Heat-resistant coxsackievirus a10 thermostable strain and use thereof

Abstract

A heat-resistant coxsackievirus A10 (CV-A10) thermostable strain and a use thereof. The heat-resistant CV-A10 thermostable strain has the protein amino acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, and is obtained by adaptively passaging a virus parent strain, which is isolated from an HEV3108 sample of a patient with the hand, foot and mouth disease in Xiangyang city, Hubei province, China 2017, under the selective pressure of thermal treatment at 52°C for half an hour for five consecutive rounds and carrying out plaque cloning purification. The thermal stability of the strain is significantly improved compared with that of the parent strain, the antigen yield and immunogenicity of the strain are not inferior to those of a thermolabile strain, and thus the strain can be used as an inactivated vaccine candidate strain, and the sequence of the strain can be used for subsequent research and development of enterovirus vaccines and related research of enterovirus stability.

Description

A heat-resistant coxsackievirus group A type 10 heat-stable strain and its application Technical Field

[0001] The present invention relates to the technical field of virology, and in particular to a heat-stable strain of heat-resistant Coxsackievirus group A type 10 (CV-A10) and applications thereof. Background Art

[0002] 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.

[0003] CV-A10 belongs to the genus Enterovirus in the family Picornaviridae. Enterovirus particles are icosahedral and spherically symmetrical, with a diameter of approximately 27-30 nm. The genome consists of approximately 7400 nt of single-stranded positive-strand RNA. The CV-A10 viral structural proteins consist of a precursor protein, which is cleaved by proteases into VP4, VP2, VP3, and VP1. VP1-VP3 are located on the surface of the virion, while VP4 is located within the virion, linked to the viral RNA. Enterovirus particles exist with two different sedimentation coefficients: hollow particles (EP) and solid particles (FP). EP particles contain three structural proteins, VP0, VP2, and VP3, and are non-infectious. FP particles contain four structural proteins, VP1, VP2, and VP4. After VP0 matures and cleaves into VP4 and VP2, the virion (FP) contains viral RNA, making it an infectious mature virus particle.

[0004] Studies have shown that thermostable strains are more immunogenic than thermounstable strains and can stimulate animals to produce higher levels of neutralizing antibodies.

[0005] Summary of the Invention

[0006] Based on this, the present invention uses the strain CV-A10-3108 / CHN XY / 2017 (CV-A10-3018-TS isolated from the HEV3108 sample of a hand, foot and mouth disease patient in Xiangyang, Hubei Province, China in 2017) to identify the pathogen. - , referred to as TS - ) was used as the mother strain and isolated on Vero cells. After multiple passages, a multi-genotype quasi-population library was formed. After five consecutive rounds of heat treatment at 52°C for half an hour, the cells were adapted to the selection pressure and then purified by plaque cloning to obtain four thermostable strains TS + , respectively CV-A10-3108-TS + Ⅰ (abbreviated as TS + Ⅰ), CV-A10-3108-TS+ Ⅱ (abbreviated as TS + Ⅱ)CV-A10-3108-TS + Ⅲ (abbreviated as TS + Ⅲ) and CV-A10-3108-TS + IV (abbreviated as TS + IV), its stability is significantly improved compared with the heat-labile strain, and its antigen production and immunogenicity are better than or not inferior to those of the heat-labile parent strain. It can be used as a candidate strain for inactivated vaccine, and its sequence can be used for the subsequent development of enterovirus VLP vaccines and basic research and applications related to enterovirus stability.

[0007] One of the purposes of the present invention is to provide a virus strain that can still proliferate on cells after being treated at 52°C for 30 minutes and whose virus particle yield and humoral immunogenicity are better than or not inferior to those of a heat-unstable virus strain, and to locate the nucleotide and amino acid sites that affect the heat stability of the virus strain, specifically to protect a heat-resistant Coxsackievirus group A type 10 heat-stable strain, the heat-resistant Coxsackievirus group A type 10 stable strain can withstand high temperature treatment below 52°C, and its protein sequence is shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

[0008] That is, compared with the wild-type strain, there is a site mutation described in any of the following (1) to (4):

[0009] (1) N69S in VP4 protein, L194V in VP2 protein, E40G in VP1 protein, I57V in 2B protein, and I44M in 3A protein;

[0010] (2) L194V in VP2 protein, E40G in VP1 protein, I57V in 2B protein, and I44M in 3A protein;

[0011] (3) L194V in VP2 protein, E40G in VP1 protein, E85G in 2A protein, I57V in 2B protein, L141S in 2A protein, V232I in 2C protein, and I44M in 3A protein;

[0012] (4) L194V in VP2 protein, E40G in VP1 protein, T103A in VP1 protein, I57V in 2B protein, L141S in 2A protein, and I44M in 3A protein.

