Thermostable coxsackievirus a6 strain and use thereof

Abstract

Provided are a thermostable Coxsackievirus A6 strain and a use thereof. The thermostable Coxsackievirus A6 strain can withstand high-temperature treatment below 55°C, and has a protein amino acid sequence as shown in any one of SEQ ID NOs: 1-4. The virus strain is obtained from a parent strain, which is separated from human rhabdomyoma cells (RD) and undergoes passage to be adapted to Vero cells, undergoing adaptive passage under a thermal selection pressure of 52-55°C and undergoing limited dilution and purification. The virus strain has thermal stability, high yield, and strong immunogenicity and can be used as an inactivated vaccine candidate strain, and a sequence of the virus strain can be used for subsequent research and development of enterovirus vaccines and related research of enterovirus stability.

Description

A heat-stable coxsackievirus A group 6 virus strain and its application Technical Field The present invention relates to the technical field of biological products, and in particular to a heat-stable Coxsackievirus A group 6 virus strain and an application thereof. Background Art Hand-foot-and-mouth disease (HFMD) is caused by a variety of enteroviruses, among which enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), coxsackievirus A6 (CV-A6), and coxsackievirus A10 (CV-A10) are the most common. CV-A6 belongs to the genus Enterovirus of the Picornaviridae family. Enterovirus particles are icosahedral and spherically symmetrical, with a diameter of about 27-30nm, and the genome is a single-stranded positive-strand RNA of about 7400nt. The structural protein of CV-A6 virus is composed of the precursor protein P1, which is cleaved into VP4, VP2, VP3, and VP1 by protease. VP1-VP3 are located on the surface of the virus particle, and VP4 is connected to the viral RNA and is located inside the virus particle. Enterovirus has two types of virus particles with different sedimentation coefficients: hollow particles (EP) and solid particles (FP). EP contains three structural proteins, VP0, VP2, and VP3, and is not infectious. FP contains four structural proteins, VP1 to VP4. After VP0 is maturely cleaved into VP4 and VP2, the virus particle (Virion, FP) contains viral RNA inside, which is a mature virus particle with infectious properties. Studies have shown that thermostable strains are more immunogenic than thermounstable strains and can stimulate animals to produce higher levels of neutralizing antibodies. Summary of the invention The present invention uses CV-A6 / CHN XY / 2017 isolated from sample No. 3391 of hand, foot and mouth disease patient in Xiangyang City, Hubei Province, China in 2017 as the mother strain, adapts it to passage under heat selection pressure, and obtains it after dilution and purification, and names them as stable strains TS + [1] TS + [4] TS + [9] TS +

[0012] At the same time, the virus liquid of the mother strain that had undergone the same number of passages on Vero cells and had not been heat-treated was directly purified by three rounds of limiting dilution to obtain multiple clones, which were then inoculated into Vero cells after being treated at 52°C for 30 minutes. The strain that could not proliferate was selected and named the heat-labile strain TS -

