TARGET SEQUENCE OF RNA VIRUS AND USE THEREOF

Abstract

The present invention provides a target sequence of an RNA virus. The target sequence is a nucleic acid sequence fragment in the gene sequence in the RNA virus containing 20-40 bases and having not less than 95% similarity to genome sequence of human or related species such as livestock and poultry. The above-mentioned target sequence of the RNA virus is selected from SEQ ID NO. 1 - SEQ ID NO. 615. The present invention also relates to a primer composition for constructing the above-mentioned target sequence, biomaterials such as antisense RNA related to the above-mentioned target sequence, and related uses such as design of a vaccine lacking the target sequence. The virus fragment with the above-mentioned sequence constructed in the present invention has the function of interacting with human genomic DNA and is similar to viral miRNA. Moreover, the effect of overexpression of the target sequence of the RNA virus on the expression level of surrounding genes is verified, and a new concept that the above-mentioned target fragment is an important pathogenic substance of the RNA virus is proposed. The above-mentioned target sequence has important application value for the detection and diagnosis of RNA viruses, drug screening, as well as the treatment of diseases caused by RNA viruses and the design/optimization of vaccines and methods.

Claims

1. A target sequence of an RNA virus, wherein the target sequence is a nucleic acid sequence fragment in the gene sequence in the RNA virus containing 20-40 bases and having not less than 95% similarity to human genome sequence.

2. The target sequence of the RNA virus according to claim 1, wherein the target sequence is a nucleic acid sequence fragment in the gene sequence in the RNA virus containing 20-28 bases and having 100% similarity to human genome sequence.

3. The target sequence of the RNA virus according to claim 1, wherein the RNA virus comprises severe acute respiratory syndrome-related coronavirus 2, severe acute respiratory syndrome-related coronavirus, middle east respiratory syndrome coronavirus, zika virus, ebola virus, HIV, norwalk virus, alkhurma virus, enterovirus, kemerovo virus, coxsackievirus, hepatitis A virus, dengue virus 2, rubella virus, marburg marburgvirus, poliovirus, respiratory syncytial virus, mumps virus, australian bat lyssavirus, andes virus, powassan virus, langat virus, eyach virus, colorado tick fever virus, lassa virus, omsk hemorrhagic fever virus, machupo virus, junin virus, guanarito virus, sin nombre virus, hantaan virus, puumala virus, dobrava virus, seoul virus, crimean-congo hemorrhagic fever virus, sabia virus, thogoto virus, black creek canal virus, european bat lyssavirus 1, european bat lyssavirus 2, chapare virus, rotavirus, tai forest ebolavirus, bundibugyo ebolavirus, rift valley fever virus, irkut virus, influenza A virus, bayou virus, kyasanur forest disease virus, black creek canal virus, japanese encephalitis virus, duvenhage lyssavirus, Lujo mammarenavirus, measles morbillivirus, tick-borne encephalitis virus, avian influenza virus, swine influenza virus and rabies virus.

4. The target sequence of the RNA virus according to claim 1, wherein the target sequence of the RNA virus is selected from any one or more of SEQ ID NO. 1 - SEQ ID NO. 615.

