Coxsackie virus A10 type strain as well as construction method and application thereof

Abstract

The invention provides a coxsackie virus A10 type strain and a construction method and application thereof, the coxsackie virus A10 type strain rCV-A10-H comprises a coxsackie virus A10 type strain VP1 capsid protein and a VP2 capsid protein, the amino acid sequence of the VP1 capsid protein is shown as SEQ ID NO: 1, and the amino acid sequence of the VP2 capsid protein is shown as SEQ ID NO: 2. The amino acid sequence of the VP2 capsid protein is as shown in SEQ ID NO: 2. According to the invention, the humoral immunogenicity of the CV-A10 strain can be improved by introducing specific amino acid substitution into the virus structural protein, and a technical basis is provided for breeding coxsackie virus A10 and other enterovirus vaccine strains which can be used for vaccine production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a Coxsackievirus A10 strain, a construction method and an application thereof. Background Art

[0002] CV-A10 is a member of the Coxsackievirus group A, primarily associated with hand, foot, and mouth disease (HFMD), particularly affecting children. CV-A10 is a single-stranded, positive-sense RNA virus with an icosahedral capsid composed of 60 capsid proteins (VP1, VP2, VP3, and VP4). Currently, there is no vaccine against CV-A10. To develop a CV-A10 vaccine, candidate strains must be screened. Research has shown that candidate strains generally exhibit poor humoral immunogenicity. Whether developed as a polyvalent inactivated vaccine or a polyvalent virus-like particle vaccine, the levels of neutralizing antibodies induced by CV-A10 candidate strains are several times lower than those of other serotypes, severely hindering the development of polyvalent HFMD vaccines, including those targeting CV-A10. Summary of the Invention

[0003] In view of this, the present invention provides a Coxsackievirus A10 strain and a construction method and application, which can improve the humoral immunogenicity of the CV-A10 strain and provide a technical basis for the selection of Coxsackievirus A10 and other enterovirus vaccine strains that can be used for vaccine production.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a Coxsackievirus A10 strain rCV-A10-H, wherein the strain rCV-A10-H comprises a VP1 capsid protein and a VP2 capsid protein, wherein the amino acid sequence of the VP1 capsid protein is shown in SEQ ID NO: 1; and the amino acid sequence of the VP2 capsid protein is shown in SEQ ID NO: 2.

[0005] Preferably, the strain rCV-A10-H also includes VP3 capsid protein and VP4 capsid protein, the amino acid sequence of the VP3 capsid protein is shown in SEQ ID NO: 3; the amino acid sequence of the VP4 capsid protein is shown in SEQ ID NO: 4.

[0006] In a second aspect, the present invention provides a genome of a Coxsackievirus A10 strain rCV-A10-H, wherein the nucleotide sequence of the strain rCV-A10-H is shown in SEQ ID NO:5.

[0007] In a third aspect, the present invention provides a virus-like particle of a Coxsackievirus A10 strain rCV-A10-H, comprising a VP1 capsid protein, a VP2 capsid protein, a VP3 capsid protein and a VP4 capsid protein, wherein the VP1 capsid protein has an amino acid sequence as shown in SEQ ID NO: 1; the VP2 capsid protein has an amino acid sequence as shown in SEQ ID NO: 2; the VP3 capsid protein has an amino acid sequence as shown in SEQ ID NO: 3; and the VP4 capsid protein has an amino acid sequence as shown in SEQ ID NO: 4.

[0008] In a fourth aspect, the present invention provides a use of the virus-like particles in the preparation of medicines for preventing hand, foot and mouth disease.

[0009] In a fifth aspect, the present invention provides a pharmaceutical composition for preventing hand, foot and mouth disease, which comprises the virus-like particles and a pharmaceutically acceptable carrier.

[0010] Preferably, the pharmaceutical composition is a vaccine composition.

[0011] In a sixth aspect, the present invention further provides a method for constructing a Coxsackievirus A10 strain rCV-A10-H, comprising the steps of replacing key amino acid sites in the structural protein region of the Coxsackievirus A10 strain CV-A10-L, or introducing nucleotide mutations in the structural protein region of the Coxsackievirus A10 strain CV-A10-L to achieve the replacement of key amino acid sites; The key amino acid sites are sites that determine humoral immunogenicity.

[0012] Preferably, the determination of key sites comprises the following steps: By comparing and screening the binding sites of the structural protein region of the Coxsackievirus A10 strain CV-A10-FJ-01 and the neutralizing monoclonal antibody, and the amino acid sites of the corresponding structural protein region of the low humoral immunogenic Coxsackievirus A10 strain CV-A10-L, the sites with different amino acids are found to be potential key amino acid sites.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a Coxsackievirus A10 strain rCV-A10-H that can improve the humoral immunogenicity of the CV-A10 strain, providing a technical basis for the selection of Coxsackievirus A10 and other enterovirus vaccine strains that can be used for vaccine production.

