein the polymerase activity of influenza virus is lost, wherein one or more of the amino acid segments of ANP32 protein are deleted or substituted with alanine, wherein the amino acid segment is selected from the following: amino acids at positions 71-80, amino acids at positions 81-90, amino acids at positions 91-100, amino acids at positions 101-110, amino acids at positions 111-120, amino acids at positions 121-130, amino acids at positions 131-140, amino acids at positions 141-150, or amino acids at positions 151-160.

35. A method of reducing the polymerase activity of influenza virus, comprising deleting or substituting one or more of the amino acid segments of human ANP32B protein with alanine, wherein the one or more of the amino acid segments are selected from the following: amino acids at positions 21-30, amino acids at positions 41-50, amino acids at positions 51-60 or amino acids at positions 161-170, or the amino acid positions, which correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.

36. A method of reducing the polymerase activity of influenza virus, comprising deleting or substituting one or more of the amino acid segments of chicken ANP32A protein with alanine, wherein the one or more of the amino acid segments are selected from the following: amino acids at positions 161-170, amino acids at positions 171-180, amino acids at positions 191-200, or the amino acid positions, which correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3, wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.

37. The method of claim 36, wherein the polymerase activity of influenza virus is lost, wherein, one or more of the amino acid segments of chicken ANP32A protein are deleted or substituted with alanine, wherein the one or more of the amino acid segments are selected from amino acids at positions 161-170, or amino acids at positions 171-180.

38. A kit comprising at least one reagent or a set of reagents, wherein the at least one reagent or a set of reagents is used to determine the type of amino acid at one or more positions of an ANP32 protein selected from the following: amino acid at position 129, amino acid at position 130, amino acid at position 149, amino acid at position 151, amino acid at position 60, amino acid at position 63, amino acid at position 87, amino acid at position 90, amino acid at position 93, amino acid at position 95, amino acid at position 112, amino acid at position 115, or amino acid at position 118, wherein when the amino acid at position 129 is isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, when the amino acid at position 130 is asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, when the amino acid at position 149 is alanine A, when the amino acid at position 151 is alanine A, when the amino acids at positions 60 and 63, at positions 87, 90, 93 and 95, at positions 112, 115, and 118 are alanine, the ability of the ANP32 protein to support the activity of influenza polymerase is decreased, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1.

39. An oligonucleotide primer for determining the type of amino acid of ANP32 protein selected from the following: amino acid at position 129, amino acid at position 130, amino acid at position 149, amino acid at position 151, amino acid at position 60, amino acid at position 63, amino acid at position 87, amino acid at position 90, amino acid at position 93, amino acid at position 95, amino acid at position 112, amino acid at position 115, or amino acid at position 118, wherein: when the amino acid at position 129 is isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, when the amino acid at position 130 is asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, when the amino acid at position 149 is alanine A, when the amino acid at position 151 is alanine A, when the amino acids at positions 60, 63, or the amino acids at positions 87, 90, 93 and 95, or the amino acids at positions 112, 115 and 118 are alanine, the ability of ANP32 protein to support the activity of influenza polymerase is decreased, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, wherein the amino acids at positions 87, 90, 93 and 95 are from a mammalian ANP32B protein, wherein the oligonucleotide primer is preferably at least 20 bases in length and, wherein the oligonucleotide primer is selected from SEQ ID NOs: 155-156, 163-166, 167-256, or 375-380.

40. A method of screening for a candidate drug for treating an influenza virus infection, comprising: (1) knocking out the ANP32A and/or ANP32B protein from a cell line containing ANP32A and/or ANP32B protein, to obtain a cell line in which ANP32A protein and/or ANP32B protein are knocked out, (2) transfecting the knockout cell line obtained in step (1) with a plasmid encoding ANP32A and/or ANP32B protein and a plasmid encoding influenza virus polymerase, (3) contacting the knockout cell line with a candidate, wherein the contacting can be performed simultaneously with or separately from the transfection of step (2), wherein the cell line treated in step (3) does not express influenza virus polymerase or has reduced expression of influenza virus polymerase compared to a cell line containing ANP32A and/or ANP32B, indicating that the candidate is a candidate drug for treating influenza virus infection, wherein the ANP32A and/or ANP32B protein is derived from chicken, human, zebra finch, duck, turkey, pig, mouse or horse; preferably, the influenza virus is selected from human, canine, avian or equine influenza virus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1 shows the map of PCAGGS vector.

[0103] FIG. 2 shows the map of PCAGGS-huANP32A vector.

[0104] FIG. 3 shows the detection of expression of ANP32 protein from various species.

[0105] FIG. 4 shows the detection of the knockout cell line by fluorescent quantitative PCR: A is the quantitative detection of huANP32A on each cell line, and B is the quantitative detection of huANP32B on each cell line.

[0106] FIG. 5 shows the detection of endogenous ANP32 protein in 293T wild-type and knockout cell lines.

[0107] FIG. 6 shows the detection of activity of H1N1.sub.SC09 polymerase on 293T wild-type and knockout cell lines.

[0108] FIG. 7 shows the detection of activity of different polymerases on 293T wild-type and knockout cell lines. FIG. 7A is the detection of activity of H7N9.sub.AH13 polymerase on 293T wild-type and knockout cell lines; FIG. 7B is the detection of activity of WSN polymerase on 293T wild-type and knockout cell lines; FIG. 7C is the detection of activity of H3N2.sub.G11 polymerase on 293T wild-type and knockout cell lines; FIG. 7D is the detection of activity of H3N8.sub.JL89 polymerase on 293T wild-type and knockout cell lines; FIG. 7E is the detection of activity of H3N8.sub.XJ07 polymerase on 293T wild-type and knockout cell lines.

[0109] FIG. 8 shows the activity assay of H1N1.sub.SC09 polymerase on DKO cell line when supplemented with different doses of huANP32A or huANP32B.

[0110] FIG. 9 shows that the activity of H1N1.sub.SC09 polymerase (A) and H7N9.sub.AH13 polymerase (B) on DKO cells is inhibited when supplemented with excessive huANP32A or huANP32B.

[0111] FIG. 10 shows the influence of huANP32A on the RNA synthesis of H1N1.sub.SC09 influenza virus by fluorescent quantitative detection.

[0112] FIG. 11 shows the influence of ANP32 protein of different species on the replication of different influenza viruses. FIG. 11A shows the influence of ANP32 protein of different species on the replication of H1N1.sub.SC09 influenza virus; FIG. 11B shows the influence of ANP32 protein of different species on the replication of H7N9.sub.AH13 influenza virus; FIG. 11C shows the influence of ANP32 protein of different species on the replication of H3N2.sub.GD11 influenza virus; FIG. 11D shows the influence of ANP32 protein of different species on the replication of H3N8.sub.XJ07 influenza virus; FIG. 11E shows the influence of ANP32 protein of different species on the replication of H3N8.sub.JL89 influenza virus; FIG. 11F shows the influence of ANP32 protein of different species on the replication of H9N2.sub.ZJ12 influenza virus; FIG. 11G shows the influence of ANP32 protein of different species on the replication of WSN influenza virus.

[0113] FIG. 12 shows the detection of H1N1.sub.SC09 influenza virus; FIG. 12A shows the amount of virus from different cell lines transfected with H1N1.sub.SC09 influenza virus; FIG. 12B shows the influence of supplementation with ANP32 protein on the amount of H1N1.sub.SC09 influenza virus from DKO cells.

[0114] FIG. 13 shows the detection of WSN influenza virus; FIG. 13A shows the virus titers of different cell lines transfected with WSN influenza virus;

[0115] FIG. 13B shows the influence of supplementation with ANP32 protein on the virus titer of WSN influenza virus from DKO cells.

[0116] FIG. 14 shows the adaptation of point mutation of PB2 gene of H7N9 subtype influenza virus to ANP32.

[0117] FIG. 15 shows the ANP32B sequences alignment, truncation and fragment interchange; FIG. 15A is an alignment of ANP32B sequences;

[0118] FIG. 15B shows an ANP32B truncation strategy; FIG. 15C shows the first round of interchanging huANP32B with chANP32B fragments; FIG. 15D shows the second round of interchanging huANP32B with chANP32B fragments.

[0119] FIG. 16 shows the ANP32B point mutation; FIG. 16A is the sequence alignment of ANP32B 110-161 regions; FIG. 16B shows the influence of the point mutation of huANP32B on H1N1.sub.SC09 polymerase activity.

[0120] FIG. 17 shows the influence of single point mutation on position 129 or 130 of huANP32B on H1N1.sub.SC09 polymerase activity.

[0121] FIG. 18 shows the influence of single point mutation of huANP32B on H7N9.sub.AH13 polymerase activity.

[0122] FIG. 19 shows the influence of point mutation at position 129 or 130 of huANP32A on H1N1.sub.SC09 polymerase activity.

[0123] FIG. 20 shows the influence of point mutation of huANP32A on H7N9.sub.AH13 polymerase activity.

[0124] FIG. 21 shows the influence of chANP32A point mutation and chANP32B point mutation on H7N9.sub.AH13 polymerase activity.

[0125] FIG. 22 shows the influence of point mutant at position 129 of chANP32A on H7N9.sub.ZJ13 polymerase activity.

[0126] FIG. 23 shows the influence of point mutant at position 129 of chANP32A on H7N9.sub.AH13 polymerase activity.

[0127] FIG. 24 shows the influence of point mutant at position 129 of chANP32A on the activity of WSN polymerase.

[0128] FIG. 25 shows the influence of point mutant at position 130 of chANP32A on H7N9.sub.ZJ13 polymerase activity.

[0129] FIG. 26 shows the influence of point mutant at position 130 of chANP32A on H7N9.sub.AH13 polymerase activity.

[0130] FIG. 27 shows the influence of point mutant at position 130 of chANP32A on the activity of WSN polymerase.

[0131] FIG. 28 shows the influence of huANP32B truncated mutant on H7N9.sub.AH13 polymerase activity.

[0132] FIG. 29 shows the influence of chANP32A truncated mutant on H7N9.sub.ZJ13 polymerase activity.

[0133] FIG. 30 shows the influence of chANP32A point mutant on H7N9.sub.AH13 polymerase activity.

[0134] FIG. 31 shows the influence of huANP32B point mutant on H7N9.sub.AH13 polymerase activity.

[0135] FIG. 32 shows the identification and sequencing result of site-directed mutant cell line of amino acid at position 129/130 of huANP32A and huANP32B.

[0136] FIG. 33 shows the protein detection result of site-directed mutant cell line of amino acid at position 129/130 of huANP32A and huANP32B.

[0137] FIG. 34 shows the detection of activity of H1N1.sub.SC09 polymerase on different cell lines.

[0138] FIG. 35 shows the detection of activity of H7N9.sub.AH13 polymerase on different cell lines.

[0139] FIG. 36 shows the alignment of amino acid sequences of avian-derived ANP32A proteins.

[0140] FIG. 37 shows the alignment of amino acid sequences between avian-derived ANP32B protein and huANP32B protein.

[0141] FIG. 38 shows the alignment of amino acid sequences between chicken ANP32A protein and mammalian ANP32A protein.

[0142] FIG. 39 shows the alignment of amino acid sequences of mammalian ANP32B protein.

[0143] FIG. 40 shows the detection of murine ANP32B protein expression.

[0144] FIG. 41 shows the influence of murine ANP32B protein point mutant on H7N9.sub.AH13 polymerase activity.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

[0145] The present invention is described in detail below with reference to the examples and the accompanying drawings. It will be understood by those skilled in the art that the following examples are for illustrative purposes and should not be construed as limiting the present invention in any way. The protection scope of the present invention is defined by the appended claims.

Example 1. Construction of ANP32 Protein Expression Vector

[0146] The nucleotide sequences of ANP32 proteins from chicken, human, zebra finch, duck, turkey, pig, mouse, horse, etc. are as follows:

[0147] chicken ANP32A (chANP32A) (Gallus gallus, XM_413932.5), human ANP32A (huANP32A) (Homo sapiens, NM 006305.3), zebra finch ANP32A (zfANP32A) (Taeniopygia guttata, XM_012568610.1), duck ANP32A (dkANP32A) (Anas platyrhynchos, XM_005022967.1), turkey ANP32A (tyANP32A) (Meleagris gallopavo, XM_010717616.1), pig ANP32A (pgANP32A) (Sus scrofa, XM_003121759.6), murine ANP32A (muANP32A) (Mus musculus, NM 009672.3), equine ANP32A (eqANP32A) (Equus caballus, XM_001495810.5), chicken ANP32B (chANP32B) (Gallus gallus, NM_001030934.1), human ANP32B (huANP32B) (Homo sapiens, NM_006401.2).

[0148] The amino acid sequences of ANP32 proteins from chicken, human, zebra finch, duck, turkey, pig, mouse, horse, etc. are as follows:

[0149] chicken ANP32A (chANP32A) (Gallus gallus, XP_413932.3), human ANP32A (huANP32A) (Homo sapiens, NP_006296.1), zebra finch ANP32A (zfANP32A) (Taeniopygia guttata, XP_012424064.1), duck ANP32A (dkANP32A) (Anas platyrhynchos, XP_005023024.1), turkey ANP32A (tyANP32A) (Meleagris gallopavo, XP_010715918.1), pig ANP32A (pgANP32A) (Sus scrofa, XP_003121807.3), mouse ANP32A (muANP32A) (Mus musculus, NP_033802.2), equine ANP32A (eqANP32A) (Equus caballus, XP_001495860.2), chicken ANP32B (chANP32B) (Gallus gallus, NP_001026105.1), human ANP32B (huANP32B) (Homo sapiens, NP_006392.1).

[0150] First, a PCAGGS-Flag recombinant plasmid was constructed. A start codon (ATG) was introduced at the N-terminus of the Flag-tag (GGCAGCGGAGACTACAAGGATGACGATGACAAG, SEQ ID NO:1), and a stop codon (TGA) was introduced at the C-terminus; NotI (GCGGCCGC, SEQ ID NO:2) restriction site was introduced upstream of the start codon, and XhoI (CTCGAG, SEQ ID NO:3) restriction site was introduced downstream of the stop codon, and a 15 bp homologous arm (underlined) of PCAGGS vector was introduced at the outer ends of the two restriction sites, and two primers Flag-S(SEQ ID NO: 4) and Flag-A (SEQ ID NO: 5) complementary to the Flag-tag gene fragment were synthesized.

TABLE-US-00001 Flag-S:SEQIDNO:4 5-AAAGAATTCGAGCTCGCGGCCGCATGGGCAGCGGAGACTACAAGGAT GACGATGACAAGTGACTCGAGCTAGCAGATCTTTTT-3 Flag-A:SEQIDNO:5 5-AAAAAGATCTGCTAGCTCGAGTCACTTGTCATCGTCATCCTTGTAGT CTCCGCTGCCCATGCGGCCGCGAGCTCGAATTCTTT-3

[0151] The designed upstream and downstream primers Flag-S and Flag-A were diluted respectively to 100 uM (diluted with TE buffer); 10 ul of each diluted primers were taken and then mixed uniformly, and placed at 95 C. for 5 min; the PCR instrument was turned off, and the temperature was naturally reduced to room temperature (about 2 h), and the obtained product is an annealed synthetic sample. Commercially available PCAGGS vector (see FIG. 1, purchased from Fenghui Bio, product number V00514, cat # JM004, www.fenghbio.cn) was subjected to double enzyme digestion by Thermo rapid restriction endonuclease Not I and Xho I under a water bath condition at 37 C. for 1.5 h, and the enzyme digested fragments were recovered by using a gel recovery kit (OMEGA, cat # D2500-01), and the obtained product is recovered for future use. The PCAGGS double-enzyme digested product and the annealed synthetic sample were ligated by In-Fusion ligase (purchased from Clontech, cat #639648) according to the instructions, and then transformed into DH5a competent cells. The next day, a single clone was selected and sequenced, and the plasmid which was verified correct by sequencing was named as PCAGGS-Flag plasmid which was used as a control plasmid for subsequent experiments and was extracted in large-scale for later use.

[0152] According to the nucleotide sequences of ANP32A and ANP32B of each species mentioned above, the sequences of ANP32A and ANP32B of each species mentioned above were synthesized respectively. During the synthesis, the stop codon was removed at the C-terminus of the gene fragment and the a Flag-tag (SEQ ID NO: 1) was added in tandem; a stop codon (TGA) was added at the end of the Flag-tag; Not I (SEQ ID NO: 2) and Xho I (SEQ ID NO: 3) restriction sites were introduced at both ends of the synthesized fragment. The PCAGGS-Flag vector was digested by Thermo rapid restriction endonucleases Not I and Xho I at 37 C. for 1.5 h, and the digested fragments were recovered using a gel recovery kit (OMEGA, cat # D2500-01). The PCAGGS-Flag double-digested product and each gene fragment were ligated by In-Fusion ligase (purchased from Clontech, cat #639648) according to the instructions, and then transformed into DH5a competent cells. The next day, a single clone was selected and sequenced, and the fragment was finally inserted into the PCAGGS vector. For example, the plasmid map of PCAGGS-huANP32A is shown in FIG. 2. The plasmid maps of ANP32A and ANP32B genes of other species are similar to that of FIG. 2, and only the corresponding gene sequences are replaced. For example, the plasmid map of PCAGGS-huANP32B is a sequence in which huANP32A is replaced by huANP32B.

[0153] After the recombinant plasmids were sequenced correctly, 1 ug of the recombinant plasmids were respectively transfected into 293T cells by lipofectamine 2000 reagent, and the cell lysate was taken after 48 hours; the protein expression was detected by Flag-antibody (purchased from Sigma, cat # F1804-1MG) by utilizing western blotting; intracellular -actin was used as an internal control gene; the antibody Monoclonal Anti--Actin antibody produced in mouse (purchased from Sigma, cat # A1978-200UL) was used and the result was shown in FIG. 3 showing that ANP32A and ANP32B proteins from various species were well expressed.

Example 2: Construction of a Cell Line

[0154] We performed the construction of cell line by using CRISPR-Cas9 technology. According to NCBI published reference nucleotide sequences of human ANP32A (NM_006305.3) and human ANP32B (NM_006401.2), sgRNAs for the two proteins were designed by using the online software http://crispr.mit.edu/ (see Table 1 for sequences).

TABLE-US-00002 TABLE1 sgRNAsequences primername primersequence(5-3) humanANP32A-sgRNA, TCTTAAGTACAATCAACGT SEQIDNO:6 humanANP32B-sgRNA, GCCTACATTTATTAAACTG SEQIDNO:7

[0155] The pMD18T-U6 recombinant plasmid, which contains a human U6 promoter sequence+sgRNA sequence (huANP32A or huANP32B)+sgRNA scaffold sequence+TTTTTT, was constructed as follows.

[0156] First, a gene fragment is synthesized, wherein the fragment contains a human U6 promoter sequence+huANP32A-sgRNA sequence+sgRNA scaffold sequence+TTTTTT, and the sequence is SEQ ID NO: 8. The synthesized fragment was directly ligated into a pMD-18T vector (TaKaRa, Cat. No. D101A), and a pMD18T-U6-huANPsgRNA-1 recombinant plasmid (containing huANP32AsgRNA) was successfully constructed. Using this plasmid as a template, sgRNA primers for huANP32B were designed, and amplified by KOD-FX Neo high-efficiency DNA polymerase (Cat. No.: KFX-201, purchased from Toyobo) using the overlapping PCR method (the reaction condition and reaction system were based on the instructions of said polymerase; unless otherwise specified, except for the quantitative PCR reaction, KOD-FX Neo high-efficiency DNA polymerase was used in the PCR reactions of the following examples, and the reaction system and reaction condition were based on the instructions of said polymerase), to construct a plasmid containing sgRNA of huANP32B; and the primer sequences were shown in Table 2, and the PCR system and procedure were performed with reference to the KOD-FX Neo instructions. The obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, and then 5 ul of the digested product was taken to be transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and the plasmid which was verified correct by sequencing, namely pMD18T-U6-huANPsgRNA-2 (containing huANP32BsgRNA), was used for subsequent transfection experiment.

TABLE-US-00003 TABLE2 primersequences primername primersequence(5-3) huANP32B-sgRNA-F, CGGGTCCGGTTCCTCAGCTCCGGTGTTTCGT SEQIDNO:9 CC huANP32B-sgRNA-R, GGAGCTGAGGAACCGGACCCGTTTTAGAGCT SEQIDNO:10 AG

[0157] 1 ug of eukaryotic plasmid pMJ920 (Addge plasmid #42234) expressing Cas9-GFP protein and pMD18T-U6-huANPsgRNA-1 or pMD18T-U6-huANPsgRNA-2 recombinant plasmids were taken respectively, and mixed with lipofectamine 2000 at a ratio of 1:2.5, and then transfected into 293T cells. After 48 hours, GFP-positive cells were screened by an ultra-speed flow cytometry sorting system, and plated in a 96-well plate at a single cell/well for about 10 days; single-cell clones were picked for expansion and culture, and then cellular RNA was extracted according to the procedure using a SimplyP total RNA extraction Kit (purchased from Bioflux, cat # BSC52M1), and cDNA was synthesized using a reverse transcription Kit of Takara Co., Ltd (PrimneScript RT reagent Kit with gDNA Eraser (Perfect read Time), Cat.RR047A); and sgRNA-targeting fragments of huANP32A and huANP32B were amplified by KOD Fx Neo polymerase using the cDNA as the template, and the amplification primers were shown in Table 3, wherein the size of huANP32A amplified fragment was 390 bp, and the size of huANP32B amplified fragment was 362 bp. Single-cell clones that were verified as gene deletion by sequencing were subject to western blotting and fluorescent quantitative identification. The huANP32A and huANP32B double-knockout cell lines were obtained after the first round of obtaining the huANP32B single-knockout cell line, followed by another round of knockout screening, and the transfection system and screening steps were as described above.