5. The target sequence of the RNA virus according to claim 4, wherein the target sequence of severe acute respiratory syndrome-related coronavirus 2 comprises SEQ ID NO. 1 - SEQ ID NO. 6; and/or, the target sequence of severe acute respiratory syndrome-related coronavirus comprises SEQ ID NO. 7 - SEQ ID NO. 9; and/or, the target sequence of middle east respiratory syndrome coronavirus comprises SEQ ID NO. 10, SEQ ID NO. 11; and/or, the target sequence of zika virus comprises SEQ ID NO. 12 - SEQ ID NO. 14; and/or, the target sequence of ebola virus comprises SEQ ID NO. 15 - SEQ ID NO. 17; and/or, the target sequence of HIV comprises SEQ ID NO. 18 - SEQ ID NO. 26; and/or, the target sequence of norwalk virus comprises SEQ ID NO. 27; and/or, the target sequence of alkhurma virus comprises SEQ ID NO. 28 - SEQ ID NO. 30; and/or, the target sequence of enterovirus comprises SEQ ID NO. 31, SEQ ID NO. 32; and/or, the target sequence of kemerovo virus comprises SEQ ID NO. 33, SEQ ID NO. 34; and/or, the target sequence of coxsackievirus comprises SEQ ID NO. 35; and/or, the target sequence of hepatitis A virus comprises SEQ ID NO. 36 - SEQ ID NO. 46; and/or, the target sequence of dengue virus 2 comprises SEQ ID NO. 47 - SEQ ID NO. 50; and/or, the target sequence of rubella virus comprises SEQ ID NO. 51; and/or, the target sequence of marburg marburgvirus comprises SEQ ID NO. 52 - SEQ ID NO. 56; and/or, the target sequence of poliovirus comprises SEQ ID NO. 57; and/or, the target sequence of respiratory syncytial virus comprises SEQ ID NO. 58 - SEQ ID NO. 85; and/or, the target sequence of mumps virus comprises SEQ ID NO. 86; and/or, the target sequence of australian bat lyssavirus comprises SEQ ID NO. 87; and/or, the target sequence of andes virus comprises SEQ ID NO. 88 - SEQ ID NO. 95; and/or, the target sequence of powassan virus comprises SEQ ID NO. 96, SEQ ID NO. 97; and/or, the target sequence of langat virus comprises SEQ ID NO. 98 - SEQ ID NO. 102; and/or, the target sequence of eyach virus comprises SEQ ID NO. 103 - SEQ ID NO. 113; and/or, the target sequence of colorado tick fever virus comprises SEQ ID NO. 114 - SEQ ID NO. 134; and/or, the target sequence of lassa virus comprises SEQ ID NO. 135, SEQ ID NO. 136; and/or, the target sequence of omsk hemorrhagic fever virus comprises SEQ ID NO. 137, SEQ ID NO. 138; and/or, the target sequence of machupo virus comprises SEQ ID NO. 139 - SEQ ID NO. 140; and/or, the target sequence of junin virus comprises SEQ ID NO. 141; and/or, the target sequence of guanarito virus comprises SEQ ID NO. 142 - SEQ ID NO. 147; and/or, the target sequence of sin nombre virus comprises SEQ ID NO. 148 - SEQ ID NO. 152; and/or, the target sequence of hantaan virus comprises SEQ ID NO. 153 - SEQ ID NO. 161; and/or, the target sequence of puumala virus comprises SEQ ID NO. 162 - SEQ ID NO. 173; and/or, the target sequence of dobrava virus comprises SEQ ID NO. 174 - SEQ ID NO. 185; and/or, the target sequence of seoul virus comprises SEQ ID NO. 186 - SEQ ID NO. 199; and/or, the target sequence of crimean-congo hemorrhagic fever virus comprises SEQ ID NO. 200 - SEQ ID NO. 204; and/or, the target sequence of sabia virus comprises SEQ ID NO. 205 - SEQ ID NO. 212; and/or, the target sequence of thogoto virus comprises SEQ ID NO. 213 - SEQ ID NO. 227; and/or, the target sequence of european bat lyssavirus 1 comprises SEQ ID NO. 228 - SEQ ID NO. 232; and/or, the target sequence of european bat lyssavirus 2 comprises SEQ ID NO. 233; and/or, the target sequence of chapare virus comprises SEQ ID NO. 234; and/or, the target sequence of rotavirus comprises SEQ ID NO. 235 - SEQ ID NO. 277; and/or, the target sequence of tai forest ebolavirus comprises SEQ ID NO. 278, SEQ ID NO. 279; and/or, the target sequence of bundibugyo ebolavirus comprises SEQ ID NO. 280; and/or, the target sequence of rift valley fever virus comprises SEQ ID NO. 281; and/or, the target sequence of irkut virus comprises SEQ ID NO. 282 - SEQ ID NO. 285; and/or, the target sequence of influenza A virus comprises SEQ ID NO. 286 - SEQ ID NO. 313; and/or, the target sequence of bayou virus comprises SEQ ID NO. 314 - SEQ ID NO. 327; and/or, the target sequence of kyasanur forest disease virus comprises SEQ ID NO. 328; and/or, the target sequence of black creek canal virus comprises SEQ ID NO. 329 - SEQ ID NO. 334; and/or, the target sequence of japanese encephalitis virus comprises SEQ ID NO. 335 - SEQ ID NO. 337; and/or, the target sequence of duvenhage lyssavirus comprises SEQ ID NO. 338 - SEQ ID NO. 344; and/or, the target sequence of Lujo mammarenavirus comprises SEQ ID NO. 345; and/or, the target sequence of measles morbillivirus comprises SEQ ID NO. 346; and/or, the target sequence of tick-borne encephalitis virus comprises SEQ ID NO. 347; and/or, the target sequence of avian influenza virus comprises SEQ ID NO. 348 - SEQ ID NO. 420; and/or, the target sequence of swine influenza virus comprises SEQ ID NO. 421 - SEQ ID NO. 521; and/or, the target sequence of rabies virus comprises SEQ ID NO. 522 - SEQ ID NO. 615.