[0016] Compared with the heat-unstable strain, the stability and virus particle yield of the stable strain are significantly improved, and the humoral immunogenicity is better than or equivalent to that of the heat-unstable strain. The heat-stable strain can be used as a candidate strain for inactivated vaccine, and its sequence can be used for the subsequent development of enterovirus VLP vaccine and basic research and application related to enterovirus stability. One of the purposes of the present invention is to protect a heat-stable Coxsackievirus A group 6 virus strain, which can withstand high-temperature treatment below 55°C, and whose protein amino acid sequence is shown in any one of SEQ ID NOs: 1 to 4. These four strains and the heat-unstable strain are composed of structural proteins VP4, VP2, VP3, VP1 and non-structural proteins 2A, 2B, 2C and 3A, 3B, 3C, 3D. Specifically, the VP4 protein is the amino acid sequence shown at positions 1 to 69 of the sequence shown in SEQ ID NOs: 1 to 4, the VP2 protein is the amino acid sequence shown at positions 70 to 325 of the sequence shown in SEQ ID NOs: 1 to 4, the VP3 protein is the amino acid sequence shown at positions 326 to 565 of the sequence shown in SEQ ID NOs: 1 to 4, the VP1 protein is the amino acid sequence shown at positions 566 to 870 of the sequence shown in SEQ ID NOs: 1 to 4, the 2A protein is the amino acid sequence shown at positions 871 to 1029 of the sequence shown in SEQ ID NOs: 1 to 4, the 2B protein is the amino acid sequence shown at positions 1021 to 1119 of the sequence shown in SEQ ID NOs: 1 to 4, the 2C protein is the amino acid sequence shown at positions 1120 to 1448 of the sequence shown in SEQ ID NOs: 1 to 4, the 3A protein is the amino acid sequence shown at positions 1449 to 1534 of the sequence shown in SEQ ID NOs: 1 to 4, and the 3B protein is the amino acid sequence shown in SEQ ID NOs: 1 to 4. The amino acid sequence is shown at positions 1535 to 1556 of the sequence shown in SEQ ID NOs: 1 to 4, the 3C protein is the amino acid sequence shown at positions 1557 to 1739 of the sequence shown in SEQ ID NOs: 1 to 4, and the 3D protein is the amino acid sequence shown at positions 1740 to 2201 of the sequence shown in SEQ ID NOs: 1 to 4. The second object of the present invention is to protect the nucleotide sequence encoding the above-mentioned thermostable Coxsackievirus group A type 6 virus strain. Furthermore, the nucleotide sequence is shown in any one of SEQ ID NOs: 6 to 9. The above four heat-stable virus strains TS + [1] TS + [4] TS + [9] TS + With mother strain or heat unstable virus strain TS -