[0014] (2) The present invention introduces amino acid substitutions in the P1 region of the structural protein coding region of the CV-A10-L strain. The modified strain's cell proliferation and stability are not affected, but its humoral immunogenicity is significantly improved. In addition, the cross-protection effect of inactivated immunization against other CV-A10 strains within the same serotype is better than that of other vaccine candidate strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the comparison of the 2G8 monoclonal antibody binding region between the CV-A10-L and CV-A10-FJ-01 strains provided in Example 1 of the present invention; Figure 2 Schematic diagram of the structure of the infectious clones pBR322-rCV-A10-L and pBR322-rCV-A10-H provided in Example 2 of the present invention; Figure 3 This is the growth curve of the recombinant viruses rCV-A10-L and rCV-A10-H provided in Example 3 of the present invention on Vero cells; Figure 4 The heat resistance curves of rCV-A10-L and rCV-A10-H provided in Example 4 of the present invention; Figure 5 This is a comparison of the neutralizing antibody levels induced by rCV-A10-L and rCV-A10-H provided in Example 5 of the present invention; Figure 6 This is a comparison of the cross-neutralization levels of neutralizing antibodies induced in mice by the three CV-A10 vaccine candidate strains provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0016] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0017] Viral materials: Coxsackievirus A10 strain CV-A10-L can adapt to the growth of Vero cells, but the level of neutralizing antibodies induced is low and is preserved in this laboratory.

[0018] Coxsackievirus A10 strains CV-A10-C1 and CV-A10-C2 can adapt to the growth of Vero cells and are the vaccine candidate strains preserved in this experiment.

[0019] Virus strain and monoclonal antibody structure information: The coxsackievirus A10 strain CV-A10-FJ-01 sequence was downloaded from NCBI (GenBank accession no. KY012321).

[0020] The structural information of monoclonal antibodies 2G8 and CV-A10-FJ-01 was downloaded from the PDB database (PDB accession numbers: 6AD0).

[0021] Cell culture: Vero cells were cultured in DMEM supplemented with 10% newborn calf serum, with no serum added for maintenance. RD cells were cultured in MEM supplemented with 10% newborn calf serum, with no serum added for maintenance.

[0022] Main reagents: Viral RNA extraction kits, DNA gel recovery kits, and plasmid extraction kits were purchased from QIAGEN, Germany. The reverse transcription kit HiScript® II, DNA polymerase Phanta®, and seamless ligation kit ClonExpress II were purchased from Nanjing Novozymes. The transfection reagent Lipofectamine™ 2000 was purchased from Thermo Fisher Scientific, USA.

[0023] Example 1: Sequence determination and sequence alignment of CV-A10 strain Genomic RNA from the CV-A10-L strain was extracted using the QIAamp viral RNA Mini Kit (Qiagen, Germany) and reverse transcribed to cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Viral cDNA was amplified by PCR using Phanta Master (Vazyme, Nanjing, China) and sent to Shanghai Bioengineering for sequencing. Sequences were assembled using Seqman (DNAStar, Wisconsin, USA) software.

[0024] Combining the structural analysis results in the database (PDB: 6AD0) and literature reports, the main binding amino acid sites of the neutralizing monoclonal antibody 2G8 on the structural protein of the CV-A10-FJ strain were found, mainly in the EF loop of the structural protein VP2, the AB loop of VP3, and the C-terminus of VP1. Using Megalign (DNAStar, Wisconsin, USA) software, the amino acid sites of the structural proteins of CV-A10-L and CV-A10-FJ-01 that bind to the monoclonal antibody 2G8 were compared, and differences were found at position 162 of VP2 and position 283 of VP1 ( Figure 1 ).

[0025] Example 2: Construction of recombinant virus Although the CV-A10-FJ-01 strain can be used to isolate the neutralizing monoclonal antibody 2G8, it is a strain cultured in RD cells and cannot be used for human vaccine production. CV-A10-L can adapt to Vero cell growth, but its humoral immunogenicity is poor. In an attempt to optimize the CV-A10-L strain to improve its humoral immunogenicity, a reverse genetics system for this strain was constructed using the CV-A10-L genome as a template. The infectious cDNA plasmid is pBR322-rCV-A10-L ( Figure 2 ), and rescued CV-A10-L on Vero cells. A nucleotide mutation replacing histidine (H) with tyrosine (Y) was introduced at position 162 of the structural protein VP2, and a nucleotide mutation replacing valine (V) with isoleucine (I) was introduced at position 283 of VP1 to construct the infectious cDNA plasmid pBE322-rCV-A10-H ( Figure 2 The rescued virus was propagated to P3 on Vero cells, and the genome was extracted for RT-PCR and sequenced to verify the correctness of the rescued virus sequence.

[0026] Example 3: Growth characteristics of recombinant virus on Vero cells Virus titration was performed using a 96-well cell culture plate with 4 rows × 9 columns as a sample area. Each plate had two partitions. The virus stock solution was diluted by 10-fold dilution method. Each dilution was repeated 4 times. Different dilutions were added to the sample area in sequence at 100 μL / well. 5 × 10 4 The RD cells were incubated at 37°C, 5% CO2 for 7 days and the cytopathic effect was observed under a microscope. The virus titer was calculated according to the Reed-Muench method and recorded as CCID 50 / mL (50% cell cultureinfectious dose per milliliter).

