Polypeptide carrier for presenting target polypeptide and uses thereof

Abstract

The invention relates to a polypeptide carrier for presenting a target polypeptide, and use thereof. In particular, the invention relates to a nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide carrier, and being used for insertion of a nucleotide sequence encoding a target polypeptide. In addition, the invention further relates to a recombinant protein comprising the polypeptide carrier and a target polypeptide. Furthermore, the invention further relates to use of the nucleic acid molecule and the recombinant protein. In addition, the invention further relates to a vaccine or a pharmaceutical composition useful for preventing, alleviating or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B), comprising a recombinant protein comprising the polypeptide carrier of the invention and an epitope from HBV.

Claims

1. A nucleic acid molecule, comprising: a nucleotide sequence encoding a polypeptide carrier, or a variant thereof, wherein the variant has at least 90% identity to the nucleotide sequence, or is capable of hybridizing to the nucleotide sequence under a stringent condition or a high stringent condition, and wherein the polypeptide carrier is selected from the group consisting of: (1) RBHBcAg carrier, which differs from a core antigen protein from roundleaf bat HBV (RBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 1 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the RBHBcAg protein are deleted; (2) TBHBcAg carrier, which differs from a core antigen protein from tent-making bat HBV (TBHBcAg protein) in that (a) 1 to 5 amino acid residues corresponding to the amino acid residues at positions 80-84 of SEQ ID NO: 2 are deleted or substituted with a linker, and (b) optionally, 1-35 amino acid residues at C-terminus of the TBHBcAg protein are deleted; and (3) HBHBcAg carrier, which differs from a core antigen protein from horseshoe bat HBV (HBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 3 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the HBHBcAg protein are deleted.

2. A vector, comprising the nucleic acid molecule of claim 1.

3. A host cell, comprising the nucleic acid molecule of claim 1 or a vector comprising the nucleic acid molecule.

4. A method for presenting a target polypeptide, comprising: (1) inserting a nucleotide sequence encoding the target polypeptide into the nucleic acid molecule of claim 1 to obtain a nucleic acid molecule encoding a recombinant protein, wherein the nucleotide sequence encoding the target polypeptide is inserted at a position of nucleotides encoding the deleted amino acid residues, inserted in the nucleotide sequence encoding the linker, or inserted at one or both termini of the nucleotide sequence encoding the linker; and (2) expressing the nucleic acid molecule encoding the recombinant protein to produce the recombinant protein.

5. A recombinant protein, comprising: a polypeptide carrier; and a target polypeptide, wherein the polypeptide carrier is selected from the group consisting of: (1) RBHBcAg carrier, which differs from a core antigen protein from roundleaf bat HBV (RBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 1 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the RBHBcAg protein are deleted; (2) TBHBcAg carrier, which differs from a core antigen protein from tent-making bat HBV (TBHBcAg protein) in that (a) 1 to 5 amino acid residues corresponding to the amino acid residues at positions 80-84 of SEQ ID NO: 2 are deleted or substituted with a linker, and (b) optionally, 1-35 amino acid residues at C-terminus of the TBHBcAg protein are deleted; and (3) HBHBcAg carrier, which differs from a core antigen protein from horseshoe bat HBV (HBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 3 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the HBHBcAg protein are deleted, and wherein the target polypeptide is inserted at a position of the deleted amino acid residues, inserted in the linker, or inserted at one or both of the termini of the linker.

6. A virus-like particle, comprising the recombinant protein of claim 5.

7. A pharmaceutical composition, comprising: the recombinant protein of claim 5 or a virus-like particle comprising the recombinant protein; and at least one pharmaceutically acceptable vehicle or excipient.

8. A polynucleotide, encoding the recombinant protein of claim 5.

9. A vector, comprising the polynucleotide of claim 8.

10. A host cell, comprising the polynucleotide of claim 8 or a vector comprising the polynucleotide.

11. A method for preparing the recombinant protein of claim 5, comprising: culturing a host cell comprising a polynucleotide encoding the recombinant protein under a condition suitable for expressing the recombinant protein; and recovering the recombinant protein.

12. The nucleic acid molecule of claim 1, wherein at least one of (i) to (xi) is satisfied: (i) the RBHBcAg protein has the amino acid sequence of SEQ ID NO: 1; (ii) the RBHBcAg carrier differs from the RBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 78-82 of SEQ ID NO: 1 are deleted or substituted with a linker, and (b) optionally, 5-35 amino acid residues at C-terminus of the RBHBcAg protein are deleted; (iii) the TBHBcAg protein has the amino acid sequence of SEQ ID NO: 2; (iv) the TBHBcAg carrier differs from the TBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 80-83 of SEQ ID NO: 2 are deleted or substituted with a linker, and (b) optionally, 5-30 amino acid residues at C-terminus of the TBHBcAg protein are deleted; (v) the HBHBcAg protein has the amino acid sequence of SEQ ID NO: 3; (vi) the HBHBcAg carrier differs from the HBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 78-82 of SEQ ID NO: 3 are deleted or substituted with a linker, and (b) optionally, 5-35 amino acid residues at C-terminus of the HBHBcAg protein are deleted; (vii) the linker is a flexible linker; (viii) a restriction enzyme cleavage site is introduced at a position of nucleotides encoding the amino acid residues that are deleted; (ix) a restriction enzyme cleavage site is introduced in the nucleotide sequence encoding the linker, and/or at either or both of the termini thereof; (x) the nucleic acid molecule is used to insert a nucleotide sequence encoding a target polypeptide; and (xi) the nucleic acid molecule further comprises a nucleotide sequence encoding a target polypeptide, wherein the target polypeptide is heterologous relative to the polypeptide carrier, and the nucleotide sequence encoding the target polypeptide is inserted at a position of nucleotides encoding the deleted amino acid residues, inserted in the nucleotide sequence encoding the linker, or inserted at one or both termini of the nucleotide sequence encoding the linker.

13. The nucleic acid molecule of claim 1, wherein the polypeptide carrier has the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8 or 9, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 12, 13, 14, 15, 16 or 17.

14. The method of claim 4, wherein the target polypeptide is an epitope peptide, and optionally, the epitope peptide satisfies at least one of (i) to (iii): (i) the epitope peptide comprises an epitope of HBsAg from human HBV, or an epitope of HIV GP120 protein, or an epitope of human PD-L1; (ii) the epitope peptide comprises amino acids corresponding to the amino acids at positions 113-135 of SEQ ID NO: 44 (HBsAg protein), the amino acids at positions 361-375 of an amino acid sequence of HIV GP120 protein, or the amino acids at positions 147-160 of an amino acid sequence of human PD-L1 protein; and (iii) the epitope peptide has the amino acid sequence of SEQ ID NO: 20, 21, 22, 60, 61, or 62.