TABLE-US-00004 TABLE3 theprimersequencesforidentificationof huANP32AandhuANP32Bknockoutcellline primersequence primername (5-3) QhuANP32A-F180, GGGCAGACGGATTCATTTAGAG SEQIDNO:11 QhuANP32A-R570, TTCTCGGTAGTCGTTCAGGTTG SEQIDNO:12 QhuANP32B-F312, GCGGAAAGTTAAGTTTGAAGAG SEQIDNO:13 G QhuANP32B-R674, GCGGAAAGTTAAGTTTGAAGAG SEQIDNO:14 G

[0158] Anti-PHAP1 antibody (purchased from Abcam, cat # ab51013) and Anti-PHAPI2/APRIL antibody [EPR14588] (purchased from Abcam, cat # ab200836) were used in Western bloting; -actin was used as the internal control gene, and the antibody of Monoclonal Anti--Actin antibody produced in mouse (purchased from Sigma, cat # A1978-200UL) was used, and the results were shown in FIG. 4. The primers for the fluorescent quantitative identification of the knockout cell line were shown in Table 4; -actin was used as an internal control gene; the results of the fluorescent PCR identification were shown in FIG. 5. Based on the above, we successfully constructed a huANP32A single-knockout cell line (AKO), a huANP32B single-knockout cell line (BKO), and a huANP32A and ANP32B double-knockout cell line (DKO), which were used for subsequent experiments. Fluorescent quantitative PCR was performed using SYBRPremix Ex Taq II (Tli RnaseH plus) (Cat. # RR820A) produced by TAKARA according to the instructions (the subsequent fluorescent quantitative PCR was also performed using the kit produced by TAKARA).

TABLE-US-00005 TABLE4 theprimersequencesforfluorescence quantitationofhuANP32AandhuANP32B primersequence primername (5-3) qhu32A-F1, GGCAGACGGATTCATTTAGAGC SEQIDNO:15 qhu32A-R, CTTTGGTAAGTTTGCGATTGA SEQIDNO:16 qhu32B-F, CTGCCCCAGCTTACCTACTTG SEQIDNO:17 qhu32B-R, ATCCTCATCGTCCTCGTCTTC SEQIDNO:18 actin-F, CATCTGCTGGAAGGTGGACAA SEQIDNO:19 actin-R, CGACATCCGTAAGGACCTGTA SEQIDNO:20

Example 3: Detection of Influenza Polymerase Activity

[0159] The influenza polymerase reporter system involved in the present invention includes influenza polymerases PB2, PB1 and PA proteins, and a nuclear protein NP. These proteins are derived from human influenza H1N1 subtype A/Sichuan/01/2009 (H1N1.sub.SC09) and A/WSN/1933(WSN), human influenza H7N9 subtype A/Anhui/01/2013 (H7N9.sub.AH13), and canine influenza H3N2 subtype A/canine/Guangdong/1/2011 (H3N2.sub.GD11), avian influenza H9N2 subtype A/chicken/Zhejiang/B2013/2012 (H9N2.sub.ZJ12) and H7N9 subtype A/chicken/Zhejiang/DTID-ZJU01/2013(H7N9.sub.ZJ13), equine influenza A/equine/Jilin/1/1989 (H3N8.sub.JL89) and A/equine/Xinjiang/3/2007 (H3N8.sub.XJ07). The sequences of PB2, PB1, PA and NP proteins of these influenza subtypes are shown in Table 5.

TABLE-US-00006 TABLE 5 The nucleotide sequences of PB2, PB1, PA and NP proteins NP of human H1N1.sub.sc09 (Genebank: GQ166225.1) PA of human H1N1.sub.sc09 (Genebank: GQ166226.1) PB1 of human H1N1.sub.sc09 (Genebank: GQ166227.1) PB2 of human H1N1.sub.sc09 (Genebank: GQ166228.1) PB2 of human H1N1.sub.WSN (Genebank: CY034139.1) PB1 of human H1N1.sub.WSN (Genebank: CY034138.1) PA of human H1N1.sub.WSN (Genebank: CY034137.1) NP of human H1N1.sub.WSN (Genebank: CY034135.1) PB2 of human H7N9.sub.AH13 (Genebank: EPI439504) PB1 of human H7N9.sub.AH13 (Genebank: EPI439508) PA of human H7N9.sub.AH13 (Genebank: EPI439503) NP of human H7N9.sub.AH13 (Genebank: EPI439505) PB2 of canine H3N2.sub.GD11 (Genebank: JX195347.1) PB1 of canine H3N2.sub.GD11 (Genebank: JX195346.1) PA of canine H3N2.sub.GD11 (Genebank: JX195340.1) NP of canine H3N2.sub.GD11 (Genebank: JX195341.1) PB2 of avian H9N2.sub.ZJ12 (Genebank: KP865886.1) PB1 of avian H9N2.sub.ZJ12 (Genebank: KP865839.1) PA of avian H9N2.sub.ZJ12 (Genebank: KP865793.1) NP of avian H9N2.sub.ZJ12 ( Genebank: KP865771.1) PB2 of equine H3N8.sub.JL89 (Genebank: KF285454.1) PB1 of equine H3N8.sub.JL89 (Genebank: KF285455.1) PA of equine H3N8.sub.JL89 (Genebank: KF285456.1) NP of equine H3N8.sub.JL89 (Genebank: M63786.1) PB2 of equine H3N8.sub.XJ07 (Genebank: EU794556.1) PB1 of equine H3N8.sub.XJ07 (Genebank: EU794557.1) PA of equine H3N8.sub.XJ07 ( Genebank: EU794558.1) NP of equine H3N8.sub.XJ07 (Genebank: EU794560.1) PB2 of avian H7N9.sub.ZJ13 (Genebank: KC899666.1) PB1 of avian H7N9.sub.ZJ13 (Genebank: KC899667.1) PA of avian H7N9.sub.ZJ13 (Genebank: KC899668.1) NP of avian H7N9.sub.ZJ13 (Genebank: KC899670.1)

[0160] A plasmid containing the above-mentioned proteins of each influenza virus subtype was constructed, for example, H1N1.sub.SC09 polymerase contained PB2, PB1 and PA proteins and a nuclear protein NP derived from human influenza H1N1 subtype A/Sichuan/01/2009 (H1N1.sub.SC09), and was named as the H1N1.sub.SC09 polymerase reporter system; plasmids were constructed with the vector PCAGGS for PB2, PB1, PA and NP, respectively, and were named as PB2 plasmid, PB1 plasmid, PA plasmid, and NP plasmid. The same is for others.

[0161] Taking the H1N1.sub.SC09 polymerase reporter system as an example, the construction process was as follows:

[0162] mRNA of H1N1.sub.SC09 strain (Master's thesis of Zhang Qianyi, Establishment of reverse genetic operating system for H1N1 influenza virus A/Sichuan/01/2009 strain, Gansu Agricultural University, 2011) was extracted according to the operation manual of QIAamp Viral RNA Mini Kit (purchased from QIAGEN, cat #52904), and then cDNA was synthesized according to the instruction of M-MLV reverse transcriptase kit (purchased from Invitrogen, cat #28025-013) using Uni12 (AGCAAAAGCAGG, SEQ ID NO:21) as reverse transcription primer. Based on the sequence information of each gene fragment, PCR primers were designed (see Table 7); a 15 bp PCAGGS homologous arm was respectively introduced at both ends; the gene of interest was synthesized using KOD FX Neo high-efficiency polymerase; and then the amplified fragment of each gene of H1N1.sub.SC09 and the double-digested PCAGGS vector in Example 1 were ligated at room temperature for 30 minutes using the seamless cloning kit, ClonExpress II One Step Cloning Kit (purchased from Vazyme, cat # C112-01) according to the instructions; the ligation product was transformed into 20 ul DH5 competent cells, and the next day a single clone was picked for sequencing. The plasmid which were verified correct by sequencing were respectively named as PB2 plasmid, PB1 plasmid, PA plasmid and NP plasmid, and used for subsequent experiments. The same is for others. See Table 6 for the sources of other various strains.

TABLE-US-00007 TABLE 6 Human H1N1 Neumann G; Watanabe T; Ito H; Watanabe S; Goto H; WSN Gao P; Hughes M; Perez D R; Donis R; Hoffmann E; Hobom G; Kawaoka Y. Generation of influenza A viruses entirely from cloned cDNAs. [J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(16): 9345-50 human Zhang, Q., Shi, J., Deng, G., Guo, J., Zeng, X., A/Anhui/01/2013 He, X., Kong, H., Gu, C., Li, X., Liu, J., et al. (H7N9.sub.AH13) (2013). H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science. 341(6144), 410-414 canine influenza Su S, Li H T, Zhao F R, et al. Avian-origin H3N2 H3N2 subtype canine influenza virus circulating in farmed dogs in A/canine/ Guangdong, China[J]. Infection Genetics & Evolution, Guangdong/ 2013, 14(2): 444-449 1/2011 (H3N2.sub.GD11) avian influenza Teng Q, Xu D, Shen W, et al. A Single Mutation at H9N2 subtype Position 190 in Hemagglutinin Enhances Binding A/chicken/ Affinity for Human Type Sialic Acid Receptor and Zhejiang/ Replication of H9N2 Avian Influenza Virus in B2013/2012 Mice[J], Journal of Virology, 2016, 90(21): (H9N2.sub.ZJ12) 9806 avian Li C, Li C, Zhang A J, et al. Avian influenza A H7N9 H7N9 subtype virus induces severe pneumonia in mice without prior A/chicken/ adaptation and responds to a combination of zanamivir Zhejiang/ and COX-2 inhibitor[J]. Plos One, 2014, 9(9): DTID-ZJU01/ e107966 2013(H7N9.sub.ZJ13) equine Zhang Xiang, Guo Wei, Wang Xiaojun. Construction A/equine/ of Two-way Transcription/Expression Vector and Jilin/1/1989 Its Application in Reverse Genetic System of (H3N8.sub.JL89) Equine Influenza Virus [J]. Chinese Journal of Preventive Veterinary Medicine, 2016, 38 (11): 860-864 equine master's thesis A/equine/ Dai Lingli. Sequence analysis of HA gene of equine Xinjiang/3/ influenza virus A/Equine/Xinjiang/3/07 (H3N8) and 2007 establishment of two PCR detection methods [D]. (H3N8.sub.XJ07) Chinese Academy of Agricultural Sciences, 200

TABLE-US-00008 TABLE7 Polymeraseconstructionprimers: primername primersequence H1N1 TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG SC09-PB2-F AACT SEQIDNO:22 H1N1 ATCTGCTAGCTCGAGCTAATTGATGGCCATCCGA SC09-PB2-R A SEQIDNO:23 H1N1 TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC SC09-PB1-F TCT SEQIDNO:24 H1N1 ATCTGCTAGCTCGAGTTATTTTTGCCGTCTGAGT SC09-PB1-R T SEQIDNO:25 H1N1 TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG SC09-PA-F AC SEQIDNO:26 H1N1 ATCTGCTAGCTCGAGCTACTTCAGTGCATGTGTG SC09-PA-R A SEQIDNO:27 H1N1 TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA SC09-NP-F CCAA SEQIDNO:28 H1N1 ATCTGCTAGCTCGAGTCAACTGTCATACTCCTCT SC09-NP-R G SEQIDNO:29 H1N1 TTCGAGCTCGCGGCCGCATGGAAAGAATAAAAG WSN-PB2-F AAC SEQIDNO:30 H1N1 ATCTGCTAGCTCGAGCTATTCGACACTAATTGAT WSN-PB2-R G SEQIDNO:31 H1N1 TTCGAGCTCGCGGCCGCATTTGAATGGATGTCAA WSN-PB1-F TCCGAC SEQIDNO:32 H1N1 ATCTGCTAGCTCGAGTCATGAAGGACAAGCTAA WSN-PB1-R ATTCA SEQIDNO:33 H1N1 TTCGAGCTCGCGGCCGCCTGATTCAAAATGGAA WSN-PA-F GATT SEQIDNO:34 H1N1 ATCTGCTAGCTCGAGTTTTTGGACAGTATGGATA WSN-PA-R GCAAA SEQIDNO:35 H1N1 TTCGAGCTCGCGGCCGCTCACTCACAGAGTGAC WSN-NP-F ATCGA SEQIDNO:36 H1N1 ATCTGCTAGCTCGAGTTCTTTAATTGTCGTACTC WSN-NP-R CT SEQIDNO:37 H7N9 TTCGAGCTCGCGGCCGCATGGAAAGAATAAAAG AH13-PB2-F AAC SEQIDNO:38 H7N9 ATCTGCTAGCTCGAGTTAATTGATGGCCATCCGA AH13-PB2-R AT SEQIDNO:39 H7N9 TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC AH13-PB1-F TTT SEQIDNO:40 H7N9 ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAGC AH13-PB1-R TC SEQIDNO:41 H7N9 TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG AH13-PA-F AC SEQIDNO:42 H7N9 ATCTGCTAGCTCGAGCTATCTTAGTGCATGTGTG AH13-PA-R A SEQIDNO:43 H7N9 TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA AH13-NP-F CCA SEQIDNO:44 H7N9 ATCTGCTAGCTCGAGTCAATTGTCATACTCCTCT AH13-NP-R GC SEQIDNO:45 H3N2 TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG GD12-PB2-F AATT SEQIDNO:46 H3N2 ATCTGCTAGCTCGAGCTAATTGATGGCCATCCGA GD12-PB2-R A SEQIDNO:47 H3N2 TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC GD12-PB1-F TTT SEQIDNO:48 H3N2 ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAGC GD12-PB1-R TC SEQIDNO:49 H3N2 TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG GD12-PA-F ACAA SEQIDNO:50 H3N2 ATCTGCTAGCTCGAGCTATTTCAGTGCATGTGTG GD12-PA-R AGG SEQIDNO:51 H3N2 TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA GD12-NP-F CCAAAC SEQIDNO:52 H3N2 ATCTGCTAGCTCGAGTTAATTGTCATACTCCTCT GD12-NP-R GC SEQIDNO:53 H3N8 TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG JL89-PB2-F AATT SEQIDNO:54 H3N8 ATCTGCTAGCTCGAGCTAATTGATGGCCATCCGA JL89-PB2-R AT SEQIDNO:55 H3N8 TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC JL89-PB1-F TTT SEQIDNO:56 H3N8 ATCTGCTAGCTCGAGTCACTGTTTTTGCCGTCTG JL89-PB1-R AG SEQIDNO:57 H3N8 TTCGAGCTCGCGGCCGCATGGAAGATTTTGTGCG JL89-PA-F ACAA SEQIDNO:58 H3N8 ATCTGCTAGCTCGAGCTATTTCAGTGCATGTGTG JL89-PA-R A SEQIDNO:59 H3N8 TTCGAGCTCGCGGCCGCAGCAAAAGCAGGGTAG JL89-NP-F ATAAT SEQIDNO:60 H3N8 ATCTGCTAGCTCGAGAGTAGAAACAAGGGTATT JL89-NP-R TTTC SEQIDNO:61 H3N8 TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG XJ07-PB2-F AACT SEQIDNO:62 H3N8 ATCTGCTAGCTCGAGTTAATTGATGGCCATCCGA XJ07-PB2-R AT SEQIDNO:63 H3N8 TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC XJ07-PB1-F TCT SEQIDNO:64 H3N8 ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAGC XJ07-PB1-R SEQIDNO:65 H3N8 TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG XJ07-PA-F ACA SEQIDNO:66 H3N8 ATCTGCTAGCTCGAGTTACTTCAGTGCATGTGTA XJ07-PA-R AGG SEQIDNO:67 H3N8 TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA XJ07-NP-F CCAAA SEQIDNO:68 H3N8 ATCTGCTAGCTCGAGTTAACTGTCAAATTCCTCA XJ07-NP-R GC SEQIDNO:69 H9N2 TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA ZJ12-NP-F C SEQIDNO:70 H9N2 ATCTGCTAGCTCGAGTCAATTGTCATACTCCT Z112-NP-R SEQIDNO:71 H9N2 TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG Z112-PA-F SEQIDNO:72 H9N2 ATCTGCTAGCTCGAGCTATCTTAGTGCATGTG Z112-PA-R SEQIDNO:73 H9N2 TTCGAGCTCGCGGCCGCATGGATGTCAATCCGA Z112-PB1-F SEQIDNO:74 H9N2 ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAG Z112-PB1-R SEQIDNO:75 H9N2 TTCGAGCTCGCGGCCGCATGGAAAGAATAAAAG ZI12-PB2-F A SEQIDNO:76 H9N2 ATCTGCTAGCTCGAGTTAATTGATGACCATCCG Z112-PB2-R SEQIDNO:77

[0163] We constructed a reporter plasmid (pMD18T-vLuc) for detecting the activity of polymerase; the reporter plasmid uses the pMD18T plasmid as the backbone and contains the sequence of interest; the sequence of interest is characterized in that the 5 end non-coding region (sequence: agcaaaagcagggg, SEQ ID NO:78) and the 3 end non-coding region (sequence: gtatactaataattaaaaacacccttgtttctact, SEQ ID NO:79) of HA gene of H3N8.sub.JL89 strain were introduced into both ends of the protein coding sequence of Firefly luciferase; a human polI promoter was introduced into the 3 end of the sequence, and a murine pol I terminator sequence was introduced into the 5end of the sequence. The sequence of interest is as follows: murine pol I terminator sequence (bold underlined)+5 non-coding region (red italics) of HA gene+gene sequence of Firefly luciferase+3 non-coding region (green italics) of HA gene+human polI promoter (bold underlined), SEQ ID NO: 80. The synthesized fragment was directly ligated into the pMD18-T vector, obtaining the reporter plasmid pMD18T-vLuc.

[0164] After co-transfecting this reporter plasmid and the influenza polymerase system into 293T, the polymerase complex can recognize the non-coding sequences of the virus at both ends of Firefly luciferase, thereby starting the synthesis of Firefly luciferase gene vRNA, cRNA and mRNA. In order to make the polymerase system more stable and stringent, we introduced a polymerase dual fluorescence reporter system, wherein Renilla luciferase (pRL-TK) was further added as an internal control into the above influenza polymerase reporter system, and we established a stable transfection system: taking a 12-well plate as an example, adding PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng) and pRL-TK plasmid (10 ng, Promega cat # E2241, GenBank number: AF025846) into each well, and then transfecting with the transfection reagent lipo2000. After 24 hours of transfection, the cell supernatant was discarded; the cells were lysed by 100 ul of cell lysis buffer/well (passive lysis buffer, derived from the dual luciferase kit (Promega)), and then measured by the dual luciferase kit (Promega) using Centro. XS LB 960 luminometer (Berthold technologies). Renilla luciferase was used as an internal control, and the ratio can represent the activity of the polymerase.

[0165] We transfected the H1N1.sub.SC09 polymerase into the cell lines AKO, BKO, DKO constructed in Example 2 and wild-type 293T cell by the above system; each group was set up with triplicate wells and then detected 24 hours after transfection, the activity of H1N1.sub.SC09 polymerase in AKO and BKO was not different from that of wild-type 293T cells, while the activity of H1N1.sub.SC09 polymerase in DKO cells decreased by more than 10,000 times, see FIG. 6. The results were processed by the biological software GraphPad Prism 5 (https://www.graphpad.com) and analyzed by one-way ANOVA and Dunnett's t-test; the difference between each experimental group and the control group is shown in the chart: ns means no difference, * means P<0.05, ** means P<0.01, *** means P<0.001, **** means P<0.0001. The symbols ns, *, **, *** and * * * * in other figures relating to the detection of polymerase activity have the same meanings as above, and processed as above.

[0166] H7N9.sub.AH13, WSN, H3N2.sub.GD11, H3N8.sub.JL89, H3N8.sub.XJ07 polymerase were transfected into different cell lines AKO, BKO, DKO and wild-type 293T cells in the same way as H1N1.sub.SC09 polymerase; the result is that the activity in AKO and BKO was not different from that in wild-type 293T cells, while the activity in DKO cells decreased by about 10,000 times; the results are shown in FIG. 7A-E.

[0167] The huANP32A and huANP32B expression plasmids constructed in Example 1 PCAGGS-huANP32A and PCAGGS-huANP32B were co-transfected with H1N1.sub.SC09 polymerase into DKO cells; the specific transfection system was: taking a 12-well plate as an example, each well was added with H1N1.sub.SC09 PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng) and different doses of ANP32A and ANP32B proteins, and the specific doses of ANP32A and ANP32B protein plasmids were respectively selected from the following doses: 0 pg, 10 pg, 100 pg, 1 ng, 5 ng, 10 ng, 20 ng, 100 ng, 500 ng, 1 ug, and a PCAGGS-Flag empty vector control was set at the same time; the total amount of plasmid was made up by the PCAGGS-Flag empty vector, and each group was provided with triplicate wells. Cells were lysed 24 h after transfection, and the polymerase activity was detected as described above.

[0168] The results showed that the supplementation of huANP32A and ANP32B proteins can restore the activity of polymerase in a dose-dependent manner, reaching a plateau phase at 20 ng, and the activity of the polymerase can be restored by about 3000 times. The activity curves of huANP32A and huANP32B proteins have the same trend, and there was no additive effect during the plateau phase, as shown in FIG. 8. Therefore, the polymerase was dose-dependent on huANP32A and ANP32B proteins, and the dose requirement was low. Whereas, the activity of polymerase was inhibited when the amount of the huANP32A and huANP32B proteins was excessive, as shown in FIG. 9A. DKO cells were co-transfected with the huANP32A and huANP32B expression plasmids constructed in Example 1 at different doses described above as well as H7N9.sub.AH13 polymerase, and the result was similar to H1N1.sub.SC09, as shown in FIG. 9B.