[0027] Vero cells were infected with the virus at a multiplicity of infection of 10 (MOI = 10), and the cell cultures were harvested at 0 h, 6 h, 12 h, 18 h, 24 h, and 48 h after infection. The cells were frozen and thawed three times and the supernatant was collected by centrifugation. Virus titration was performed, and the titers of the viruses at different time points were recorded, and the virus growth curve was drawn ( Figure 3 The results showed that rCV-A10-H and rCV-A10-L had similar proliferation abilities on Vero cells, and substitution of the two amino acid sites did not affect the proliferation of rCV-A10-H strain on Vero cells.

[0028] Example 4: Stability of recombinant virus Dilute rCV-A10-H and rCV-A10-L to 107 CCID50 / mL, 500μL / tube were dispensed into 1.5mL centrifuge tubes, and treated at 37℃, 40℃, 43℃, 46℃, 49℃, 52℃, 54℃, 56℃, 58℃ for 30min, and then transferred to ice. The titer of each tube of virus solution was determined, and a heat resistance curve was drawn ( Figure 4 The results showed that the viral titers of rCV-A10-H and rCV-A10-L decreased to a similar extent as the temperature increased, and the substitution of the two amino acid sites did not affect the stability of the rCV-A10-H strain.

[0029] Example 5: Neutralizing antibody assay in mice immunized with recombinant virus intraperitoneally Twenty-three 6-week-old female Balb / c mice were divided into three groups. Five mice were injected with PBS as blank control, nine mice were injected with rCV-A10-L, and nine mice were injected with rCV-A10-H. Immunization was performed intraperitoneally at week 0 and week 2 for 10 8 CCID50 / mL virus solution. Blood was collected from the eye sockets at week 3. Serum was centrifuged and inactivated at 56°C for 30 min. Aliquots were stored at -20°C pending neutralization testing.

[0030] Serum samples were diluted 2-fold with MEM. Two replicate wells were set for each sample. 100 μL / well was added to column A of a 96-well plate. 50 μL of MEM was added to each well of columns B to H. Serum samples in column A were serially diluted 2-fold to column H. 50 μL of the last column was discarded after dilution. CV-A10-L was used as a neutralizing virus seed and diluted to 100 CCID 50 / 50μL, draw 50μL vertically suspended dropwise into the diluted serum sample. Place the 96-well plate in a 37℃ incubator for 2h. After neutralization, the digested RD cells were plated at 1×10 5 / mL density was added to 96-well plate. At the same time, the titer of the diluted neutralization poison was determined by ten-fold dilution to ensure that the neutralization poison was diluted to 30-300 CCID 50 / hole.

[0031] Balb / c mice were immunized with rCV-A10-H and rCV-A10-L virus supernatants of the same titer, and the neutralizing antibody levels against CV-A10-L in mouse serum were measured ( Figure 5 The results showed that rCV-A10-H induced significantly higher levels of neutralizing antibodies in mice than rCV-A10-L, and substitution of two amino acid sites could significantly enhance the humoral immunogenicity of CV-A10 strain.

[0032] Example 6: Evaluation of cross-neutralization ability within immune serotypes after inactivation of recombinant virus Vaccine candidate strains CV-A10-C1, CV-A10-C2, and rCV-A10-H were purified and inactivated with formaldehyde. Thirty-six experimental animals were divided into six groups and immunized with 1 or 4 μg of inactivated CV-A10-C1, CV-A10-C2, and rCV-A10-H via intraperitoneal injection at weeks 0 and 2, respectively. Orbital blood was collected at week 4, and serum was centrifuged and inactivated at 56°C for 30 minutes. The serum was aliquoted and stored at -20°C. Neutralization testing was performed using the same method as in Example 5, with rCV-A10-L, CV-A10-C1, and CV-A10-C2 as neutralizing agents. All serum samples were tested for neutralization.

[0033] After mice were immunized with 1μg and 4μg antigen amounts of the candidate CV-A10 vaccine strains rCV-A10-H, CV-A10-C1 and CV-A10-C2, respectively, the levels of neutralizing antibodies against different strains in the mouse serum were measured. Using CV-A10-L as the neutralizing toxin, the level of neutralizing antibodies induced in mice by rCV-A10-H was significantly higher than that of CV-A10-C1 and CV-A10-C2. Using CV-A10-C1 as the neutralizing toxin, the level of neutralizing antibodies induced in mice by rCV-A10-H was similar to that of CV-A10-C1, and both were significantly higher than CV-A10-C2. Using CV-A10-C2 as the neutralizing toxin, the level of neutralizing antibodies induced in mice by rCV-A10-H was slightly higher than that of CV-A10-C2, and both were significantly higher than CV-A10-C1 ( Figure 6 The results showed that the recombinant virus rCV-A10-H obtained by replacing two amino acid sites as an inactivated vaccine had better cross-neutralization ability against strains within the CV-A10 serotype than other vaccine candidate strains.