15. The recombinant protein of claim 5, wherein at least one of (i) to (x) is satisfied: (i) the RBHBcAg protein has the amino acid sequence of SEQ ID NO: 1; (ii) the RBHBcAg carrier differs from the RBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 78-82 of SEQ ID NO: 1 are deleted or substituted with a linker, and (b) optionally, 5-35 amino acid residues at C-terminus of the RBHBcAg protein are deleted; (iii) the TBHBcAg protein has the amino acid sequence of SEQ ID NO: 2; (iv) the TBHBcAg carrier differs from the TBHBcAg protein in that (a) amino acid residues from corresponding to the amino acid residues at positions 80-83 of SEQ ID NO: 2 are deleted or substituted with a linker, and (b) optionally, 5-30 amino acid residues at C-terminus of the TBHBcAg protein are deleted; (v) the HBHBcAg protein has the amino acid sequence of SEQ ID NO: 3; (vi) the HBHBcAg carrier differs from the HBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 78-82 of SEQ ID NO: 3 are deleted or substituted with a linker, and (b) optionally, 5-35 amino acid residues at C-terminus of the HBHBcAg protein are deleted; (vii) the linker is a flexible linker; (viii) the polypeptide carrier has the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, or 9; (ix) the target polypeptide is an epitope peptide, and optionally, the epitope peptide comprises an epitope of HBsAg from human HBV, an epitope of HIV GP120 protein, or an epitope of human PD-L1, or comprises amino acids at positions 113-135 of an amino acid sequence of HBsAg protein, amino acids at positions 361-375 of an amino acid sequence of HIV GP120 protein, or amino acids at positions 147-160 of an amino acid sequence of human PD-L1 protein, or comprises the amino acid sequence of SEQ ID NO: 20, 21, 22, 60, 61, or 62; and (x) the recombinant protein comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOS: 23-40 and 69-74.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a scheme of cloning solutions in which recombinant proteins are constructed by inserting a target polypeptide (a target antigen peptide fragment) into RBHBcAg carrier, TBHBcAg carrier and HBHBcAg carrier of the invention.

(2) FIG. 2 shows the SDS-PAGE results of 18 recombinant proteins constructed in Example 2, and the Transmission Electron Microscope (TEM) results of the virus-like particles formed by the recombinant proteins.

(3) FIG. 3 shows changes in titer of antibodies against the target polypeptides in the recombinant proteins in mouse sera over time, after the immunization of BALB/C mice with the virus-like particles formed by 18 recombinant proteins constructed in Example 2. Longitudinal axis: antibody titer (log 10); horizontal axis: time (week). FIG. 3A: the target polypeptide used was SEQ ID NO: 20, and the titer of anti-GP120 antibodies was determined; FIG. 3B: the target polypeptide used was SEQ ID NO: 21, and the titer of anti-PD-L1 antibodies was determined; FIG. 3C: the target polypeptide used was SEQ ID NO: 22, and the titer of anti-HBsAg antibodies was determined.

(4) FIG. 4 shows changes in HBsAg level in mouse sera over time, after the treatment of HBV transgenic male (FIG. 4A) and female (FIG. 4B) mice with different virus-like particles presenting the same epitope peptide (SEQ ID NO: 22). Longitudinal axis: HBsAg level (IU/ml); horizontal axis: time (week). The arrows indicate the time points of administering virus-like particles to mice.

(5) FIG. 5 shows changes in HBV DNA level in mouse sera over time, after the treatment of HBV transgenic male mice with different virus-like particles presenting the same epitope peptide (SEQ ID NO: 22). Longitudinal axis: HBV DNA level (Log 10 IU/ml); horizontal axis: time (week). The arrows indicate the time points of administering virus-like particles to mice.

(6) FIG. 6 shows changes in titer of anti-HBsAg antibodies in mouse sera over time, after the treatment of HBV transgenic male (FIG. 6A) and female (FIG. 6B) mice with different virus-like particles presenting the same epitope peptide (SEQ ID NO: 22). Longitudinal axis: the titer of anti-HBsAg antibodies; horizontal axis: time (week).

(7) FIG. 7 shows the TEM results of the virus-like particles formed by 6 recombinant proteins constructed in Example 5.

(8) FIG. 8 shows the titers of antibodies against the corresponding target polypeptides (SEQ ID NO: 60, 22, 61, and 62) in mouse sera, three weeks after the immunization of BALB/C mice with the virus-like particles formed by 8 recombinant proteins; wherein the epitope peptide (SEQ ID NO: 60) of HBsAg protein from HBV genotype A was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ60 and TBHBcAg153-SEQ60; the epitope peptide (SEQ ID NO: 22) of HBsAg protein from HBV genotype B was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ22 and TBHBcAg153-SEQ22; the epitope peptide (SEQ ID NO: 61) of HBsAg protein from HBV genotype C was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ61 and TBHBcAg153-SEQ61; and the epitope peptide (SEQ ID NO: 62) of HBsAg protein from HBV genotype D was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ62 and TBHBcAg153-SEQ62. The results show that all the virus-like particles formed by the 8 recombinant proteins have good immunogenicity, and can induce generation of high-titer antibodies that specifically bind to target antigens in mice.

(9) FIG. 9 shows changes in HBsAg level in mouse sera, after the treatment of HBV transgenic male (FIG. 9A) and female (FIG. 9B) mice with the virus-like particles formed by 4 recombinant proteins (SEQ ID NO: 36, 69, 70, and 71), wherein, longitudinal axis: HBsAg level (IU/ml); horizontal axis: time (week).

SEQUENCE INFORMATION

(10) Information on a part of sequences (SEQ ID NO: 1-44) involved in the invention is provided in the following Table 1.