Example 4: Fluorescent Quantitative Detection of the Influence of ANP32 Protein on RNA Synthesis of Influenza Virus

[0169] Real-time PCR was used to detect the differences in the synthesis of cRNA, vRNA and mRNA of influenza virus on wild-type 293T and the double-knockout cell line DKO. First, wild-type 293T and double-knockout cell line DKO cell were plated in a 12-well plate, and the 293T cells were transfected with the H1N1.sub.SC09 polymerase dual fluorescence reporter system of Example 3. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng), and empty vector PCAGGS-Flag plasmid (20 ng); the DKO cells were transfected with H1N1.sub.SC09 polymerase dual fluorescence reporter system of Example 3. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng), and PCAGGS-huANP32A plasmid (20 ng), while the empty vector PCAGGS-Flag plasmid (20 ng) was set as a negative control. After 24 h, total cellular RNA was extracted, and reverse transcription was performed using particular primers (see Table 8) to synthesize the first cDNAs of cRNA, vRNA and mRNA, respectively, followed by fluorescent quantitative PCR using specific primers (see Table 9); using random primers (contained in the kit) as reverse transcription primers for internal control gene -actin, and the fluorescent quantitative primers of -actin were shown in Table 4 of Example 2. The results showed that compared to the wild type, the synthesis of viral cRNA, vRNA and mRNA was significantly reduced (about 30-50 times) on the double-knockout cell line, indicating that RNA of influenza virus was hardly replicated in cell lines lacking ANP32A and ANP32B. When human ANP32A was supplemented to the double-knockout cell line, the replication and synthesis of RNA could be restored, as shown in FIG. 10. The above experimental procedure was repeated with the huANP32B plasmid instead of the huANP32A plasmid; it was found that human ANP32B also had the same function (data is similar to DKO+huANP32A in FIG. 10). This showed that the ANP32A and ANP32B proteins were involved in the synthesis and replication of influenza virus RNA and played a decisive role therein.

TABLE-US-00009 TABLE8 Reversetranscriptionprimers primername primersequence Luc-vRNA, CATTTCGCAGCCTACCGTGGTGT SEQIDNO:81 T Luc-cRNA, AGTAGAAACAAGGGTG SEQIDNO:82 Luc-mRNA, oligo-dT20 SEQIDNO:83

TABLE-US-00010 TABLE9 fluorescentquantitativePCRprimers primername primersequence Luc-F,SEQIDNO:84 GATTACCAGGGATTTCAGTCG Luc-R,SEQIDNO:85 GACACCTTTAGGCAGACCAG

Example 5: Influence of ANP32 Protein on the Replication of Influenza Virus

[0170] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the ANP32A and ANP32B protein plasmids of different species constructed in Example 1 were co-transfected with 6 plasmids of the H1N1.sub.SC09 polymerase reporter system. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD 18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng), and ANP32A or ANP32B protein plasmid (20 ng), and the empty vector PCAGGS-Flag (20 ng) was set as a negative control, and each group was provided with triplicate wells. 24 h after transfection, the cells were lysed as described in Example 3 to detect the activity of polymerase, showing that: compared to the empty vector, avian ANP32A such as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such as huANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity of H1N1.sub.SC09 polymerase, whereas the two proteins chANP32B and muANP32A did not have the ability to support the activity of H1N1.sub.SC09 polymerase, as shown in FIG. 11A.

[0171] The above experiment was repeated with the H7N9.sub.AH13 polymerase reporter system instead of the H1N1.sub.SC09 polymerase reporter system, and the result showed that: compared to the empty vector, avian ANP32A such as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such as huANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity of H7N9.sub.AH13 polymerase, whereas the two proteins chANP32B and muANP32A did not have the ability to support the activity of H7N9.sub.AH13 polymerase, as shown in FIG. 11B.

[0172] The above experiment was repeated with the H3N2.sub.Gm1 polymerase reporter system instead of the H1N1.sub.SC09 polymerase reporter system, and the result showed that: compared to the empty vector, avian ANP32A such as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such as huANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity of H3N2.sub.Gm1 polymerase, whereas the two proteins chANP32B and muANP32A did not have the ability to support the activity of H3N2.sub.Gm1 polymerase, as shown in FIG. 11C.

[0173] The above experiment was repeated with the H3N8.sub.XJ07 polymerase reporter system instead of the H1N1.sub.SC09 polymerase reporter system, and the result showed that: compared to the empty vector, avian ANP32A such as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such as huANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity of H3N8.sub.XJ07 polymerase, whereas the two proteins chANP32B and muANP32A did not have the ability to support the activity of H3N8.sub.XJ07 polymerase, as shown in FIG. 11D.

[0174] The above experiment was repeated with the H3N8.sub.JL89 polymerase reporter system instead of the H1N1.sub.SC09 polymerase reporter system, and the result showed that: compared to the empty vector, avian ANP32A such as chANP32A, dkANP32A, zfANP32A, tyANP32A supported the activity of H3N8.sub.JL89 polymerase, whereas chANP32B and mammalian ANP32 such as huANP32A, pgANP32A, eqANP32A, huANP32B, muANP32A did not support the activity of H3N8.sub.JL89 polymerase, as shown in FIG. 11E.

[0175] The above experiment was repeated with the H9N2.sub.JL12 polymerase reporter system instead of the H1N1.sub.SC09 polymerase reporter system, and the result showed that: compared to the empty vector, avian ANP32A such as chANP32A, dkANP32A, zfANP32A, tyANP32A supported the activity of H9N2.sub.JL12 polymerase, whereas chANP32B and mammalian ANP32 such as huANP32A, eqANP32A, huANP32B, muANP32A did not support the activity of H9N2.sub.JL12 polymerase, and pgANP32A substantially did not support the activity of H9N2.sub.ZJ12 polymerase. The results were shown in FIG. 11F.

[0176] The above experiment was repeated with the WSN polymerase reporter system instead of the H1N1.sub.SC09 polymerase reporter system, and the result showed that: compared to the empty vector, avian ANP32A such as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such as huANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity of WSN polymerase, whereas the two proteins chANP32B and muANP32A did not have the ability to support the activity of WSN polymerase, as shown in FIG. 11G.

Example 6: H1N1.SUB.SC09 .Influenza Virus Transfected Cell Line

[0177] The influence of ANP32A and ANP32B proteins on virus replication was further investigated using the influenza virus reverse genetic system. Eight gene fragments of influenza H1N1.sub.SC09 (PB2, PB1, PA, NP, HA, NA, M and NS) were ligated into a pBD vector with a double promoter (Journal of Virology, September 2005, p12058-12064, Molecular Basis of Replication of Duck H5N1 influencza Viruses in a Mammalian Mouse Model; Establishment of reverse genetic operating system for H1N1 influenza virus A/Sichuan/01/2009 strain, Zhang Qianyi et al, Veterinary Science in China, 2011, 41 (05): 448-452).

[0178] The steps for constructing the pBD vector were as follows: the Pol-HDVR expression cassette was inserted in reverse orientation into the XbaI cleavage site, using pCI vector (purchased from Promega, Cat. No. E1841, GenBank No. U47120) as the backbone. The sequence of the Pol-HDVR expression cassette is artificially synthesized as SEQ ID NO: 86. That is, the pCI vector was digested with XbaI restriction enzyme (NEB, cat # R0145S), and then the linearized vector was recovered using a gel recovery kit (OMEGA, cat # D2500-01), treated with dephosphorylating enzyme CIAP (purchased from TAKARA, cat # D2250) according to the instructions, and then recovered for use. The artificially synthesized Pol-HDVR expression cassette and the digested fragments of pCI vector have a 15 bp homologous arm on both the left arm and the right arm, then the Pol-HDVR expression cassette fragment and the pCI linearized vector were ligated for 30 min at room temperature by using a seamless cloning Kit of Clonexpress II One Step Cloning Kit (purchased from Vazyme, cat # C112-01) according to the instructions; the ligation product was transformed into 20 ul of DH5 competent cells, and the next day a single clone was picked for sequencing. The plasmid which was verified correct by sequencing was the pBD two-way expression vector.

[0179] According to the methods described in Zhang Qianyi et al, Establishment of reverse genetic operating system for H1N1 influenza virus A/Sichuan/01/2009 strain, Veterinary Science in China, 2011, 41(05): 448-452 and Master's thesis of Zhang Qianyi, Establishment of reverse genetic operating system for H1N1 influenza virus A/Sichuan/01/2009 strain, Gansu Agricultural University, 2011, the H1N1.sub.SC09 pBD 8 plasmid system was constructed as follows:

[0180] mRNA of H1N1.sub.SC09 strain was extracted according to the operation manual of QIAamp Viral RNA Kit Manual, and then cDNA was synthesized using Uni12 (AGCAAAAGCAGG, SEQ ID NO:21) as a reverse transcription primer according to the instructions of Invitrogen M-MLV Kit. Based on the sequence information of each gene fragment (the sequence of PB2 is SEQ ID NO: 87, the sequence of PB1 is SEQ ID NO: 88, the sequence of PA is SEQ ID NO: 89, the sequence of NP is SEQ ID NO: 90, and the sequence of HA is SEQ ID NO: 91, the sequence of NA is SEQ ID NO: 92, the sequence of M is SEQ ID NO: 93 and the sequence of NS is SEQ ID NO: 94), PCR primers were designed (see Table 10); a 15 bp pBD homologous arm was respectively introduced at both ends; the gene of interest and linearized pBD vector were amplified using KOD FX Neo high-efficiency polymerase; and then the amplified fragment of each gene of H1N1.sub.SC09 and the linearized pBD vector were ligated at room temperature for 30 minutes by using the seamless cloning kit of ClonExpress II One Step Cloning Kit (purchased from Vazyme, cat # C112-01) according to the instructions; the ligation product was transformed into 20 ul DH5 competent cells, and the next day a single clone was picked for sequencing. The plasmid which were verified correct by sequencing were respectively named as pBD-H1N1.sub.SC09-PB2 plasmid, pBD-H1N1.sub.SC09-PB1 plasmid, pBD-H1N1.sub.SC09-PA plasmid, pBD-H1N1.sub.SC09-NP plasmid, pBD-H1N1.sub.SC09-HA plasmid, pBD-H1N1.sub.SC09-NA plasmid, pBD-H1N1.sub.SC09-M plasmid and pBD-H1N1.sub.SC09-NS plasmid for subsequent experiments.

TABLE-US-00011 TABLE10 TheprimersforconstructingH1N1SC09 pBDeightplasmids primername primersequence(5-3) pBD-up, GGCCGGCATGGTCCCAGCCTCCTC SEQIDNO:95 GC pBD-down, AATAACCCGGCGGCCCAAAATGCC SEQIDNO:96 GACTCG pBD-PB2-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:97 CAGGTCAAATATAT pBD-PB2-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:98 AAGGTCGTTTTTAA pBD-PB1-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:99 CAGGCAAACCATT pBD-PB1-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:100 AAGGCATTTTTTCA pBD-PA-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:101 CAGGTACTGATCCA pBD-PA-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:102 AAGGTACTTTTTTGG pBD-NP-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:103 CAGGGTAGAT pBD-NP-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:104 AAGGGTATTTTTC pBD-HA-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:105 CAGGGGAAAA pBD-HA-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:106 AAGGGTGT pBD-NA-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:107 CAGGAGTTTAA pBD-NA-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:108 AAGGAGTTT pBD-M-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:109 CAGGTAGATA pBD-M-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:110 AAGGTAGTTT pBD-NS-F, GGGACCATGCCGGCCAGCAAAAG SEQIDNO:111 CAGGGTGACAAAG pBD-NS-R, GGCCGCCGGGTTATTAGTAGAAAC SEQIDNO:112 AAGGGTGTTTTTTAT

[0181] The cell lines AKO, BKO, DKO constructed in Example 2 and wild type 293T cell line were counted respectively and plated in a 6-well plate at 410.sup.5/well. After cultured in an incubator at 37 C. for 20 h, the system of H1N1.sub.SC09 pBD 8 plasmids was transfected into a wild type 293T cell line and a knockout cell line at 0.5 ug per plasmid; the cell supernatant was collected at 0 h, 12 h, 24 h, 36 h, 48 h and 60 h after transfection, and the virus yield was determined by using an NP double antibody sandwich ELISA method: the 96-well ELISA plate was first coated with NP monoclonal antibody 2B8A11 at 1 ug/well (Wang Yadi, Wen Kun, Qiu Liwen, etc., the establishment of ELISA capture method of influenza A virus nucleocapsid protein and the clinical application thereof, 2009, Guangdong Medicine. 30(5): 703-705) at 4 C. overnight. The plate was rinsed with 1PBST washing solution for 3 times (5 minutes each time) and shaked, and then patted clean, added with 5% calf serum as a blocking solution, and blocked at 37 C. for 2 hr. The plate was again rinsed with 1PBST washing solution for 3 times (5 minutes each time) and shaked, and then patted clean; the sample to be tested was added. The sample dilution used for the sample to be tested was 10% fetal bovine serum+0.1% TritonX-100 wherein the NP protein (NP protein construction plasmid pET30a-NP is cited from Master's thesis: Ji Yuanyuan, the establishment of capture ELISA detection method of the equine influenza virus antigen and the primary application thereof [D]. Chinese Academy of Agricultural Sciences, 2011) was used as a standard sample with a 2-fold dilution, and the collected cell supernatant was diluted 5-fold. Each sample was set with 3 gradients, 2 replicate wells. The plate was incubated at 37 C. for 2 h, rinsed with 1PBST washing solution for 3 times (5 minutes each time), and then patted clean; added with NP monoclonal antibody C16A15 strain (Wang Yadi, Wen Kun, Qiu Liwen, etc., the establishment of ELISA capture method of influenza A virus nucleocapsid protein and the clinical application thereof, 2009, Guangdong Medicine. 30(5): 703-705) by 1 ug/well, incubated at 37 C. for 1 h, discarded, rinsed with 1PBST washing solution for 5 times (5 minutes each time) and shaked. After the washing, 1:1 mixed AB color developing solution (purchased from Beijing Taitianhe Biotech Co., Ltd., cat # ME142) was added by 100 ul/well, and the color development was performed for 10 min. The color development was stopped by adding 2M H2504 at 50 ul/well, and OD450 nm was detected by using a biotech Ex150 microplate reader. A standard curve was drawn by using the NP protein standard sample and the concentration of the sample to be tested was calculated. The results showed that: compared to 293T cells, the amount of virus in the supernatant of AKO/BKO cells had a similar growth curve after transfection, and almost no virus particles were detected in the supernatant of DKO cells. This indicated that DKO cells did not supported the replication and growth of virus, and the results were shown in FIG. 12A. It was shown that the knockout of ANP32A or ANP32B alone did not affect the replication and growth of the virus.

[0182] The DKO cell lines constructed in Example 2 were counted respectively and plated in a 6-well plate at 310.sup.5/well. After incubated in an incubator at 37 C. for 20 h, transfection wells were set: 1 ug empty vector PCAGGS-Flag, 1 ug PCAGGS-huANP32A, 1 ug PCAGGS-huANP32B, 0.5 ug PCAGGS-huANP32A+0.5 ug PCAGGS-huANP32B. 24 hours after transfection, the system of H1N1.sub.SC09 pBD 8 plasmids was transfected into cells of different treatment groups at 0.5 ug of each plasmid; after transfection, the cell supernatant was respectively collected at 0 h, 12 h, 24 h, 36 h, 48 h and 60 h, and virus yield was determined by NP double antibody sandwich ELISA method, in which the specific steps were described above. The results showed that: compared to the empty vector of PCAGGS-Flag, the supplementation of huANP32A, the supplementation of huANP32B, and the simultaneous supplementation of both huANP32A and huANP32B proteins all supported virus replication very well, and the results were shown in FIG. 12B. In summary, the huANP32A or huANP32B proteins was essential in the replication of H1N1.sub.SC09.

Example 7: The Experiment of H1N1/WSN Influenza Virus Infection

[0183] The cell lines AKO, BKO, DKO constructed in Example 2 and wild type 293T cell line were counted respectively and plated in a 6-well plate at 410.sup.5/well. After incubated in an incubator at 37 C. for 20 hours, the cells were infected with 0.01 MOI of WSN virus (Neumann G; et al. Generation of influenza A viruses entirely from cloned cDNAs.[J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(16):9345-50); after 2 hours of virus adsorption, the virus-infected solution was discarded and rinsed twice with 1PBS buffer, and then 2 ml of a cell maintenance solution containing 1% pancreatin (sigma)+1% double antibody (gibco)+0.5% fetal bovine serum (sigma) was added to each well, and cell infection supernatants were taken at 0 h, 12 h, 24 h, 36 h, 48 h after infection and frozen at 80 C. for use. MDCK cells (canine kidney cell line, purchased from China Institute of Veterinary Drug Control) were plated in a 96-well plate at 1.510.sup.4/well, and the above-obtained supernatant was 10-fold diluted with culture solution DMEM (hyclone) and then added into a 96-well plate at 100 ul/well with 8 replicates per gradient. After 2 hours of virus adsorption, the virus infection solution was discarded and the wells were rinsed twice by 1PBS buffer, then 100 ul of cell maintenance solution containing 2% fetal bovine serum (sigma)+1% double antibody (gibco)+1% pancreatin (sigma) was added into each well; after 48 hours, the cell lesion was observed and counted; virus TCID50 at different time points of different treatment groups was calculated according to a Reed-Muench method, and finally, the virus growth curve was drawn by Graphpad prism 5 software. The result showed that: the virus growth curves of AKO and BKO cells were consistent with that of wild-type 293T cells, whereas DKO cells hardly supported virus growth. The result was shown in FIG. 13A.

[0184] Double-knockout cell lines (DKO) were plated in a 6-well plate at 410.sup.5/well; 20 hours later, four transfection groups were set: PCAGGS-huANP32A (1 ug) plasmid, PCAGGS-huANP32B (1 ug) plasmid, PCAGGS-huANP32A+ PCAGGS-huANP32B (0.5 ug+0.5 ug), and PCAGGS-Flag empty vector (1 ug). After 24 hours of transfection, cells of different treatment groups were infected with 0.01 MOI of WSN virus; after 2 hours of virus adsorption, the virus infection solution was discarded and the wells were rinsed twice with 1PBS buffer, and then 2 ml of a cell maintenance solution containing 1% pancreatin (sigma)+1% double antibody (gibco)+0.5% fetal bovine serum (sigma) was added to each well, and virus infection supernatants were taken at 0 h, 12 h, 24 h, 36 h, 48 h after infection and frozen at 80 C. for use. MDCK cells were plated in a 96-well plate at 1.510.sup.4/well, and the above-obtained supernatant was 10-fold diluted and then added into a 96-well plate at 100 ul/well with 8 replicates per gradient. After 2 hours of virus adsorption, the virus infection solution was discarded and rinsed twice by 1PBS buffer, then the cell maintenance solution was added; after 48 hours, the cell lesion was observed and counted; virus TCID50 at different time points of different treatment groups was calculated according to a Reed-Muench method, and finally, the virus growth curve was drawn by Graphpad prism 5 software. The result showed that: compared with the empty vector, supplementation of huANP32A or huANP32B alone can restore the growth of virus in DKO cells, which was consistent with the virus growth curves of supplementation of both huANP32A and huANP32B. The result was shown in FIG. 13B.

[0185] It was shown that the knockout of huANP32A or huANP32B alone did not affect the replication and growth of the virus, and that huANP32A and huANP32B had a functional compensation effect on the replication and growth of influenza virus.

Example 8: Influence of ANP32A and ANP32B Proteins on Polymerase Replication after Mutation of Homologous or Heterologous Virus

Construction of Point Mutation Vector of PB2 Gene of H7N9 Subtype Influenza Virus

[0186] The analysis of some key amino acid sites on PB2 gene of a human-derived H7N9 isolated strain showed that compared with an avian-derived isolates, the human-derived isolates had some reported point mutations related to host adaptability, such as A588V, Q591K, Q591R, V598I, E627K, D701N and the like (Hu et al., 2017, PB2 substitutions V598T/I increase the virulence of H7N9 influenza A virus in mammals. Virology. 501, 92-101.; Mok et al., 2014, Amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel A/H7N9 influenza virus in mammalian hosts. Journal of virology. 88(6), 3568-3576; Xiao et al., 2016, PB2-588 V promotes the mammalian adaptation of H10N8, H7N9 and H9N2 avian influenza viruses. Sci Rep. 6, 19474.; Yamayoshi et al., 2015, Amino acids substitutions in the PB2 protein of H7N9 influenza A viruses are important for virulence in mammalian hosts[J]. Sci Rep, 2015, 5:8039.; Zhang et al., 2014, The PB2 E627K mutation contributes to the high polymerase activity and enhanced replication of H7N9 influenza virus. J Gen Virol. 95(Pt 4), 779-786.), that is, after the point mutations of A588V, Q591K, Q591R, V598I, E627K or D701N were performed on the avian-derived influenza strain, the strain became adaptive to a human body.

(1) Mutant PB2 Gene and Plasmid Construction

[0187] We performed a single point mutation at the above six positions on PB2 of avian H7N9 influenza A/chicken/Zhejiang/DTID-ZJU01/2013(H7N9.sub.ZJ13); the mutation primers were shown in Table 11 (the underlined parts were mutant bases); the avian H7N9 (H7N9.sub.ZJ13) PB2 plasmid was used as a template, and KOD-FX Neo high-efficiency DNA polymerase was used for amplification; the obtained PCR product was digested with Dpn I for 30 minutes in a constant temperature water bath at 37 C., then 5 ul of the digested product was transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and a large amount of plasmid which was verified correct by sequencing was extracted for later use. PB2(A588V), PB2(Q591K), PB2(Q591R), PB2(V598I), PB2(E627K) and PB2(D701N) mutant genes were obtained, respectively.