6. A primer composition for constructing a target sequence of an RNA virus, characterized in that, the primers of the target sequence SEQ ID NO. 1 are SEQ ID NO. 616 - SEQ ID NO. 619; and/or, the primers of the target sequence SEQ ID NO. 2 are SEQ ID NO. 620 - SEQ ID NO. 623; and/or, the primers of the target sequence SEQ ID NO. 3 are SEQ ID NO. 624 - SEQ ID NO. 627; and/or, the primers of the target sequence SEQ ID NO. 4 are SEQ ID NO. 628 - SEQ ID NO. 631; and/or, the primers of the target sequence SEQ ID NO. 5 are SEQ ID NO. 632 - SEQ ID NO. 635; and/or, the primers of the target sequence SEQ ID NO. 7 are SEQ ID NO. 636 - SEQ ID NO. 639; and/or, the primers of the target sequence SEQ ID NO. 8 are SEQ ID NO. 640 - SEQ ID NO. 643; and/or, the primers of the target sequence SEQ ID NO. 10 are SEQ ID NO. 644 - SEQ ID NO. 647; and/or, the primers of the target sequence SEQ ID NO. 11 are SEQ ID NO. 648 - SEQ ID NO. 651; and/or, the primers of the target sequence SEQ ID NO. 12 are SEQ ID NO. 652 - SEQ ID NO. 655; and/or, the primers of the target sequence SEQ ID NO. 13 are SEQ ID NO. 656 - SEQ ID NO. 659; and/or, the primers of the target sequence SEQ ID NO. 14 are SEQ ID NO. 660 - SEQ ID NO. 663; and/or, the primers of the target sequence SEQ ID NO. 15 are SEQ ID NO. 664 - SEQ ID NO. 667; and/or, the primers of the target sequence SEQ ID NO. 16 are SEQ ID NO. 668 - SEQ ID NO. 671; and/or, the primers of the target sequence SEQ ID NO. 17 are SEQ ID NO. 672 - SEQ ID NO. 675; and/or, the primers of the target sequence SEQ ID NO. 18 are SEQ ID NO. 676 - SEQ ID NO. 679; and/or, the primers of the target sequence SEQ ID NO. 19 are SEQ ID NO. 680 - SEQ ID NO. 683; and/or, the primers of the target sequence SEQ ID NO. 20 are SEQ ID NO. 684 - SEQ ID NO. 687; and/or, the primers of the target sequence SEQ ID NO. 21 are SEQ ID NO. 688 - SEQ ID NO. 691; and/or, the primers of the target sequence SEQ ID NO. 22 are SEQ ID NO. 692 - SEQ ID NO. 695; and/or, the primers of the target sequence SEQ ID NO. 23 are SEQ ID NO. 696 - SEQ ID NO. 699; and/or, the primers of the target sequence SEQ ID NO. 24 are SEQ ID NO. 700 - SEQ ID NO. 703; and/or, the primers of the target sequence SEQ ID NO. 25 are SEQ ID NO. 704 - SEQ ID NO. 707; and/or, the primers of the target sequence SEQ ID NO. 26 are SEQ ID NO. 708 - SEQ ID NO. 711.

7. An RNA drug against a virus, wherein the RNA drug comprises the reverse complementary sequence of the target sequence of the RNA virus according to claim 1, cholesterol modification and four phosphorothioate backbone modifications are made at the 3′ end of the reverse complementary sequence of the target sequence of the RNA virus, two phosphorothioate backbone modifications are made at the 5′ end, and methoxy modification is made on the whole chain, or, cholesterol modification and four phosphorothioate backbone modifications are made at the 3′ end of the target sequence of the RNA virus, or two phosphorothioate backbone modifications are made at the 5′ end, and methoxy modification is made on the whole chain.

8. The RNA drug according to claim 7, wherein the reverse complementary sequence of the target sequence of the RNA virus comprises reverse complementary RNA sequence or reverse complementary DNA sequence.

9. The RNA drug according to claim 7, further comprising a pharmaceutically acceptable carrier or excipient.

10. The RNA drug against the virus according to claim 7, wherein the dosage form of the RNA drug comprises powder, tablet, granule, capsule, solution, aerosol, injection, emulsion or suspension.

11. A biomaterial related to the target sequence of the RNA virus according to claim 1, wherein the biomaterial is : A) a DNA and/or RNA molecule that is complementary and paired to the target sequence of the RNA virus according to claim 1; or B) an expression cassette, a recombinant vector, a recombinant microorganism, a recombinant cell line containing the target sequence of the RNA virus according to claim 1 or the DNA molecule in A).

12. The biomaterial according to claim 11, wherein the biomaterial is a recombinant vector, and whose construction steps comprise: 1) designing a primer, and amplifying the target sequence of the RNA virus by PCR; 2) digesting the amplified sequence fragment and an expression vector, and ligating a sequence fragment of interest and the expression vector; 3) transferring the ligated product into Escherichia coli and cultivating the Escherichia coli; and 4) after identification, extracting recombinant plasmid and packaging the recombinant plasmid.

13. The biomaterial according to claim 11, wherein the recombinant vector has target sequences expressing severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome-related coronavirus (SARS-CoV), or middle east respiratory syndrome coronavirus (MERS-CoV).

14. A method of preventing or treating a condition caused by the RNA virus comprising administering the RNA virus according to claim 1 to a subject in need thereof.

15. The method according to claim 14, wherein the condition comprise a human disease, an animal disease and zoonosis.

16. The method according to claim 14, wherein the RNA virus is administered as a vaccine and an effective substance that regulates the target sequence is directly screened; alternatively, according to the effect of the gene regulated by the target sequence, an effective substance against the gene and gene product regulated by the target sequence is screened.

17. The method according to claim 14, wherein the target sequence is knocked out.

18. The method according to claim 17, wherein the method for knocking out the target sequence comprises: CRISPR system and/or ribozyme technology.

19. The method according to claim 16, wherein the vaccine is a live attenuated vaccine.

20. A live attenuated vaccine, wherein the target sequence of the RNA virus according to claim 1 is deleted or mutated in the whole genome of the live attenuated vaccine.

21. A method of activating related genes at the cellular level and screening therapeutic drugs against the related genes comprising contacting the RNA virus according to claim 1 to genes.