[0013] Wherein, the protein sequence of the wild-type strain is as shown in SEQ ID NO: 1, wherein the VP4 protein is the amino acid sequence shown at positions 1 to 69 of the sequence shown in SEQ ID NO: 1, the VP2 protein is the amino acid sequence shown at positions 70 to 324 of the sequence shown in SEQ ID NO: 1, the VP3 protein is the amino acid sequence shown at positions 325 to 564 of the sequence shown in SEQ ID NO: 1, the VP1 protein is the amino acid sequence shown at positions 565 to 862 of the sequence shown in SEQ ID NO: 1, the 2A protein is the amino acid sequence shown at positions 863 to 1012 of the sequence shown in SEQ ID NO: 1, the 2B protein is the amino acid sequence shown at positions 1013 to 1111 of the sequence shown in SEQ ID NO: 1, the 2C protein is the amino acid sequence shown at positions 1112 to 1440 of the sequence shown in SEQ ID NO: 1, the 3A protein is the amino acid sequence shown at positions 1441 to 1526 of the sequence shown in SEQ ID NO: 1, the 3B protein is the amino acid sequence shown at positions 1527 to 1548 of the sequence shown in SEQ ID NO: 1, and the 3C protein is the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence is shown at positions 1549 to 1731 of the sequence shown in NO: 1, and the 3D protein is the amino acid sequence shown at positions 1732 to 2194 of the sequence shown in SEQ ID NO: 1.

[0014] The second purpose of the present invention is to protect the coding gene of the above-mentioned heat-resistant Coxsackievirus A group 10 heat-stable strain.

[0015] Furthermore, the nucleotide sequence of the encoding gene is as described in any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10.

[0016] Specifically, the heat-labile strain CV-A10-3018-TS - Strain compared: CV-A10-3108-TS + A total of 9 nucleotide mutations occurred in the I coding region, including 5 missense mutations and 4 synonymous mutations. The 5 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP4-A952G / N69S, VP2-C1533G / L194V, VP1-A2557G / E40G, 2B-A3951G / I57V, 3A-A5198G / I44M; the nucleotide positions of the 4 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 3D-T7205G.

[0017] CV-A10-3108-TS +A total of 9 nucleotide mutations occurred in the II coding region, including 4 missense mutations and 5 synonymous mutations. The 4 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP2-C1533G / L194V, VP1-A2557G / E40G, 2B-A3951G / I57V, 3A-A5198G / I44M; the nucleotide positions of the 5 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 2C-T4148C; 3D-T7205G.

[0018] CV-A10-3108-TS + A total of 11 nucleotide mutations occurred in the III coding region, including 7 missense mutations and 4 synonymous mutations. The 7 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP2-C1533G / L194V, VP1-A2557G / E40G, 2A-A3586G / E85G, 2B-A3951G / I57V, 2A-T3754C / L141S, 2C-G4773A / V232I, 3A-A5198G / I44M; the nucleotide positions of the 4 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 3D-T7205G.

[0019] CV-A10-3108-TS + A total of 11 nucleotide mutations occurred in the IV coding region, including 6 missense mutations and 5 synonymous mutations. The 6 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP2-C1533G / L194V, VP1-A2557G / E40G, VP1-A2745G / T103A, 2B-A3951G / I57V, 2A-T3754C / L141S, 3A-A5198G / I44M; the nucleotide positions of the 5 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 2C-T4838C; 3D-T7205G.

[0020] The present invention also aims to protect the method for culturing the above-mentioned heat-resistant Coxsackievirus A10 type heat-stable strain, using Vero cells as hosts to culture the Coxsackievirus A10 type strain.