TABLE-US-00012 TABLE11 Mutationprimers primername primersequence ZJ13-PB2A588V-S, CTAAAGCTGTCAGAGGCCAATAT SEQIDNO:113 AGTG ZJ13-PB2A588V-A, TATTGGCCTCTGACAGCTTTAGG SEQIDNO:114 CACT ZJ13-PB2Q591K-S, TGCCAGAGGCAAATATAGTGGG SEQIDNO:115 TTCGTG ZJ13-PB2Q591K-A, CCCACTATATTTGCCTCTGGCAG SEQIDNO:116 CTTTA ZJ13-PB2Q591R-S, TGCCAGAGGCAGATATAGTGGG SEQIDNO:117 TTCGTG ZJ13-PB2Q591R-A, CCCACTATATCTGCCTCTGGCAG SEQIDNO:118 CTTTA ZJ13-PB2V598I-S, AGTGGGTTCGTGAGGATTCTATT SEQIDNO:119 CCAACAGATG ZJ13-PB2V5981-A, CATCTGTTGGAATAGAATCCTC SEQIDNO:120 ACGAACCCACT ZJ13-PB2E627K-S, GCAGCCCCGCCGAAGCAGAGTA SEQIDNO:121 GGATGCA ZJ13-PB2E627K-A, ATCCTACTCTGCTTCGGCGGGGC SEQIDNO:122 TGCTGCA ZJ13-PB2D701N-S, GGGCAAAGAAAATAAAAGATAT SEQIDNO:123 GGGCCA ZJ13-PB2D701N-A, CCATATCTTTTATTTTCTTTGCCC SEQIDNO:124 AGAATC
(2) Influence of huANP32A and huANP32B on Polymerase Replication

[0188] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the plasmids of PCAGGS-chANP32A, PCAGGS-huANP32A, pCAGGS-huANP32B constructed in Example 1 and the empty vector PCAGGS-Flag were respectively co-transfected with the plasmids of H7N9.sub.ZJ13 polymerase reporter system. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng, that is, PB2(A588V), PB2(Q591K), PB2(Q591R), PB2(V598I), PB2(E627K) and PB2(D701N) were respectively used), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng), and ANP32A protein plasmid (20 ng), and the empty vector PCAGGS-Flag (20 ng) was set as a negative control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed for detecting the activity of polymerase, and the result showed that: compared with the empty vector, chANP32A, huANP32A and huANP32B can effectively promote the activity of polymerase with point mutations of A588V, Q591K, Q591R, V598I, D701N and E627K; in the absence of huANP32A and huANP32B, none of these point mutations allowed the polymerase to acquire the ability to replicate on 293T; these results indicated that: for the replication in humans of the human-derived influenza strain or mutant strain which was obtained by mutation of avian-derived influenza strain to adapt to the human body, huANP32A or huANP32B was a prerequisite for the replication of H7N9 polymerase in 293T. The result was shown in FIG. 14.

Example 9: Determination of Functional Domain of ANP32 Protein

[0189] According to the results in Example 5, it was shown that chANP32B protein did not support the activity of polymerase.

[0190] By aligning the protein sequences of chANP32B, huANP32B and pgANP32B, it was found that there are differences in the sequence of ANP32B (FIG. 15A). According to the UniProtKB database, the functional domains of huANP32B protein were displayed: 1-41aa was the LRR1 region; 42-110aa was the LRR2, 3 &4 region; 111-161aa was the LRRCT region; and 162-251aa was the LCAR region. According to the functional domain of the huANP32B protein, the huANP32B gene fragment and the chANP32B gene fragment were replaced, and the replacement strategy of the gene fragment was shown in FIG. 15B.

[0191] Firstly, the huANP32B gene sequence was divided into two fragments: 1-161aa and 162-262aa; by using a homologous recombination PCR method, primers were designed to replace the corresponding fragment with a chANP32B fragment to construct a recombinant plasmid.

[0192] For example, the replacement of 1-161aa fragment was performed as follows: using a PCAGGS-chANP32B plasmid as a template, a PCAGGS-chANP32B1-161 gene (SEQ ID NO:125 and SEQ ID NO:126 as primers) lacking the 1-161aa gene fragment was amplified by KOD-FX Neo high-efficiency DNA polymerase; then by using a PCAGGS-huANP32B plasmid as a template, a huANP32B (1-161) fragment was amplified (SEQ ID NO:127 and SEQ ID NO:128 as primers), wherein both ends of the primers amplifying the huANP32B (1-161) fragment respectively contain 15 bp bases which are the same as the left and right arms of the PCAGGS-chANP32B1-161 gene. After the fragments were amplified and recovered, the two fragments were ligated by In-Fusion ligase (purchased from Clontech) according to the instructions, and then transformed into DH5 competent cells. The next day, a single clone was picked and sequenced, and the plasmid which was verified correct by sequencing was named as chANP32B(162-262) and was extracted in large-scale for later use. The primers were shown in Table 12.

[0193] The chANP32B(1-161) recombinant plasmid was constructed according to the method described above. By using a PCAGGS-chANP32B plasmid as a template, a PCAGGS-chANP32B162-262 gene lacking the 162-262aa gene fragment was amplified by KOD-FX Neo high-efficiency DNA polymerase (ca #KFX-201, purchased from Toyobo) (SEQ ID NO:129 and SEQ ID NO:130 as primers); then by using a PCAGGS-huANP32B plasmid as a template, a huANP32B(162-262) fragment was amplified(SEQ ID NO:131 and SEQ ID NO:132 as primers), wherein both ends of the primers used for amplifying the huANP32B (162-262) fragment respectively contain 15 bp bases which are the same as the left and right arms of the PCAGGS-chANP32B162-262 gene. After the fragments were amplified and recovered, the two fragments were ligated by In-Fusion ligase (purchased from Clontech) according to the instructions, and then transformed into DH5 competent cells. The next day, a single clone was picked and sequenced, and the plasmid which was verified correct by sequencing was named as chANP32B(1-161) and was extracted in large-scale for later use. The primers were shown in Table 12.

TABLE-US-00013 TABLE12 primers primername primersequence pCAGGSVector_up, GCGGCCGCGAGCTCGAATTCTTTGCCAA SEQIDNO:125 AA pCAGGS_ch32B GAGGCAGATGGGGATGGACTGGAAGAC (162-262)-down GAG SEQIDNO:126 hu32B(1-161)_F, GAATTGTGCGGCCGCATGGACATGAAG SEQIDNO:127 AGGAGGATCCA hu32B(1-161)_R, ATCCCCATCTGCCTCGGCATCTGAGTCA SEQIDNO:128 GGTGCTTCCT pCAGGS_ch32B AGGGTCTGAGTCAGGGGCTTCCTGCTCA (1-161)-vectorup TC SEQIDNO:129 pCAGGS GGCAGCGGAGACTACAAGGATGACGAT Vector_down, GAC SEQIDNO:130 hu32B(162-262)_F, CCTGACTCAGACCCTGAGGTGGATGGTG SEQIDNO:131 TGGATGAAGA hu32B(162-262)_R. GTAGTCTCCGCTGCCATCATCTTCTCCTT SEQIDNO:132 CATCATCTG

[0194] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the plasmids of PCAGGS-huANP32B, PCAGGS-chANP32B, PCAGGS-chANP32B(1-161) and PCAGGS-chANP32B(162-262) were respectively co-transfected with the H1N1.sub.SC09 polymerase reporter system into the DKO cell line. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng) and ANP32A protein plasmid (20 ng), and each group was provided with triplicate wells. 24 hours after transfection, the cells were lysed and the activity of polymerase was detected; the result was shown in FIG. 15C: the ability of chANP32B(1-161) to support H1N1.sub.SC09 polymerase activity was lost, while chANP32B(162-262) retained the ability to support H1N1.sub.SC09 polymerase activity. Therefore, the key region of the protein ANP32B that supports polymerase activity is located within 1-161aa.

[0195] The homologous recombination PCR was performed as described above; by using huANP32B as a backbone, chANP32B 1-161aa was further divided into three regions of 1-41aa, 42-110aa and 111-161aa to replace the corresponding fragments of PCAGGS-huANP32B, to construct the recombinant plasmid PCAGGS-chANP32B(1-41) (using plasmid PCAGGS-huANP32B as a template, using the primer pair SEQ ID NO:133 and SEQ ID NO: 134 to amplify the PCAGGS-hu32A(1-41) gene fragment; using the PCAGGS-chANP32B plasmid as a template, using the primer pair SEQ ID NO: 135 and SEQ ID NO: 136 to amplify the chANP32B (1-41) gene fragment), PCAGGS-chANP32B (42-110) (using the PCAGGS-huANP32B plasmid as a template, using the primer pair SEQ ID NO: 137 and SEQ ID NO: 138 to amplify the PCAGGS-hu32BA(42-110) gene fragment; using the PCAGGS-chANP32B plasmid as a template, using the primer pair SEQ ID NO: 139 and SEQ ID NO: 140 to amplify the chANP32B (42-110) gene fragment) and PCAGGS-chANP32B (111-161) (using the PCAGGS-huANP32B plasmid as a template, using primer pair SEQ ID NO: 141 and SEQ ID NO: 142 to amplify the PCAGGS-hu32BA(111-161) gene fragment; using the PCAGGS-chANP32B plasmid as a template; using the primer pair SEQ ID NO: 143 and SEQ ID NO: 144 to amplify the chANP32B (111-161) gene fragment). The primers were shown in Table 13.

TABLE-US-00014 TABLE13 primers primername primersequence pCAGGSVector_up, GCGGCCGCGAGCTCGAATTCTTTGCCAA SEQIDNO:133 pCAGGS_hu32B GTGAACTTAGAGTTCCTCAGTTTAATAAA (42-262)-down T SEQIDNO:134 ch32B(1-41)_F, GAATTGTGCGGCCGCATGGAGATGAAAA SEQIDNO:135 AGCGGCTCAC ch32B(1-41)_R, GAACTCTAAGTTCACAAAATCTGAAGAGA SEQIDNO:136 GCCCAACGA pCAGGS_hu32B AAATTCAGCTGTTAAGCCCTCAATTTTTCC (1-41)_up, SEQIDNO:137 pCAGGS_hu32B AAGTTAGAATGTCTGAAAAGCCTGGACCT (111-262)_down C SEQIDNO:138 ch32B(42-110)_F, TTAACAGCTGAATTTGAGAACCTGGAGTT SEQIDNO:139 CCTCAGCAT ch32B(42-110)_R, CAGACATTCTAACTTTTTCAAGGGTTCCA SEQIDNO:140 GGGTATTGA pCAGGS_hu32B TTTCAAAGGTTCCAAGGTGCTGATATCTTT (1-110)-up SEQIDID:141 pCAGGS_hu32B CTCCTCCTCTTCATCCACACCATCCACCTC (162-262)_down SEQIDNO:142 ch32B(111-161)_F, TTGGAACCTTTGAAAAAGTTGCCAAACCT SEQIDNO:143 GCATAGTCT ch32B(111-161)_R, CACACCATCCACCTCAGGGTCTGAGTCAG SEQIDNO:144 GGGCTTCCT

[0196] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the plasmids of PCAGGS-huANP32B, PCAGGS-chANP32B, PCAGGS-chANP32B (1-41), PCAGGS-chANP32B(42-110) and PCAGGS-chANP32B(111-161) were respectively co-transfected with the H1N1.sub.SC09 polymerase reporter system into the DKO cell line. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng) and ANP32A protein plasmid (20 ng), and each group was provided with triplicate wells. 24 hours after transfection, the cells were lysed as described in Example 3 and the activity of polymerase was detected; the result was shown in FIG. 15D: the recombinant plasmid PCAGGS-chANP32B(111-161) lost the ability to support the activity of H1N1.sub.SC09 polymerase, while the recombinant plasmids PCAGGS-chANP32B(1-41) and PCAGGS-chANP32B(42-110) still maintained the support for the activity of H1N1.sub.SC09 polymerase.

[0197] The homologous recombination PCR was performed as described above, and the corresponding fragment of PCAGGS-chANP32B was replaced with the corresponding 111-161aa sequence of PCAGGS-huANP32B (using the PCAGGS-huANP32B plasmid as a template and using the primer pair of SEQ ID NO: 145 and SEQ ID NO:146 to amplify the PCAGGS-ch32BA(111-161) gene fragment; using the PCAGGS-huANP32B plasmid as a template and using the primer pair of SEQ ID NO:147 and SEQ ID NO:148 to amplify the huANP32B (111-162) gene fragment), to construct the recombinant plasmid PCAGGS-chANP32B(hu111-161); the primers were shown in Table 14. DKO cell line was co-transfected with the H1N1.sub.SC09 polymerase reporter system according to the above system, and the polymerase activity was detected as described in Example 3; the result was shown in FIG. 15D (column of chANP32B(hu111-161)), indicating that chANP32B(hu111-161) acquired the ability to support the activity of H1N1.sub.SC09 polymerase, which further indicated that the key region of the ANP32B protein to support polymerase activity was located within 111-161aa.

TABLE-US-00015 TABLE14 primers primername primersequence pCAGGS_ch32B CTTTTTCAAGGGTTCCAGGGTATTGATGTC (1-110)_up SEQIDNO:145 pCAGGS_ch32B GAGGCAGATGGGGATGGACTGGAAGACG (162-262)_down AG SEQIDNO:146 hu32B(111-161)_F GAACCCTTGAAAAAGTTAGAATGTCTGAA SEQIDNO:147 AAGCCTGGA hu32B(111-161)_R ATCCCCATCTGCCTCGGCATCTGAGTCAG SEQIDNO:148 GTGCTTCCT

[0198] To further identify the key regions, alignment of the protein sequences of chANP32B, huANP32B and pgANP32B revealed that amino acids in the 111-161aa region of huANP32B and pgANP32B proteins were relatively conserved, while there were differences of mainly eight amino acids between chANP32B and the above two proteins (positions 113, 116, 127, 129, 130, 137, 150 and 160, respectively), as shown in FIG. 16A. Mutant primers were designed for these 8 amino acids in the huANP32B sequence (see Table 15 for the primers, and the mutant bases were underlined); by using the homologous recombination method as described above, the following point mutants were respectively constructed by using KOD FX Neo polymerase with PCAGGS-huANP32B plasmid as template: huANP32B E113P (using primer pair of SEQ ID NO: 149 and SEQ ID NO: 150), K116H (using primer pair of SEQ ID NO: 151 and SEQ ID NO: 152), N127M (using primer pair of SEQ ID NO: 153 and SEQ ID NO: 154), N129I/D130N (using primer pair SEQ ID NO: 155 and SEQ ID NO: 156), K137T (using primer pair of SEQ ID NO: 157 and SEQ ID NO: 158), R150A (using primer pair of SEQ ID NO: 159 and SEQ ID NO: 160), A160P (using primer pair of SEQ ID NO: 161 and SEQ ID NO: 162), which were respectively named as PCAGGS-huANP32B E113P, PCAGGS-huANP32B K116H, PCAGGS-huANP32B N127M, PCAGGS-huANP32B N129I/D130N, PCAGGS-huANP32B K137T, PCAGGS-huANP32B R150A, PCAGGS-huANP32B A160P, for use in the next step of transfection after confirming by sequencing.

TABLE-US-00016 TABLE15 primersforaminoacidmutation primername primersequence(5-3) huB_E113P_F CCTTTGAAAAAGTTACCCTGTCTGAAAAGCC SEQIDNO:149 TG huB_E113P_R CAGGCTTTTCAGACAGGGTAACTTTTTCAAA SEQIDNO:150 GG huB_K116H_F AAGTTAGAATGTCTGCACAGCCTGGACCTCT SEQIDNO:151 TT huB_K116H_R AAAGAGGTCCAGGCTGTGCAGACATTCTAAC SEQIDNO:152 TT huB_N127M_F AACTGTGAGGTTACCATGCTGAATGACTACC SEQIDNO:153 GA huB_N127M_R TCGGTAGTCATTCAGCATGGTAACCTCACAGT SEQIDNO:154 T huB_N129I/ GAGGTTACCAACCTGATTAACTACCGAGAGA D130N_F GTGTC SEQIDNO:155 huB_N129I/ GACACTCTCTCGGTAGTTAATCAGGTTGGTAA D130N_R CCTC SEQIDNO:156 huB_K137T_F CGAGAGAGTGTCTTCACCCTCCTGCCCCAGC SEQIDNO:157 TT huB_K137T_R AAGCTGGGGCAGGAGGGTGAAGACACTCTC SEQIDNO:158 TCG huB_R150A_F TTGGATGGCTATGACGCTGAGGACCAGGAAG SEQIDNO:159 CA huB_R150A_R TGCTTCCTGGTCCTCAGCGTCATAGCCATCCA SEQIDNO:160 A huB_A160P_F GCACCTGACTCAGATCCGGAGGTGGATGGTG SEQIDNO:161 TG huB_A160P_R CACACCATCCACCTCCGGATCTGAGTCAGGT SEQIDNO:162 GC

[0199] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the point mutant plasmids of PCAGGS-Flag, PCAGGS-huANP32B, PCAGGS-chANP32B and PCAGGS-huANP32B, namely, PCAGGS-huANP32B E113P, PCAGGS-huANP32B K116H, PCAGGS-huANP32B N127M, PCAGGS-huANP32B N291/D130N, PCAGGS-huANP32B K137T, PCAGGS-huANP32B R150A, PCAGGS-huANP32B A160P were respectively co-transfected with the H1N1.sub.SC09 polymerase reporter system into the DKO cell line. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng) and ANP32A mutant protein plasmid (20 ng), and each group was provided with triplicate wells. 24 hours after transfection, the cells were lysed as described in Example 3 and the polymerase activity were detected; the result was shown in FIG. 16B: compared to huANP32B, chANP32B and huANP32B N129I/D130N completely lost support for the activity of H1N1.sub.SC09 polymerase, while the point mutants of huANP32B E113P, huANP32B K116H, huANP32B N127M, huANP32B K137T, huANP32B R150A and huANP32B A160P still retained support for the activity of H1N1.sub.SC09 polymerase.

[0200] For the two sites 129/130, single point mutations of huANP32B N129I (using primer pair of SEQ ID NO:163 and SEQ ID NO: 164) and D130N (using primer pair of SEQ ID NO: 165 and SEQ ID NO: 166) were designed (see Table 16 for primers, and the mutated bases were underlined), and the resulting plasmids were named as PCAGGS-huANP32B N129I and PCAGGS-huANP32B D130N, wherein the PCAGGS-huANP32B plasmid was used as a template and the procedure was as described in this Example for the point mutation of 8 amino acids in the huANP32B sequence. After verification by sequencing, the plasmids were extracted in large amount for further transfection.

TABLE-US-00017 TABLE16 primersforsinglepointmutationhuANP32B N129IandD130N primername primersequence(5-3) huB_N129I_F GAGGTTACCAACCTGATTGACTACCGAGAGAGT SEQIDNO:163 huB_N129I_R ACTCTCTCGGTAGTCAATCAGGTTGGTAACCTC SEQIDNO:164 huB_D130N_F GTTACCAACCTGAATAACTACCGAGAGAGTGTC SEQIDNO:165 huB_D130N_R GACACTCTCTCGGTAGTTATTCAGGTTGGTAAC SEQIDNO:166

[0201] Double-knockout cell line (DKO) was plated in a 12-well plate at 310.sup.5/well and transfected as described above after 20 h, and the result showed that: compared to huANP32B, huANP32B N129I almost lost support for H1N1.sub.SC09 polymerase activity, while the support of huANP32B D130N for H1N1.sub.SC09 polymerase activity was reduced by about 5 times. This showed that the two sites of 129/130 were important for the activity of the ANP32 protein. The result was shown in FIG. 17.

[0202] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the point mutation plasmids of PCAGGS-Flag empty vector, PCAGGS-huANP32B and PCAGGS-huANP32B were respectively co-transfected with the H7N9.sub.AH13 polymerase reporter system into the DKO cell line. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng) and ANP32A mutant protein plasmid (20 ng), and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed as described in Example 3 and the polymerase activity were detected; the result showed that: compared to huANP32B, huANP32B N129I and huANP32B N129I/D130N completely lost support for the activity of H7N9.sub.AH13 polymerase, while the point mutants of huANP32B K116H, huANP32B N127M, huANP32B R150A and huANP32B A160P still retained support for the activity of H7N9.sub.AH13 polymerase, and the ability of huANP32B E113P, huANP32B D130N and huANP32B K137T to support the activity of H7N9.sub.AH13 polymerase was reduced by about 3-8 times. The result was shown in FIG. 18.

[0203] According to the screening results of huANP32B point mutation, huANP32A was also subjected to the point mutant construction of N129I (using primer pair of SEQ ID NO: 167 and SEQ ID NO: 168), D130N (using primer pair of SEQ ID NO: 169 and SEQ ID NO: 170) and ND129/130IN (using primer pair of SEQ ID NO: 171 and SEQ ID NO: 172) (see Table 17 for primers, and mutated bases were underlined) by using overlapping PCR with the PCAGGS-huANP32A plasmid as a template, wherein the procedure is as described in the construction of a point mutant of 8 amino acids on the huANP32B sequence. As described above, the obtained plasmids were named as PCAGGS-huANP32A N129I, PCAGGS-huANP32A D130N and PCAGGS-huANP32A N129I/D130N. After verification by sequencing, the plasmids were extracted in large amount for further transfection.

TABLE-US-00018 TABLE17 PrimersforpointmutationsofN129I,D130N, N129I/D130NonhuANP32A primername primersequence(5-3) huA_N129I_F GAGGTAACCAACCTGATTGACTACCGAGAAA SEQIDNO:167 AT huA_N129I_R ATTTTCTCGGTAGTCAATCAGGTTGGTTACCT SEQIDNO:168 C huA_D130N_F GTAACCAACCTGAACAACTACCGAGAAAATG SEQIDNO:169 TG huA_D130N_R CACATTTTCTCGGTAGTTGTTCAGGTTGGTTA SEQIDNO:170 C huA_N129I/ GAGGTAACCAACCTGATTAACTACCGAGAAA D130N_F ATGTG SEQIDNO:171 huA_N129I/ CACATTTTCTCGGTAGTTAATCAGGTTGGTTA D130N_R CCTC SEQIDNO:172

[0204] Double-knockout cell line (DKO) was plated in a 12-well plate at 310.sup.5/well and co-transfected with the H1N1.sub.SC09 polymerase reporter system as described above after 20 h, and the result showed that: compared to huANP32A, huANP32A N129I/D130N completely lost support for H1N1.sub.SC09 polymerase activity, huANP32A N129I almost lost support for H1N1.sub.SC09 polymerase activity, while the ability of huANP32B D130N to support H1N1.sub.SC09 polymerase activity was reduced by more than 100 times, as shown in FIG. 19.