(11) TABLE-US-00001 TABLE1 SequenceinformationofSEQIDNO:1-44 SEQIDNO Name Sequenceinformation 1 RBHBcAg MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGPLIQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQ PTVIEFLVSFGTWIRTPQAYRPPNAPILSTLPEHTIVRRRGGSRATRSPRRRTPSP RRRRSQSPRRRRSQSPASSNC 2 TBHBcAg MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGLSPDADALLAGYLRSKYLKHITKAIWYHLSCL TFGKQTVHEYLVSFGTWIRTPAAYRPVNAPILTTLPETSVIRRRPASRRSTPSPRR RRSQSPRRRRSPSPRPASNC 3 HBHBcAg MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGQPVQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQ QTVIEFLVSFGTWMRTPAAYRPPNAPILSTLPEHTVIRRRGNPRAPRSPRRRTPSP RRRRSQSPRRRRSQSPAPSNC 4 RBHBcAg189 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIVQQVQA SVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAYRPPNAPILSTLPEHT IVRRRGGSRATRSPRRRTPSPRRRRSQSPRRRRSQSPASSNC 5 RBHBcAg149 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIVQQVQA SVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAYRPPNAPILSTLPEHT IV 6 TBHBcAg188 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSEFGGGGSGGGGSDADALLAG YLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRTPAAYRPVNAPILTTL PETSVIRRRPASRRSTPSPRRRRSQSPRRRRSPSPRPASNC 7 TBHBcAg153 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSEFGGGGSGGGGSDADALLAG YLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRTPAAYRPVNAPILTTL PETSVI 8 HBHBcAg189 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIIGYVQT TVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAYRPPNAPILSTLPEHT VIRRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSPAPSNC 9 HBHBcAg149 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIIGYVQT TVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAYRPPNAPILSTLPEHT VI 10 HBcAg183 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALR QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSEFGGGGSGGGGSRELVVSYVNV NMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPQNAPILSTLPETT VVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 11 HBcAg149 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALR QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSEFGGGGSGGGGSRELVVSYVNV NMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPQNAPILSTLPETT VV 12 RBHBcAg189 ATGGACATTGATCCTTATAAAGAATTTGGAGCTTCATCTCAACTGATCTCTTTCTT GCCTGAGGACTTTTTCCCAAACCTTGCAGAATTGGTCGAGACCACCACAGCTCTCT ATGAAGAAGAATTAGTAGGTAAGGAGCATTGCTCCCCTCACCATACTGCTTTACGA TCCTTGCTAAATTGCTGGGGAGAGACTGTTAGATTAATAACTTGGGTCAGGAACTC TGTGGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAATTCGGTG GTGGAGGTTCAGGAGGAGGTGGTTCCCAAGATGCCATTGTCCAGCAAGTTCAGGCC TCGGTGGGCCTGCGCATGAGACAGTTAATGTGGTTCCATCTCTCATGCCTAACATT TGGACAGCCCACTGTCATAGAATTTCTGGTCTCTTTTGGAACATGGATCAGAACCC CGCAAGCTTACAGACCCCCTAATGCACCCATTCTCTCGACTCTTCCGGAGCATACA ATCGTTAGGAGAAGAGGAGGTTCACGCGCTACTAGGTCCCCCCGAAGGCGCACTCC CTCTCCTCGCCGACGCAGATCTCAATCGCCGCGTCGCCGCAGATCTCAGTCTCCAG CTTCCTCCAACTGCTAA 13 RBHBcAg149 ATGGACATTGATCCTTATAAAGAATTTGGAGCTTCATCTCAACTGATCTCTTTCTT GCCTGAGGACTTTTTCCCAAACCTTGCAGAATTGGTCGAGACCACCACAGCTCTCT ATGAAGAAGAATTAGTAGGTAAGGAGCATTGCTCCCCTCACCATACTGCTTTACGA TCCTTGCTAAATTGCTGGGGAGAGACTGTTAGATTAATAACTTGGGTCAGGAACTC TGTGGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAATTCGGTG GTGGAGGTTCAGGAGGAGGTGGTTCCCAAGATGCCATTGTCCAGCAAGTTCAGGCC TCGGTGGGCCTGCGCATGAGACAGTTAATGTGGTTCCATCTCTCATGCCTAACATT TGGACAGCCCACTGTCATAGAATTTCTGGTCTCTTTTGGAACATGGATCAGAACCC CGCAAGCTTACAGACCCCCTAATGCACCCATTCTCTCGACTCTTCCGGAGCATACA ATCGTT 14 TBHBcAg188 ATGGAAAACCTTGAAAGACTTGACATCTATAAAGAATTTGGAGTCTCTGATGTCTT GGTGTCTTTCTTACCTGATGATTTCTTTCCAACTTTACAGCAACTTTTGGAATCAG TGAATGCCCTATATGAGGATGAACTCACTGGGCCTAATCACTGTTCTCCCCATCAT ACTGCCTTAAGGCACTTGATTATGTGTGGGGTAGAATTAAGAGATTTTATTGATTG GATGCATGAACAGGGGGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAAT TCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCGATGCAGACGCTCTTTTGGCTGGT TACCTTCGATCCAAATATCTTAAACATATTACCAAGGCTATTTGGTATCATTTAAG CTGTTTGACCTTTGGTAAGCAAACAGTGCATGAATACCTGGTATCCTTTGGCACCT GGATCAGAACCCCAGCTGCATATAGACCAGTGAATGCACCCATTCTCACCACTCTT CCGGAAACTTCAGTTATCAGAAGAAGGCCTGCCTCCAGAAGATCTACTCCCTCTCC TCGCAGACGCCGATCTCAATCACCGCGCCGCCGCCGCTCTCCATCTCCAAGACCAG CAAGCAATTGCTGA 15 TBHBcAg153 ATGGAAAACCTTGAAAGACTTGACATCTATAAAGAATTTGGAGTCTCTGATGTCTT GGTGTCTTTCTTACCTGATGATTTCTTTCCAACTTTACAGCAACTTTTGGAATCAG TGAATGCCCTATATGAGGATGAACTCACTGGGCCTAATCACTGTTCTCCCCATCAT ACTGCCTTAAGGCACTTGATTATGTGTGGGGTAGAATTAAGAGATTTTATTGATTG GATGCATGAACAGGGGGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAAT TCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCGATGCAGACGCTCTTTTGGCTGGT TACCTTCGATCCAAATATCTTAAACATATTACCAAGGCTATTTGGTATCATTTAAG CTGTTTGACCTTTGGTAAGCAAACAGTGCATGAATACCTGGTATCCTTTGGCACCT GGATCAGAACCCCAGCTGCATATAGACCAGTGAATGCACCCATTCTCACCACTCTT CCGGAAACTTCAGTTATC 16 HBHBcAg189 ATGGACATTGATCCTTATAAAGAGTTCGGTGCTTCATCTCAACTTGTCTCCTTTTT GCCTGCTGACTTCTTTCCCGCCTTGAACGACCTGGTGGAAACTTCGGTGGCCTTAT ATGAGGAAGACCTTGTAGGTAAGGAGCATTGCTCCCCTCATCATGCAGCCTTAAGG GCCCTACTTAATTGCTGGGAGGAAACAGTCAGACTGATTACCTGGGTCCGTGCCAC AGTAGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAATTCGGTG GTGGAGGTTCAGGAGGAGGTGGTTCCCAGGATGCCATCATCGGTTATGTCCAGACT ACGGTGGGCCTACGCATGAGACAACAGATCTGGTTCCATCTCTCATGCCTTACTTT TGGGCAGCAGACTGTGATAGAGTTCCTGGTCTCATTTGGGACATGGATGAGAACTC CAGCCGCCTATAGACCCCCCAATGCACCCATTTTATCAACTCTTCCAGAGCACACA GTCATTAGGAGAAGAGGAAATCCGCGTGCTCCTAGGTCCCCCAGAAGGCGCACTCC CTCTCCTCGCCGACGCAGATCTCAATCTCCGCGTCGCCGGAGATCTCAATCTCCAG CTCCCTCCAACTGCTAA 17 HBHBcAg149 ATGGACATTGATCCTTATAAAGAGTTCGGTGCTTCATCTCAACTTGTCTCCTTTTT GCCTGCTGACTTCTTTCCCGCCTTGAACGACCTGGTGGAAACTTCGGTGGCCTTAT ATGAGGAAGACCTTGTAGGTAAGGAGCATTGCTCCCCTCATCATGCAGCCTTAAGG GCCCTACTTAATTGCTGGGAGGAAACAGTCAGACTGATTACCTGGGTCCGTGCCAC AGTAGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAATTCGGTG GTGGAGGTTCAGGAGGAGGTGGTTCCCAGGATGCCATCATCGGTTATGTCCAGACT ACGGTGGGCCTACGCATGAGACAACAGATCTGGTTCCATCTCTCATGCCTTACTTT TGGGCAGCAGACTGTGATAGAGTTCCTGGTCTCATTTGGGACATGGATGAGAACTC CAGCCGCCTATAGACCCCCCAATGCACCCATTTTATCAACTCTTCCAGAGCACACA GTCATT 18 HBcAg183 ATGGACATTGATCCATATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTT GCCTTCCGACTTCTTTCCTTCTATCCGAGATCTCCTCGACACCGCCTCTGCTCTGT ATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACGGCACTCAGG CAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTAGCCACCTGGGTGGGAAGTAA TTTGGAAGATGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAATTCGGTG GTGGAGGTTCAGGAGGAGGTGGTTCCAGGGAACTAGTAGTCAGCTATGTCAACGTT AATATGGGCCTAAAAATCAGACAACTATTGTGGTTTCACATTTCCTGTCTTACTTT TGGGAGAGAAACTGTTCTTGAATATTTGGTGTCTTTTGGAGTGTGGATTCGCACTC CTCCTGCATATAGACCACAAAATGCCCCTATCTTATCAACACTTCCGGAAACTACT GTTGTTCGTCGCCGAGGCCGTAGCCCGCGACGACGTACCCCGAGCCCGCGTCGACG TCGCAGCCAGAGCCCGCGCCGTCGTCGCAGCCAGAGCCGTGAAAGCCAGTGCTAA 19 HBcAg149 ATGGACATTGATCCATATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTT GCCTTCCGACTTCTTTCCTTCTATCCGAGATCTCCTCGACACCGCCTCTGCTCTGT ATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACGGCACTCAGG CAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTAGCCACCTGGGTGGGAAGTAA TTTGGAAGATGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGGATCCGAATTCGGTG GTGGAGGTTCAGGAGGAGGTGGTTCCAGGGAACTAGTAGTCAGCTATGTCAACGTT AATATGGGCCTAAAAATCAGACAACTATTGTGGTTTCACATTTCCTGTCTTACTTT TGGGAGAGAAACTGTTCTTGAATATTTGGTGTCTTTTGGAGTGTGGATTCGCACTC CTCCTGCATATAGACCACAAAATGCCCCTATCTTATCAACACTTCCGGAAACTACT GTTGTT 20 HIV-GP120-aa FKQSSGGDPEIVTHS 361-375 21 hPDL1-aa147-160 TSEHELTCQAEGYP 22 HBsAg-aa113-135 SSTTSTGPCKTCTTPAQGTSMFP 23 RBHBcAg189-SEQ20 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGGGGS GGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAY RPPNAPILSTLPEHTIVRRRGGSRATRSPRRRTPSPRRRRSQSPRRRRSQSPASSN C 24 RBHBcAg149-SEQ20 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGGGGS GGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAY RPPNAPILSTLPEHTIV 25 TBHBcAg188-SEQ20 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGGG GSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRT PAAYRPVNAPILTTLPETSVIRRRPASRRSTPSPRRRRSQSPRRRRSPSPRPASNC 26 TBHBcAg153-SEQ20 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGGG GSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRT PAAYRPVNAPILTTLPETSVI 27 HBHBcAg189-SEQ20 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGGGGS GGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAY RPPNAPILSTLPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSPAPSN C 28 HBHBcAg149-SEQ20 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGGGGS GGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAY RPPNAPILSTLPEHTVI 29 RBHBcAg189-SEQ21 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGGGSG GGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAYR PPNAPILSTLPEHTIVRRRGGSRATRSPRRRTPSPRRRRSQSPRRRRSQSPASSNC 30 RBHBcAg149-SEQ21 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGGGSG GGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAYR PPNAPILSTLPEHTIV 31 TBHBcAg188-SEQ21 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGGG SGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRTP AAYRPVNAPILTTLPETSVIRRRPASRRSTPSPRRRRSQSPRRRRSPSPRPASNC 32 TBHBcAg153-SEQ21 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGGG SGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRTP AAYRPVNAPILTTLPETSVI 33 HBHBcAg189-SEQ21 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGGGSG GGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAYR PPNAPILSTLPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSPAPSNC 34 HBHBcAg149-SEQ21 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGGGSG GGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAYR PPNAPILSTLPEHTVI 35 RBHBcAg189-SEQ22 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTSMF PEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGT WIRTPQAYRPPNAPILSTLPEHTIVRRRGGSRATRSPRRRTPSPRRRRSQSPRRRR SQSPASSNC 36 RBHBcAg149-SEQ22 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTSMF PEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGT WIRTPQAYRPPNAPILSTLPEHTIV 37 TBHBcAg188-SEQ22 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTS MFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLV SFGTWIRTPAAYRPVNAPILTTLPETSVIRRRPASRRSTPSPRRRRSQSPRRRRSP SPRPASNC 38 TBHBcAg153-SEQ22 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTS MFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLV SFGTWIRTPAAYRPVNAPILTTLPETSVI 39 HBHBcAg189-SEQ22 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTSMF PEFGGGGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGT WMRTPAAYRPPNAPILSTLPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQSPRRRR SQSPAPSNC 40 HBHBcAg149-SEQ22 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAALR ALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTSMF PEFGGGGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGT WMRTPAAYRPPNAPILSTLPEHTVI 41 HBcAg183-SEQ22 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALR QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTSMF PEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGV WIRTPPAYRPQNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRE SQC 42 HBcAg149-SEQ22 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALR QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTSMF PEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGV WIRTPPAYRPQNAPILSTLPETTVV 43 Linker GGGGGSGGGGTGSEFGGGGSGGGGS 44 HBsAg MENIASGLLGPLLVLQAGFFLLTKILTIPQSLDSWWTSLNFLGGTPVCLGQNSQSQ ISSHSPTCCPPICPGYRWMCLRRFIIFLCILLLCLIFLLVLLDYQGMLPVCPLIPG SSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASV RFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWFWGPSLYNILSPFMPLLPIFFCLWV YI