[0021] The third purpose of the present invention is to protect the application of the above-mentioned heat-resistant Coxsackievirus A group 10 heat-stable strain and encoding gene in studying the pathogenic mechanism of Coxsackievirus.

[0022] The fourth purpose of the present invention is to protect the use of the above-mentioned heat-resistant Coxsackievirus A group 10 heat-stable strain and encoding gene in the preparation of vaccines against hand, foot and mouth disease.

[0023] A fifth object of the present invention is to protect the use of the above-mentioned heat-resistant Coxsackievirus group A type 10 heat-stable strain and encoding gene in testing the protective efficacy of Coxsackievirus vaccines.

[0024] The present invention has at least the following beneficial technical effects:

[0025] (1) The present invention obtained four thermostable strains through cell culture virus technology;

[0026] (2) By sequencing and comparing the entire genome of the virus strain, we discovered nucleotide and amino acid sites that affect the proliferation of the CV-A10 strain after treatment at 52°C. These mutation sites are different from the sites associated with the thermal stability of poliovirus reported in existing literature, i.e., they are not located within the VP1 hydrophobic pocket, and the VP1 amino acids outside the hydrophobic pocket are not located within the thermal stability sites of other enteroviruses reported in the literature.

[0027] (3) The heat-labile and heat-stable strains were further cultured in cell factories and purified, and the viral titer, particle production, and humoral immunogenicity were compared and analyzed. It was found that the titer of the heat-stable strain was not lower than that of the heat-labile strain, and the viral particle production and humoral immunogenicity were better than or not inferior to those of the heat-labile strain.

[0028] (4) Quantitative analysis of virus particles after Vero cell culture and purification showed that after two amino acid changes in the VP1 sequence, the particle yield of the virus after cell culture was higher than that of the parent strain (heat-unstable strain). This feature is opposite to the decrease in virus yield after amino acid changes at multiple sites in the heat-stable polio strain, which is conducive to the high yield requirement of inactivated vaccine strains;

[0029] (5) The obtained thermostable strains can be used as candidate strains for inactivated vaccines, and can also be used to construct VLP vaccines based on the discovered thermostable sites. They 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

[0030] FIG1 is a comparison of the growth status of the original strain and the screened thermostable strain on Vero cells;

[0031] FIG2 is a comparison of the growth curves of the original strain and the screened thermostable strain on Vero cells;

[0032] Figure 3 shows the comparison of the growth status of the original strain and the selected thermostable strain after inoculation into Vero cells after treatment at 52°C for 30 min;

[0033] FIG4 is a comparison of the heat resistance of the original strain and the screened thermostable strain;

[0034] FIG5 shows the centrifugation results of the separation and purification of virus particles of the original strain and the screened thermostable strain;

[0035] Figure 6 shows the results of virus purification and identification of the original strain and the screened thermostable strain;

[0036] FIG7 is a comparison of the heat resistance of virus particles of the original strain and the screened thermostable strain;

[0037] FIG8 is a comparison of the humoral immunogenicity of the original strain and the screened heat-stable strain. DETAILED DESCRIPTION

[0038] 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 those of ordinary skill in the art without making 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.

[0039] Unless otherwise specified, all reagents used in the examples of the present invention are commercially available. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0040] Example 1 Screening of heat-stable strains

[0041] (1) Virus sample CV-A10-3108-TS - (isolated from sample 3108 of hand, foot and mouth disease patient in Xiangyang, Hubei Province, China in 2017) treated at 52°C for 30 min, inoculated with Vero cells, cultured in a 37°C incubator until the cells showed pathological changes, and then stored at -20°C;

[0042] (2) The virus solution was harvested by repeated freezing and thawing three times, centrifuged at 6000 g for 5 min, the cell debris pellet was removed, and the supernatant was collected;

[0043] (3) Repeat (1) to (2) for the collected virus solution, and heat-treat and passage it 5 times.