[0205] Double-knockout cell line (DKO) was plated in a 12-well plate at 310.sup.5/well and after 20 h was co-transfected with the H7N9.sub.SC09 polymerase reporter system as described above, and the result showed that: huANP32A N129I, huANP32A D130N and huANP32A N129I/D130N completely lost support for H7N9.sub.AH13 polymerase activity as compared with huANP32A. The result was shown in FIG. 20.

[0206] According to the screening results of huANP32B point mutation, chANP32A was subjected to the point mutations of N129I (using primer pair of SEQ ID NO: 173 and SEQ ID NO: 174), D130N (using primer pair of SEQ ID NO: 175 and SEQ ID NO: 176) and N129I/D130N (using primer pair of SEQ ID NO: 177 and SEQ ID NO: 178) by overlapping PCR using PCAGGS-chANP32A plasmid as a template; at the same time, chANP32B was subjected to the point mutations of I129N (using primer pair of SEQ ID NO: 179 and SEQ ID NO: 180), N130D (using primer pair of SEQ ID NO: 181 and SEQ ID NO: 182) and I129N/N130D (using primer pair of SEQ ID NO: 183 and SEQ ID NO: 184) by using PCAGGS-chANP32B plasmid as a template (see Table 18 for primers, and the mutated bases were underlined.) After verification by sequencing, the plasmids were extracted in large amount for further transfection.

TABLE-US-00019 TABLE18 PrimersforpointmutationsofchANP32Aand chANP32B primername primersequence(5-3) chA_N129I_F GAGGTAACCAACTTGATTGATTATAGAGAAA SEQIDNO:173 AC chA_N129I_R GTTTTCTCTATAATCAATCAAGTTGGTTACCTC SEQIDNO:174 chA_D130N_F GTAACCAACTTGAATAACTATAGAGAAAACG SEQIDNO:175 TA chA_D130N_R TACGTTTTCTCTATAGTTATTCAAGTTGGTTAC SEQIDNO:176 chA_ND129/ GAGGTAACCAACTTGATTAACTATAGAGAAA 130IN_F ACGTA SEQIDNO:177 chA_ND129/ TACGTTTTCTCTATAGTTAATCAAGTTGGTTAC 130IN_R CTC SEQIDNO:178 chB_I129N_F GAGGTGACGATGCTCAATAACTACCGGGAGA SEQIDNO:179 GT chB_I129N_R ACTCTCCCGGTAGTTATTGAGCATCGTCACCT SEQIDNO:180 C chB_N130D_F GTGACGATGCTCATCGACTACCGGGAGAGTG SEQIDNO:181 TG chB_N130D_R CACACTCTCCCGGTAGTCGATGAGCATCGTC SEQIDNO:182 AC chB_IN129/ GAGGTGACGATGCTCAATGACTACCGGGAGA 130ND_F GTGTG SEQIDNO:183 chB_IN129/ CACACTCTCCCGGTAGTCATTGAGCATCGTCA 130ND_R CCTC SEQIDNO:184

[0207] Double-knockout cell line (DKO) was plated in a 12-well plate at 310.sup.5/well and co-transfected with the H7N9.sub.AH13 polymerase reporter system as described above after 20 h, and the result showed that: compared with chANP32A, chANP32A N129I/D130N lost the support for H7N9.sub.AH13 polymerase activity, the ability of chANP32A N129I to support H7N9.sub.AH13 polymerase activity was decreased by more than 100 times, the ability of chANP32A D130N to support H7N9.sub.AH13 polymerase activity was decreased by about 5 times; compared with chANP32B, chANP32B I129N and chANP32B I129N/N130D had the ability to support H7N9.sub.AH13 polymerase activity, while chANP32B N130D still did not have the ability to support H7N9.sub.AH13 polymerase activity. The result was shown in FIG. 21.

Example 10: Construction of the 129-Site Mutant of chANP32A Protein

[0208] Specifically, the primers for point mutation were shown in Table 19 (mutated bases were underlined), using PCAGGS-chANP32A plasmid as a template, the following point mutants of chANP32A were constructed by KOD-FX Neo high-efficiency DNA polymerase: N129A (using primer pair SEQ ID NO: 185 and SEQ ID NO: 186), N129C (using primer pair SEQ ID NO: 187 and SEQ ID NO: 188), N129D (using primer pair SEQ ID NO: 189 and SEQ ID NO: 190), N129E (using primer pair SEQ ID NO: 191 and SEQ ID NO: 192), N129F (using primer pair SEQ ID NO: 193 and SEQ ID NO: 194), N129G (using primer pair SEQ ID NO: 195 and SEQ ID NO: 196) N129H (using primer pair SEQ ID NO: 197 and SEQ ID NO: 198), N129K (using primer pair SEQ ID NO: 199 and SEQ ID NO: 200), N129L (using primer pair SEQ ID NO: 201 and SEQ ID NO: 202), N129M (using primer pair SEQ ID NO: 203 and SEQ ID NO: 204), N129I (using primer pair SEQ ID NO: 173 and SEQ ID NO: 174), N129P (using primer pair SEQ ID NO: 205 and SEQ ID NO: 206), N129Q (using primer pair SEQ ID NO: 207 and SEQ ID NO: 208), N129R (using primer pair SEQ ID NO: 209 and SEQ ID NO: 210), N129S (using primer pair SEQ ID NO: 211 and SEQ ID NO: 212), N129T (using primer pair SEQ ID NO: 213 and SEQ ID NO: 214), N129V (using primer pair SEQ ID NO: 215 and SEQ ID NO: 216), N129W (using primer pair SEQ ID NO: 217 and SEQ ID NO: 218), N129Y (using primer pair SEQ ID NO: 219 and SEQ ID NO: 220).

[0209] The obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, and then 5 ul of the digested product was taken and transformed into 20 ul of DH5 competent cells; the next day, a single clone was selected for sequencing, and the plasmid which was verified correct by sequencing was used for subsequent transfection experiment.

TABLE-US-00020 TABLE19 primersforpointmutation primername primersequence(5-3) chA_N129A_F GAGGTAACCAACTTGGCAGATTATAGAGAAAAC SEQIDNO:185 chA_N129A_R GTTTTCTCTATAATCTGCCAAGTTGGTTACCTC SEQIDNO:186 chA_N129C_F GAGGTAACCAACTTGTGTGATTATAGAGAAAAC SEQIDNO:187 chA_N129C_R GTTTTCTCTATAATCACACAAGTTGGTTACCTC SEQIDNO:188 chA_N129D_F GAGGTAACCAACTTGGACGATTATAGAGAAAAC SEQIDNO:189 chA_N129D_R GTTTTCTCTATAATCGTCCAAGTTGGTTACCTC SEQIDNO:190 chA_N129E_F GAGGTAACCAACTTGGAAGATTATAGAGAAAAC SEQIDNO:191 chA_N129E_R GTTTTCTCTATAATCTTCCAAGTTGGTTACCTC SEQIDNO:192 chA_N129F_F GAGGTAACCAACTTGTTCGATTATAGAGAAAAC SEQIDNO:193 chA_N129F_R GTTTTCTCTATAATCGAACAAGTTGGTTACCTC SEQIDNO:194 chA_N129G_F GAGGTAACCAACTTGGGAGATTATAGAGAAAAC SEQIDNO:195 chA_N129G_R GTTTTCTCTATAATCTCCCAAGTTGGTTACCTC SEQIDNO:196 chA_N129H_F GAGGTAACCAACTTGCACGATTATAGAGAAAAC SEQIDNO:197 chA_N129H_R GTTTTCTCTATAATCGTGCAAGTTGGTTACCTC SEQIDNO:198 chA_N129K_F GAGGTAACCAACTTGAAGGATTATAGAGAAAAC SEQIDNO:199 chA_N129K_R GTTTTCTCTATAATCCTTCAAGTTGGTTACCTC SEQIDNO:200 chA_N129L_F GAGGTAACCAACTTGCTAGATTATAGAGAAAAC SEQIDNO:201 chA_N129L_R GTTTTCTCTATAATCTAGCAAGTTGGTTACCTC SEQIDNO:202 chA_N129M_F GAGGTAACCAACTTGATGGATTATAGAGAAAAC SEQIDNO:203 chA_N129M_R GTTTTCTCTATAATCCATCAAGTTGGTTACCTC SEQIDNO:204 chA_N129P_F GAGGTAACCAACTTGCCAGATTATAGAGAAAAC SEQIDNO:205 chA_N129P_R GTTTTCTCTATAATCTGGCAAGTTGGTTACCTC SEQIDNO:206 chA_N129Q_F GAGGTAACCAACTTGCAAGATTATAGAGAAAAC SEQIDNO:207 chA_N129Q_R GTTTTCTCTATAATCTTGCAAGTTGGTTACCTC SEQIDNO:208 chA_N129R_F GAGGTAACCAACTTGAGAGATTATAGAGAAAAC SEQIDNO:209 chA_N129R_R GTTTTCTCTATAATCTCTCAAGTTGGTTACCTC SEQIDNO:210 chA_N129S_F GAGGTAACCAACTTGAGCGATTATAGAGAAAAC SEQIDNO:211 chA_N129S_R GTTTTCTCTATAATCGCTCAAGTTGGTTACCTC SEQIDNO:212 chA_N129T_F GAGGTAACCAACTTGACAGATTATAGAGAAAAC SEQIDNO:213 chA_N129T_R GTTTTCTCTATAATCTGTCAAGTTGGTTACCTC SEQIDNO:214 chA_N129V_F GAGGTAACCAACTTGGTAGATTATAGAGAAAAC SEQIDNO:215 chA_N129V_R GTTTTCTCTATAATCTACCAAGTTGGTTACCTC SEQIDNO:216 chA_N129W_F GAGGTAACCAACTTGTGGGATTATAGAGAAAAC SEQIDNO:217 chA_N129W_R GTTTTCTCTATAATCCCACAAGTTGGTTACCTC SEQIDNO:218 chA_N129Y_F GAGGTAACCAACTTGTACGATTATAGAGAAAAC SEQIDNO:219 chA_N129Y_R GTTTTCTCTATAATCGTACAAGTTGGTTACCTC SEQIDNO:220

Example 11: Influence of the 129-Site Mutant of chANP32A Protein on the Replication of Influenza Virus H7N9.SUB.ZJ13

[0210] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the 129-site mutant of chANP32A constructed in Example 10 were respectively co-transfected with the 6 plasmids of H7N9.sub.ZJ13 polymerase reporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng), PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng), pRL-TK (10 ng) and the plasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng) was set as a negative control, chANP32A (20 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; The result showed that: compared with chANP32A, the two-point mutant of chANP32A N129I/D130N and the single-point mutants of chANP32A N129I, chANP32A N129R, chANP32A N129K, chANP32A N129D and chANP32A N129E did not have the ability to support H7N9.sub.ZJ13 polymerase activity; chANP32A N129P, chANP32A N129Q, chANP32A N129G almost completely lost the ability to support H7N9.sub.ZJ13 polymerase activity, while chANP32A N129L, chANP32A N129F, chANP32A N129A, chANP32A N129M, chANP32A N129S, chANP32A N129T, chANP32A N129C and chANP32A N129Y all supported H7N9.sub.ZJ13 polymerase activity; the ability of chANP32A N129V, chANP32A N129W and chANP32A N129H to support H7N9.sub.ZJ13 polymerase activity was reduced by approximately 100 times; the result was shown in FIG. 22.

Example 12: Influence of the 129-site mutant of chANP32A protein on the replication of influenza virus H7N9.SUB.AH13

[0211] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the 129-site mutant of chANP32A constructed in Example 10 were co-transfected with the 6 plasmids of H7N9.sub.AH13 polymerase reporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng), PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng), pRL-TK (10 ng) and the plasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng) was set as a negative control, chANP32A (20 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; The result showed that: compared with chANP32A, the two-point mutant of chANP32A N129I/D130N and the single-point mutants of chANP32A N129P, chANP32A N129R, chANP32A N129K, chANP32A N129Q, chANP32A N129D and chANP32A N129E did not have the ability to support H7N9.sub.AH13 polymerase activity; chANP32A N129I has little ability to support H7N9.sub.AH13 polymerase activity; chANP32A N129F, chANP32A N129A, chANP32A N129M, chANP32A N129S, chANP32A N129G, chANP32A N129T, chANP32A N129C and chANP32A N129Y all supported H7N9.sub.AH13 polymerase activity; the ability of chANP32A N129L and chANP32A N129W to support H7N9.sub.AH13 polymerase activity was reduced by 3-10 times, and the ability of chANP32A N129V and chANP32A N129H to support H7N9.sub.AH13 polymerase activity was reduced by approximately 20-100 times; the result was shown in FIG. 23.

Example 13: Influence of the 129-Site Mutant of chANP32A Protein on the Replication of Influenza Virus WSN

[0212] Double-knockout cell lines (DKO) were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the chANP32A 129-site mutant constructed in Example 10 were co-transfected with the 6 plasmids of WSN polymerase reporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng), PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng), pRL-TK (10 ng) and the plasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng) was set as a negative control, chANP32A (20 ng) was set as positive control, and each group was provided with triplicate wells. 24 hours after transfection, the cells were lysed and the activity of polymerase was detected; The result showed that: compared with chANP32A, the two-point mutant of chANP32A N129I/D130N and the single-point mutants of chANP32A N129K and chANP32A N129D did not have the ability to support WSN polymerase activity; while chANP32A N129F, chANP32A N129A, chANP32A N129M, chANP32A N129S, chANP32A N129G, chANP32A N129T and chANP32A N129C all supported WSN polymerase activity; the ability of chANP32A N129P, chANP32A N129I, chANP32A N129H, chANP32A N129R, chANP32A N129Q and chANP32A N129E to support WSN polymerase activity was reduced by approximately 100 times; the ability of chANP32A N129L, chANP32A N129W, chANP32A N129Y and chANP32A N129V to support WSN polymerase activity was reduced by approximately 5-20 times; the result was shown in FIG. 24.

Example 14: Construction of the 130-Site Mutant of chANP32A Protein

[0213] The primers for point mutation were shown in Table 20 (mutated bases were underlined), using PCAGGS-chANP32A as a template, the following point mutants of chANP32A were constructed by KOD-FX Neo high-efficiency DNA polymerase amplification: N130A (using primer pair SEQ ID NO: 221 and SEQ ID NO: 222), D130C (using primer pair SEQ ID NO: 223 and SEQ ID NO: 224), D130E (using primer pair SEQ ID NO: 225 and SEQ ID NO: 226), D130F (using primer pair SEQ ID NO: 227 and SEQ ID NO: 228), D130G (using primer pair SEQ ID NO: 229 and SEQ ID NO: 230), D130H (using primer pair SEQ ID NO: 231 and SEQ ID NO: 232), D130K (using primer pair SEQ ID NO: 233 and SEQ ID NO: 234), D130L (using primer pair SEQ ID NO: 235 and SEQ ID NO: 236), D130M (using primer pair SEQ ID NO: 237 and SEQ ID NO: 238), D130N (using primer pair SEQ ID NO: 175 and SEQ ID NO: 176), D130P (using primer pair SEQ ID NO: 239 and SEQ ID NO: 240), D130Q (using primer pair SEQ ID NO: 241 and SEQ ID NO: 242), D130R (using primer pair SEQ ID NO: 243 and SEQ ID NO: 244), D130S (using primer pair SEQ ID NO: 245 and SEQ ID NO: 246), D130T (using primer pair SEQ ID NO: 247 and SEQ ID NO: 248), D130V (using primer pair SEQ ID NO: 249 and SEQ ID NO: 250), D130W (using primer pair SEQ ID NO: 251 and SEQ ID NO: 252), D130Y (using primer pair SEQ ID NO: 253 and SEQ ID NO: 254), D1301 (using primer pair SEQ ID NO: 255 and SEQ ID NO: 256).

[0214] The obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, and then 5 ul of the digested product was taken and transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and the plasmids which were verified correct by sequencing were used for subsequent transfection experiment.

TABLE-US-00021 TABLE20 primersof130-sitepointmutation primername primersequence(5-3) chAD130A-F,SEQIDNO:221 GTAACCAACTTGAATGCATAT AGAGAAAAC chAD130A-R,SEQIDNO:222 CGTTTTCTCTATATGCATTCAA GTTGGTTACC chAD130C-F,SEQIDNO:223 GTAACCAACTTGAATTGTTAT AGAGAAAAC chAD130C-R,SEQIDNO:224 CGTTTTCTCTATAACAATTCA AGTTGGTTACCT chAD130E-F,SEQIDNO:225 GTAACCAACTTGAATGAATAT AGAGAAAAC chAD130E-R,SEQIDNO:226 CGTTTTCTCTATATTCATTCAA GTTGGTTACC chAD130F-F,SEQIDNO:227 GTAACCAACTTGAATTTCTAT AGAGAAAAC chAD130F-R,SEQIDNO:228 CGTTTTCTCTATAGAAATTCA AGTTGGTTACC chAD130G-F,SEQIDNO:229 GTAACCAACTTGAATGGCTAT AGAGAAAAC chAD130G-R,SEQIDNO:230 CGTTTTCTCTATAGCCATTCAA GTTGGTTACC chAD130H-F,SEQIDNO:231 GTAACCAACTTGAATCACTAT AGAGAAAAC chAD130H-R,SEQIDNO:232 CGTTTTCTCTATAGTGATTCAA GTTGGTTACC chAD130K-F,SEQIDNO:233 GTAACCAACTTGAATAAGTAT AGAGAAAAC chAD130K-R,SEQIDNO:234 CGTTTTCTCTATACTTATTCAA GTTGGTTACC chAD130L-F,SEQIDNO:235 GTAACCAACTTGAATCTATAT AGAGAAAAC chAD130L-R,SEQIDNO:236 CGTTTTCTCTATATAGATTCAA GTTGGTTACC chAD130M-F,SEQIDNO:237 GTAACCAACTTGAATATGTAT AGAGAAAAC chAD130M-R,SEQIDNO:238 CGTTTTCTCTATACATATTCAA GTTGGTTACC chAD130P-F,SEQIDNO:239 GTAACCAACTTGAATCCATAT AGAGAAAAC chAD130P-R,SEQIDNO:240 CGTTTTCTCTATATGGATTCAA GTTGGTTACC chAD130Q-F,SEQIDNO:241 GTAACCAACTTGAATCAATAT AGAGAAAAC chAD130Q-R,SEQIDNO:242 CGTTTTCTCTATATTGATTCAA GTTGGTTACC chAD130R-F,SEQIDNO:243 GTAACCAACTTGAATAGATAT AGAGAAAAC chAD130R-R,SEQIDNO:244 CGTTTTCTCTATATCTATTCAA GTTGGTTACC chAD130S-F,SEQIDNO:245 GTAACCAACTTGAATAGCTAT AGAGAAAAC chAD130S-R,SEQIDNO:246 CGTTTTCTCTATAGCTATTCAA GTTGGTTACC chAD130T-F,SEQIDNO:247 GTAACCAACTTGAATACATAT AGAGAAAAC chAD130T-R,SEQIDNO:248 CGTTTTCTCTATATGTATTCAA GTTGGTTACC chAD130V-F,SEQIDNO:249 GTAACCAACTTGAATGTATAT AGAGAAAAC chAD130V-R,SEQIDNO:250 CGTTTTCTCTATATACATTCAA GTTGGTTACC chAD130W-F,SEQIDNO:251 GTAACCAACTTGAATTGGTAT AGAGAAAAC chAD130W-R,SEQIDNO:252 CGTTTTCTCTATACCAATTCAA GTTGGTTACC chAD130Y-F,SEQIDNO:253 GTAACCAACTTGAATTACTAT AGAGAAAAC chAD130Y-R,SEQIDNO:254 CGTTTTCTCTATAGTAATTCAA GTTGGTTACC chAD130I-F,SEQIDNO:255 GTAACCAACTTGAATATCTAT AGAGAAAAC chAD130I-R,SEQIDNO:256 CGTTTTCTCTATAGATATTCAA GTTGGTTACC

Example 15: Influence of the 130-Site Mutant of chANP32A Protein on the Replication of Influenza Virus H7N9.SUB.ZJ13

[0215] DKO cells constructed in Example 2 were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the 130-site mutant of chANP32A constructed in Example 14 were co-transfected with the 6 plasmids of H7N9.sub.ZJ13 polymerase reporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng), PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng), pRL-TK (10 ng) and the plasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng) was set as a negative control, chANP32A (20 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; The result showed that: compared with chANP32A, the two-point mutant of chANP32A N129I/D130N and the single-point mutants of chANP32A D130V, chANP32A D130F, chANP32A D130W, chANP32A D130H, chANP32A D130R, chANP32A D130K and chANP32A D130Y did not have the ability to support H7N9.sub.ZJ13 polymerase activity; while chANP32A D130A, chANP32A D130G, chANP32A D130C and chANP32A D130E all supported H7N9.sub.ZJ13 polymerase activity; the ability of chANP32A D130S and chANP32A D130T to support polymerase activity was reduced by approximately 3 times; the ability of chANP32A D130L, chANP32A D130P, chANP32A D1301, chANP32A D130M, chANP32A D130Q and chANP32A D130N to support H7N9.sub.ZJ13 polymerase activity was reduced by approximately 10-50 times; the result was shown in FIG. 25.