22. The method according to claim 21, wherein the RNA virus is a coronavirus, and the related genes comprise ACE2 gene, the coding genes of the hyaluronic acid synthase family HAS1, HAS2, and HAS3, and/or genes within 200 k around the fragment.

23. (canceled)

24. A method of identifying drug targets against diseases caused by an RNA virus comprising analyzing the target sequence of the RNA virus according to claim 1 which is found in the cells of the diseases caused by the RNA virus, and the drug targets are found within 200 k around the target sequence of the RNA virus or the drug targets are found beyond 200 k using the prediction software blast 2.2.30 or bedtools 2.29.2.

25. The method according to claim 23, wherein the drug comprises a miRNA antagonist.

26. A method for detecting a virus, comprising detecting the target sequence of the RNA virus according to of claim 1.

27. The method according to claim 26, wherein the detection of the target sequence comprises PCR amplification and nucleotide sequencing.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a running gel electropherogram of 6 target viral vectors related to the coronavirus SARS-CoV-2 amplified by PCR in an embodiment of the present invention.

[0066] FIG. 2 is a schematic diagram of the result of the mRNA level after overexpression of the target fragments of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗∗, p<0.001.

[0067] FIG. 3 is a schematic diagram of the result of the mRNA level of the gene ACE2 after overexpression of the target fragments of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01, .sup.∗∗∗, p<0.001.

[0068] FIG. 4 is a schematic diagram of the result of the mRNA level of the gene HAS1 after overexpression of the target fragments of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01, .sup.∗∗∗, p<0.001.

[0069] FIG. 5 is a schematic diagram of the result of the mRNA level of the gene HAS2 after overexpression of the target fragments of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01.

[0070] FIG. 6 is a schematic diagram of the result of the mRNA level of the gene HAS3 after overexpression of the target fragments of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01, .sup.∗∗∗, p<0.001.

[0071] FIG. 7 is a schematic diagram of the result of the mRNA level of the surrounding gene FBXO15 after overexpression of the target fragment SARS-CoV-2-HIS-4 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗∗, p<0.001.

[0072] FIG. 8 is a schematic diagram of the result of the mRNA level of the surrounding gene MYL9 after overexpression of the target fragment SARS-CoV-2-HIS-3 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗∗, p<0.001.

[0073] FIG. 9 is a schematic diagram of the result of the mRNA level of the surrounding gene ATP8B1 after overexpression of the target fragment SARS-CoV-2-HIS-1 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01.

[0074] FIG. 10 is a schematic diagram of the result of the mRNA level of the surrounding gene KALRN after overexpression of the target fragment SARS-CoV-2-HIS-5 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01.

[0075] FIG. 11 is a schematic diagram of the result of the mRNA level of the surrounding genes after overexpression of the target fragment SARS-CoV-2-HIS-6 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention.

[0076] FIG. 12 is a schematic diagram of the result of the mRNA level of the surrounding genes after overexpression of the target fragment SARS-CoV-HIS-2 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01..sup.∗∗∗, p<0.001.

[0077] FIG. 13 is a schematic diagram of the result of the mRNA level of the surrounding gene after overexpression of the target fragment MERS-CoV-HIS-2 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention; wherein, .sup.∗∗, p<0.01.

[0078] FIG. 14 is a schematic diagram of the result of the mRNA level of the surrounding genes after overexpression of target fragments of zika virus in 293T cells by qPCR detection in an embodiment of the present invention.

[0079] FIG. 15 is a schematic diagram of the result of the mRNA level of the surrounding genes after overexpression of target fragments of ebola virus in 293T cells by qPCR detection in an embodiment of the present invention.

[0080] FIG. 16 is a schematic diagram of the result of the mRNA level of the surrounding genes after overexpression of HIV-2 target fragments in 293T cells by qPCR detection in an embodiment of the present invention.

[0081] FIG. 17 is a schematic diagram of the result of antagomir on the mRNA level of the surrounding genes after overexpression of the target fragment SARS-CoV-HIS-2 of coronavirus in 293T cells by qPCR detection in an embodiment of the present invention.

[0082] FIG. 18 is a schematic diagram of the result of the inhibitory effect of antagomir on the mRNA level of genes activated by the target fragment MERS-CoV-HIS-2 of coronavirus by qPCR detection in an embodiment of the present invention; wherein, .sup.∗<0.05.

[0083] FIG. 19 is a schematic diagram of the result of the inhibitory effect of antagomir on the mRNA level of genes activated by the target fragment SARS-CoV-2-HIS-4 of coronavirus by qPCR detection in an embodiment of the present invention; wherein, .sup.∗<0.05.

[0084] FIG. 20 is a schematic diagram of the result of the inhibitory effect of antagomir on the mRNA level of genes activated by the target fragment SARS-CoV-2-HIS-3 of coronavirus by qPCR detection in an embodiment of the present invention; wherein, .sup.∗<0.05.