[0016] By comparison, 23 nucleotide mutations were produced in the coding regions of the four heat-stable strains, including 9 missense mutations and 14 synonymous mutations. The 9 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP2-T1052C / Y041H; VP2-G1359C / G143A; VP3-A1709G / T004A; VP3-T1727A / S010T; VP1-C2580T / A054V; VP1-A2823C / N135T; 2C-G4319A / V080I; 3A-G5235A / S56N; 3D-C7275T / A445V. The nucleotide positions of the 14 synonymous mutations in the coding region are: VP4-T799C; VP4-C874T; VP2-C977T; VP2-T1612C; VP1-C2896T; VP1-A3034G; VP1-C3199T; VP1-G3211A; 2C-A4396G; 2C-C4675T; 3A-T5296C; 3C-C5765T; 3D-T6037C; 3D-T6176C. The present invention also aims to protect the method for culturing the above-mentioned heat-stable Coxsackievirus A6 strain, specifically: using Vero cells as hosts to culture the Coxsackievirus A6 strain. The present invention also aims to protect the application of the above-mentioned heat-stable Coxsackievirus A group 6 virus strain and nucleotide sequence in studying the pathogenic mechanism of Coxsackievirus. The purpose of the present invention is also to protect the use of the above-mentioned heat-stable Coxsackievirus group A type 6 virus strain and nucleotide sequence in the preparation of a vaccine against hand, foot and mouth disease. The present invention also aims to protect the use of the above-mentioned heat-stable Coxsackievirus Group A Type 6 virus strain and nucleotide sequence in testing the protective efficacy of Coxsackievirus vaccines. The present invention uses cell culture virus technology to screen and separate 4 CV-A6 thermostable strains and 1 thermounstable strain from the wild-type strain CV-A6 / CHN XY / 2017. Compared with the prior art, the present invention has the following beneficial effects: (1) The thermostable and thermolabile strains were cultured in large quantities in cell factories and then purified. The virus titer, particle yield, and humoral immunogenicity after rat live virus immunization were compared. The titer and humoral immunogenicity of the four thermostable strains after rat live virus immunization were better than or equivalent to those of the thermolabile strains, and the stability and virus particle yield were better than those of the thermolabile strains. The four thermostable strains can meet the high-yield requirements of inactivated vaccine strains, extend the shelf life of vaccines, and reduce the cost of cold chain transportation of vaccines; (2) By determining the whole genome sequence of the thermostable strain and the thermolabile strain, we compared and found the nucleotide and amino acid mutation sites that affect the continued proliferation of the CV-A6 virus after being treated with a heat pressure gradient of 52-55°C. These sites are different from the sites related to enterovirus thermostability reported in the existing literature. The nucleotide and amino acid sites of the genes related to thermostability can be applied to the construction of high-yield and thermostable strains of other enteroviruses besides CV-A6. Based on the changes in the structural protein gene sequence sites, we can construct thermostable virus-like particle candidate vaccines for CV-A6 and other enteroviruses. This can also provide a theoretical and application basis for the study of other enterovirus production, particle stability and immunogenicity-related sites. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the growth status of the wild-type strain and the heat-labile strain after inoculation into Vero cells after being treated at 52°C to 56°C for 30 min; FIG2 is a comparison of the growth status of thermostable strains and thermolabile strains on Vero cells; FIG3 is a comparison of heat resistance between thermostable and thermolabile strains; FIG4 is a comparison of the humoral immunogenicity of live strains of thermostable and thermolabile strains; FIG. 5 shows the results of purification and identification of thermostable and thermolabile strains. DETAILED DESCRIPTION The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly. The following embodiments are only used to illustrate the present invention, but are not used to limit the scope of the present invention. Based on the specific embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, if not specifically specified, the technical means used are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the examples of the present invention are all commercially available. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989) compiled by Sambrook et al., or under the conditions recommended by the manufacturer. Example 1: Screening of heat-stable high-yield strains (1) Virus sample CV-A6 / CHN XY / 2017 (isolated from hand, foot and mouth disease patient sample No. 3391 in Xiangyang City, Hubei Province, China (CHN XY) in 2017) was treated at 52°C for 30 min and immediately stored at 4°C; (2) After the treated sample is thawed, inoculate Vero cells, place in a 37°C, 5% CO2 incubator until the cells show pathological changes, and store at -20°C; (3) The virus solution was harvested by repeated freezing and thawing three times, centrifuged at 6000 g for 5 min, the cell debris precipitate was removed, and the supernatant was collected; (4) Repeat (1) to (3) for the harvested virus solution, heat-treat and subculture for 5 times, and gradually increase the heat treatment temperature by 1°C to 56°C; (5) Select a batch of virus harvest fluid that can withstand the highest temperature treatment (the fourth batch in this case, i.e., the screening temperature reaches 55°C), package it and freeze it as a quasi-species library of heat-stable high-yield strains. Among them, the CV-A6 / CHN XY / 2017 wild-type strain still showed obvious lesions after being treated at 55°C for 30 minutes, and no visible lesions appeared until the temperature was raised to 56°C, as shown in Figure 1. The virus supernatant that still had lesions after being treated at 55°C was selected for limited dilution purification to obtain the heat-stable strain TS + [1] TS + [4] TS + [9] TS +

[0012] Multiple clones were obtained by direct limiting dilution from the CV-A6 / CHN XY / 2017 wild-type strain, and were inoculated into Vero cells after being treated at 52°C for 30 min. The clones that failed to cause obvious pathological changes in Vero cells were taken as the heat-unstable strain TS. -

[0016] . Example 2: Limiting dilution purification of thermostable strains (1) After the Vero cells with good growth status were digested, they were mixed with appropriate ratio (5×10 5 / mL) were inoculated into a 96-well cell culture plate and placed in a 37°C, 5% CO2 cell culture incubator for later use. (2) Add cell maintenance medium DMEM to a clean 24-well cell culture plate, 900 μL per well, take 100 μL of virus sample into the first well, repeatedly pipette and mix, pipette 100 μL into the next well, and perform ten-fold gradient dilutions in sequence. Replace the pipette tip for each dilution, and add the diluted virus solution to a 96-well plate in order from high to low according to the dilution gradient. Set 8 replicate wells (100 μL / well) for each dilution, and culture at 37°C, 5% CO2 for 7 days. The results of observation under a microscope are shown in Figure 2. (3) Virus harvesting: Observe the cytopathic effect (CPE) of each well under a microscope, pick the clone from the duplicate well corresponding to the last CPE-positive dilution, and transfer 200 μL of the virus harvest solution from the well to a 1.5 mL centrifuge tube. Take 100 μL of the harvest solution and dilute it tenfold. Repeat the above steps for three consecutive limiting dilutions to finally obtain the purified virus clone TS. + [1] TS + [4] TS + [9] TS +