Example 16: Influence of the 130-Site Mutant of chANP32A Protein on the Replication of Influenza Virus H7N9.SUB.AH13

[0216] DKO cells constructed in Example 2 were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the 130-site mutant of chANP32A constructed in Example 14 were co-transfected with the 6 plasmids of H7N9.sub.AH13 polymerase reporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng), PA (40 ng), NP (160 ng), pMD18T-vLuc (80 ng), pRL-TK (10 ng) and the plasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng) was set as a negative control, chANP32A (20 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; The result showed that: compared with chANP32A, the two-point mutant of chANP32A N129I/D130N and the single-point mutants of chANP32A D130F and chANP32A D130K did not have the ability to support H7N9.sub.AH13 polymerase activity; while chANP32A D130A, chANP32A D130S, chANP32A D130G and chANP32A D130E all supported H7N9.sub.AH13 polymerase activity; the ability of chANP32A D130V and chANP32A D130R to support polymerase activity was reduced by more than 100 times, and almost did not have the ability to support polymerase activity; the ability of chANP32A D130L, chANP32A D130P, chANP32A D1301, chANP32A D130M, chANP32A D130W, chANP32A D130H, chANP32A D130Q and chANP32A D130Y to support H7N9.sub.AH13 polymerase activity was reduced by approximately 10-100 times; the ability of chANP32A D130T, chANP32A D130C and chANP32A D130N to support H7N9.sub.AH13 polymerase activity was reduced by approximately 3-5 times; the result was shown in FIG. 26.

Example 17: Influence of the 130-Site Mutant of chANP32A Protein on the Replication of Influenza Virus WSN

[0217] DKO cells constructed in Example 2 were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the 130-site mutant of chANP32A constructed in Example 14 was co-transfected with the 6 plasmids of WSN polymerase reporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng), PA (40 ng), NP (160 ng), pMD18T-vLuc (80 ng), pRL-TK (10 ng) and the plasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng) was set as a negative control, chANP32A (20 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; The result showed that: compared with chANP32A, the two-point mutant of chANP32A N129I/D130N and the single-point mutants of chANP32A D130F, chANP32A D130R and chANP32A D130K did not have the ability to support WSN polymerase activity; while chANP32A D130S and chANP32A D130G, chANP32A D130E all supported WSN polymerase activity; chANP32A D130V, chANP32A D130W, chANP32A D130H and chANP32A D130Y almost did not have the ability to support polymerase activity; the ability of chANP32A D130L, chANP32A D130P, chANP32A D1301, chANP32A D130M, chANP32A D130Q and chANP32A D130N to support WSN polymerase activity was reduced by approximately 10-50 times; the ability of chANP32A D130A, chANP32A D130T and chANP32A D130C to support WSN polymerase activity was reduced by approximately 2-3 times; the result was shown in FIG. 27.

Example 18: Construction of the Vector of huANP32B Protein Segmented Mutation and Determination of Polymerase Activity

[0218]

TABLE-US-00022 TABLE21 primersequencesofhuANP32Bprotein segmentedmutation primername sequence(5-3) huANP_B1_F, TCGCGGCCGCATGGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCCTGAGGAACCGGACCCCG 257 Human_B1_R, GGTTCCTCAGGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCCATGCGGCCGCGAGCTCGAA 258 Human_B2_F, CCACCTGGAGGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGAACTTGTCTTGGACAAT 259 Human_B2_R, AGACAAGTTCGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCCTCCAGGTGGATCCTCCT 260 Human_B3_F, AGCTGTTCGAGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGATGGAAAAATTGAGGGC 261 Human_B3_R, TTTTTCCATCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCTCGAACAGCTGCCGGGGT 262 Human_B4_F, CAAATCAAATGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCTTTGTGAACTTAGAGTTC 263 Human_B4_R, AGTTCACAAAGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCATTTGATTTGCAATTGTC 264 Human_B5_F, AACAGCTGAAGCCGCCGCCGCCGCCGCCGC SEQIDNO: CGCCGCCGCCAATGTAGGCTTGATCTCA 265 Human_B5_R, AGCCTACATTGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCTTCAGCTGTTAAGCCCTC 266 Human_B6_F, CAGTTTAATAGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCCCCAAGCTGCCTAAATTG 267 Human_B6_R, GCAGCTTGGGGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCTATTAAACTGAGGAACTC 268 Human_B7_F, TTCAAATCTCGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCCTCAGTGAAAATAGAATC 269 Human_B7_R, TTTCACTGAGGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCGAGATTTGAAACTGAGAT 270 Human_B8_F, AAAGCTTGAAGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGACATGTTAGCTGAAAAA 271 Human_B8_R, CTAACATGTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCTTCAAGCTTTTTCAATTT 272 Human_B9_F, TGGAGGTCTGGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCACACATCTAAACTTAAGT 273 Human_B9_R, TTAGATGTGTGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCCAGACCTCCAAAGATTCT 274 Human_B10_F, TCCAAATCTCGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCAAAGATATCAGCACCTTG 275 Human_B10_R, TGATATCTTTGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCGAGATTTGGAAGTTTTTC 276 Human_B11_F, AAATAAACTGGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCAAGTTAGAATGTCTGAAA 277 Human_B11_R, ATTCTAACTTGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCCAGTTTATTTCCACTTAA 278 Human_B12_F, ACCTTTGAAAGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCTTTAACTGTGAGGTTACC 279 Human_B12_R, CACAGTTAAAGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCTTTCAAAGGTTCCAAGGT 280 Human_B13_F, CCTGGACCTCGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCTACCGAGAGAGTGTCTTC 281 Human_B13_R, TCTCTCGGTAGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCGAGGTCCAGGCTTTTCAG 282 Human_B14_F, CCTGAATGACGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCCAGCTTACCTACTTGGAT 283 Human_B14_R, AGGTAAGCTGGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCGTCATTCAGGTTGGTAAC 284 Human_B15_F, GCTCCTGCCCGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGAGGACCAGGAAGCACCT 285 Human_B15_R, CCTGGTCCTCGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCGGGCAGGAGCTTGAAGAC 286 Human_B16_F, CTATGACCGAGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGAGGTGGATGGTGTGGAT 287 Human_B16_R, CATCCACCTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCTCGGTCATAGCCATCCAA 288 Human_B17_F, CTCAGATGCCGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGACGAAGAAGGAGAAGAT 289 Human_B17_R, CTTCTTCGTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCGGCATCTGAGTCAGGTGC 290 Human_B18_F, AGAGGAGGAGGCCGCCGCCGCCGCCGCCGC SEQIDNO: CGCCGCCGCCGACGATGAGGATGGTGAA 291 Human_B18_R, CCTCATCGTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCCTCCTCCTCTTCATCCAC 292 Human_B19_F, GGAAGACGAGGCCGCCGCCGCCGCCGCCGC SEQIDNO: CGCCGCCGCCGATGAAGAAGATGATGAA 293 Human_B19_R, CTTCTTCATCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCCTCGTCTTCCTCATCTTC 294 Human_B20_F, AGAGGAGTTTGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGAAGGGGATGAGGACGAC 295 Human_B20_R, CATCCCCTTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCAAACTCCTCTTCTTCACC 296 Human_B21_F, TGAAGATGTAGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGAGGAGGAAGAAGAATTT 297 Human_B21_R, CTTCCTCCTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCTACATCTTCATCTTCATC 298 Human_B22_F, TGAAGTCAGTGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGAAGATGAAGATGAGGAT 299 Human_B22_R, CTTCATCTTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCACTGACTTCATCGTCGTC 300 Human_B23_F, ACTTGATGAAGCCGCCGCCGCCGCCGCCGCC SEQIDNO: GCCGCCGCCGAGGAAGAAGGTGGGAAA 301 Human_B23_R, CTTCTTCCTCGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCTTCATCAAGTCCAAATTC 302 Human_B24_F, GGATGAAGAGGCCGCCGCCGCCGCCGCCGC SEQIDNO: CGCCGCCGCCAAGAGAGAAACAGATGA 303 Human_B24_R, TTTCTCTCTTGGCGGCGGCGGCGGCGGCGGC SEQIDNO: GGCGGCGGCCTCTTCATCCTCATCCTC 304 Human_B25_F, TGAAAAGAGGGCCGCCGCCGCCGCCGCCGC SEQIDNO: CGCCGCCGCCGCCGGCAGCGGAGACTACA 305 Human_B25_R, CTCCGCTGCCGGCGGCGGCGGCGGCGGCGG SEQIDNO: CGGCGGCGGCGGCCCTCTTTTCACCTTT 306

[0219] The huANP32B protein was subjected to a segmented mutation, wherein every 10 amino acids as a group were uniformly mutated to alanine, and the primers for point mutation were shown in Table 21 (the mutated bases were underlined). For example, the huANP32B B1-10A mutant was resulted from the mutation of amino acid segment DMKRRIHLE at positions 2-9 of huANP32B protein to AAAAAAAAA, and the huANP32B B11-20A mutant was resulted from the mutation of amino acid segment LRNRTPAAVR at positions 11-20 of huANP32B protein to AAAAAAAAAA, and so on. Using KOD-FX high-efficiency DNA polymerase and using PCAGGS-huANP32B as a template, the following segmented mutants were respectively constructed: huANP32B B1-10A (using primer pair SEQ ID NO: 257 and SEQ ID NO: 258), huANP32B B11-20A (using primer pair SEQ ID NO: 259 and SEQ ID NO: 260), huANP32B B21-30A (using primer pair SEQ ID NO: 261 and SEQ ID NO: 262), huANP32B B31-40A (using primer pair SEQ ID NO: 263 and SEQ ID NO: 264), huANP32B B41-50A (using primer pair SEQ ID NO: 265 and SEQ ID NO: 266), huANP32B B51-60A (using primer pair SEQ ID NO: 267 and SEQ ID NO: 268), huANP32B B61-70A (using primer pair SEQ ID NO: 269 and SEQ ID NO: 270), huANP32B B71-80A (using primer pair SEQ ID NO: 271 and SEQ ID NO: 272), huANP32B B81-90A (using primer pair SEQ ID NO: 273 and SEQ ID NO: 274), huANP32B B91-100A (using primer pair SEQ ID NO: 275 and SEQ ID NO: 276), huANP32B B101-110A (using primer pair SEQ ID NO: 277 and SEQ ID NO: 278), huANP32B B111-120A (using primer pair SEQ ID NO: 279 and SEQ ID NO: 280), huANP32B B121-130A (using primer pair SEQ ID NO: 281 and SEQ ID NO: 282), huANP32B B131-140A (using primer pair SEQ ID NO: 283 and SEQ ID NO: 284), huANP32B B141-150A (using primer pair SEQ ID NO: 285 and SEQ ID NO: 286), huANP32B B151-160tA (using primer pair SEQ ID NO: 287 and SEQ ID NO: 288), huANP32B B161-170A (using primer pair SEQ ID NO: 289 and SEQ ID NO: 290), huANP32B B171-180A (using primer pair SEQ ID NO: 291 and SEQ ID NO: 292), huANP32B B181-190A (using primer pairs SEQ ID NO: 293 and SEQ ID NO: 294), huANP32B B191-200A (using primer pair SEQ ID NO: 295 and SEQ ID NO: 296), huANP32B B201-210A (using primer pairs SEQ ID NO: 297 and SEQ ID NO: 298), huANP32B B211-220A (using primer pair SEQ ID NO: 299 and SEQ ID NO: 300), huANP32B B221-230A (using primer pair SEQ ID NO: 301 and SEQ ID NO: 302), huANP32B B231-240A (using primer pair SEQ ID NO: 303 and SEQ ID NO: 304), huANP32B B241-251A (using primer pair SEQ ID NO: 305 and SEQ ID NO: 306), and were respectively named as huANP32B B1-10A, huANP32B B11-20A, huANP32B B21-30A, huANP32B B31-40A, huANP32B B41-50A, huANP32B B51-60A, huANP32B B61-70A, huANP32B B71-80A, huANP32B B81-90A, huANP32B B91-100A, huANP32B B101-110A, huANP32B B111-120A, huANP32B B121-130A, huANP32B B131-140A, huANP32B B141-150A, huANP32B B151-160A, huANP32B B161-170A, huANP32B B171-180A, huANP32B B181-190A, huANP32B B191-200A, huANP32B B201-210A, huANP32B B211-220A, huANP32B B221-230A, huANP32B B231-240A and huANP32B B241-251A. The obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, and then 2.5 ul of the digested product was taken and transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and the plasmid which was verified correct by sequencing was used for subsequent transfection experiment.

Example 19: Influence of the Segmented Mutant of huANP32B Protein on the Replication of Influenza Virus H7N9.SUB.AH13

[0220] Double-knockout cell lines (DKO) were plated in a 24-well plate at 110.sup.5/well; after 20 hours, the segmented mutant of huANP32B constructed in Example 18 were co-transfected with the 6 plasmids of H7N9.sub.AH13 polymerase reporter system. The transfection system was: PB1 (40 ng), PB2 (40 ng), PA (20 ng), NP (80 ng), pMD18T-vLuc (40 ng), pRL-TK (5 ng) and the plasmid of ANP32 mutant protein (10 ng); and the empty vector (10 ng) was set as a negative control, huANP32B (10 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; the result showed that: compared with huANP32B, the segmented mutants of huANP32B B51-60A, huANP32B B61-70A, huANP32B B71-80A, huANP32B B81-90A, huANP32B B91-100A, huANP32B B101-110A, huANP32B B111-120A, huANP32B B121-130A, huANP32B B131-140A, huANP32B B141-150A and huANP32B B151-160A did not have the ability to support H7N9.sub.AH13 polymerase activity; while huANP32B B1-10A, huANP32B B11-20A, huANP32B B21-30A, huANP32B B31-40A, huANP32B B171-180A, huANP32B B181-190A, huANP32B B191-200A, huANP32B B201-210A, huANP32B B211-220A, huANP32B B221-230A, huANP32B B231-240A and huANP32B B241-251A all had the ability to support H7N9.sub.AH13 polymerase activity; the ability of huANP32B B41-50A and huANP32B B161-170A to support H7N9.sub.AH13 polymerase activity was reduced by about 200 times; the result was shown in FIG. 28.

Example 20: Construction of the Vector of chANP32A Protein Segmented Mutation and Determination of Polymerase Activity

[0221]

TABLE-US-00023 TABLE22 primersequencesusedforchANP32Aprotein segmentedmutation name sequence(5-3) CK32A_B1_F,SEQID TCGCGGCCGCATGGCCGCCGCCGCCG NO:307 CCGCCGCCGCCGCCCTGCGGAACAGG ACGCCCT CK32A_B1_R,SEQID TCCTGTTCCGCAGGGCGGCGGCGGCG NO:308 GCGGCGGCGGCGGCCATGCGGCCGCG AGCTCGAA CK32A_B2_F,SEQID GGATCCACTTAGAGGCCGCCGCCGCC NO:309 GCCGCCGCCGCCGCCGCCGAACTTGTT CTTGAC CK32A_B2_R,SEQID AAGAACAAGTTCGGCGGCGGCGGCGG NO:310 CGGCGGCGGCGGCGGCCTCTAAGTGG ATCCTT CK32A_B3_F,SEQID CAGATGTTAAGGCCGCCGCCGCCGCC NO:311 GCCGCCGCCGCCGCCGAAGGCAAAAT TGAAGG CK32A_B3_R,SEQID AATTTTGCCTTCGGCGGCGGCGGCGGC NO:312 GGCGGCGGCGGCGGCCTTAACATCTG AGGGC CK32A_B4_F,SEQID CTGTAGGTCATACGCCGCCGCCGCCGC NO:313 CGCCGCCGCCGCCGCCTTTGAAGAGC TGGAAT CK32A_B4_R,SEQID AGCTCTTCAAAGGCGGCGGCGGCGGC NO:314 GGCGGCGGCGGCGGCGTATGACCTAC AGTTGT CK32A_B5_F,SEQID TTACAGATGAGGCCGCCGCCGCCGCC NO:315 GCCGCCGCCGCCGCCAACGTAGGCTTA GCCTC CK32A_B5_R,SEQID TAAGCCTACGTTGGCGGCGGCGGCGG NO:316 CGGCGGCGGCGGCGGCCTCATCTGTA AGGCCT CK32A_B6_F,SEQID TGAGTACAATCGCCGCCGCCGCCGCCG NO:317 CCGCCGCCGCCGCCCCAAAGTTAAAC AAACT CK32A_B6_R,SEQID TTAACTTTGGGGCGGCGGCGGCGGCG NO:318 GCGGCGGCGGCGGCGATTGTACTCAA GAATTCC CK32A_B7_F,SEQID TGCAAACTTAGCCGCCGCCGCCGCCG NO:319 CCGCCGCCGCCGCCCTAAGTGACAAC AGAGTC CK32A_B7_R,SEQID TTGTCACTTAGGGCGGCGGCGGCGGC NO:320 GGCGGCGGCGGCGGCTAAGTTTGCAA CTGAGG CK32A_B8_F,SEQID GAAGCTCGAAGCCGCCGCCGCCGCCG NO:321 CCGCCGCCGCCGCCGAAGTGTTGGCA GAAAAG CK32A_B8_R,SEQID CCAACACTTCGGCGGCGGCGGCGGCG NO:322 GCGGCGGCGGCGGCTTCGAGCTTCTTA AGTTT CK32A_B9_F,SEQID AGGAGGACTGGCCGCCGCCGCCGCCG NO:323 CCGCCGCCGCCGCCACGCATCTAAATC TAAGT CK32A_B9_R,SEQID TTTAGATGCGTGGCGGCGGCGGCGGC NO:324 GGCGGCGGCGGCGGCCAGTCCTCCTG AGACT CK32A_B10_F,SEQ TCCAAACCTCGCCGCCGCCGCCGCCG IDNO:325 CCGCCGCCGCCGCCAAAGATCTTGGTA CAATA CK32A_B10_R,SEQ CCAAGATCTTTGGCGGCGGCGGCGGC IDNO:326 GGCGGCGGCGGCGGCGAGGTTTGGAC ACTTTT CK32A_B11_F,SEQ GGCAACAAAATAGCCGCCGCCGCCGC IDNO:327 CGCCGCCGCCGCCGCCAAGTTAGAAA ACCTGA CK32A_B11_R,SEQ TTTTCTAACTTGGCGGCGGCGGCGGCG IDNO:328 GCGGCGGCGGCGGCTATTTTGTTGCCA CTTA CK32A_B12_F,SEQ ACCTCTGAAAGCCGCCGCCGCCGCCG IDNO:329 CCGCCGCCGCCGCCTTCAATTGCGAGG TAACC CK32A_B12_R,SEQ CGCAATTGAAGGCGGCGGCGGCGGCG IDNO:330 GCGGCGGCGGCGGCTTTCAGAGGTTC TATTGT CK32A_B13_F,SEQ TTTAGATCTTGCCGCCGCCGCCGCCGC IDNO:331 CGCCGCCGCCGCCTATAGAGAAAACGT ATTC CK32A_B13_R,SEQ TTTCTCTATAGGCGGCGGCGGCGGCGG IDNO:332 CGGCGGCGGCGGCAAGATCTAAACTC TTCAG CK32A_B14_F,SEQ ACTTGAATGATGCCGCCGCCGCCGCCG IDNO:333 CCGCCGCCGCCGCCCAACTCACATACC TCGA CK32A_B14_R,SEQ ATGTGAGTTGGGCGGCGGCGGCGGCG IDNO:334 GCGGCGGCGGCGGCATCATTCAAGTTG GTTAC CK32A_B15_F,SEQ GCTCCTCCCAGCCGCCGCCGCCGCCGC IDNO:335 CGCCGCCGCCGCCGATGACAAAGAAG CACCA CK32A_B15_R,SEQ TCTTTGTCATCGGCGGCGGCGGCGGCG IDNO:336 GCGGCGGCGGCGGCTGGGAGGAGCTT GAATA CK32A_B16_F,SEQ CTACGATCGGGCCGCCGCCGCCGCCGC IDNO:337 CGCCGCCGCCGCCGAGGGCTACGTGG AGGGC CK32A_B16_R,SEQ CGTAGCCCTCGGCGGCGGCGGCGGCG IDNO:338 GCGGCGGCGGCGGCCCGATCGTAGCC ATCGAG CK32A_B17_F,SEQ CTCTGATGCAGCCGCCGCCGCCGCCGC IDNO:339 CGCCGCCGCCGCCGAGGAAGATGAAG ATGTC CK32A_B17_R,SEQ CATCTTCCTCGGCGGCGGCGGCGGCG IDNO:340 GCGGCGGCGGCGGCTGCATCAGAGTC TGGTGC CK32A_B18_F,SEQ AGACGATGAGGCCGCCGCCGCCGCCG IDNO:341 CCGCCGCCGCCGCCAAAGATCGGGAT GACAAA CK32A_B18_R,SEQ CCCGATCTTTGGCGGCGGCGGCGGCG IDNO:342 GCGGCGGCGGCGGCCTCATCGTCTAA GCCCTC CK32A_B19_F,SEQ ATCTCTAGTGGCCGCCGCCGCCGCCGC IDNO:343 CGCCGCCGCCGCCTCTGATGCAGAGG GCTAC CK32A_B19_R,SEQ CTGCATCAGAGGCGGCGGCGGCGGCG IDNO:344 GCGGCGGCGGCGGCCACTAGAGATAA GACATC CK32A_B20_F,SEQ AGCACCGGACGCCGCCGCCGCCGCCG IDNO:345 CCGCCGCCGCCGCCGACGACGAGGAG GAAGAT CK32A_B20_R,SEQ CCTCGTCGTCGGCGGCGGCGGCGGCG IDNO:346 GCGGCGGCGGCGGCGTCCGGTGCTTC TTTGTC CK32A_B21_F,SEQ GGAAGGCTTAGCCGCCGCCGCCGCCG IDNO:347 CCGCCGCCGCCGCCGAGTATGACGATG ATGCT CK32A_B21_R,SEQ CGTCATACTCGGCGGCGGCGGCGGCG IDNO:348 GCGGCGGCGGCGGCTAAGCCTTCCAC GTAGCC CK32A_B22_F,SEQ AGACGAAGAGGCCGCCGCCGCCGCCG IDNO:349 CCGCCGCCGCCGCCGATGAAGAGGAT GAGGAG CK32A_B22_R,SEQ CCTCTTCATCGGCGGCGGCGGCGGCG IDNO:350 GCGGCGGCGGCGGCCTCTTCGTCTTCA TCTTC CK32A_B23_F,SEQ GGTAGTAGAAGCCGCCGCCGCCGCCG IDNO:351 CCGCCGCCGCCGCCGGAGAAGAGGAG GACGTA CK32A_B23_R,SEQ CCTCTTCTCCGGCGGCGGCGGCGGCG IDNO:352 GCGGCGGCGGCGGCTTCTACTACCTGA GCATC CK32A_B24_F,SEQ GGAAGAGGAAGCCGCCGCCGCCGCCG IDNO:353 CCGCCGCCGCCGCCGAGGAGGATGAG GAAGGC CK32A_B24_R,SEQ CATCCTCCTCGGCGGCGGCGGCGGCG IDNO:354 GCGGCGGCGGCGGCTTCCTCTTCCTCC TCCTC CK32A_B25_F,SEQ CGGAGAGGAAGCCGCCGCCGCCGCCG IDNO:355 CCGCCGCCGCCGCCGACGTAGATGATG ATGAA CK32A_B25_R,SEQ CATCTACGTCGGCGGCGGCGGCGGCG IDNO:356 GCGGCGGCGGCGGCTTCCTCTCCGCTT ACGTC CK32A_B26_F,SEQ TAATGATGGTGCCGCCGCCGCCGCCGC IDNO:357 CGCCGCCGCCGCCCCCGATGAAGAAC GGGGA CK32A_B26_R,SEQ CTTCATCGGGGGCGGCGGCGGCGGCG IDNO:358 GCGGCGGCGGCGGCACCATCATTATAG CCTTC CK32A_B27_F,SEQ TGAAGAAGAAGCCGCCGCCGCCGCCG IDNO:359 CCGCCGCCGCCGCCCGAGAACCCGAA GACGAA CK32A_B27_R,SEQ CGGGTTCTCGGGCGGCGGCGGCGGCG IDNO:360 GCGGCGGCGGCGGCTTCTTCTTCATCT TCATC CK32A_B28_F,SEQ GAAGAGGAAAGCCGCCGCCGCCGCCG IDNO:361 CCGCCGCCGCCGCCGCCGGCAGCGGA GACTAC CK32A_B28_R,SEQ CTCCGCTGCCGGCGGCGGCGGCGGCG IDNO:362 GCGGCGGCGGCGGCGGCTTTCCTCTTC TGTCC