[0034] By comparing the amino acid sequences of the structural protein regions of the CV-A10-FJ-01 strain used to isolate the coxsackievirus A10 neutralizing monoclonal antibody 2G8 with the Vero cell-adapted CV-A10-L strain with low humoral immunogenicity, this study identified key amino acid sites potentially determining the humoral immunogenicity of CV-A10. Using the low humoral immunogenic CV-A10 strain as the parent strain, a reverse genetic manipulation system was constructed to obtain the rescued virus rCV-A10-L. Furthermore, two amino acid substitutions (H162Y in VP2 and V283I in VP1) were introduced into the structural protein regions of rCV-A10-L to generate the recombinant virus rCV-A10-H. This strain maintains its Vero cell proliferation and viral stability, while significantly enhancing its humoral immunogenicity. The present invention transforms the rCV-A10-L strain with low humoral immunogenicity into the rCV-A10-H strain with enhanced humoral immunogenicity by introducing substitutions of two amino acid sites in the structural protein coding region, providing a technical method for the design and optimization of CV-A10 vaccine candidate strains.

[0035] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0036] Gene sequences: all are the corresponding amino acid and nucleotide sequences of the strains mentioned in the above specific embodiments.

[0037] rCV-A10-H amino acid sequence Amino acid sequence of the CV-A10-FJ structural protein: MGAQVSTQKSGSHETGNVATGGSTINFTNINYYKDSYAASATRQDFTQDPKKFTQPVLDSIRELSAPLNSPSVEACGYSDRVAQLTVGNSSITTQEAANIVLAYGEWPEYCPDTDATAVDKPTRPDVSVNRFYTLDSKMWQENSTGWYWKFPDVLNKTGVFGQNAQFHYLYRSGFCLHVQCNASKFHQGALLVAVIPEFVIAGRGSNTKPNEAPHPGFTTTFPGTTGATFYDPYVLDSGVPLSQALIYPHQWINLRTNNCATVIVPYINAVPFDSAINHSNFGLIVIPVSPLKYSSGATTAIPITITIAPLNSEFGGLRQAVSQGIPAELRPGTNQFLTTDDGTAAPILPGFTPTPTIHIPGEVHSLLELCRVETILEVNNTTEATGLTRLLIPVSSQNKADELCAAFMVDPGRIGPWQSTLVGQICRYYTQWSGSLKVTFMFTGSFMATGKMLVAYSPPGSAQPANRETAMLGTHVIWDFGLQSSVSLVIPWISNTHFRTAKTGGNYDYYTAGVVTLWYQTNYVVPPETPGEAYIIAMGAAQDNFTLKICKDTDEVTQQAVLQGDPVEDIIHDALGNTARRAISSATNVESAANTTPSSHRLETGRVPALQAAETGATSNATDENMIETRCVVNRNGVLETTINHFFSRSGLVGVVNLTDGGTDTTGYATWDIDIMGFVQLRRKCEMFTYMRFNAEFTFVTTTENGGARPYMLQYMYVPPGAPKPTGRDAFQWQTATNPSVFVKLTDPPAQVSVPFMSPASAYQWFYDGYPTFGQHPETSNTTYGLCPNNMMGTFAVRVVSREASQLKLQTRVYMKLKHVRAWVPRPIRSQPYLLKNFPNYDSSKITNSARDRSSIKQANM Amino acid sequence of the CV-A10-L structural protein: MGAQVSTQKSGSHETGNVATGGSTINFTNINYYKDSYAASATRQDFTQDPKKFTQPVLDSIRELSAPLNSPSVEACGYSDRVAQLTVGNSSITTQEAANIVLAYGEWPEYCPDTDATAVDKPTRPDVSVNRFYTLDSKMWQENSTGWYWKFPDVLNKTGVFGQNAQFHYLYRSGFCLHVQCNASKFHQGALLVAVIPEFVIAGRGSNTKPNKAPHPGFTTTFPGTTGATFHDPYVLDSGVPLSQALIYPHQWINLRTNNCATVIVPYINAVPFDSAINHSNFGLIVIPVSPLKYSSGATTAIPITITIAPLNSEFGGLRQAVSQGIPAELRPGTNQFLTTDDDTAAPILPGFTPTPTIHIPGEVHSLLELCRVETILEVNNTTEATGLTRLLIPVSSQNKADELCAAFMVDPGRIGPWQSTLVGQICRYYTQWSGSLKVTFMFTGSFMATGKMLVAYSPPGSAQPANRETAMLGTHVIWDFGLQSSVSLVIPWISNTHFRTAKTGGNYDYYTAGVVTLWYQTNYVVPPETPGEAYIIAMGAAQDNFTLKICKDTDEVTQQAVLQGDPVEDIIHDALGNTARRAISSVTNVESAANTTPSSHRLETGRVPALQAAETGATSNATDENMIETRCVVNRNGVLETTINHFFSRSGLVGVVNLTDGGTDTTGYATWDIDIMGFVQLRRKCEMFTYMRFNAEFTFVTTTKNGEARPYMLQYMYVPPGAPKPTGRDAFQWQTATNPSVFVKLTDPPAQVSVPFMSPASAYQWFYDGYPTFGQHPETSNTTYGLCPNNVMGTFAVRVVSKRASQLKLQTRVYMKLKHVRAWVPRPIRSQPYLLKNFPNYDSSKVTNSARDRSSVKQANM