[0044] Example 2 Plaque Purification of Thermostable Strains

[0045] (1) The prepared Vero cells were digested and then plated at 5×10 5 The cells were seeded into 6-well cell culture plates at a density of 100 cells / mL;

[0046] (2) When the cells grew into a dense monolayer, the culture medium was discarded and the cells were washed twice with 0.01 M PBS;

[0047] (3) The fifth-generation heat-treated virus solution was treated again at 52°C for 30 min and then serially diluted 10-fold for a total of 6 dilution gradients;

[0048] (4) Add the virus solution to a 6-well plate and adsorb at 37°C for 2 h before discarding the virus solution;

[0049] (5) Mix 2% low-melting-point agarose and 2× DMEM maintenance solution in a 1:1 ratio of equal volumes, add 2 ml to each well of a 6-well plate, wait for it to solidify, and place in an incubator;

[0050] (6) Observe the results after 2-3 days and pick out plaques at the lowest dilution;

[0051] (7) Repeat plaque purification three times to obtain cloned strains;

[0052] (8) The clones obtained from three rounds of plaque purification were blind-passaged three times on Vero cells at a low MOI to obtain a heat-stable virus harvest.

[0053] Example 3 Virus titer determination

[0054] (1) Vero cells were cultured at a rate of 1×10 5 / mL of cells were plated into 96-well plates, with each 100 μL;

[0055] (2) The harvested virus solution was serially diluted 10-fold using DMEM maintenance medium, for a total of 8 dilution gradients;

[0056] (3) After the cells in the 96-well plate adhered to the wall, the diluted virus solution was added. Each gradient was replicated to 8 wells, with 100 μl per well. After adding the virus solution, the plate was placed in a 37°C incubator for incubation.

[0057] (4) After 5 days, the pathological changes in the 96-well plate were observed and the viral titer was calculated according to the Reed-Mench formula;

[0058] (5) Repeat the titration 3-6 times to determine the virus titer.

[0059] Example 4 Comparison of growth ability between thermostable strain and wild type

[0060] (1)CV-A10-3108-TS + strains (thermostable strains purified in Example 2, respectively named CV-A10-3108-TS+ Ⅰ.CV-A10-3108-TS + II. CV-A10-3108-TS + III. CV-A10-3108-TS + IV) and CV-A10-3018-TS - The strains were seeded at the same MOI (MOI=5) into 6-well plates filled with Vero cells and cultured in a 37°C incubator. The pathological changes were observed. The results are shown in FIG1 .

[0061] (2) titrating the treated virus solution according to the virus titer determination method in Example 3;

[0062] (3) Replace CV-A10-3018-TS - 、CV-A10-3108-TS + Ⅰ.CV-A10-3108-TS + II. CV-A10-3108-TS + Ⅲ and CV-A10-3108-TS + After the IV strain was inoculated into Vero cells at the same inoculum volume, the virus was harvested at different times and the CCID 50 The titer of the strains was detected on Vero cells by the method, and the growth curve was drawn to compare and analyze the growth ability of the two strains. The results are shown in Figure 2. There was no difference in growth ability between the heat-stable strain and the heat-unstable strain.

[0063] Example 5 Comparison of heat resistance between thermostable strains and wild-type strains

[0064] (1)CV-A10-3108-TS + CV-A10-3018-TS - The strains were treated at 37℃, 42℃, 45℃, 48℃, 50℃, 52℃ and 54℃ for 30min, and immediately stored at 4℃; then inoculated into Vero cells and the pathological state was observed. 4 days after virus inoculation, CV-A10-3108-TS + Strain 1, CV-A10-3018-TS + Strain II, CV-A10-3108-TS + strain III and CV-A10-3108-TS + IV strain is completely lesional, while CV-A10-3018-TS - No obvious lesions were observed 4 days after inoculation. The specific results are shown in Figure 3.

[0065] (2) titrating the treated virus solution according to the virus titer determination method in Example 3;

[0066] (3) Replace CV-A10-3018-TS - 、CV-A10-3108-TS + Ⅰ.CV-A10-3108-TS + II. CV-A10-3108-TS + Ⅲ and CV-A10-3108-TS + After strain IV was treated at 37-54℃ for 30min, it was immediately cooled to 4℃ and the CCID 50 The titer of the strains was detected on Vero cells by the method, and the heat resistance curve was drawn to compare and analyze the heat resistance of the two strains. The results are shown in Figure 4. The heat-unstable strain (CV-A10-3018-TS - ) Most viruses were inactivated and lost their infectivity when treated at 48°C, but the heat-stable strain (CV-A10-3108-TS + Ⅰ.CV-A10-3108-TS + II. CV-A10-3108-TS + Ⅲ and CV-A10-3108-TS + IV) After treatment at 50℃, the titer of the strain can still be maintained at a high level, and its thermal stability is significantly better than that of the thermally unstable strain.