DETAILED DESCRIPTION OF THE INVENTION

[0085] The specific implementations of the present invention will be further described below in conjunction with the drawings and examples. The following examples are only used to illustrate the technical solutions of the present invention more clearly, and cannot be used to limit the scope of protection of the present invention. In experimental methods in the following examples where no specific conditions are indicated, choices can be made according to conventional methods and conditions in the art or commodity instructions; the relevant reagents and biomaterials in the following examples are all commercially available products; The molecular cloning technology in the following examples provides a method for purifying and amplifying specific DNA fragments at the molecular level in the prior art. The coronavirus, zika virus, ebola virus and HIV are mainly used as examples for discussion in the following examples.

Example 1 - Construction of an Overexpression Vector of the Target of the RNA Virus

[0086] This example is the construction of an overexpression vector of the target of the RNA virus, and the steps comprise: [0087] 1. Sequence acquisition and primer design SARS-CoV-2 gene sequences were found from Nucleotide database Genbank of NCBI, and then the whole genome nucleotide sequences of the virus were Blast-aligned with the whole genome sequence in human, and finally, the virus nucleotide sequence fragments with a similarity of not less than 95% were screened as viral RNA target sequences (hereinafter referred to as targets). 5 sequences that were completely complementary and paired to the human genome and 1 sequence that was not completely complementary to human genes were screened from SARS-CoV-2. For zika virus, ebola virus, HIV, SARS-CoV, MERS-CoV, and other RNA viruses, the same method was used to obtain target sequences. The screened target sequences are shown in Table 1 above. The upstream and downstream primers were determined using primer5 software, respectively, and the protective base and EcoRI restriction site sequence (CGGAATTC) were added to 5′ end of the upstream primer, and the protective base and BamHI restriction site sequence (CGGGATCC) were added to 5′ end of the downstream primer. The primers were synthesized by Shanghai Sunny Biotechnology Co., Ltd. The primer sequences of some targets are shown in Table 3 above. [0088] 2. Obtainment of the target fragment sequence of interest of the RNA virus Taking severe acute respiratory syndrome-related coronavirus 2 target sequence as an example, the viral target fragment was artificially synthesized by means of homologous recombination. After the primers of F123 and R1 designed according to the sequence were annealed, two rounds of nested PCR were performed using F123 and R2 and F123 and R3, and the gene fragments of interest were amplified with Q5 enzyme. The amplification system and program were as follows:

TABLE-US-00004 PCR system Total volume 50 .Math.l 5 × Reaction buffer 10 .Math.l dNTPs (10 mM) 1 .Math.l Upstream primer (10 .Math.M) 2.5 .Math.l Downstream primer (10 .Math.M) 2.5 .Math.l cDNA template 1 .Math.l Q5 polymerase 0.5 .Math.l ddH2O 32.5 .Math.l

[0089] PCR program: 98° C. for 30 s; [0090] 98° C. for 10 s, 55-72° C. for 30 s, 72° C. for 30 s/kb, 35 cycles; and 72° C. for 2 min. For severe acute respiratory syndrome-related coronavirus and middle east respiratory syndrome coronavirus, F1 and R1 primers were used to anneal, and then F2 and R2 and the annealed product were subjected to nested PCR to obtain the fragments of interest. [0091] 3. Recovery, restriction digestion and purification of PCR products The PCR products were detected by electrophoresis in 1% agarose gel, the gel was cut and recovered, and the fragments of interest were recovered using a ordinary agarose gel DNA recovery kit (Tiangen Biotech Co., Ltd.); the enzyme digestion process referred to the enzyme digestion system on NEB website, and the enzyme digestion was carried out at 37° CoVemight, and a PCR product recovery kit (Tiangen Biotech Co., Ltd.) was used for purification and recovery. [0092] 4. Ligation The digested PCR product and the digested pCDH vector were ligated with T4 ligase according to the following ligation system at 16° CoVemight.

TABLE-US-00005 Ligation system Reagents Volume PCR product 1 .Math.l Digested pCDH vector 1 .Math.l T4DNA ligase buffer 1 .Math.l T4DNA ligase 1 .Math.l H.sub.2O 6 ul Total 10 ul

[0093] 5. Transforming and picking monoclonal ligation [0094] (1) 10 .Math.l of ligation product was added to 50 .Math.l of DH5α competent cells, and incubated on ice for 30 min. [0095] (2) The competent cells were heat shocked at 42° C. for 90 s, and then immediately placed on ice for 5 min. [0096] (3) 300 .Math.l of LB liquid medium without antibiotics was added on a clean bench, and the bacteria was shaken on a constant temperature shaker at 37° C. for 30 min. [0097] (4) 1000 g of bacterial solution was centrifuged for 5 min and the supernatant was discarded. The remaining 50 .Math.l of bacterial solution was spread evenly on the LB solid plate supplemented with ampicillin, and the plate was incubated in a constant temperature incubator at 37° CoVemight. [0098] (5) An appropriate amount of monoclonal colonies was picked from the overnight-cultured plate, and put into EP tubes containing 200 .Math.l of LB liquid medium supplemented with ampicillin. The bacteria were shaken in a constant temperature shaker at 37° C. for 2 hours, and then subjected to sequencing and identification. Finally, the target band can be obtained by vector PCR (FIG. 1).