Nucleotide sequence of rCV - A10 - L 1 TTAAAACAGC CTGTGGGTTG TGCCCACCCA CAGGGCCCAC TGGGCGCTAG CACTCCGATT 61 CTGCGGAATC CTTGTGCGCC TGTTTTATAA CCCCCCCCCG AAACTTGTAA CTTAGAAGTT 121 ATGTACGTTA CCGATCAGCA GCAGGCGTGG CACACCAGCC ATGTCTTGAT CAAGCACTTC 181 TGTACCCCCG GACCGAGTAT CAATAGACTG CTCACGTGGT CGAAGGAGAA AACGTTCGTT 241 ATCCGGCTAA CTACTTCGAG AAGCCTAGTA GCGCCACTGA AACTGCGGAG TGTTTCGCTC 301 AGCACTTCCC CCGTGTAGAT CAGGTCGATG AGTCACTGCT CACCCCACGG GTGACCGTGG 361 CAGTGGCTGC GTTGGCGGCC TGCCTATGGG GCAACCCATA GGACGCTCTA AAGTGGACAT 421 GGTGCGAAGA GTCTATTGAG CTAGTTAGTA GTCCTCCGGC CCCTGAATGC GGCTAATCCT 481 AACTGCGGAG CGCATGCCCC CAAACCAGAG GGTGGTGCGT CGTAACGGGT AACTCTGCAG 541 CGGAACCGAC TACTTTGGGT GTCCGTGTTT CCTTTATTCT TATAATGGCT GCTTATGGTG 601 ACAATTGAGG AATTGTTACC ATATAGCTAT TGGATTGGCC ATCCGGTGTG CAACAGAGCT 661 ATTATCTACT TGTTTGTTGG ATACATTCCA TTAACACCTA AATCCTTCAA TACATTGTAC 721 TATATTCTAA CGCTGAACGC GAGAAAATGG GAGCTCAAGT CTCGACGCAA AAATCCGGCA 781 GTCACGAGAC TGGTAACGTA GCCACTGGAG GATCTACAAT AAACTTCACT AACATCAATT 841 ACTATAAAGA TTCTTACGCC GCGTCAGCTA CTCGGCAAGA CTTCACACAA GATCCAAAGA 901 AGTTCACACA ACCAGTGTTA GACTCCATTA GAGAACTATC AGCTCCTTTG AATTCCCCTT 961 CTGTGGAGGC TTGCGGCTAT AGTGATAGGG TCGCTCAGCT TACCGTTGGG AACTCCTCCA 1021 TTACTACCCA AGAGGCTGCT AATATTGTAT TAGCTTATGG AGAGTGGCCTGAATACTGCC 1081 CTGACACAGA CGCAACTGCC GTGGATAAGC CAACTCGCCC GGACGTGTCTGTCAACAGGT 1141 TCTACACTTT GGACTCTAAA ATGTGGCAAG AAAACTCGAC TGGTTGGTATTGGAAGTTCC 1201 CTGACGTGCT GAACAAGACG GGGGTGTTCG GACAGAATGC CCAGTTTCATTACTTGTACC 1261 GGTCAGGTTT CTGCTTGCAT GTACAGTGCA ACGCTAGTAA ATTCCACCAGGGGGCTCTTC 1321 TTGTGGCTGT GATACCAGAA TTTGTGATTG CTGGCAGAGG ATCTAACACAAAACCAAATG 1381 AAGCACCCCA CCCAGGGTTT ACTACAACTT TCCCTGGTAC CACCGGTGCTACATTCCACG 1441 ATCCATACGT TCTGGATTCC GGGGTACCAT TGAGTCAGGC TCTAATATACCCCCATCAAT 1501 GGATCAATCT CCGTACCAAC AACTGTGCAA CTGTTATAGT CCCGTACATCAATGCTGTTC 1561 CGTTTGACTC AGCTATCAAT CATAGCAACT TTGGGCTAAT AGTGATACCAGTTAGTCCGC 1621 TGAAGTATTC GTCCGGGGCA ACCACTGCAA TCCCAATCAC TATCACTATAGCCCCCTTGA 1681 ATTCGGAGTT CGGAGGACTA CGACAAGCCG TTAGCCAAGG CATCCCAGCTGAGCTCAGGC 1741 CCGGGACTAA TCAATTCCTG ACCACAGATG ACGATACCGC AGCGCCCATCCTCCCAGGAT 1801 TCACCCCCAC ACCCACAATT CATATACCAG GGGAAGTACA CTCTTTGCTGGAGTTGTGTA 1861 GGTGGGAGAC TATTTTGGAA GTGAACAACA CCACAGAAGC AACAGGATTAACAAGGCTCT 1921 TAATACCAGT GTCCTCGCAA AACAAAGCCG ATGAGTTGTG TGCTGCGTTTATGGTTGATC 1981 CAGGCCGGAT TGGACCTTGG CAATCTACCT TAGTTGGACA GATTTGCCGTTATTACACAC 2041 AATGGTCCGG GTCTTTGAAG GTGACCTTTA TGTTCACAGG ATCCTTTATGGCAACAGGCA 2101 AGATGCTGGT GGCCTACTCC CCGCCCGGAA GCGCTCAACC AGCTAACAGAGAAACCGCCA 2161 TGCTGGGCAC GCACGTCATT TGGGACTTTG GACTACAATC ATCGGTCTCTTTGGTGATAC 2221 CGTGGATCAG TAACACTCAC TTTCGCACTG CCAAGACAGG TGGGAATTACGATTACTACA 2281 CGGCAGGTGT AGTGACCTTG TGGTATCAAA CAATTACGT GGTCCCGCCAGAAACCCCTG 2341 GAGAGGCGTA TATTATAGCA ATGGGGGCAG CACAAGACAA CTTCACTTTGAAGATTTGCA 2401 AGGATACTGA TGAAGTGACG