[0067] Example 6 Comparison of heat resistance between thermostable strains and wild-type virus particles

[0068] (1) The harvested virus solution was concentrated by ultrafiltration, centrifuged on a sucrose cushion, and centrifuged on a CsCl density gradient. The results are shown in FIG5 . Three opalescent bands appeared in the centrifuge tube. After removing CsCl by centrifugation, the virus was identified by WB. The results are shown in FIG6 . After purification, the EP, FP, and AP virus particles of the virus were obtained.

[0069] (2) Preparation buffer (2 mM HEPES, 200 mM NaCl, 1× SYBR green II, pH = 8);

[0070] (3) Place 1 μg of purified FP in a qPCR tube and fill the tube to 50 μL with buffer;

[0071] (4) A qPCR instrument was used to draw its melting curve; the temperature was raised from 25 to 95°C at a rate of 30 s / °C to draw a heat resistance curve, and the heat resistance of the virus particles was compared and analyzed. The results are shown in Figure 7. The RNA release temperature of the heat-unstable strain is lower than that of the heat-stable strain, and the heat stability of the heat-stable strain is significantly better than that of the heat-unstable strain.

[0072] Example 7 Whole genome sequence determination of thermostable and thermolabile strains

[0073] (1) Viral RNA extraction: Viral RNA was extracted and purified using a Sangon column-based viral RNA extraction and purification kit (Cat. No. B518667);

[0074] (2) Reverse transcription and cDNA synthesis: using Takara's RNA reverse transcription kit PrimeScript TM cDNA was synthesized by reverse transcription using II 1st Strand cDNA Synthesis Kit (Cat. No. 6210A);

[0075] (3) PCR amplification: PCR amplification was performed using 2× Phanta Flash Master Mix (Dye Plus) (Cat. No.: P520) produced by Novozymes, 98°C for 30 sec → 98°C for 10 sec, 61°C for 5 sec, 72°C (extension rate 10 sec / kb), cycle number 30-35. The primers for full sequence determination are shown in Table 1 below:

[0076] Table 1 Primers used for full sequence determination Note: F, forward primer; R, reverse primer.

[0077] (4) Agarose gel electrophoresis: After the PCR reaction is completed, the product is subjected to 1% agarose gel electrophoresis to identify the PCR amplification bands. After the PCR product is correctly identified, the amplified product is sent to Sangon Biotechnology Service Co., Ltd. for sequencing, and the fragments are spliced ​​to obtain the full genome sequence.

[0078] Sequencing results showed that CV-A10-3018-TS - The complete genome sequence and polyprotein amino acid sequence of the strain are shown in SEQ ID NO: 2 and SEQ ID NO: 1, respectively. + The complete genome sequence and polyprotein amino acid sequence of strain I are shown in SEQ ID NO: 4 and SEQ ID NO: 3, respectively. + The complete genome sequence and polyprotein amino acid sequence of strain II are shown in SEQ ID NO:6 and SEQ ID NO:5, respectively. + The complete genome sequence and polyprotein amino acid sequence of strain III are shown in SEQ ID NO:8 and SEQ ID NO:7, respectively. + The complete genome sequence and polyprotein amino acid sequence of strain IV are shown in SEQ ID NO: 10 and SEQ ID NO: 9, respectively.

[0079] Among them, the nucleotides at positions 1 to 746 in the whole genome sequence are 5'UTR, the nucleotides at positions 747 to 953 encode VP4 protein, the nucleotides at positions 954 to 1718 encode VP2 protein, the nucleotides at positions 1719 to 2438 encode VP3 protein, the nucleotides at positions 2439 to 3332 encode VP1 protein, the nucleotides at positions 3333 to 3782 encode 2A protein, and the nucleotides at positions 3783 to 4079 encode VP3 protein. Nucleotides 4080-5066 encode protein 2C, nucleotides 5067-5324 encode protein 3A, nucleotides 5325-5390 encode protein 3B, nucleotides 5391-5939 encode protein 3C, nucleotides 5940-7328 encode protein 3D, and nucleotides 7329-7439 are 3'UTR, which is followed by a poly A tail of undetermined length.