[0099] The results showed that: The length of each target-vector is200-250 bp. FIG. 1 shows the electrophoresis results of the target-vectors containing 6 targets from severe acute respiratory syndrome-related coronavirus 2, respectively. Specifically, HIS1 is the target-vector containing SARS-CoV-2-HIS-1, and HIS2 is the target-vector containing SARS-CoV-2-HIS-2, HIS3 is the target-vector containing SARS-CoV-2-HIS-3, HIS4 is the target-vector containing SARS-CoV-2-HIS-4, HIS5 is the target-vector containing SARS-CoV-2-HIS-5, and HIS6 is the target-vector containing SARS-CoV-2-HIS-6.

[0100] The same operation as above applied to SARS-CoV, MERS-CoV, zika virus, ebola virus and HIV.

[0101] Example 2 The effect of overexpression of the target sequences of the RNA virus in cells on the expression level of surrounding genes In this example, the effect of the overexpression of the target sequences of the RNA virus in 293T cells on the expression level of surrounding genes was detected. The steps are briefly described as follows: [0102] 1. Preparation of lentivirus by liposome method: According to molecular cloning, SARS-CoV-2, SARS-CoV, MERS-CoV overexpression plasmid, virus packaging plasmid psPAX2 and capsid plasmid pMD2.G-VSVG were transferred into 293T cells, and the supernatant was collected after 48 hr and 72 hr, respectively. The cell debris was filtered through a 0.45 .Math.m filter to obtain the lentivirus stock solution. [0103] 2. Cell infection: 200,000 cells to be infected (lentiviral stock solution) was spread in a 6 cm culture dish in advance, after the cells adhered on the second day, the first infection was carried out, and the infection was repeated again on the third day; on the fourth day, the cells were allowed to recover for one day without adding any stimulation; on the fifth day, drug screening was started to perform based on corresponding markers carried by the plasmid that reduce the potency of the drug. [0104] 3. Real-time fluorescence quantitative PCR

Total RNA Extraction

[0105] 10.sup.6-10.sup.7 cells were prepared, resuspended in PBS, and then centrifuged to remove the supernatant, 1 ml of Trizol was added for lysis at room temperature for 5 min, then 0.2 ml of chloroform was added. The mixture was shaken in a vortex shaker for 15 s, and left to stand at room temperature for 2 min. The mixture was centrifuged in a centrifuge at 4° C. for 15 min at 13,300 rpm. The upper colorless water phase was transferred into another EP tube. An equal volume of isopropanol was added, mixed thoroughly in a vortex shaker, and the mixture was centrifuged in a centrifuge at 4° C. at 13,300 rpm for 10 min. The supernatant was discarded, and 1 ml of 75% ethanol prepared with DEPC water was added, turned upside down until the precipitate was suspended, and centrifuged in a centrifuge at 4° C. at 13.300 rpm for 5 min. The supernatant was aspirated with a pipette, during the period of drying at room temperature for 5-20 min, the morphology of the precipitation was observed. When just being transparent, 40-100 .Math.l of DEPC water was used for dissolution according to the amount of precipitation. 1 .Math.l was taken and the concentration and OD260/OD280 was measured on Nanodrop. The extracted RNA was stored in a refrigerator at -80° C.

Reverse Transcription Synthesis of cDNA

[0106] Takara (D2680A) reverse transcription PCR kit was used, the PCR reaction system and program were as follows:

TABLE-US-00006 Reverse transcription PCR system Total volume 20 .Math.l 5 × PrimeScript Buffer 4 .Math.l dNTP Mixture (2.5 mM each) 4 .Math.l Random 6 mers (100 .Math.M) 1 .Math.l OligodT Primer (50 .Math.M) 1 .Math.l PrimeScript Reverse Transcriptase (200 U/.Math.l) 0.5 .Math.l RNase Inhibitor (40 U/.Math.l) 0.5 .Math.l Total RNA 1 .Math.g RNase Freed H2O up to 20 .Math.l

Reverse transcription PCR program: 42° C. for 10 min, 95° C. for 2 min.

RT-qPCR

[0107] The expression of the gene of interest at the transcription level was detected using Takara real-time fluorescent quantitative PCR kit.

TABLE-US-00007 Real-time fluorescence quantitative PCR system Total volume 10 .Math.l Sybr Green Mix 5 .Math.l Forward (10 .Math.m) 1 .Math.l Reverse (10 um) 1 .Math.l cDNA 3 .Math.l

[0108] Experimental results: After overexpression of the target sequence fragment, the expression level of the fragment was up-regulated tens of thousands of times (FIG. 2). Specifically, the ACE2 gene, which is very related to the coronavirus, was activated after overexpression of the SARS-CoV-HIS, SARS-COV-2-HIS-3 and SARS-COV-2-HIS-4 fragments (FIG. 3). The HAS1 (FIG. 4), HAS2 (FIG. 5) and HAS3 (FIG. 6) genes of the hyaluronic acid synthase family related to severe acute respiratory syndrome-related coronavirus 2 were also significantly activated by SARS-CoV-HIS, MERS-CoV-HIS, SARS-COV-2-HIS-3 and SARS-COV-2-HIS-4 fragments. Finally, it can be seen from the detection that the genes within 200 k around the SARS-COV-2-HIS-4 (FIG. 7), SARS-COV-2-HIS-3 (FIG. 8), SARS-COV-2-HIS-1 (FIG. 9) and SARS-COV-2-HIS-5 (FIG. 10) fragments were all significantly activated. The same results were obtained for the fragment SARS-CoV-2-HIS-6, which was not completely complementary (FIG. 11). The specific genes comprised: FBXO15, MYL9, KALRN, ATP8B1, C5AR1, EPAS1, etc. The same results were also obtained for SARS-COV-HIS-2 (FIG. 12) and MERS-COV-HIS-2 (FIG. 13).