(12) Specific Modes for Carrying Out the Invention

(13) The invention is illustrated by reference to the following examples (which are intended to describe the invention rather than limiting the protection scope of the present invention).

(14) Unless indicated otherwise, the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes are used under the conditions recommended by manufacturers of the products. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the present invention.

EXAMPLE 1

Construction of Plasmids Encoding Polypeptide Carriers

(15) In the Example, plasmids encoding polypeptide carriers were constructed.

(16) 1.1 Preparation of Nucleotide Sequences Encoding Polypeptide Carriers

(17) Based on three bat-derived HBV core antigens (i.e., RBHBcAg protein, TBHBcAg protein, and HBHBcAg protein), the following polypeptide carriers were designed:

(18) RBHBcAg189 carrier, which differs from RBHBcAg protein (SEQ ID NO: 1) in that the amino acid residues from positions 78-81 of RBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of RBHBcAg189 carrier is set forth in SEQ ID NO: 4, and the nucleotide sequence of RBHBcAg189 carrier is set forth in SEQ ID NO: 12;

(19) TBHBcAg188 carrier, which differs from TBHBcAg protein (SEQ ID NO: 2) in that the amino acid residues from positions 80-83 of RBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of TBHBcAg188 carrier is set forth in SEQ ID NO: 6, the nucleotide sequence of TBHBcAg188 carrier is set forth in SEQ ID NO: 14;

(20) HBHBcAg189 carrier, which differs from HBHBcAg protein (SEQ ID NO: 3) in that the amino acid residues from positions 78-81 of RBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of HBHBcAg189 carrier is set forth in SEQ ID NO: 8, and the nucleotide sequence of HBHBcAg189 carrier is set forth in SEQ ID NO: 16.