[0012] and TS -

[0016] . (4) Expansion culture of virus clones: The virus clones obtained after three consecutive purifications were inoculated into Vero cells at a low MOI for expansion culture. Example 3: Virus titer determination (1) RD cells grown to a dense monolayer were digested and then plated at 1×10 5 / mL of cells were plated into a 96-well plate, 100 μL per well; (2) The harvested virus solution was diluted in DMEM maintenance medium in a 10-fold gradient, for a total of 8 dilution gradients; (3) After the cells in the 96-well plate adhered, the diluted virus solution was added, with 8 replicate wells per gradient, 100 μL per well, the virus solution and cells were mixed and placed in a 37°C, 5% CO2 incubator for culture; (4) After 7 days, the pathological changes in the 96-well plate were observed and the virus titer was calculated according to the Reed-Mench formula; (5) Repeat the titration 3-6 times to determine the virus titer. Example 4: Thermostable strain TS + [1] TS + [4] TS + [9] TS +

[0012] and heat unstable strain TS - Comparison of heat treatment capability (1) The purified clone CV-A6 TS obtained in Example 2 was + [1] TS + [4] TS + [9] TS +

[0012] strain and TS -

[0016] strains were treated at 37°C, 42°C, 45°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C and 60°C for 30 min and immediately stored at 4°C; (2) Each treated strain was seeded into a 6-well plate filled with Vero cells at the same MOI (calculated according to the titer measured before heat treatment) and cultured in a 37°C, 5% CO2 incubator to observe the pathological changes. The results showed that the heat-labile strain TS -

[0016] After treatment at 52°C, most viruses were inactivated and lost their infectivity, but the heat-stable strain TS + [1] TS + [4] TS + [9] TS +

[0012] After treatment at 56°C, the titer can still be maintained at a high level, and the thermal stability is significantly better than that of the thermally unstable strain; (3) titrating the treated virus solution according to the virus titer determination method in Example 3; (6) A heat resistance curve was drawn to compare and analyze the heat resistance of each strain. The results are shown in FIG3 . Example 5: Thermostable strain TS + [1] TS + [4] TS + [9] TS +

[0012] and heat unstable strain TS -

[0016] Full genome sequence determination (1) Viral RNA extraction: CV-A6 viral RNA was extracted and purified using QIAGEN's viral RNA extraction and purification kit QIAamp Viral RNA Mini Kit (Cat. No. 52906); (2) Reverse transcription to synthesize cDNA: Add the reagents listed in Table 1 to a PCR tube, mix well, and place in a PCR instrument. React at 65°C for 5 min, and then quickly cool on ice. Add the reagents listed in Table 2 to the above reaction solution, mix well, and place in a PCR instrument. The reaction conditions are 30°C for 15 min; 42°C for 60 min; and 95°C for 15 min. Table 1 Reaction system for the first step of reverse transcription synthesis of cDNA Table 2 Reaction system for the second step of reverse transcription synthesis of cDNA (3) PCR amplification: The primers for determining the full sequence of CV-A6 are shown in Table 3. KOD-Plus-Neo (Cat. No.: KOD-401) produced by Toyobo was used for PCR amplification. The reaction conditions were 98°C, 10 min; 98°C, 30 sec; 55°C, 30 sec; 68°C (extension rate 30 sec / kb); and the number of cycles was 30-35. Table 3 Whole genome sequencing primers (4) Agarose gel electrophoresis: After the PCR reaction is completed, the product is subjected to 1% agarose gel electrophoresis to identify the PCR amplification band. After the PCR product is correctly identified, the amplified product is sent to Sangon Biotechnology Technology Service Co., Ltd. for sequencing, and the fragments are spliced ​​to obtain the whole genome sequence. Sequencing results showed that the four CV-A6 thermostable strains TS + [1] TS + [4] TS + [9] TS +