[0109] Specifically, the expression of the gene IGF2R around the target fragment of SARS virus was increased, and the expression of the gene IGF2R around the target fragment of MERS virus was increased. In addition, zika virus (FIG. 14), ebola virus (FIG. 15), and HIV-2 (FIG. 16) were also detected in this example, and the results were also the same, specifically: after overexpression of the target fragment of zika virus in 293T cells, the expression of surrounding 16 genes such as CNMD and VPS36 was increased; after overexpression of the target fragment of ebola virus in 293T cells, the expression of surrounding 15 genes such as VGLL4 and TAMM41 was all increased; after overexpression of HIV target fragment in 293T cells, the expression of surrounding genes BMP5, MMP1 and ADCYAP1 was increased; after overexpression of HIV2 target fragment in 293T cells, the expression of surrounding 8 genes such as LAPTM4A and LRRC14B was increased.

[0110] The above results prove that the constructed vector plays a certain function in the expression of miRNA related to SARS-CoV-2, and provides a research basis for subsequent research.

Example 3 - Inhibitory Effect of miRNA Inhibitor (antagomiR) or Antisense Sequence For the Target of the RNA Virus on Activated Target Genes

[0111] This example verifies the inhibitory effect of the inhibitor antagomir for the target of the RNA virus on activated target genes, and comprises the following steps: [0112] step one: preparation of the inhibitor antagomir for the viral target: cholesterol modification and four phosphorothioate backbone modifications were made at the 3′ end of the reverse complementary sequence of the target sequence of the RNA virus, two phosphorothioate backbone modifications were made at the 5′ end, and methoxy modification was made on the whole chain to obtain the corresponding inhibitor antagomir for the target of the virus. [0113] step two: the virus stock solution was prepared by the method of example 2, and the cells were infected with the virus stock solution. The infected cells were divided into two groups: an experimental group and a control group, wherein the experimental group was: 10 .Math.M of virus-infected cell solution added with corresponding inhibitor for the viral target; the control group was: 10 .Math.M of virus-infected cell solution. After 48 hours, the cell solution of the experimental group and the control group were tested according to the method of real-time fluorescent quantitative PCR in example 4.

[0114] The results of the test were shown in FIGS. 18-21. The inhibitor for the viral target can specifically inhibit the replication of the target sequence, and the antagomir can target the target sequence well, so that the surrounding genes activated by SARS-CoV-HIS-2 (FIG. 17), MERS-CoV -HIS-2 (FIG. 18), SARS-CoV-2-HIS-4 (FIG. 19) and SARS-CoV-2-HIS-3 (FIG. 20) shown a significant tendency to decrease, further verifying the therapeutic value of targets in RNA virus.

[0115] This experiment further verified the inhibitory effect of the reverse complementary sequence of the target sequence of the RNA virus (comprising antisense DNA sequence and antisense RNA sequence), as well as cholesterol modification and four phosphorothioate backbone modifications made at the 3′ end of the target sequence of the RNA virus, two phosphorothioate backbone modifications made at the 5′ end, and methoxy modification made on the whole chain as an inhibitor on the activated target genes was verified. The test results were similar to that of the inhibitor antagomiR. It can be seen that the above-mentioned three inhibitors all had an inhibitory effect on activated target genes. Antisense RNA or antisense DNA of the target sequence of the RNA virus can be used to inhibit RNA virus nucleic acid and block important pathogenic pathways of RNA virus. The different modified or unmodified products of the antisense RNA or antisense DNA provided an important material basis for the treatment of RNA virus diseases. The detailed sequences of the antisense RNA or antisense DNA are shown in Table 2.

Example 4 - The Increase in Hyaluronic Acid Affected by the Target Can Be Reduced by the Hyaluronic Acid Inhibitor 4-MU

[0116] This example verifies that the increase in hyaluronic acid affected by the target can be reduced by the hyaluronic acid inhibitor 4-MU and comprises the following steps:

[0117] the lentivirus and infected cells were prepared by the method of example 2;