CAACAAGCTG TGTTGCAAGG TGACCCTGTGGAAGATATAA 2461 TCCATGACGC TCTGGGAAAT ACAGCGCGCA GGGCTATTAG CAGTGTTACAAATGTCGAAT 2521 CCGCAGCTAA CACCACCCCC AGTTCACACC GACTGGAGAC TGGACGCGTACCAGCGCTAC 2581 AGGCTGCAGA AACGGGTGCC ACTTCTAATG CCACAGATGA GAACATGATTGAGACCCGTT 2641 GTGTGGTTAA CAGAAATGGG GTGCTGGAAA CCACTATTAA TCATTTCTTCTCCCGCTCTG 2701 GATTAGTGGG AGTGGTTAAC CTCACAGATG GGGGGACGGA CACCACTGGGTATGCTACAT 2761 GGGATATAGA CATTATGGGC TTTGTCCAAC TCCGCAGAAA GTGCGAGATGTTCACATACA 2821 TGAGATTCAA CGCGGAATTC ACGTTTGTCA CAACGACTAA GAATGGGGAGGCTCGCCCGT 2881 ACATGCTGCA ATACATGTAT GTGCCCCCTG GCGCCCCTAA ACCGACGGGAAGGGATGCCT 2941 TCCAATGGCA AACAGCAACT AACCCGTCAG TCTTTGTCAA ACTCACTGACCCCCCTGCAC 3001 AAGTCTCAGT CCCTTTCATG TCACCAGCTA GTGCATATCA GTGGTTTTATGATGGTTATC 3061 CCACTTTCGG CCAGCACCCG GAGACCTCAA ACACAACATA CGGGTTGTGCCCAAACAATG 3121 TGATGGGCAC TTTTGCGGTG AGAGTTGTTA GTAGAGAGGC AAGTCAACTAAAACTACAGA 3181 CTAGAGTGTA CATGAAGCTT AAGCATGTGA GGGCTTGGGT CCCAAGACCGATCAGGTCTC 3241 AGCCATACTT GCTTAAAAAC TTCCCCAATT ACGACAGTAG CAAAATTACCAACAGTGCAC 3301 GGGACCGATC TAGTATCAAG CAAGCTAATA TGGGTAAATT TGGGCAACAATCTGGCGCCA 3361 TCTACGTTGG TAACTACAGA GTGGTCAATA GACACTTGGC CACCCACAATGACTGGGCCA 3421 ACCTGGTGTG GGAAGATAGT TCTCGAGACT TGCTTGTCTC GTCTACCACTGCCCAGGGTT 3481 GTGATACGAT TGCCCGTTGT GAGTGTCAGA CAGGGGTGTA CTATTGTAACTCAAGGAGGA 3541 AGCATTATCC AGTTAGCTTC TCAAAACCTA GCCTCGTTTT CATAGAGGCCAGTGAATATT 3601 ACCCTGCTAG ATATCAATCT CACTTAATGC TTGCTGCAGG CCACTCTGAACCTGGGGATT 3661 GCGGGGGTAT CTTGAGGTGT CAACATGGTG TAGTCGGCAT AGTATCTACTGGTGGCAACG 3721 GTCTTGTCGG TTTTGCAAAC GTGAGGGACC TCTTATGGTT GGATGAAGAAGCCATGGAAC 3781 AGGGAGTGTC TGACTACATT AAGGGACTCG GCGACGCTTT TGGTACTGGTTTCACTGACG 3841 CAGTGTCTAG GGAAGTGGAG GCTTTGAAAA ATTACCTGAT TGGTTCCGAGGGAGCGGTGG 3901 AGAAGATCCT GAAGAATTTG GTGAAACTCA TATCAGCTTT GGTCATAGTTATCAGAAGCG 3961 ACTATGACAT GGTCACCCTT ACCGCAACTC TAGCTCTGAT TGGGTGTCACGGGAGCCCAT 4021 GGGCGTGGAT TAAAGCAAAG ACGGCATCCA TCTTAGGTAT TCCTATGGCACAGAAGCAGA 4081 GTGCATCTTG GCTTAAGAAG TTCAGTGATA TGGCGAACGC CGCAAAGGGGTTGGAGTGGA 4141 TCTCTAACAA AATCAGTAAA TTTATTGACT GGCTTAAGGA AAAGATCATTCCAGCTGCAA 4201 AGGAAAAAGT TGAGTTTCTT AACAATCTCA AGCAACTGCC CTTGATGGAAAACCAAATTG 4261 CTAACTTAGA ACAGTCTGCT GCTTCACAAG AAGACCTCGA AGTCATGTTTGGTAATGTGT 4321 CATACCTAGC TCACTTTTGT CGCAAGTTCC AGCCACTCTA TGCAACTGAAGCGAAGAGAG 4381 TGTACGCCTT GGAGAAGAGA ATGAATAATT ACATGCAGTT CAAGAGCAACACCGTATTG 4441 AACCTGTATG TTTAATTATC AGGGGCTCAC CAGGAACTGG CAAGTCACTCGCCACAGGTA 4501 TAATAGCAAG AGCCATTGCT GACAAATACC ACTCTAGTGT GTACTCCCTTCCGCCAGATC 4561 CAGATCACTT TGATGGATAC AAGCAGCAGG TGGTGACAGT CATGGATGATTTGTGTCAAA 4621 ACCCAGATGG CAAAGACATG TCATTGTTTT GCCAAATGGT GTCCACTGTCGATTTTATAC 4681 CCCCAATGGC TTCACTGGAA GAGAAAGGCG TATCCTTCAC ATCTAAATTTGTTATTGCTT 4741 CAACTAACGC TAACAACATC ATTGTCCCCA CGGTCTCCAGA CTCTGACGCTATTGAAGGA 4801 GATTCTTCAT GGATTGTGAC ATCGAAGTGA CTGACTCTTA CAAGACAGACTTAGGCCGCT 4861 TAGATGCGGG TAGGGCCGCA AAGCTCTGCT CAGAGAATAA CACCGCCAATTTCAAGAGGT 4921 GCAGCCCGTT AGTGTGCGGT AAAGCCATCC AACTGAGAGA TAGGAAGTCCAAAGTCAGGT 4981 ATAGTGTAGA TACTGTGGTA TCAGAGTTGG TTAGGGAGTA TAGTAACAGGTCTGCTATAG 5041 GAAATACTAT