[0012] The amino acid sequences of the polyproteins are shown in SEQ ID NOs: 1 to 4, respectively, and the complete genome sequences are shown in SEQ ID NOs: 6 to 9, respectively. The heat-unstable strain TS - The amino acid sequence of the polyprotein of

[0016] is shown in SEQ ID NO:5, and the full genome sequence is shown in SEQ ID NO:10. It is composed of non-coding region 5'-UTR, 3'-UTR and structural proteins VP4, VP2, VP3, VP1 and non-structural protein 2A, 2B, 2C, 3A, 3B, 3C, 3D coding regions, and the genome length is 7430 nucleotide residues. The nucleotide positions of non-coding region 5'UTR and 3'UTR in the genome are 1 to 724 and 7331 to 7430, respectively, and the 3'UTR is followed by a poly A tail of undetermined length, and the encoded polyprotein length is 2201 amino acid residues, wherein the amino acid sequences and positions of each structural protein and non-structural protein of the virus are respectively: The VP4 protein is the amino acid sequence at positions 1 to 69 shown in SEQ ID NOs: 1 to 4; The VP2 protein is the amino acid sequence at positions 70 to 325 shown in SEQ ID NOs: 1 to 4; The VP3 protein is the amino acid sequence at positions 326-565 shown in SEQ ID NOs: 1-4; The VP1 protein is the amino acid sequence at positions 566-870 shown in SEQ ID NOs: 1-4; The 2A protein is the amino acid sequence at positions 871 to 1020 shown in SEQ ID NOs: 1 to 4; The 2B protein is the amino acid sequence at positions 1021 to 1119 shown in SEQ ID NOs: 1 to 4; The 2C protein is the amino acid sequence at positions 1120-1448 shown in SEQ ID NOs: 1-4; The 3A protein is the amino acid sequence at positions 1449-1534 shown in SEQ ID NOs: 1-4; The 3B protein is the amino acid sequence at positions 1535-1556 shown in SEQ ID NOs: 1-4; The 3C protein is the amino acid sequence at positions 1557-1739 shown in SEQ ID NOs: 1-4; The 3D protein is the amino acid sequence at positions 1740-2201 shown in SEQ ID NOs: 1-4. Example 6: Analysis of key sites causing differences in stability and yield among strains Four CV-A6 thermostable strains TS + [1] TS + [4] TS + [9] TS +

[0012] and heat unstable strain TS - The whole genome sequence of

[0016] was compared using SnapGene software. Heat labile strain TS -

[0016] The 41st amino acid of the VP2 protein is tyrosine (Y); the 143rd amino acid of the VP2 protein is glycine (G); the 4th amino acid of the VP3 protein is threonine (T); the 10th amino acid of the VP3 protein is serine (S); the 54th amino acid of the VP1 protein is alanine (A); the 135th amino acid of the VP1 protein is asparagine (N); the 80th amino acid of the 2C protein is valine (V); the 56th amino acid of the 3A protein is serine (S); and the 445th amino acid of the 3D protein is alanine (A). CV-A6 thermostable strain TS + [1] TS + [4] TS + [9] TS + Compared to TS -