(21) In addition, based on HBcAg protein of human HBV, HBcAg183 carrier was also designed, as a control. HBcAg183 carrier differs from HBcAg protein of human HBV in that the amino acid residues from positions 79-81 of HBcAg protein of human HBV are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of HBcAg183 carrier is set forth in SEQ ID NO: 10, and the nucleotide sequence of HBcAg183 carrier is set forth in SEQ ID NO: 18.

(22) With respect to the nucleotide sequences of said four carriers, their whole gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd.

(23) 1.2 Preparation of Plasmids Encoding Polypeptide Carriers

(24) By using the synthesized nucleotide sequences as templates, and using the primers in Table 2, the full-length genes and truncates (i.e., gene fragments truncated at C-terminus) of said 4 carriers were amplified by PCR, respectively. 8 PCR products were obtained, i.e., the gene encoding RBHBcAg189 carrier (SEQ ID NO: 12; the amino acid sequence encoded thereby is SEQ ID NO: 4), the gene encoding RBHBcAg149 carrier (SEQ ID NO: 13; the amino acid sequence encoded thereby is SEQ ID NO: 5), the gene encoding TBHBcAg188 carrier (SEQ ID NO: 14; the amino acid sequence encoded thereby is SEQ ID NO: 6), the gene encoding TBHBcAg153 (SEQ ID NO: 15; the amino acid sequence encoded thereby is SEQ ID NO: 7), the gene encoding HBHBcAg189 carrier (SEQ ID NO: 16; the amino acid sequence encoded thereby is SEQ ID NO: 8), the gene encoding HBHBcAg149 carrier (SEQ ID NO: 17; the amino acid sequence encoded thereby is SEQ ID NO: 9), the gene encoding HBcAg183 carrier (SEQ ID NO: 18; the amino acid sequence encoded thereby is SEQ ID NO: 10), and the gene encoding HBcAg149 carrier (SEQ ID NO: 19; the amino acid sequence encoded thereby is SEQ ID NO: 11).

(25) pTO-T7 vector (Luo Wenxin, Zhang Jun, Yang Haijie, et al., Construction and Application of an Escherichia coli High Effective Expression Vector with an Enhancer [J], Chinese Journal of Biotechnology, 2000, 16(5): 578-581) was subjected to double enzyme digestion by NdeI and HindIII, to obtain a linear vector. By Gibson assembly cloning method (New England Biolabs (UK) Ltd), 8 PCR products obtained were ligated to the linear vector, and transformed into DH5a competent bacteria. The transformed bacteria were spread on a plate and cultured, monoclonal colonies were then selected, and the plasmids were extracted and sequenced. It was confirmed by sequencing that 8 plasmids comprising the nucleotide sequences encoding the polypeptide carriers were obtained.

(26) The primers involved in the PCR are shown in Table 2.

(27) TABLE-US-00002 TABLE2 Primersequences SEQIDNO: Primername Sequence 45 RBHBcAg149/189F ACTTTAAGAAGGAGATATACATATGATGGACATTGATCCTTATAAAG 46 RBHBcAg149R GTGGTGCTCGAGGCGGCCGCAAGCTTTTAAACGATTGTATGCTCCGGAAG AGTCGA 47 RBHBcAg189R GTGGTGCTCGAGGCGGCCGCAAGCTTTTAGCAGTTGGAGGAAGCTGGAGA CTGAGATCTGCGGCGAC 48 TBHBcAg153/188F ACTTTAAGAAGGAGATATACATATGATGGAAAACCTTGAAAGACTTG 49 TBHBcAg153R GTGGTGCTCGAGGCGGCCGCAAGCTTTTAGATAACTGAAGTTTCCGGAAG AGTG 50 TBHBcAg188R GTGGTGCTCGAGGCGGCCGCAAGCTTTTAGCAATTGCTTGCTGGTCTTG 51 HBHBcAg149/189F ACTTTAAGAAGGAGATATACATATGATGGACATTGATCCTTATAAAG 52 HBHBcAg149R GTGGTGCTCGAGGCGGCCGCAAGCTTTTAAATGACTGTGTGCTCTGGAAG AGTTGA 53 HBHBcAg189R GTGGTGCTCGAGGCGGCCGCAAGCTTTTAGCAGTTGGAGGGAGCTGGAGA TTGAGATCTCCGGCGAC

EXAMPLE 2

Preparation of Recombinant Proteins

(28) In the Example, a nucleotide sequence encoding a target polypeptide was inserted into the plasmid constructed in Example 1, and a recombinant protein comprising the target polypeptide and the polypeptide carrier was obtained. The scheme of cloning solutions, in which recombinant proteins are constructed by inserting a target polypeptide (a target antigen peptide fragment) into RBHBcAg carrier, TBHBcAg carrier and HBHBcAg carrier of the invention, is shown in FIG. 1.

(29) 2.1 Construction of Expression Plasmids of Recombinant Proteins Comprising a Target Polypeptide and a Polypeptide Carrier

(30) In the Example, 3 target polypeptides were used to verify the versatility of the polypeptide carrier of the invention for presenting peptide fragments. Said 3 target polypeptides were: polypeptide HIV-GP120-aa361-375 (i.e., the amino acids from positions 361-375 of HIV GP120 protein, its amino acid sequence is set forth in SEQ ID NO: 20); polypeptide hPDL1-aa147-160 (i.e., the amino acids from positions 147-160 of human PD-L1 protein, its amino acid sequence is set forth in SEQ ID NO: 21); and polypeptide HBsAg-aa113-135 (i.e., the amino acids from positions 113-135 of hepatitis B surface antigen (HBsAg) from human HBV, its amino acid sequence is set forth in SEQ ID NO: 22).

(31) The sense and antisense sequences coding said 3 target polypeptides (as shown in Table 3) were synthesized directly, and annealed, so as to obtain the gene fragments having cohesive end and encoding the target polypeptides.

(32) TABLE-US-00003 TABLE3 Senseandantisensesequencescoding3targetpolypeptides SEQIDNO: Primername Sequence 54 hPDL1-aa147-160F GATCCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCG 55 hPDL1-aa147-160R AATTCGGGGTAGCCCTCAGCCTGACATGTCAGTTCATGTTCAGAGGTG 56 HIV-GP120-aa361-375F GATCCTTCAAACAGTCTTCTGGTGGTGACCCGGAAATCGTTACCCACTCTG 57 HIV-GP120-aa361-375R AATTCAGAGTGGGTAACGATTTCCGGGTCACCACCAGAAGACTGTTTGAAG 58 HBsAg-aa113-135F GATCCTCATCAACAACCAGCACCGGACCATGCAAAACCTGCACAACTCCTG CTCAAGGAACCTCTATGTTTCCCG 59 HBsAg-aa113-135R AATTCGGGAAACATAGAGGTTCCTTGAGCAGGAGTTGTGCAGGTTTTGCAT GGTCCGGTGCTGGTTGTTGATGAG

(33) The 6 plasmids (RBHBcAg189, RBHBcAg149, TBHBcAg188, TBHBcAg153, HBHBcAg189 and HBHBcAg149) obtained in Example 1 were subjected to double enzyme digestion by BamHI and EcoRI, to obtain 6 linear vectors. Then, the 3 gene fragments having cohesive end and encoding the target polypeptides, as prepared above, were ligated to the linear vectors, to obtain the expression plasmids encoding recombinant proteins (18 in total: RBHBcAg189-SEQ20, RBHBcAg149-SEQ20, TBHBcAg188-SEQ20, TBHBcAg153-SEQ20, HBHBcAg189-SEQ20, HBHBcAg149-SEQ20, RBHBcAg189-SEQ21, RBHBcAg149-SEQ21, TBHBcAg188-SEQ21, TBHBcAg153-SEQ21, HBHBcAg189-SEQ21, HBHBcAg149-SEQ21, RBHBcAg189-SEQ22, RBHBcAg149-SEQ22, TBHBcAg188-SEQ22, TBHBcAg153-SEQ22, HBHBcAg189-SEQ22, and HBHBcAg149-SEQ22).