[0080] The VP4 protein is the amino acid sequence from positions 1 to 69, the VP2 protein is the amino acid sequence from positions 70 to 324, the VP3 protein is the amino acid sequence from positions 325 to 564, the VP1 protein is the amino acid sequence from positions 565 to 862, the 2A protein is the amino acid sequence from positions 863 to 1012, the 2B protein is the amino acid sequence from positions 1013 to 1111, the 2C protein is the amino acid sequence from positions 1112 to 1440, the 3A protein is the amino acid sequence from positions 1441 to 1526, the 3B protein is the amino acid sequence from positions 1527 to 1548, the 3C protein is the amino acid sequence from positions 1549 to 1731, and the 3D protein is the amino acid sequence from positions 1732 to 2193.

[0081] Example 8: Analysis of key sites causing differences in strain stability, yield, and immunogenicity

[0082] CV-A10-3108-TS + CV-A10-3018-TS - The whole genome sequences of the strains were aligned using SnapGene software.

[0083] CV-A10-3018-TS - The 69th amino acid of the VP4 protein of the strain is asparagine (N); the 194th amino acid of the VP2 protein is leucine (L); the 40th amino acid of the VP1 protein is glutamic acid (E); the 103rd amino acid of the VP1 protein is threonine (T); the 85th amino acid of the 2A protein is glutamic acid (E); the 141st amino acid of the 2A protein is leucine (L); the 57th amino acid of the 2B protein is isoleucine (I); the 232nd amino acid of the 2C protein is valine (V); and the 44th amino acid of the 3A protein is isoleucine (I).

[0084] CV-A10-3108-TS + The 69th amino acid of VP4 protein of strain I is serine (S); the 194th amino acid of VP2 protein is valine (V); the 40th amino acid of VP1 protein is glycine (G); the 57th amino acid of 2B protein is valine (V); and the 44th amino acid of 3A protein is methionine (M).

[0085] CV-A10-3018-TS + The 194th amino acid of VP2 protein of strain II is valine (V); the 40th amino acid of VP1 protein is glycine (G); the 57th amino acid of 2B protein is valine (V); and the 44th amino acid of 3A protein is methionine (M).

[0086] CV-A10-3108-TS + The 194th amino acid of the VP2 protein of strain III is valine (V); the 40th amino acid of the VP1 protein is glycine (G); the 85th amino acid of the 2A protein is glycine (G); the 141st amino acid of the 2A protein is serine (S); the 57th amino acid of the 2B protein is valine (V); the 232nd amino acid of the 2C protein is isoleucine (I); and the 44th amino acid of the 3A protein is methionine (M).

[0087] The 194th amino acid of the VP2 protein of the CV-A10-3108-TS+IV strain is valine (V); the 40th amino acid of the VP1 protein is glycine (G); the 103rd amino acid of the VP1 protein is alanine (A); the 141st amino acid of the 2A protein is serine (S); the 57th amino acid of the 2B protein is valine (V); and the 44th amino acid of the 3A protein is methionine (M).

[0088] and heat-labile strain CV-A10-3018-TS - Strain compared: CV-A10-3108-TS + A total of 9 nucleotide mutations occurred in the I coding region, including 5 missense mutations and 4 synonymous mutations. The 5 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP4-A952G / N69S, VP2-C1533G / L194V, VP1-A2557G / E40G, 2B-A3951G / I57V, 3A-A5198G / I44M; the nucleotide positions of the 4 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 3D-T7205G.

[0089] CV-A10-3108-TS +A total of 9 nucleotide mutations occurred in the II coding region, including 4 missense mutations and 5 synonymous mutations. The 4 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP2-C1533G / L194V, VP1-A2557G / E40G, 2B-A3951G / I57V, 3A-A5198G / I44M; the nucleotide positions of the 5 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 2C-T4148C; 3D-T7205G.

[0090] CV-A10-3108-TS + A total of 11 nucleotide mutations occurred in the coding region of gene III, including 7 missense mutations and 4 synonymous mutations. The 7 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP2-C1533G / L194V, VP1-A2557G / E40G, 2A-A3586G / E85G, 2B-A3951G / I57V, 2A-T3754C / L141S, 2C-G4773A / V232I, 3A-A5198G / I44M; the nucleotide positions of the 4 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 3D-T7205G.