[0222] The chANP32A protein was subjected to a segmented mutation, wherein every 10 amino acids as a group were uniformly mutated to alanine, and the primers for point mutation were shown in Table 22 (the mutated bases were underlined). For example, the chANP32A 1-10A mutant was resulted from the mutation of amino acid segment DMKKRIHLE at positions 2-9 of chANP32A protein to AAAAAAAAA, and the chANP32A 11-20A mutant is the mutation of amino acid segment LRNRTPSDVK at positions 11-20 of chANP32A protein to AAAAAAAAAA, and so on. Using KOD-FX high-efficiency DNA polymerase and using PCAGGS-chANP32A as a template, the following segmented mutants were respectively constructed: chANP32A 1-10 mutA (using primer pair SEQ ID NO: 307 and SEQ ID NO: 308), chANP32A 11-20 mutA (using primer pair SEQ ID NO: 309 and SEQ ID NO: 310), chANP32A 21-30mutA (using primer pair SEQ ID NO: 311 and SEQ ID NO: 312), chANP32A 31-40mutA (using primer pair SEQ ID NO: 313 and SEQ ID NO: 314), chANP32A 41-50mutA (using primer pair SEQ ID NO: 315 and SEQ ID NO: 316), chANP32A 51-60mutA (using primer pair SEQ ID NO: 317 and SEQ ID NO: 318), chANP32A 61-70mutA (using primer pair SEQ ID NO: 319 and SEQ ID NO: 320), chANP32A 71-80mutA (using primer pair SEQ ID NO: 321 and SEQ ID NO: 322), chANP32A 81-90mutA (using primer pair SEQ ID NO: 323 and SEQ ID NO: 324), chANP32A 91-100mutA (using primer pair SEQ ID NO: 325 and SEQ ID NO: 326), chANP32A 101-110mutA (using primer pair SEQ ID NO: 327 and SEQ ID NO: 328), chANP32A 111-120mutA (using primer pair SEQ ID NO: 329 and SEQ ID NO: 330), chANP32A 121-130mutA (using primer pair SEQ ID NO: 331 and SEQ ID NO: 332), chANP32A 131-140mutA (using primer pair SEQ ID NO: 333 and SEQ ID NO: 334), chANP32A 141-150mutA (using primer pair SEQ ID NO: 335 and SEQ ID NO: 336), chANP32A 151-160mutA (using primer pairs SEQ ID NO: 337 and SEQ ID NO: 338), chANP32A 161-170mutA (using primer pair SEQ ID NO: 339 and SEQ ID NO: 340), chANP32A 171-180mutA (using primer pair SEQ ID NO: 341 and SEQ ID NO: 342), chANP32A 181-190mutA (using primer pairs SEQ ID NO: 343 and SEQ ID NO: 344), chANP32A 191-200mutA (using primer pairs SEQ ID NO: 345 and SEQ ID NO: 346), chANP32A 201-210mutA (using primer pairs SEQ ID NO: 347 and SEQ ID NO: 348), chANP32A 211-220mutA (using primer pairs SEQ ID NO: 349 and SEQ ID NO: 350), chANP32A 221-230mutA (using primer pairs SEQ ID NO: 351 and SEQ ID NO: 352), chANP32A 231-240mutA (using primer pair SEQ ID NO: 353 and SEQ ID NO: 354), chANP32A 241-250mutA (using primer pair SEQ ID NO: 355 and SEQ ID NO: 356), chANP32A 251-260mutA (using primer pair SEQ ID NO: 357 and SEQ ID NO: 358), chANP32A 261-270mutA (using primer pair SEQ ID NO: 359 and SEQ ID NO: 360), chANP32A 271-281mutA (using primer pair SEQ ID NO: 361 and SEQ ID NO: 362), and were respectively named as chANP32A 1-10mutA, chANP32A 11-20mutA, chANP32A 21-30mutA, chANP32A 31-40mutA, chANP32A 41-50mutA, chANP32A 51-60mutA, chANP32A 61-70mutA, chANP32A 71-80mutA, chANP32A 81-90mutA, chANP32A 91-100mutA, chANP32A 101-110mutA, chANP32A 111-120mutA, chANP32A 121-130mutA, chANP32A 131-140mutA, chANP32A 141-150mutA, chANP32A 151-160mutA, chANP32A 161-170mutA, chANP32A 171-180mutA, chANP32A 181-190mutA, chANP32A 191-200mutA, chANP32A 201-210mutA, chANP32A 211-220mutA, chANP32A 221-230mutA, chANP32A 231-240mutA, chANP32A 241-250mutA, chANP32A 251-260mutA, chANP32A 261-270mutA and chANP32A 271-281mutA. The obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, and then 2.5 ul of the digested product was taken and transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and the plasmid which was verified correct by sequencing was used for subsequent transfection experiment.

Example 21: Influence of the Segmented Mutant of chANP32A Protein on the Replication of Influenza Virus H7N9.SUB.ZJ13

[0223] Influence of the segmented mutant of chANP32A protein on the replication of influenza virus H7N9.sub.ZJ13: double-knockout cell lines (DKO) were plated in a 24-well plate at 110.sup.5/well; after 20 hours, the segmented mutant of chANP32A constructed in Example 20 were co-transfected with the 6 plasmids of H7N9.sub.ZJ13 polymerase reporter system. The transfection system was: PB1 (40 ng), PB2 (40 ng), PA (20 ng), NP (80 ng), pMD18T-vLuc (40 ng), pRL-TK (5 ng) and the plasmid of ANP32 mutant protein (10 ng); and the empty vector (10 ng) was set as a negative control, chANP32A (10 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected, and the results showed that: compared with chANP32A, the segmented mutants chANP32A 71-80mutA, chANP32A 81-90mutA, chANP32A 91-100mutA, chANP32A 101-110mutA, chANP32A 111-120mutA, chANP32A 121-130mutA, chANP32A 131-140mutA, chANP32A 141-150mutA, chANP32A 151-160mutA, chANP32A 161-170mutA and chANP32A 171-180mutA did not have the ability to support H7N9.sub.ZJ13 polymerase activity; while chANP32A 1-10mutA, chANP32A11-20mutA, chANP32A 21-30mutA, chANP32A 31-40mutA, chANP32A 41-50mutA, chANP32A 51-60mutA, chANP32A 181-190mutA, chANP32A 201-210mutA, chANP32A 211-220mutA, chANP32A 221-230mutA, chANP32A 231-240mutA, chANP32A 241-250mutA, chANP32A 251-260mutA, chANP32A 261-270mutA, chANP32A 271-281mutA all supported H7N9.sub.ZJ13 polymerase activity; the ability of chANP32A 61-70mutA and chANP32A 191-200mutA to support H7N9.sub.ZJ13 polymerase activity was reduced by about 100 times; the result was shown in FIG. 29.

Example 22: Construction of the Amino Acid Site Mutation Vector of chANP32A Protein and Determination of Polymerase Activity

[0224]

TABLE-US-00024 TABLE23 primersequencesofchANP32Aproteinaminoacid sitemutation name sequence(5-3) chA_D149A_F,SEQID TTATCTCTAGTGAAAGCCCGGGATGA NO:363 CAAAGAA chA_D149A_R,SEQID TTCTTTGTCATCCCGGGCTTTCACTAG NO:364 AGATAA chA_R150A_F,SEQID TCTCTAGTGAAAGATGCCGATGACAA NO:365 AGAAGCA chA_R150A_R,SEQID TGCTTCTTTGTCATCGGCATCTTTCAC NO:366 TAGAGA chA_D151A_F,SEQID CTAGTGAAAGATCGGGCCGACAAAG NO:367 AAGCACCG chA_D151A_R,SEQID CGGTGCTTCTTTGTCGGCCCGATCTTT NO:368 CACTAG chA_D152A_F,SEQID GTGAAAGATCGGGATGCCAAAGAAG NO:369 CACCGGAC chA_D152A_R,SEQID GTCCGGTGCTTCTTTGGCATCCCGAT NO:370 CTTTCAC chA_K153A_F,SEQID AAAGATCGGGATGACGCCGAAGCAC NO:371 CGGACTCT chA_K153A_R,SEQID AGAGTCCGGTGCTTCGGCGTCATCCC NO:372 GATCTTT chA_E154A_F,SEQID GATCGGGATGACAAAGCCGCACCGG NO:373 ACTCTGAT chA_E154A_R,SEQID ATCAGAGTCCGGTGCGGCTTTGTCAT NO:374 CCCGATC

[0225] The primers of point mutation were shown in Table 23 (mutated bases were underlined);. using KOD-FX high-efficiency DNA polymerase and using PCAGGS-chANP32A as a template, the following point mutants of chANP32A were constructed: D149A (using primer pair SEQ ID NO: 363 and SEQ ID NO: 364), R150A (using primer pair SEQ ID NO: 365 and SEQ ID NO: 366), D151A (using primer pair SEQ ID NO: 367 and SEQ ID NO: 368), D152A (using primer pair SEQ ID NO: 369 and SEQ ID NO: 370), K153A (using primer pair SEQ ID NO: 371 and SEQ ID NO: 372) and E154A (using primer pair SEQ ID NO: 373 and SEQ ID NO: 374), and were respectively named as chANP32A D149A, chANP32A R150A, chANP32A D151A, chANP32A D152A, chANP32A K153A and chANP32A E154A. The obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, and then 2.5 ul of the digested product was taken and transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and the plasmid which was verified correct by sequencing was used for subsequent transfection experiment.

Example 23: Influence of the chANP32A Protein Point Mutant on the Replication of Influenza Virus H7N9.SUB.AH13

[0226] Double-knockout cell lines (DKO) were plated in a 24-well plate at 110.sup.5/well; after 20 hours, the amino acid mutant of chANP32A constructed in Example 22 were co-transfected with the 6 plasmids of H7N9.sub.AH13 polymerase reporter system. The transfection system was: PB1 (40 ng), PB2 (40 ng), PA (20 ng), NP (80 ng), pMD18T-vLuc (40 ng), pRL-TK (5 ng) and the plasmid of ANP32 mutant protein (10 ng); and the empty vector (10 ng) was set as a negative control, chANP32A (10 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; the result showed that: compared with chANP32A, the ability of chANP32A D149A to support H7N9.sub.AH13 polymerase activity was reduced by about 1000 times, and almost did not have the ability to support the polymerase activity; the ability of chANP32A D151A to support H7N9.sub.AH13 polymerase activity was reduced by about 50 times; chANP32A R150A, chANP32A D152A and chANP32A K153A all supported the H7N9.sub.AH13 polymerase activity; the ability of chANP32A E154A to support polymerase activity was reduced by about 5 times; the result was shown in FIG. 30.

Example 24: Construction of the Amino Acid Site Mutation Vector of huANP32B Protein and Determination of Polymerase Activity

[0227]

TABLE-US-00025 TABLE24 primersequencesofhuANP32Bproteinamino acidsitemutation name sequence(5-3) huB_NES1_F,SEQID TGATCTCAGTTTCAAATGCCCCCAAGG NO:375 CCCCTAAATTGAAAAAGCTTGAACTC AGTGA huB_NES1_R,SEQID AAGCTTTTTCAATTTAGGGGCCTTGGG NO:376 GGCATTTGAAACTGAGATCAAGCCTAC ATTT huB_NES2_F,SEQID AGCTGAAAAAGCCCCAAATGCCACAC NO:377 ATGCCAACGCCAGTGGAAATAAACTG AAAGA huB_NES2_R,SEQID TTATTTCCACTGGCGTTGGCATGTGTG NO:378 GCATTTGGGGCTTTTTCAGCTAACATG TCCA huB_NES3_F,SEQID GAACCTTTGAAAAAGGCCGAATGTGC NO:379 CAAAAGCGCCGACCTCTTTAACTGTG AGGTT huB_NES3_R,SEQID TTAAAGAGGTCGGCGCTTTTGGCACAT NO:380 TCGGCCTTTTTCAAAGGTTCCAAGGTG CTG

[0228] The protein sequence of huANP32B was analyzed and it contained 3 known Nuclear Export Signals (NES), which were NSE1(LPKLPKLKKL, located at positions 60-71), NSE2 (LPNLTHLNL, located at positions 87-95) and NES3 (LEPLKKLECLKSLDL, located at positions 106-120). To determine whether the nuclear export domain of huANP32B was correlated with its ability to support polymerase activity, mutations were made to these three nuclear export regions, respectively. For the NES1 region, leucines at positions 60 and 63 were both mutated to alanine, and the mutant was named as huANP32B NES1mut. For the NES2 region, leucines at positions 87, 90, 93 and 95 were all mutated to alanine, and the mutant was named as huANP32B NES2mut. For the NES3 region, leucines at positions 112, 115 and 118 were all mutated to alanine, and the mutant was named as huANP32B NES3mut. The primers of point mutation were shown in Table 24 (mutated bases were underlined);. using KOD-FX high-efficiency DNA polymerase for amplification and using PCAGGS-huANP32B as a template, the following point mutants of huANP32B were constructed: NES1mut (using primer pair SEQ ID NO: 375 and SEQ ID NO: 376), NES2mut (using primer pair SEQ ID NO: 377 and SEQ ID NO: 378) and NES3mut (using primer pair SEQ ID NO: 379 and SEQ ID NO: 380), and were respectively named as huANP32B NES1mut, huANP32B NES2mut and huANP32B NES3mut. The obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, and then 2.5 ul of the digested product was taken and transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and the plasmid which was verified correct by sequencing was used for subsequent transfection experiment.

Example 25: Influence of the Point Mutant of huANP32B Protein on the Replication of Influenza Virus H7N9.SUB.AH13

[0229] Double-knockout cell lines (DKO) were plated in a 24-well plate at 110.sup.5/well; after 20 hours, the amino acid mutant of huANP32B constructed in Example 24 were co-transfected with the 6 plasmids of H7N9.sub.AH13 polymerase reporter system. The transfection system was: PB1 (40 ng), PB2 (40 ng), PA (20 ng), NP (80 ng), pMD18T-vLuc (40 ng), pRL-TK (5 ng) and the plasmid of ANP32 mutant protein (10 ng); and the empty vector (10 ng) was set as a negative control, huANP32B (10 ng) was set as positive control, and each group was provided with triplicate wells.24 hours after transfection, the cells were lysed and the activity of polymerase was detected; the result showed that: compared with huANP32B, the ability of huANP32B NES1mut to support H7N9.sub.AH13 polymerase activity was reduced by about 1000 times, and almost did not have the ability to support the polymerase activity; huANP32B NES2mut and huANP32B NES3mut did not have the ability to support H7N9.sub.AH13 polymerase activity; the result was shown in FIG. 31.

Example 26: Construction of a Site-Directed Mutant Cell Line

[0230] We performed the construction of a site-directed mutant cell line by using CRISPR-Cas9 technology. According to NCBI published reference nucleotide sequences human ANP32A (NM_006305.3) and human ANP32B (NM_006401.2), sgRNAs for positions 129/130 of the two proteins were designed by using the online software http://crispr.mit.edu/ (see Table 25 for sequences).

TABLE-US-00026 TABLE25 sgRNAsequences primername primersequence(5-3) huANP32A-129/130-sgRNA, CCAACCTGAACGACTACCGA SEQIDNO:381 huANP32B-129/130-sgRNA, CTCTCGGTAGTCATTCAGGT SEQIDNO:382

[0231] sgRNA primers for the huANP32A 129/130 amino acid site and the huANP32B129/130 amino acid site were designed; using the recombinant plasmid pMD18T-U6-huANPsgRNA-1 constructed in Example 2 as a template and using KOD-FX Neo high-efficiency DNA polymerase (cat # KFX-201, purchased from Toyobo) for amplification, recombinant plasmids pMD18T-U6-huANP32A-129/130-sgRNA (using primer pair SEQ ID NO:383 and SEQ ID NO: 384) and pMD18T-U6-huANP32B-129/130-sgRNA (using primer pair SEQ ID NO:386 and SEQ ID NO: 387) were respectively constructed by a point mutation PCR method, and the obtained PCR product was digested with Dpn I in a 37 C. constant temperature water bath for 30 minutes, then 5 ul of the digested product was taken and transformed into 20 ul of DH5 competent cells; the next day, a single clone was picked for sequencing, and the plasmids which were verified correct by sequencing, pMD18T-U6-huaNP32A-129/130-sgRNA (containing huANP 32A-129/130-sgRNA) and pMD18T-U6-huANP32B-129/130-sgRNA (containing huANP 32B-129/130-sgRNA), were used for subsequent transfection experiment. At the same time, a donor sequence huANP32A-sgRNA-ssODN (SEQ ID NO: 385) for intracellular huANP32A N129I/D130N point mutation and a donor sequence huANP32B-sgRNA-ssODN (SEQ ID NO: 388) for huANP32B N129I/D130N point mutation were synthesized. The synthesized single nucleotide sequence was diluted to 10 uM with distilled water for future use.

TABLE-US-00027 TABLE26 primersequences primername primersequence(5-3) huANP32A-sgRNA-F, TCTCGGTAGTCGTTCAGGTGTTT SEQIDNO:383 TAGAGCTAGAAAT huANP32A-sgRNA-R, ACCTGAACGACTACCGAGACGG SEQIDNO:384 TGTTTCGTCCTTTC huANP32A-sgRNA-ssODN, TGAGTTGCGGGAGGAGCTTGAA SEQIDNO:385 CACATTTTCTCGGTAGTTAATCA GGTTGGTTACCTCGCAATTGAA AAGGTCTAAGCTC huANP32B-sgRNA-F, TCTCGGTAGTCATTCAGGTGTTT SEQIDNO:386 TAGAGCTAGAAATAGC huANP32B-sgRNA-R, ACCTGAATGACTACCGAGACGG SEQIDNO:387 TGTTTCGTCCTTTC huANP32B-sgRNA-ssODN, TAAGCTGGGGCAGGAGCTTGAA SEQIDNO:388 GACACTCTCTCGGTAGTTAATCA GGTTGGTAACCTCACAGTTAAA GAGGTCCAGGCTT

[0232] 1 ug of eukaryotic plasmid pMJ920 (Addge plasma #42234) expressing Cas9-GFP protein, 1 ug of pMD18T-U6-huANP32A-129/130-sgRNA recombinant plasmid and 0.5 L of diluted huANP32A-sgRNA-ssODN (SEQ ID NO: 385) were mixed with lipofectamine 2000 at a ratio of 1:2.5, and then transfected into 293T cells; 1 ug of eukaryotic plasmid pMJ920 (Addge plasma #42234) expressing Cas9-GFP protein, 1 ug of pMD18T-U6-huANP32B-129/130-sgRNA recombinant plasmid and 0.5 L of diluted huANP32B-sgRNA-ssODN (SEQ ID NO: 388) were mixed with lipofectamine 2000 at a ratio of 1:2.5, and then transfected into 293T cells. After 48 hours, GFP-positive cells were screened by an ultra-speed flow cytometry sorting system, and plated in a 96-well plate at a single cell/well for about 10 days; single-cell clones were picked for expansion and culture, and then cellular RNA was extracted according to the procedure using a SimplyP total RNA extraction Kit (purchased from Bioflux, cat # BSC52M1), and cDNA was synthesized using a reverse transcription Kit of Takara Co., Ltd (PrimneScript RT reagent Kit with gDNA Eraser (Perfect read Time), Cat.RR047A); and sgRNA-targeting fragments of huANP32A (using primer pair SEQ ID NO:389 and SEQ ID NO: 390) and huANP32B (using primer pair SEQ ID NO:391 and SEQ ID NO: 392) were amplified by KOD Fx Neo polymerase using the cDNA as a template, and the amplification primers were shown in Table 27, wherein the size of huANP32A amplified fragment was 570 bp, and the size of huANP32B amplified fragment was 572 bp. Single-cell clones that were verified to have correct gene mutations by sequencing were subject to western blotting identification and subsequent experimental studies. The cell lines of mutations of both the 129/130 amino acid sites of huANP32A and huANP32B were obtained after the first round of obtaining the huANP32B single-knockout cell line, followed by another round of knockout screening, and the transfection system and screening steps were as described above. For the constructed cell lines, the fragments of interest of huANP32A and huANP32B were amplified; the effect of single mutation and double mutation were verified; the identification and sequencing results of cell lines were shown in FIG. 32, wherein FIG. 32A showed the sequencing results of huANP32A and huANP32B129/130-site amino acids of the huANP32A N129I/D130N single-mutant cell line (named as A21 IN), FIG. 32B showed the sequencing results of huANP32A and huANP32B129/130-site amino acids of the huANP32B N129I/D130N single-mutant cell line (named as B5 IN), and FIG. 32C showed the sequencing results of huANP32A and huANP32B 129/130-site amino acids of the double-mutant cell line (named as AB IN).