(34) 2.2 Expression, purification and assembly of recombinant proteins The 18 expression plasmids constructed in the previous step, were used to express and purify the recombinant proteins encoded by the expression plasmids via the same method. RBHBcAg149-SEQX (SEQX represents SEQ20, SEQ21 or SEQ22) was used as an example to describe the expression and purification of the recombinant proteins.

(35) (2.2.1) Preparation of bacterial strains for expressing recombinant proteins: the expression plasmid RBHBcAg149-SEQX obtained in 2.1 was transformed into E. coli strain ER2566, so as to obtain the expression bacterial strain.

(36) (2.2.2) Expression of the recombinant protein RBHBcAg149-SEQX: the expression bacterial strain was seeded in a 500 mL triangular flask, and was cultured at 37 C. on a shaking table until OD was about 1.0; later, isopropyl-beta-D-thiogalactoside (IPTG) was added at a final concentration of 0.5 mM, and the expression was further performed at 25 C. for 6 h.

(37) (2.2.3) Purification of the recombinant protein RBHBcAg149-SEQX:

(38) (2.2.3.1) Ultrasonic disruption of bacteria: the bacteria in 2.2.2 were harvested by centrifugation, and were subjected to ultrasonic disruption. Sonication buffer: 20 mM phosphate buffer (PH6.0)+300 mM NaCl.

(39) (2.2.3.2) Primary purification of the recombinant protein: the mixture obtained after ultrasonic disruption was incubated in a 65 C. water bath for 30 min, and the supernatant was then collected by centrifugation; saturated ammonium sulfate was added to the supernatant at a volume ratio of 1:1, and the precipitate was collected by centrifugation; a suitable volume of buffer (20 mM phosphate buffer (pH=7.4)+150 mM NaCl) was added to resuspend the precipitate, so as to obtain the primarily purified recombinant protein RBHBcAg149-SEQX.

(40) (2.2.3.3) Purification of the recombinant protein by chromatography: in accordance with the instructions of manufacturer, the protein obtained in 2.2.3.2 was further purified by Sepharose 4FF(GE) molecular sieve column chromatography, so as to obtain the purified recombinant protein. The purified target protein was detected by SDS-PAGE, and the VLP formed by the recombinant protein was observed by Transmission Electron Microscope (TEM).

(41) FIG. 2 shows the SDS-PAGE results of the 18 recombinant proteins as constructed, and the TEM results of the virus-like particles formed by the recombinant proteins. The results show that all the 18 recombinant proteins as obtained had a purity of above 85%, and could be assembled into virus-like particles with a diameter of about 30 nm. These results show that the polypeptide carriers constructed in the invention have a broad versatility, can be used to present various target polypeptides, and can form VLPs.

EXAMPLE 3

Evaluation on Immunogenicity of Virus-Like Particles

(42) In the Example, the inventors verified the immunogenicity of the virus-like particles formed by the recombinant proteins of the invention. All such virus-like particles can induce generation of antibodies that specifically bind to target antigens in organisms.

(43) 3.1 Immunization of Mice

(44) BALB/C mice were immunized with the 18 virus-like particles prepared in Example 2, respectively. The immunization process was as followed: the immunoadjuvant used was aluminum hydroxide adjuvant; the immunizing dose was 3 ug/dose; the immunization was performed by intramuscular injection at lateral thigh of hindlimb; the immune procedure was primary immunization+booster immunization 2 weeks later (i.e. two times in total).

(45) 3.2 Detection of Titer of Antibodies that Specifically Bind to Target Antigens in Sera

(46) 3.2.1 Preparation of Reaction Plates

(47) The antigens for coating reaction plates were the target antigens corresponding to said three target polypeptides, i.e., HIV-1 gp120 protein (purchased from Sino Biological Inc., Catalog No. 11233-V08H), human PD-L1 protein (purchased from Sino Biological Inc.), and human hepatitis B virus surface antigen recombinantly expressed in CHO cells (HBsAg, purchased from Beijing Wantai Biological Pharmacy).

(48) 3 recombinant proteins were diluted with pH9.6 50 mM CB buffer (NaHCO.sub.3/Na.sub.2CO.sub.3 buffer, at a final concentration of 50 mM, pH=9.6), respectively, at a final concentration of 2 g/mL, to obtain the coating solutions. To each well of a 96-well ELISA plate, 100 L coating solution was added, and the wells were coated at 2-8 C. for 16-24 h, and then further coated at 37 C. for 2 h. After that, PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) was used to wash wells once; and 200 L blocking solution (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer solution containing 20% bovine calf serum and 1% casein, pH=7.4) was then added to each well, and the wells were blocked at 37 C. for 2 h. The blocking solution was discarded. After that, the ELISA plate was dried, and packaged into an aluminum foil bag, which was stored at 2-8 C. for further use.

(49) 3.2.2 ELISA Detection of Anti-HBsAg Antibody Titer in Serum

(50) Collection of serum samples: blood was collected from the eye orbit of mice at Week 0, 2, and 4, the serum was separated and cryopreserved at 20 C., until detection.

(51) Sample dilution: a mouse serum was diluted with PBS solution containing 20% newborn bovine serum at 7 dilution gradients, i.e. 1:100, 1:500, 1:2500, 1:12500, 1:62500, 1:312500, and 1:1562500.

(52) ELISA detection: to each well of the coated ELISA plate, 100 L diluted serum sample was added, and incubated at 37 C. for 30 min. The ELISA plate was then washed with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for five times. After washing, to each well of the ELISA plate, 100 L GAM-HRP reaction solution was added, and incubated at 37 C. for 30 min. The ELISA plate was then washed with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for five times. After washing, to each well of the ELISA plate, 50 L TMB color developing agent (provided by Beijing Wantai Biological Pharmacy) was added, and incubated at 37 C. for 15 min. After the incubation, to each well of the ELISA plate, 50 L stop solution (provided by Beijing Wantai Biological Pharmacy) was added, and the OD450/630 value for each well was read by an ELISA instrument.

(53) Calculation of antibody titer: samples, the read values of which were within 0.2-2.0, were analyzed; a regression curve was plotted with the dilution fold and the read value, and the dilution fold of the sample, at which the read value was 2-fold of the background value, was calculated; and the dilution fold of the sample was used as the titer of the specific antibody in serum.