[0091] CV-A10-3108-TS + A total of 11 nucleotide mutations occurred in the IV coding region, including 6 missense mutations and 5 synonymous mutations. The 6 missense mutation nucleotides and corresponding amino acid positions in the coding region were: VP2-C1533G / L194V, VP1-A2557G / E40G, VP1-A2745G / T103A, 2B-A3951G / I57V, 2A-T3754C / L141S, 3A-A5198G / I44M; the nucleotide positions of the 5 synonymous mutations in the coding region were: VP2-C1532T; VP3-A1910G; 2A-C3377T; 2C-T4838C; 3D-T7205G.

[0092] The nucleotide variations and nonsense nucleotide variations that cause the above amino acid changes are shown in Table 2. All the nucleotide and amino acid variations listed are potentially related to the growth ability of the two virus strains in a heat-treated environment, the production of viral particles, and the immunogenicity of the virus.

[0093] Table 2 Key sites affecting the proliferation of strains in Vero cells after treatment at 52℃

[0094] Example 9: Virus particle purification and antigen preparation

[0095] (1) When the Vero cells in the ten-layer cell factory grow to a dense monolayer state, the CV-A10 strain is inoculated into the cell factory at the same MOI and cultured in a 37°C incubator;

[0096] (2) After more than 90% of the cells have become pathological, the virus is harvested and concentrated by ultrafiltration, centrifuged on a sucrose cushion, and centrifuged on a CsCl density gradient. The virus particles are then harvested after centrifugal filtration and desalting.

[0097] (3) The protein concentration of the purified virus particles was detected by BCA method, and the total protein content of the harvested virus was calculated. The results of the harvested EP, FP, and AP antigen content are shown in Table 3:

[0098] Table 3 Comparison of antigen production between heat-stable and heat-unstable strains

[0099] The virus particle yield is the yield after purification of the virus supernatant harvested from two 10-layer cell factories.

[0100] CV-A10-3018-TS - The yield of a single ten-layer cell factory of the strain virus particles was 0.29 mg;

[0101] CV-A10-3108-TS + The yield of a single ten-layer cell factory for strain 1 virus particles was 0.18 mg;

[0102] CV-A10-3018-TS + The yield of a single ten-layer cell factory for strain II virus particles was 0.25 mg;

[0103] CV-A10-3108-TS + The yield of a single ten-layer cell factory for strain III virus particles was 0.26 mg;

[0104] CV-A10-3108-TS + The amount of antigen harvested from a single ten-layer cell factory of strain IV was 0.29 mg.

[0105] (4) 4% formaldehyde was added to the EP and FP particle suspensions of the above strains to inactivate them respectively, inactivated at 37°C for 48 h, and then centrifuged through a 10 kd ultrafiltration concentrator to remove the formaldehyde.

[0106] Example 10: Animal Immunization

[0107] (1) The concentration of formaldehyde-inactivated virus particles was determined by the BCA method. The inactivated EP, FP, and AP were mixed with chromatography salt buffer and aluminum adjuvant according to the yield ratio of EP:FP:AP harvested after purification to prepare three antigen dose groups of 0.1 μg, 0.5 μg, and 2.5 μg;

[0108] (2) Female Wistar rats weighing 180-200 g were selected, with 8 rats per group receiving intraperitoneal injection of 500 μl per dose. An aluminum adjuvant control group was set up. The primary immunization was performed on day 0, the booster immunization was performed on day 14, and blood was collected on day 28.

[0109] Example 11: Serum neutralizing antibody titer detection

[0110] (1) Dilute the serum sample at a ratio of 1:16 with diluent (e.g., MEM maintenance medium) and add 100 μl / well to column A of a 96-well plate. Set up two replicates. Add 50 μl of diluent to each well of columns B to H. Serially dilute the serum sample in column A by 2-fold to column H. Discard 50 μl of the last column after dilution.

[0111] (2) Dilute the neutralized virus 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 2 hours. 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, namely 100, 10, 1, and 0.1 CCID 50 / 50μl), 8 replicate wells for each dilution, as a back titration plate, and place at 4℃.