TABLE-US-00028 TABLE27 theprimersequencesforidentificationof huANP32AandhuANP32Bpoint-mutantcellline primername primersequence(5-3) hu32AgRNA-F, CCAAAGTTAAACAAACTTAAGAAGC SEQIDNO:389 TTGAACTAAGC hu32AgRNA-R, TTAGTCATCATCTTCTCCCTCATCTTC SEQIDNO:390 AGGTTCT hu32BgRNA-F, AGCTGCCTAAATTGAAAAAGCTTGAA SEQIDNO:391 CTC hu32BgRNA-R, TTAATCATCTTCTCCTTCATCATCTGTT SEQIDNO:392 TCTCTC

[0233] Anti-PHAP1 antibody (purchased from Abcam, cat # ab51013) and Anti-PHAPI2/APRIL antibody [EPR14588] (purchased from Abcam, cat # ab200836) were used in Western bloting; -actin is used as the internal control gene, and the antibody of Monoclonal Anti--Actin antibody produced in mouse (purchased from Sigma, cat # A1978-200UL) was used, and the results were shown in FIG. 33.

[0234] Based on the above, we successfully constructed a huANP32A N129I/D130N single-mutant cell line (named as A21 IN), a huANP32B N129I/D130N single-mutant cell line (named as B5 IN) and a huANP32A and huANP32B double-mutant cell line (named as AB IN), which were used for subsequent experiments.

Example 27: Influence of ANP32 Protein on the Replication of Influenza Virus

[0235] The double-knockout cell line (DKO) constructed in Example 2, the A21 IN cell line, B5 IN cell line and AB IN cell line constructed in Example 26, and the wild-type 293T cell line were plated in a 12-well plate at 310.sup.5/well; after 20 hours, the 5 plasmids of H1N1.sub.SC09 polymerase reporter system were co-transfected. The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng) and pRL-TK plasmid (10 ng), and each group was provided with triplicate wells.24 h after transfection, the cells were lysed as described in Example 3 and the activity of polymerase was detected; the result showed that: the activity of H1N1.sub.SC09 polymerase on the single-mutant cell line of A21 IN cell line and B5 IN cell line was slightly different from that of the wild type 293T cell, and was reduced by only 3-5 times, while the activity of the polymerase on the AB IN cell line and the DKO cell line was significantly reduced by about 3000-5000 times, and the result was shown in FIG. 34.

[0236] The above experiment was repeated with the H7N9.sub.AH13 polymerase reporter system instead of the H1N1.sub.SC09 polymerase reporter system; the result showed that: the activity of H7N9.sub.AH13 polymerase on the single-mutant cell line of A21 IN cell line and B5 IN cell line was slightly different from that of the wild type 293T cell, and was reduced by only about 10 times, while the activity of the H7N9.sub.AH13 polymerase on the AB IN cell line and the DKO cell line was significantly reduced by about 7000-10000 times, and the result was shown in FIG. 35.

Example 28: Alignment of Amino Acid Sequences of Avian-Derived ANP32A Proteins

[0237] The amino acid sequences of avian-derived ANP32A proteins (chANP32A, zfANP32A, dkANP32A and tyANP32A) were compared (see FIG. 36), wherein the corresponding relationship of amino acid positions 129, 130, 149, 151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118 between each avian-derived ANP32A protein and chANP32A protein was shown in Table 28.

TABLE-US-00029 TABLE 28 chANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 zfANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 dkANP32A 119 120 139 141 50 53 77 80 83 85 102 105 108 tyANP32A 128 129 148 150 59 62 86 89 92 94 111 114 117

[0238] As demonstrated in Example 21, the mutation of amino acids in the amino acid segment 71-180 of chANP32A to alanine resulted in a complete loss of the ability of the protein to support polymerase activity, while the mutation of amino acids in the amino acid segments 61-70 and 191-200 to alanine resulted in a decrease of the ability of the protein to support polymerase activity.

[0239] As can be seen from the alignment of the amino acid sequences of the zfANP32A protein and the chANP32A protein in FIG. 36, the sequences of the following amino acid segments of zfANP32A are completely identical to corresponding amino acid sequences in chANP32A: amino acid segment 61-71, amino acid segment 73-75, amino acid segment 77-99, amino acid segment 101-165, amino acid segment 167-169, amino acid segment 171-180 and amino acid segment 181-200, therefore, the mutation of amino acids in amino acid segment 61-71, amino acid segments 73-75, 77-99, 101-165, 167-169 and 171-180 of the zfANP32A protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segments 61-70 and 191-200 to alanine resulted in a decrease in the ability to support polymerase activity.

[0240] From the alignment of the amino acid sequences between the dkANP32A protein and the chANP32A protein in FIG. 36, it was found that the amino acid at position 66 of the dkANP32A protein was I, while the amino acid at position 76 of the chANP32A protein was V, and that the amino acids at positions 164-167 of dkANP32A were deleted (aligned with positions 176-179 of chANP32 protein). It can be seen that the mutation of amino acids in the amino acid segments 61-65 and 67-166 of the dkANP32A protein to alanine resulted in a complete loss of the ability of the protein to support polymerase activity, while the mutation of amino acids in the amino acid segments 51-60 and 177-186 to alanine resulted in a decrease of the ability of the protein to support polymerase activity.

[0241] From the alignment of the amino acid sequences between the tyANP32A protein and the chANP32A protein in FIG. 36, it can be seen that the amino acid sequence of the tyANP32A protein is completely identical to the amino acid sequence of the chANP32A protein. It can be seen that the mutation of amino acids in the amino acid segment 60-179 of the tyANP32A protein to alanine resulted in a complete loss of the ability of the protein to support polymerase activity, while the mutation of amino acids in the amino acid segments 60-69 and 190-199 to alanine resulted in a decrease of the ability of the protein to support polymerase activity.

Example 29: Alignment of Amino Acid Sequences Between Avian-Derived ANP32B Protein and huANP32B Protein

[0242] The amino acid sequences of avian-derived ANP32B proteins (chANP32B, dkANP32B(XP_012963723.1) and tyANP32B(XP_010723174.1)) were compared with the amino acid sequence of huANP32B protein (see FIG. 37), wherein the corresponding relationship of amino acid positions 129, 130, 149, 151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118 between each avian-derived ANP32B protein and huANP32B protein was shown in Table 29.

TABLE-US-00030 huANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118 chANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118 dkANP32B 143 144 163 165 74 77 101 103 107 109 126 129 132 tyANP32B 81 82 101 103 12 15 39 42 45 47 64 67 70

[0243] As demonstrated in Example 19, the mutation of amino acids in the amino acid segment 51-160 of huANP32B to alanine resulted in a complete loss of the ability of the protein to support polymerase activity, while the mutation of amino acids in the amino acid segments 41-50 and 161-170 to alanine resulted in a decrease of the ability of the protein to support polymerase activity.

[0244] As can be seen from the alignment of the amino acid sequences of the chANP32B protein and the huANP32B protein in FIG. 37, the sequences of the following amino acid segments of chANP32B are completely identical to corresponding amino acid sequences in huANP32B protein; amino acid segment 43-48, amino acid segment 50-52, amino acid segment 58-63, amino acid segment 68-72, amino acid segment 74-76, amino acid segment 78-80, amino acid segment 83-85, amino acid segment 88-99, amino acid segment 101-103, amino acid segment 105-112, amino acid segment 117-126, amino acid segment 131-136, amino acid segment 138-147 and amino acid segment 153-159, therefore, the mutation of amino acids in amino acid segments 50-52, 58-63, 68-72, 74-76, 78-80, 83-85, 88-99, 101-103, 105-112, 117-126, 131-136, 138-147 and 153-159 of the chANP32B protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segment 43-48 to alanine resulted in a decrease in the ability to support polymerase activity.

[0245] As can be seen from the alignment of the amino acid sequences of the dkANP32B protein and the huANP32B protein in FIG. 37, the sequences of the following amino acid segments of dkANP32B are completely identical to corresponding amino acid sequences in huANP32B protein; amino acid segment 57-62 (corresponding to the amino acid segment 43-48 of huANP32B), amino acid segment 70-77 (corresponding to the amino acid segment 56-63 of huANP32B), amino acid segment 82-86 (corresponding to amino acid segment 68-72 of huANP32B), amino acid segment 88-90 (corresponding to the amino acid segment 74-76 of huANP32B), amino acid segment 92-94 (corresponding to the amino acid segment 78-80 of huANP32B), amino acid segment 97-99 (corresponding to the amino acid segment 83-85 of huANP32B), amino acid segment 102-113 (corresponding to the amino acid segment 88-99 of huANP32B), amino acid segment 115-117 (corresponding to the amino acid segment 101-103 of huANP32B), amino acid segment 119-126 (corresponding to the amino acid segment 105-112 of huANP32B), amino acid segment 131-140 (corresponding to the amino acid segment 117-126 of huANP32B), amino acid segment 145-150 (corresponding to the amino acid segment 131-136 of huANP32B), amino acid segment 152-161 (corresponding to the amino acid segment 138-147 of huANP32B) and amino acid segment 166-173 (corresponding to the amino acid segments 152-159 of huANP32B), therefore, the mutation of amino acids in amino acid segments 70-77, 82-86, 88-90, 92-94, 97-99, 102-113, 115-117, 119-126, 131-140, 145-150, 152-161 and 166-173 of the dkANP32B protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segment 57-62 to alanine resulted in a decrease in the ability to support polymerase activity.

[0246] As can be seen from the alignment of the amino acid sequences of the tyANP32B protein and the huANP32B protein in FIG. 37, the sequences of the following amino acid segments of tyANP32B are completely identical to corresponding amino acid sequence in huANP32B protein: amino acid segment 10-15 (corresponding to the amino acid segment 58-63 of huANP32B), amino acid segment 20-24 (corresponding to amino acid segment 68-72 of huANP32B), amino acid segment 26-28 (corresponding to the amino acid segment 74-76 of huANP32B), amino acid segment 30-32 (corresponding to the amino acid segment 78-80 of huANP32B), amino acid segment 35-37 (corresponding to the amino acid segment 83-85 of huANP32B), amino acid segment 40-51 (corresponding to the amino acid segment 88-99 of huANP32B), amino acid segment 53-55 (corresponding to the amino acid segment 101-103 of huANP32B), amino acid segment 57-64 (corresponding to the amino acid segment 105-112 of huANP32B), amino acid segment 69-78 (corresponding to the amino acid segment 117-126 of huANP32B), amino acid segment 83-88 (corresponding to the amino acid segment 131-136 of huANP32B), amino acid segment 90-99 (corresponding to the amino acid segment 138-147 of huANP32B) and amino acid segment 105-111 (corresponding to the amino acid segments 153-159 of huANP32B), therefore, the mutation of amino acids in amino acid segments 20-24, 26-28, 30-32, 35-37, 40-51, 53-55, 57-64, 69-78, 83-88, 90-99 and 105-111 of the tyANP32B protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity.

Example 30: Alignment of Amino Acid Sequences Between Mammalian ANP32A Protein and chANP32A Protein

[0247] The amino acid sequences of mammalian ANP32A proteins (huANP32A, dogANP32A (NP_001003013.2), eqANP32A, muANP32A, pgANP32A) were compared with the amino acid sequence of chANP32A protein (see FIG. 38), wherein the corresponding relationship of amino acid positions 129, 130, 149, 151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118 between each mammalian ANP32A protein and chANP32A protein was shown in Table 30.

TABLE-US-00031 TABLE 30 huANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 dogANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 eqANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 muANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 pgANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118

[0248] As demonstrated in Example 21, the mutation of amino acids in the amino acid segment 71-180 of chANP32A to alanine resulted in a complete loss of the ability of the protein to support polymerase activity, while the mutation of amino acids in the amino acid segments 61-70 and 191-200 to alanine resulted in a decrease of the ability of the protein to support polymerase activity.

[0249] As can be seen from the alignment of the amino acid sequences of the huANP32A protein and the chANP32A protein in FIG. 38, the sequences of the following amino acid segments of huANP32A are completely identical to corresponding amino acid sequences in chANP32A: amino acid segment 41-54, amino acid segment 58-75, amino acid segment 77-102 and amino acid segment 104-170, therefore, the mutation of amino acids in amino acid segments 77-102 and 104-170 of the huANP32A protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segment 61-70 to alanine resulted in a decrease in the ability to support polymerase activity.

[0250] As can be seen from the alignment of the amino acid sequences of the eqANP32A protein and the chANP32A protein in FIG. 38, the sequences of the following amino acid segments of eqANP32A are completely identical to corresponding amino acid sequences in chANP32A: amino acid segment 41-54, amino acid segment 56-75, amino acid segment 77-102 and amino acid segment 104-169, therefore, the mutation of amino acids in amino acid segments 77-102 and 104-169 of the eqANP32A protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segment 61-70 to alanine resulted in a decrease in the ability to support polymerase activity.

[0251] As can be seen from the alignment of the amino acid sequences of the dogANP32A protein and the chANP32A protein in FIG. 38, the sequences of the following amino acid segments of dogANP32A are completely identical to corresponding amino acid sequences in chANP32A: amino acid segment 41-54, amino acid segment 56-75, amino acid segment 77-102 and amino acid segment 104-169, therefore, the mutation of amino acids in amino acid segments 77-102 and 104-169 of the dogANP32A protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segment 61-70 to alanine resulted in a decrease in the ability to support polymerase activity.

[0252] As can be seen from the alignment of the amino acid sequences of the pgANP32A protein and the chANP32A protein in FIG. 38, the sequences of the following amino acid segments of pgANP32A are completely identical to corresponding amino acid sequences in chANP32A: amino acid segment 41-54, amino acid segment 56-75, amino acid segment 77-102, amino acid segment 106-155 and amino acid segment 157-169, therefore, the mutation of amino acids in amino acid segments 77-102,106-155 and 157-169 of the pgANP32A protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segment 61-70 to alanine resulted in a decrease in the ability to support polymerase activity.

[0253] As can be seen from the alignment of the amino acid sequences of the muANP32A protein and the chANP32A protein in FIG. 38, the sequences of the following amino acid segments of muANP32A are completely identical to corresponding amino acid sequences in chANP32A: amino acid segment 41-54, amino acid segment 59-72, amino acid segment 80-90, amino acid segment 92-102, amino acid segment 104-129, amino acid segment 131-141, amino acid segment 144-151, amino acid segment 153-159 and amino acid segment 161-165, therefore, the mutation of amino acids in amino acid segments 80-90, 92-102, 104-129, 131-141, 144-151, 153-159 and 161-165 of the muANP32A protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segments 61-70 to alanine resulted in a decrease in the ability to support polymerase activity.

Example 31: Alignment of Amino Acid Sequence of Mammalian ANP32B Protein

[0254] The amino acid sequences of mammalian ANP32B proteins (huANP32B, dogANP32B (XP_013973354.1), eqANP32B(XP_023485491.1), muANP32B(NP_570959.1), pgANP32B(XP_020922136.1)) were compared (see FIG. 39), wherein the corresponding relationship of amino acid positions 129, 130, 149, 151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118 among various mammalian ANP32B proteins was shown in Table 31.

TABLE-US-00032 TABLE 31 huANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118 dogANP32B 136 137 156 158 67 70 94 97 100 102 119 122 125 eqANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118 muANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118 pgANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118

[0255] As demonstrated in Example 19, the mutation of amino acids in the amino acid segment 51-160 of huANP32B to alanine resulted in a complete loss of the ability of the protein to support polymerase activity, while the mutation of amino acids in the amino acid segments 41-50 and 161-170 to alanine resulted in a decrease of the ability of the protein to support polymerase activity.

[0256] As can be seen from the alignment of the amino acid sequences of the eqANP32B protein and the huANP32B protein in FIG. 39, the sequences of the following amino acid segments of eqANP32B are completely identical to corresponding amino acid sequences in huANP32B: amino acid segment 41-72, amino acid segment 74-112 and amino acid segment 115-170, therefore, the mutation of amino acids in amino acid segments 51-72, 74-112 and 115-160 of the eqANP32B protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segments 41-50 and 161-170 to alanine resulted in a decrease in the ability to support polymerase activity.

[0257] As can be seen from the alignment of the amino acid sequences of the pgANP32B protein and the huANP32B protein in FIG. 39, the sequences of the following amino acid segments of pgANP32B are completely identical to corresponding amino acid sequences in huANP32B: amino acid segment 41-72, amino acid segment 78-142, amino acid segment 144-150 and amino acid segment 154-169, therefore, the mutation of amino acids in amino acid segments 51-72, 78-142, 144-150 and 154-160 of the pgANP32B protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segments 41-50 and 161-169 to alanine resulted in a decrease in the ability to support polymerase activity.

[0258] As can be seen from the alignment of the amino acid sequences of the muANP32B protein and the huANP32B protein in FIG. 39, the sequences of the following amino acid segments of muANP32B are completely identical to corresponding amino acid sequences in huANP32B: amino acid segment 41-50, amino acid segment 60-81, amino acid segment 90-98, amino acid segment 100-110, amino acid segment 114-121, amino acid segment 123-127, amino acid segment 130-133, amino acid segment 138-142 and amino acid segment 144-159, therefore, the mutation of amino acids in amino acid segments 60-81, 90-98, 100-110, 114-121, 123-127, 130-133, 138-142 and 144-159 of the muANP32B protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segment 41-50 to alanine resulted in a decrease in the ability to support polymerase activity.

[0259] As can be seen from the alignment of the amino acid sequences of the dogANP32B protein and the huANP32B protein in FIG. 39, the sequences of the following amino acid segments of dogANP32B are completely identical to corresponding amino acid sequences in huANP32B: amino acid segment 48-79 (corresponding to the amino acid segment 41-72 of the huANP32B protein), amino acid segment 81-120 (corresponding to the amino acid segment 74-113 of the huANP32B protein), amino acid segment 122-159 (corresponding to the amino acid segment 115-152 of the huANP32B protein), amino acid segment 161-177 (corresponding to amino acid segment 154-170 of the huANP32B protein), therefore, the mutation of amino acids in amino acid segments 58-79, 81-120, 122-159 and 161-167 of the dogANP32B protein to alanine also resulted in a complete loss of the ability of the protein to support the polymerase activity; the mutation of amino acids in amino acid segments 48-57 and 168-177 to alanine resulted in a decrease in the ability to support polymerase activity.

Example 32: Construction of Murine ANP32B Protein Expression Vector and Mutation Vector

[0260] The nucleotide sequence of murine ANP32B (muANP32B) was shown in GenBank No. NM_130889.3, the amino acid sequence thereof was shown in GenBank No. NP_570959.1; the PCAGGS-muANP32B recombinant plasmid was constructed according to the construction method described in Example 1, and the protein expression was detected by Western blotting as the detection method described in Example 1, and the result was shown in FIG. 40.

[0261] According to the screening results of huANP32B point mutation described in Example 8, muANP32B was subjected to a point mutant construction of S129I/D130N (using primer pair of SEQ ID NO: 393 and SEQ ID NO: 394) (see Table 32 for primers, and mutated bases were underlined) by using overlapping PCR with the PCAGGS-muANP32B recombinant plasmid as a template, wherein the process was as described for the construction of a point mutant in Example 8. As described above, the obtained plasmid was named as PCAGGS-muANP32B S129I/D130N. After verification by sequencing, the plasmids were extracted in large amount for further transfection.

TABLE-US-00033 TABLE32 Primersforpointmutationof S129I/D130NonmuANP32B primername primersequence(5-3) muB_S129I/D130N_F CTAACCGGATTAACTACCGAGAAACTGTCTT SEQIDNO:393 muB_S129I/D130N_R TCGGTAGTTAATCCGGTTAGTGACCTCACA SEQIDNO:394

Example 33: Influence of Murine ANP32B Protein Mutant on Polymerase Activity

[0262] Double-knockout cell line (DKO) was plated in a 12-well plate at 310.sup.5/well and after 20 h was co-transfected with the H7N9.sub.AH13 polymerase reporter system according to the transfection system described in Example 8, and the result showed that: muANP32B S129I/D130N completely lost its support for H7N9.sub.AH13 polymerase activity as compared with muANP32B, as shown in FIG. 41.