(54) FIG. 3 shows changes in titer of antibodies against the target antigen in mouse sera over time, after the immunization of BALB/C mice with the virus-like particles formed by 18 recombinant proteins, respectively. FIG. 3A: the target polypeptide used was SEQ ID NO: 20, and the titer of anti-GP120 antibodies was determined; FIG. 3B: the target polypeptide used was SEQ ID NO: 21, and the titer of anti-PD-L1 antibodies was determined; FIG. 3C: the target polypeptide used was SEQ ID NO: 22, and the titer of anti-HBsAg antibodies was determined. The results show that all the virus-like particles formed by 18 recombinant proteins have good immunogenicity, and can induce the generation of high-titer antibodies that specifically bind to target antigens in mice.

EXAMPLE 4

Evaluation on Anti-HBV Therapeutic Effects of Virus-Like Particles Presenting HBsAg Epitope (SEQ ID NO: 22)

(55) In the Example, the inventors evaluated the anti-HBV therapeutic effects of the virus-like particles presenting the same epitope peptide (SEQ ID NO: 22), as constructed based on different polypeptide carriers.

(56) 4.1 Immunization of Mice

(57) According to the methods described in Example 1-2, 2 recombinant proteins (i.e., HBcAg183-SEQ22, its amino acid sequence is set forth in SEQ ID NO: 41; and HBcAg149-SEQ22, its amino acid sequence is set forth in SEQ ID NO: 42), presenting HBsAg epitope (SEQ ID NO: 22) and constructed based on HBcAg of human HBV, were prepared, and the virus-like particles formed by the 2 recombinant proteins were prepared.

(58) Later, 5 virus-like particles presenting HBsAg epitope (SEQ ID NO: 22) prepared in Example 2, and 2 virus-like particles prepared in the Example were evaluated for the anti-HBV therapeutic effects in a HBV transgenic mouse model.

(59) The immunization method was as followed: the immunoadjuvant used was aluminum hydroxide adjuvant; and the immunizing dose was 12 g/dose; the immunization was performed by intramuscular injection at lateral thigh of hindlimb; the immune procedure was immunization at Week 0, 2, 3, 4, 5, and 6, (i.e. six times in total).

(60) 4.2 Detection of Antibody Titer and Virological Index in Serum

(61) According to the method as described in Example 3.2, the Anti-HBsAg antibody titer in serum was determined, and the virological index (i.e., the level of HBV DNA and HBsAg) in mouse serum was determined.

(62) 4.3 Analysis of Therapeutic Effects of Recombinant Proteins

(63) The detection results are shown in FIG. 4-6. FIG. 4 shows changes in HBsAg level in mouse sera over time, after the treatment of HBV transgenic male (FIG. 4A) and female (FIG. 4B) mice with different virus-like particles presenting the same epitope peptide (SEQ ID NO: 22). FIG. 5 shows changes in HBV DNA level in mouse sera over time, after the treatment of HBV transgenic male mice with different virus-like particles presenting the same epitope peptide (SEQ ID NO: 22). FIG. 6 shows changes in titer of anti-HBsAg antibodies in mouse sera over time, after the treatment of HBV transgenic male (FIG. 6A) and female (FIG. 6B) mice with different virus-like particles presenting the same epitope peptide (SEQ ID NO: 22).

(64) The results show that in the groups receiving immunotherapy with VLP, Anti-HBsAg antibodies were detected in mouse sera after immunization, and the level of HBV DNA and HBsAg decreased to different extents in mouse sera. By comparison, no Anti-HBsAg antibodies were generated in the sera of control mice (which were not immunized with VLP), and no decrease in the level of HBV DNA and HBsAg in sera was observed.

(65) These results show that all the 6 polypeptide carriers, constructed based on bat hepatitis B virus core protein, can be used to effectively present the epitope peptide (e.g., HBsAg-aa113-135) of HBsAg from human HBV, can form VLPs, and induce generation of high-titer anti-HBsAg antibodies in organisms, thereby inhibiting the level of HBV DNA and HBsAg (i.e., HBV DNA and HBsAg decreased significantly) in mice. In addition, the experimental data in FIG. 4-6 also shows that the virus-like particles, based on the polypeptide carriers (e.g. RBHBcAg149 and TBHBcAg153) of the invention, have particularly significant anti-HBV therapeutic effects, better than the virus-like particle constructed based on HBcAg of human HBV.

(66) Therefore, the experimental results in the Example show: (1) the polypeptide carriers of the invention can form VLPs, are suitable for presenting various target polypeptides, and can induce generation of high-titer antibodies against target polypeptides in organisms; (2) the polypeptide carriers of the invention are particularly suitable for presenting epitopes of human HBV (e.g., an epitope of HBsAg of human HBV), can induce generation of high-titer antibodies against HBsAg in organisms, and can clean or inhibit the level of HBV DNA and HBsAg in vivo, with an efficacy better than that of the polypeptide carrier constructed based on HBcAg of human HBV. Thus, the recombinant proteins presenting human HBV epitopes according to the invention are potential in treating HBV infection, and are particularly suitable for inducing effective, specific and therapeutic anti-HBV immunization.

EXAMPLE 5

Preparation and Evaluation of Virus-Like Particles Presenting an Epitope of HBsAg from Different HBV Genotypes

(67) The HBsAg epitope (SEQ ID NO: 22) used in Example 2-4 was from HBV genotype B. In order to confirm the broad versatility of the polypeptide carrier of the invention for various HBV genotypes, the inventors also used RBHBcAg149 and TBHBcAg153 as exemplary polypeptide carriers, to construct the recombinant proteins presenting an epitope of HBsAg from different HBV genotypes (genotype A, C and D), and evaluated the ability of the constructed recombinant proteins to be assembled into virus-like particles, the immunogenicity of the virus-like particle produced, and the therapeutic effect thereof against HBV infection.

(68) 5.1 Construction of Expression Plasmids Encoding Recombinant Proteins Comprising a Target Polypeptide and a Polypeptide Carrier

(69) In the Example, in addition to the HBsAg epitope (from HBV genotype B, SEQ ID NO: 22) used in Example 2-4, the target polypeptide further includes the HBsAg epitope (amino acids from positions 113-135) from HBV genotype A, C and D, designated as: HBsAg-aa113-135-A, HBsAg-aa113-135-C and HBsAg-aa113-135-D, and their sequences (SEQ ID NO: 60-62) are shown in Table 4.

(70) TABLE-US-00004 TABLE4 Sequencesofaminoacidsfrompositions113-135 ofHBsAgproteinfromHBVgenotypeA,CandD SEQ IDNO Name Sequenceinformation 60 HBsAg-aa113-135-A STTTSTGPCKTCTTPAQGNSMFP 61 HBsAg-aa113-135-C TSTTSTGPCKTCTIPAQGTSMFP 62 HBsAg-aa113-135-D SSTTSTGPCRTCTTPAQGTSMYP

(71) The sense and antisense sequences (as shown in Table 5) coding said 3 target polypeptides were synthesized directly, and annealed, to obtain the gene fragments having cohesive end and encoding the target polypeptides.