[0016] A total of mutations are included: the 41st amino acid of VP2 protein is histidine (H); the 143rd amino acid of VP2 protein is alanine (A); the 4th amino acid of VP3 protein is alanine (A); the 10th amino acid of VP3 protein is threonine (T); the 54th amino acid of VP1 protein is valine (V); the 135th amino acid of VP1 protein is threonine (T); the 80th amino acid of 2C protein is (I); the 56th amino acid of 3A protein is asparagine (N); the 445th amino acid of 3D protein is valine (V). The nucleotide variations that cause the above amino acid changes and the variations of nonsense nucleotides in the genome are shown in Table 4. All the nucleotide and amino acid variations listed may be related to the growth ability of each virus strain under heat treatment environment, virus particle production and immunogenicity. Table 4 Key sites affecting the thermal stability of strains Example 7: Quantification of Antigen Content in Virus Harvest Fluid (1) Preparation of ELISA plate: Dilute the purified CV-A6 rabbit polyclonal antibody to 2 μg / mL in carbonate buffer (pH 9.6, 0.05 mol / L), coat the ELISA plate at 100 μL / well, and incubate at 4°C overnight. After washing the plate, add PBST buffer containing 1% bovine serum albumin, incubate at 37°C for 2 h, discard the blocking solution, pat dry, and store at 4°C for later use. (2) Sandwich ELISA: Dilute the virus harvest and CV-A6 antigen standard to be tested in a 1:4 ratio with PBST buffer containing 1% bovine serum albumin, add them to the ELISA plate, 100 μL / well, incubate at 37°C for 1 hour, wash the plate and pat dry. Add mouse anti-CV-A6 IgG-HRP conjugate diluted to the working concentration at 100 μL / well, incubate at 37°C for 1 hour, wash the plate and pat dry. Then add 1:1 mixed substrate A and B solution for color development, 50 μL / well, incubate at 37°C in the dark for 15 minutes, then add 2 mol / L H2SO4 to terminate the reaction, 50 μL / well. Read the absorbance of each well of the ELISA plate at 450nm and 630nm, make a linear fit with the antigen content of the standard and its corresponding absorbance value (A450-A630), and calculate the antigen content of the sample to be tested according to the standard curve equation. Example 8: Animal Immunization (1) After the antigen content of the virus harvest fluid of each strain was determined by ELISA method, 500 U of antigen was set as the dose for live virus immunization; (2) 175-200 g female SPF Wistar rats were selected, 8 rats per group, and 500 U of antigen per dose was injected intraperitoneally. A DMEM maintenance solution control group was set up. The first immunization was performed on day 0, the booster immunization was performed on day 14, and blood was collected on day 21. Example 9: Serum neutralizing antibody titer detection (1) Serum samples were diluted with diluent (such as DMEM maintenance medium) at a ratio of 1:8 and added to column A of a 96-well plate at 100 μL / well. Two replicate wells were set up and 50 μL of diluent was added to each well of columns B to H. The serum samples in column A were diluted 2-fold to column H. After the last column was diluted, 50 μL was discarded. (2) Dilute the neutralized virus to 100 CCID 50 / 50μL, draw 50μL vertically suspended and drop into the diluted serum sample, place the 96-well plate in a 37℃ incubator for 2h. Dilute the diluted neutralized toxin in a 10-fold gradient to 10CCID 50 / 50μL, 1CCID 50 / 50μl, 0.1CCID 50 / 50μL, add 50μL maintenance solution and 50μL virus solution to each well (a total of 4 dilutions, 100, 10, 1, 0.1CCID 50 / 50μL), 8 replicate wells for each dilution, as a back titration plate, and place at 4°C. (3) After neutralization, the digested RD cells were plated at 1×10 5 / ml density was plated on the neutralization plate and the back titration plate, and the neutralization results were determined at 37°C and 5% CO2 for 5 to 7 days. The results are shown in Figure 4. Each point in the figure represents a Wistar rat. The titer of the DMEM control group was lower than 8. The values ​​were assigned and plotted. One-way analysis of variance (ANOVA) was performed using the Sidak multiple comparison test. The statistical significance is as follows: ns, not significant (P ≥ 0.05); *, 0.01 ≤ P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001. As shown in Figure 4, 7 days after the booster immunization, the CV-A6 heat-stable strain TS + [1] TS + [4] TS + [9] TS + The serum neutralizing antibody titer of Wistar rats immunized with live virus is better than or equivalent to that of the heat-labile strain TS -