[0118] Replacement with the fresh medium was performed, 100 .Math.M of hyaluronic acid inhibitor 4-MU was added in the experimental group, and DMSO (the solvent for 4-MU) was added in the control group. After 24 hours, the cell supernatant was collected and detected with hyaluronic acid ELISA kit (R&D, DY3614-05). The steps are briefly described as follows: [0119] 1) Coating ELISA plate: The plate was coated with 100 .Math.l/well of Capture Reagent overnight. [0120] 2) Sealing: The Capture Reagent was removed by patting the plate. The plate was washed 3 times with 400 .Math.l/wellof Wash buffer and patted to dryness. The plate was sealed with 100 .Math.l/wel/welll of Dilute Reagent for 1 h. [0121] 3) Washing the plate and incubating the sample: The plate was washed with 400 .Math.l/well of Wash buffer 3 times, 100 .Math.l/well of standard and serum to be tested were added (100 .Math.l of the serum from patients with mild and severe COVID-19 was diluted with 200 .Math.l of Dilute Reagent in the kit to a total volume of 300 .Math.l, 3 replicate wells were made), and incubated at room temperature for 2 h. [0122] 4) Washing the plate and incubation with the Detect Reagent. The plate was washed with 400 .Math.l/well of Wash buffer 3 times, 100 .Math.l/well of Detect Reagent was added and incubated at room temperature for 2 h. [0123] 5) Washing the plate and incubation with HRP. The plate was washed with 400 .Math.l/wellof Wash buffer 3 times, 100 .Math.l/well of HRP was added and incubated at room temperature for 20 min. [0124] 6) Washing the plate and incubation with the substrate. The plate was washed with 400 .Math.l/well of Wash buffer 3 times, 100 .Math.l/well of mixed solution of substrates A and B was added and incubated at room temperature for 20 min. [0125] 7) Stopping color development. 50 .Math.l/well of stop solution was added.

[0126] Absorbance was read at 450 nm within 15 min. The test results are shown in Table 4 and Table 5: After overexpression of the target sequence of the virus in cell lines 293T and MRC5, the hyaluronic acid content was significantly increased (Table 4). The hyaluronic acid produced due to overexpression of the target sequence can be reduced using hyaluronic acid inhibitor 4-MU (Table 5). This example proves that the target of the virus has scientific research value and 4-MU has the potential to become a therapeutic drug targeting the target and has a therapeutic value for complications related to the target of the RNA virus.

TABLE-US-00008 Determination of hyaluronic acid content in 293T and MRC5 cells in which the target of the virus is overexpressed 293T Hyaluronic acid (ng/ml) p value p value MRC5 CTRL 7.39±0.26 - 59.55±4.73 - HIS-MERS-CoV-2 76.91±2.29 ∗∗ 106.97±4.69 ∗∗ HlS-SARS-CoV-1-2 115.60+18.10 ∗∗ 116.84±1.52 ∗∗ HIS-SARS-CoV-2-3 62.66±7.14 ∗∗ 72.40±8.75 ns HlS-SARS-CoV-2-4 113.95±13.14 ∗∗ 117.44±2.03 ∗∗

TABLE-US-00009 Determination of the inhibitory ability of hyaluronic acid inhibitor on hyaluronic acid in the case of overexpression of the target of the virus Hyaluronic acid (ng/ml) p value DMSO 4-MU (100 .Math.M) CTRL 7.39±0.26 3.20±0.39 ∗∗ HIS-MERS-CoV-2 76.91±2.29 39.72±5.75 ∗∗ HIS-SARS-CoV-1-2 115.60±18.10 23.50±3.44 ∗∗ HIS-SARS-CoV-2-3 62.68±7.14 30.02±2.00 ∗∗ H IS-SARS-CoV-2-4 113.95±13.14 19.76±11.3 ∗

Example 5 - Detection of Blood Routine Index

[0127] The blood routine index was provided by the hospital, and the hyaluronic acid in the blood was detected using the hyaluronic acid ELISA kit (R&D, DY3614-05). Specifically, the HA content in the serum of a patient with severe COVID-19 was significantly increased compared with that in a patient with mild COVID-19 (Table 6). In addition, the number of lymphocytes in a patient with severe COVID-19 was significantly lower than that in a patient with mild COVID-19, suggesting that the number of the immune cells in a patient was decreased with the disease progressing to severe; furthermore, D-dimer is a fibrin degradation product, and the increase of D-dimer level indicates the existence of hypercoagulable state and secondary hyperfibrinolysis in the body. Therefore, the mass concentration of D-dimer has diagnostic significance for thrombotic diseases. The content of D-dimer in the serum of a patient with severe COVID-19 was significantly higher than that in a patient with mild COVID-19, indicating that the risk of coagulation in a patient was increased with the condition of COVID-19 progressing to severe, and also indicating that there was a certain feasibility of subsequent anticoagulation therapy.

TABLE-US-00010 Hematological indicators of a patient with mild or severe COVID-19 HA (ng/ml) LYMPH# (10^9/L) CRP (mg/L) D-D (ug/ml) Mild (n=37) 3.77±2.86 1.79±0.50 0.77±0.68 0.28±0.12 Severe (n=22) 35.41±28.88∗∗∗ 1.40±0.43∗∗ 8.49±9.66∗∗∗ 0.49±0.36∗

[0128] The above results provide a basis for the changes in hematological indicators caused by the target sequences of the RNA virus to become clinical diagnosis, and reflects the clinical diagnostic value of the targets of the RNA virus. Moreover, the targets have the potential to become a vaccine. In addition, in the process of preparing vaccines, common attenuated live vaccines still have certain risks that need to be further optimized. The pathogenic risk of a vaccine will be greatly reduced by knockout of the targets.

[0129] The specific examples of the present invention are described in detail above and are only for illustration, and the present invention is not limited to the specific examples described above. For a person skilled in the art, any equivalent modifications and alternatives made to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should fall within the scope of the present invention.