(72) TABLE-US-00005 TABLE5 Senseandantisensesequencescoding3targetpolypeptides SEQIDNO: Primername Sequenceinformation 63 HBsAg-aa113-135-AF GATCCTCTACCACCACCTCTACCGGTCCGTGCAAAACCTGCA CCACCCCGGCTCAGGGTAACTCTATGTTCCCGG 64 HBsAg-aa113-135-AR AATTCCGGGAACATAGAGTTACCCTGAGCCGGGGTGGTGCAG GTTTTGCACGGACCGGTAGAGGTGGTGGTAGAG 65 HBsAg-aa113-135-CF GATCCACCTCTACCACCTCTACCGGTCCGTGCAAAACCTGCA CCATCCCGGCTCAGGGTACCTCTATGTTCCCGG 66 HBsAg-aa113-135-CR AATTCCGGGAACATAGAGGTACCCTGAGCCGGGATGGTGCAG GTTTTGCACGGACCGGTAGAGGTGGTAGAGGTG 67 HBsAg-aa113-135-DF GATCCTCTTCTACCACCTCTACCGGTCCGTGCCGTACCTGCA CCACCCCGGCTCAGGGTACCTCTATGTACCCGG 68 HBsAg-aa113-135-DR AATTCCGGGTACATAGAGGTACCCTGAGCCGGGGTGGTGCAG GTACGGCACGGACCGGTAGAGGTGGTAGAAGAG

(73) As described in Example 2, the 3 gene fragments having cohesive end and encoding the target polypeptides as prepared above were ligated to linear vectors RBHBcAg149 and TBHBcAg153, respectively, so as to obtain the expression plasmids encoding the recombinant proteins (6 in total: RBHBcAg149-SEQ60, RBHBcAg149-SEQ61, RBHBcAg149-SEQ62, TBHBcAg153-SEQ60, TBHBcAg153-SEQ61, and TBHBcAg153-SEQ62). The amino acid sequences of the recombinant proteins encoded by the expression plasmids are shown in Table 6.

(74) TABLE-US-00006 TABLE6 Aminoacidsequencesof6recombinantproteins SEQ Recombinant IDNO: protein Sequenceinformation 69 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ60 SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSSTTTSTGPCKTCTTPAQGNSMF PEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGT WIRTPQAYRPPNAPILSTLPEHTIV 70 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ61 SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSTSTTSTGPCKTCTIPAQGTSMF PEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGT WIRTPQAYRPPNAPILSTLPEHTIV 71 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ62 SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSSSTTSTGPCRTCTTPAQGTSMY PEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGT WIRTPQAYRPPNAPILSTLPEHTIV 72 TBHBcAg153- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH SEQ60 TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSSTTTSTGPCKTCTTPAQGNS MFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLV SFGTWIRTPAAYRPVNAPILTTLPETSVI 73 TBHBcAg153- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH SEQ61 TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSTSTTSTGPCKTCTIPAQGTS MFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLV SFGTWIRTPAAYRPVNAPILTTLPETSVI 74 TBHBcAg153- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPHH SEQ62 TALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTGPCRTCTTPAQGTS MYPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLV SFGTWIRTPAAYRPVNAPILTTLPETSVI

(75) 5.2 Expression, Purification and Assembly of Recombinant Proteins

(76) As described in Example 2, by using the 6 expression plasmids constructed in the previous step, the recombinant proteins encoded by the expression plasmids were expressed and purified. Later, the VLPs formed by the recombinant proteins were observed by Transmission Electron Microscope (TEM).

(77) FIG. 7 shows the TEM results of the virus-like particles formed by the 6 recombinant proteins constructed. The results show that all the 6 recombinant proteins obtained can be assembled into virus-like particles with a diameter of about 30 nm. These results show that the polypeptide carriers constructed in the invention have a broad versatility, can be used to present epitope peptides (e.g., aa113-135 of HBsAg protein) from various HBV genotypes, and can form VLPs well.

(78) 5.3 Evaluation of Immunogenicity of Virus-Like Particles

(79) By using the method described in Example 3, the virus-like particles, formed by the 6 recombinant proteins as constructed above and the recombinant proteins RBHBcAg149-SEQ22 and TBHBcAg153-SEQ22 in Example 2, were evaluated for their immunogenicity. The experimental results are shown in FIG. 8.

(80) FIG. 8 shows the titers of antibodies against the corresponding target polypeptides (SEQ ID NO: 60, 22, 61, and 62) in mouse sera, three weeks after the immunization of BALB/C mice with the virus-like particles formed by the 8 recombinant proteins; wherein the epitope peptide (SEQ ID NO: 60) of HBsAg protein from HBV genotype A was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ60 and TBHBcAg153-SEQ60; the epitope peptide (SEQ ID NO: 22) of HBsAg protein from HBV genotype B was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ22 and TBHBcAg153-SEQ22; the epitope peptide (SEQ ID NO: 61) of HBsAg protein from HBV genotype C was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ61 and TBHBcAg153-SEQ61; and the epitope peptide (SEQ ID NO: 62) of HBsAg protein from HBV genotype D was used to determine the antibody titers in sera of mice immunized with RBHBcAg149-SEQ62 and TBHBcAg153-SEQ62. The results show that the virus-like particles formed by the 8 recombinant proteins have good immunogenicity, and can induce generation of high-titer antibodies that specifically bind to target epitopes in mice.

(81) 5.4 Evaluation on Anti-HBV Therapeutic Effects of Virus-Like Particles

(82) By using the method as described in Example 4, the virus-like particles formed by 4 recombinant proteins (SEQ ID NO: 36, 69, 70, and 71) were evaluated for the anti-HBV therapeutic effects. The experimental results are shown in FIG. 9.

(83) FIG. 9 shows changes in HBsAg level in mouse sera over time, after the treatment of HBV transgenic male (FIG. 9A) and female (FIG. 9B) mice with the virus-like particles formed by the 4 recombinant proteins constructed above (RBHBcAg149-SEQ22, RBHBcAg149-SEQ60, RBHBcAg149-SEQ61, and RBHBcAg149-SEQ62; the sequences thereof are SEQ ID NO: 36, 69, 70, and 71, respectively), wherein, longitudinal axis: HBsAg level (IU/ml); horizontal axis: time (week). The results show that in the mice receiving immunotherapy with VLP, the HBsAg level in mouse sera decreased significantly after immunization.

(84) These experimental results show that the polypeptide carriers of the invention (e.g., RBHBcAg149 and TBHBcAg153) can be used to effectively present epitope peptides (e.g., HBsAg-aa113-135) of HBsAg from human HBV of different genotypes (e.g., genotype A, B, C and D). The recombinant proteins, constructed based on the polypeptide carriers of the invention and epitope peptides of HBsAg, can form VLPs, and can induce the generation of high-titer anti-HBsAg antibodies in organisms, thereby inhibiting the HBsAg level (i.e., the HBsAg level decreased significantly) in mice. This indicates that the recombinant proteins comprising the polypeptide carriers of the invention and epitope peptides of HBsAg can be used to prevent and treat the infection by various HBV genotypes, and therefore can be used in the development of new anti-HBV vaccines and medicaments.

(85) Although the embodiments of the invention have been described in detail, a person skilled in the art would understand that according to all the disclosed teachings, details can be amended and modified, and these alterations all fall into the protection scope of the invention. The scope of the invention is defined by the attached claims and any equivalent thereof.