[0016] The serum neutralization titer after immunization with the same antigen amount. Example 10: Virus particle purification and antigen preparation (1) When the Vero cells in the ten-layer cell factory grow to a dense monolayer state, each CV-A6 strain is inoculated into the cell factory at the same MOI and cultured in a 37°C, 5% CO2 incubator. (2) When more than 90% of the cells became diseased, the virus was harvested and concentrated by ultrafiltration, centrifuged on a sucrose cushion, and subjected to CsCl isodensity gradient centrifugation. The virus particles were harvested after centrifugal filtration and desalting. The result is shown in FIG5A . Three opalescent bands appeared in the centrifuge tube. The bands were extracted and centrifuged to remove cesium chloride and then identified by SDS-PAGE. The result is shown in FIG5B . After purification, three virus particles, CV-A6EP, FP, and AP, were obtained. (3) The protein concentration of purified virus particles was detected by BCA method, and the total protein content of the harvested virus was calculated. The results of the EP, FP, and AP antigen content of each strain are shown in Table 5. The CV-A6 thermostable strain TS + [1] TS + [4] TS + [9] TS + The total amount of antigen harvested from a single ten-layer cell factory ranged from 0.33 mg to 0.76 mg, with an average yield of 0.57 mg; the CV-A6 heat-unstable strain TS -

[0016] The amount of antigen harvested from a single ten-layer cell factory was only 0.39 mg. Table 5 Comparison of antigen production between heat-stable and heat-unstable strains It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A thermostable coxsackievirus group A type 6 strain, characterized in that: The heat-stable Coxsackievirus group A type 6 virus strain can withstand high temperature treatment below 55° C., and its protein amino acid sequence is shown in any one of SEQ ID NOs: 1 to 4.

2. The thermostable Coxsackievirus group A type 6 strain according to claim 1, characterized in that The virus strain is composed of structural proteins VP4, VP2, VP3, VP1 and non-structural proteins 2A, 2B, 2C and 3A, 3B, 3C, 3D, wherein The VP4 protein is the amino acid sequence shown at positions 1 to 69 of the sequence shown in SEQ ID NOs: 1 to 4, the VP2 protein is the amino acid sequence shown at positions 70 to 325 of the sequence shown in SEQ ID NOs: 1 to 4, the VP3 protein is the amino acid sequence shown at positions 326 to 565 of the sequence shown in SEQ ID NOs: 1 to 4, the VP1 protein is the amino acid sequence shown at positions 566 to 870 of the sequence shown in SEQ ID NOs: 1 to 4, the 2A protein is the amino acid sequence shown at positions 871 to 1020 of the sequence shown in SEQ ID NOs: 1 to 4, the 2B protein is the amino acid sequence shown at positions 1021 to 1119 of the sequence shown in SEQ ID NOs: 1 to 4, the 2C protein is the amino acid sequence shown at positions 1120 to 1448 of the sequence shown in SEQ ID NOs: 1 to 4, the 3A protein is the amino acid sequence shown at positions 1449 to 1534 of the sequence shown in SEQ ID NOs: 1 to 4, the 3B protein is the amino acid sequence shown at positions 1535 to 1556 of the sequence shown in SEQ ID NOs: 1 to 4, and the 3C protein is the amino acid sequence shown in positions 1567 to 1570 of the sequence shown in SEQ ID NOs: 1 to 4. The amino acid sequence is the amino acid sequence shown at positions 1557 to 1739 of the sequence shown in SEQ ID NOs: 1 to 4, and the 3D protein is the amino acid sequence shown at positions 1740 to 2201 of the sequence shown in SEQ ID NOs: 1 to 4.

3. A nucleotide sequence encoding the thermostable Coxsackievirus group A type 6 strain according to claim 1 or 2.

4. The nucleotide sequence according to claim 3, characterized in that The nucleotide sequence is shown in any one of SEQ ID NOs: 6 to 9.

5. The method for culturing a thermostable Coxsackievirus group A type 6 strain according to claim 1 or 2, characterized in that: Vero cells were used as hosts to culture the Coxsackievirus A6 strain.

6. Use of the thermostable coxsackievirus group A type 6 strain according to claim 1 or 2 and the nucleotide sequence according to claim 3 or 4 in studying the pathogenic mechanism of coxsackievirus.

7. Use of the heat-stable Coxsackievirus group A type 6 strain according to claim 1 or 2 and the nucleotide sequence according to claim 3 or 4 in the preparation of a vaccine against hand, foot and mouth disease.

8. Use of the thermostable coxsackievirus group A type 6 strain according to claim 1 or 2 and the nucleotide sequence according to claim 3 or 4 in testing the protective efficacy of coxsackievirus vaccines.

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