AGAAGCTTTA TTCCAAGGGC CTCCTAAATT TAGGCCTATAAGAATTAGCC 5101 TTGATGAGAA ACCCGCTCCA GATGCTATTA GTGACCTGCT TGCTAGCGTTGACAGCGAGG 5161 AGGTGCGGCA GTACTGCAGA GATCAAGGAT GGATAATACC TGAAACGCCAACCAATGTGG 5221 AGCGGCATCT CAATAGGGCA GTGTTAGTGA TGCAATCTAT CGCTACTGTAGTCGCAGTTG 5281 TGTCCCTTGT TTATGTTGTT TACAAGCTAT TTGCTGGTTT CCAAGGCGCATATTCTGGAG 5341 CGCCCAAGCA AGCTCTCAAG AAACCTGTGT TGAGAACAGC CACTGTTCAAGGGCCCAGCT 5401 TAGATTTTGC CCTGTCTCTC CTGCGACGTA ACATCAGGCA GGTGCAAACTGACCAAGGGC 5461 ACTTTACCAT GCTTGGGGTA CGTGACCGCC TTGCTATCTT GCCTCGTCACTCACAACCAG 5521 GGAAGACTAT CTGGGTTGAG CACAAGTTGG TCAATGTGCT TGATGCTGTTGAGCTAGTGG 5581 ATGAACAAGG TGTTAATTTG GAGCTTACAC TAGTAACCCT AGACACCAATGAGAAGTTTA 5641 GAGACGTCAC CAAGTTCATT CCAGAGAATA TCAGTGGAGC CAGTGATGCCACATTAGTAA 5701 TCAACACTGA ACACATGCCA TCAATGTTTG TCCCGGTAGG GGATGTTGTTCAATACGGGT 5761 TCCTAAATCT TAGTGGTAAG CCAACCCACA GGACCATGAT GTACAATTTCCCTACAAAGG 5821 CTGGACAGTG TGGAGGCGTG GTAACATCTG TCGGTAGAAT CATTGGCATCCACATTGGGG 5881 GCAATGGGCG ACAAGGCTTT TGCGCTGGCC TGAAAGGAG TTATTTCGCAAGTGAACAAG 5941 GTGAGATCCA ATGGGTGAAA CCTAACAAGG AGACCGGCAG ATTAAATATCAATGGTCCAA 6001 CACGCACCAA GTTAGAGCCC AGTGTGTTCC ATGATGTGTT TGAGGGCAATAAGGAGCCAG 6061 CAGTCCTAAC AAGCAAGGAT CCCAGATTAG AGGTTGACTT TGAGCAAGCCCTCTTTCCA 6121 AGTATGTGGG TAATGTCCTC CACGAACCTG ATGAATATGT GAAGCAGGCAGCCCTCCACT 6181 ACGCAAATCA GCTCAAGCAA CTGGATATAA ACACCAATAA GATGAGCATGGAGGAAGCGT 6241 GCTATGGCAC AGAGAACCTG GAAGCAATCG ATCTCCACAC TAGTGCAGGGTACCCATAC 6301 GTGCTCTAGG CATAAAGAAG AGGGACATAC TAGATCCTAC CACTAGGGACACAACAAAA 6361 TGAAGTTTTA CATGGATAAG TATGGTTTGG ACTTACCGTA CTCCACCTATGTCAAAAGATG 6421 AACTTAGGTC TCTGGACAAG ATTAAGAAAG GGAAATCCCG CTTAATAGAAGCTAGCAGCC 6481 TGAATGACTC AGTGTACCTT AGAATGACTT TTGGCCATCT GTATGAAGCGTTCCATGCAA 6541 ATCCAGGGAC TGTGACCGGA TCAGCGGTTG GATGCAATCC GGATGTGTTCTGGAGTAAAC 6601 TCCCAATCCT ACTCCCAGGC TCGCTATTTG CCTTTGACTA TTCAGGCTACGATGCCAGTC 6661 TTAGCCCCGT CTGGTTCAGG GCTTTGGAGA TGGTCTTGCG GGACATTGGTTACTCAGAGG 6721 AAGCAGTGTC ACTCATAGAA GGAATAAATC ATACCCATCA TGTGTACCGGAACAAAACAT 6781 ATTGTGTCCT TGGCGGGATG CCGTCAGGAT GTTCTGGCAC CTCCATTTTCAACTCGATGA 6841 TCAACAACAT CATCATCAGG ACGCTTTTGA TTAAAACATT TAAAGGGATAGATTTAGACG 6901 AGTTGAATAT GGTGGCCTAT GGGGATGATG TGCTAGCTAG TTATCCTTTCCCCATTGATT 6961 GCTTTGAACT AGCTAAAACT GGCAAAGAAT ATGGGCTGAC TATGACACCTGCAGACAAGT 7021 CACCTTGCTT TAACGAAGTG ACGTGGGAAA ACGCCACCTT TCTGAAGAGAGGGTTTTTAC 7081 CAGACCATCA ATTCCCATTC TTGATTCATC CCATAATGCC CATGAAAGAGATCCACGAAT 7141 CCATACGCTG GACCAAGGAT GCGTGCAACA CCCAAGACCA TGTACGCTCCCTGTGTTTAC 7201 TAGCTTGGCA TAACGGTAAG GATGAATATG AAAAATTTGT GAGTACAATTAGATCAGTCC 7261 CAGTTGGGAA AGCATTGGCT ATTCCGAACT TTGAAAATCT GAGAAGAAATTGGCTCGAAC 7321 TATTTTAAAT TTACAGTTGG AAGCTGAACC CCACCAGAAA TCTGGTCGTGTTAATGACTG 7381 GTGGGGGTAA ATTTGTTATA ACCAGAATAG C rCV-A10-L The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Coxsackievirus A10 strain rCV-A10-H, characterized in that: The strain rCV-A10-H includes: VP1 capsid protein, the amino acid sequence of which is shown in SEQ ID NO: 1; and VP2 capsid protein, the amino acid sequence is shown in SEQ ID NO:

2.

2. The strain rCV-A10-H according to claim 1, characterized in that The strain rCV-A10-H also includes: VP3 capsid protein, the amino acid sequence of which is shown in SEQ ID NO:3; and VP4 capsid protein, the amino acid sequence is shown in SEQ ID NO:

4.

3. A genome of a Coxsackievirus A10 strain rCV-A10-H, characterized in that: The nucleotide sequence of the strain rCV-A10-H according to any one of claims 1 to 2 is shown in SEQ ID NO:

5.

4. A virus-like particle of Coxsackievirus A10 strain rCV-A10-H, characterized in that: include: VP1 capsid protein, the amino acid sequence of which is shown in SEQ ID NO: 1; VP2 capsid protein, the amino acid sequence of which is shown in SEQ ID NO:2; VP3 capsid protein, the amino acid sequence of which is shown in SEQ ID NO:3; and VP4 capsid protein, the amino acid sequence is shown in SEQ ID NO:

4.

5. Use of the virus-like particles according to claim 4 in the preparation of medicines for preventing hand, foot and mouth disease.

6. A pharmaceutical composition for preventing hand, foot and mouth disease, characterized in that: The pharmaceutical composition comprises the virus-like particle according to claim 4 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is a vaccine composition.

8. A method for constructing a Coxsackievirus A10 strain rCV-A10-H, characterized in that: Including the step of replacing key amino acid sites in the structural protein region of the Coxsackievirus A10 strain CV-A10-L, or introducing nucleotide mutations in the structural protein region of the Coxsackievirus A10 strain CV-A10-L to achieve the step of replacing key amino acid sites; The key amino acid sites are sites that determine humoral immunogenicity.

9. The method according to claim 8, characterized in that The determination of key sites includes the following steps: By comparing and screening the binding sites of the structural protein regions of the Coxsackievirus A10 strain CV-A10-FJ-01 and the neutralizing monoclonal antibody, and the amino acid sites of the corresponding structural protein regions of the low humoral immunogenic Coxsackievirus A10 strain CV-A10-L, the sites with different amino acids are found to be potential key amino acid sites.

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