[0112] (3) After neutralization, the digested RD cells were divided into 1×10 5 / ml density was plated on neutralization plate and back titration plate, and the neutralization result was determined in 5 to 7 days. + II strain was used as a neutralizing toxin, and the humoral immunogenicity of the isolated serum was evaluated, CV-A10-3018-TS + The 2.5μg dose group of strain II showed that CV-A10-3018-TS + The neutralizing antibody titer of strain II was higher than that of CV-A10-3018-TS - strains. For specific results, see Figure 7. In Figure 7, each point represents a Wistar rat. The titer of the aluminum adjuvant-vaccinated group was less than 8, and the values ​​were assigned for plotting. 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. As shown in Figure 8, 14 days after the booster immunization, CV-A10-3018-TS + The neutralizing antibody titer of the high-dose group of strain II was higher than that of CV-A10-3018-TS- strain.

[0113] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A thermostable strain of heat-resistant Coxsackievirus A group 10, characterized in that: The thermotolerant Coxsackievirus A group 10 stable strain can withstand high temperature treatment below 52°C, and its protein sequence has a site mutation described in any one of the following (1) to (4) compared with the wild-type strain: (1) N69S in VP4 protein, L194V in VP2 protein, E40G in VP1 protein, I57V in 2B protein, and I44M in 3A protein; (2) L194V in VP2 protein, E40G in VP1 protein, I57V in 2B protein, and I44M in 3A protein; (3) L194V in VP2 protein, E40G in VP1 protein, E85G in 2A protein, I57V in 2B protein, L141S in 2A protein, V232I in 2C protein, and I44M in 3A protein; (4) L194V in VP2 protein, E40G in VP1 protein, T103A in VP1 protein, I57V in 2B protein, L141S in 2A protein, and I44M in 3A protein; Wherein, the protein sequence of the wild-type strain is as shown in SEQ ID NO:1, wherein the VP4 protein is the amino acid sequence shown at positions 1 to 69 of the sequence shown in SEQ ID NO:1, the VP2 protein is the amino acid sequence shown at positions 70 to 324 of the sequence shown in SEQ ID NO:1, the VP3 protein is the amino acid sequence shown at positions 325 to 564 of the sequence shown in SEQ ID NO:1, the VP1 protein is the amino acid sequence shown at positions 565 to 862 of the sequence shown in SEQ ID NO:1, the 2A protein is the amino acid sequence shown at positions 863 to 1012 of the sequence shown in SEQ ID NO:1, the 2B protein is the amino acid sequence shown at positions 1013 to 1111 of the sequence shown in SEQ ID NO:1, the 2C protein is the amino acid sequence shown at positions 1112 to 1440 of the sequence shown in SEQ ID NO:1, the 3A protein is the amino acid sequence shown at positions 1441 to 1526 of the sequence shown in SEQ ID NO:1, the 3B protein is the amino acid sequence shown at positions 1527 to 1548 of the sequence shown in SEQ ID NO:1, and the 3C protein is the amino acid sequence shown in SEQ ID NO:

1. The amino acid sequence is the amino acid sequence shown at positions 1549 to 1731 of the sequence shown in NO:1, and the 3D protein is the amino acid sequence shown at positions 1732 to 2194 of the sequence shown in SEQ ID NO:

1.

2. The coding gene of the thermostable strain of the heat-resistant Coxsackievirus group A type 10 according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the encoding gene is as described in any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:

10.

4. The method for culturing a thermostable strain of heat-resistant Coxsackievirus A group 10 according to claim 1, characterized in that: Vero cells were used as hosts to culture the Coxsackievirus A10 strain.

5. Use of the heat-resistant coxsackievirus group A type 10 heat-stable strain according to claim 1 and the encoding gene according to claim 2 or 3 in studying the pathogenic mechanism of coxsackievirus.

6. Use of the heat-resistant Coxsackievirus A group 10 heat-stable strain according to claim 1 and the encoding gene according to claim 2 or 3 in the preparation of a vaccine against hand, foot and mouth disease.

7. Use of the heat-resistant coxsackievirus group A type 10 heat-stable strain of claim 1 and the encoding gene of claim 2 or 3 in testing the protective efficacy of coxsackievirus vaccines.

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