CN117143836A - Heat-resistant coxsackievirus A group 10 type thermostable strain and application thereof - Google Patents
Heat-resistant coxsackievirus A group 10 type thermostable strain and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of virology, in particular to a thermostable strain of a heat-resistant coxsackievirus A group 10 (CV-A10) and application thereof, wherein the protein amino acid sequence of the thermostable strain of the heat-resistant coxsackievirus A group 10 (CV-A10) is shown as any one of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8, the heat-resistant coxsackievirus A group 10 (CV-A10) is obtained by adapting to passage under the heat treatment selection pressure of 5 consecutive rounds and 52 ℃ for half an hour by a virus parent strain separated from a sample of a hand-foot-mouth patient HEV3108 in the Xianbei province of China, and the heat stability is obviously improved compared with that of the parent strain, the antigen yield and the immunogenicity are not inferior to that of the heat-unstable strain, and the heat-resistant coxsackievirus A group 10 can be used as an inactivated vaccine candidate strain, and the sequence of the heat-resistant coxsackievirus A is used for research on the development of a subsequent enterovirus vaccine and the stability.
Description
Technical Field
The invention relates to the technical field of virology, in particular to a thermostable coxsackie virus A group 10 (CV-A10) strain and application thereof.
Background
Hand-foot-and-mouth disease (HFMD) is caused by various enteroviruses, and is most commonly seen by enterovirus type A71 (EV-A71), coxsackievirus type A16 (CV-A16), coxsackievirus type A6 (CV-A6), coxsackievirus type A10 (CV-A10), and the like.
CV-A10 belongs to the genus Enterovirus of the family Picornaviridae, and the enterovirus particles are icosahedral spherical symmetry, have a diameter of about 27-30nm and a genome of about 7400nt of single-stranded positive strand RNA. CV-A10 viral structural proteins are composed of precursor proteins sheared into VP4, VP2, VP3 and VP1 by protease, wherein VP1-VP3 is positioned on the surface of virus particles, and VP4 is connected with virus RNA and positioned in the interior of the virus particles. Enteroviruses exist in two virus particles with different sedimentation coefficients, namely hollow particles (EP) and solid particles (FP), the EP comprises three structural proteins VP0, VP2 and VP3, the enteroviruses are not infectious, the FP comprises four structural proteins VP1-VP 4, and after VP0 is mature and sheared into VP4 and VP2, virus RNA is contained in the virus particles (Virion, FP) and is an infectious mature virus particle.
Studies have shown that thermostable strains are more immunogenic than thermostable strains and can stimulate animals to produce higher levels of neutralizing antibodies.
Disclosure of Invention
Based on the above, the invention uses the strain CV-A10-3108/CHN XY/2017 (CV-A10-3018-TS) separated from the sample of HEV3108 of hand-foot-and-mouth disease patient in the Kangaang city of Hubei province in 2017 - Abbreviated as TS - ) Separating it from Vero cell as mother strain, and performing multiple passages to form quasi-population library of multiple genotypes, and heat treating at 52deg.C for half an hour for 5 consecutive roundsUnder selective pressure, the strain is adapted to passage, and 4 strains of thermostable strain TS are obtained through plaque cloning and purification + CV-A10-3108-TS, respectively + I (TS for short) + Ⅰ)、CV-A10-3108-TS + II (TS for short) + Ⅱ)、CV-A10-3108-TS + III (abbreviated as TS) + III) and CV-A10-3108-TS + IV (TS for short) + IV), the stability of the strain is obviously improved relative to a heat-labile strain, the antigen yield and the immunogenicity are superior or not inferior to those of a heat-labile parent strain, the strain can be used as an inactivated vaccine candidate strain, and the sequence of the strain can be used for the research and development of subsequent enterovirus VLP vaccines and related basic research and application of enterovirus stability.
It is an object of the present invention to provide a virus strain which can proliferate on cells after 30min of treatment at 52 ℃ and whose viral particle yield and humoral immunogenicity are superior or not inferior to those of the heat-labile strain, and to locate the nucleotide and amino acid sites which affect the heat stability of the virus strain, in particular to protect a heat-resistant coxsackievirus type a 10-type heat-stable strain which can withstand high temperature treatment at 52 ℃ or less, and whose protein sequence is represented by any one of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8.
That is, there are the site mutations described in any one of (1) to (4) below compared with the wild-type strain:
(1) I44M in VP4 protein N69S, VP2 protein L194V, VP1 protein E40G, 2B protein I57V, 3A protein;
(2) VP2 protein L194V, VP1 protein E40G, 2B protein I57V, 3A protein I44M;
(3) L194V, VP in VP2 protein, E40G in 2A protein, E85G in 2B protein, I57V in 2A protein, L141S in 2A protein, V232I in 2C protein, I44M in 3A protein;
(4) VP2 protein L194V, VP protein E40G, VP protein T103A, 2B protein I57V, 2A protein L141S, 3A protein I44M.
The protein sequence of the wild strain is shown as SEQ ID NO. 1, wherein VP4 protein is the amino acid sequence shown as 1st to 69 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP2 protein is the amino acid sequence shown as 70 th to 324 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP3 protein is the amino acid sequence shown as 325 th to 564 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP1 protein is the amino acid sequence shown as 565 th to 862 th positions of the sequence shown as 1st position of the SEQ ID NO. 2A protein is the amino acid sequence shown as 863 th to 1012 th positions of the sequence shown as 1st position of the SEQ ID NO. 2B protein is the amino acid sequence shown as 1013 th to 1111 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP 2C protein is the amino acid sequence shown as 1441 th to 1526 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, and 3B protein is the amino acid sequence shown as 1527 th to 1548 positions of the sequence shown as 1st position of the SEQ ID NO. 1732 nd sequence shown as 3C protein.
The second purpose of the invention is to protect the encoding gene of the heat-resistant coxsackievirus A group 10 thermostable strain.
Further, the nucleotide sequence of the coding gene is shown as any one of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10.
Specifically, with the heat-labile strain CV-A10-3018-TS - The plant is compared with: CV-A10-3108-TS + The coding region I generates 9 nucleotide mutations in total, wherein the missense mutations are 5, the synonymous mutations are 4, and the coding region 5 missense mutant nucleotides and corresponding amino acid positions are respectively: VP4-A952G/N69S, VP2-C1533G/L194V, VP-A2557G/E40G, 2B-A3951G/I57V, 3A-A5198G/I44M; the nucleotide positions of the 4 synonymous mutations of the coding region are respectively: VP2-C1532T; VP3-A1910G;2A-C3377T;3D-T7205G.
CV-A10-3108-TS + The coding region II generates 9 nucleotide mutations in total, wherein the missense mutations are 4, the synonymous mutations are 5, and the coding region 4 missense mutant nucleotides and corresponding amino acid positions are respectively: VP2-C1533G/L194V, VP-A2557G/E40G, 2B-A3951G/I57V, 3A-A5198G/I44M; the nucleotide positions of 5 synonymous mutations in the coding region are: VP2-C1532T; VP3-A1910G;2A-C3377T;2C-T4148C;3D-T7205G.
CV-A10-3108-TS + The III coding region produces a total of 11 nucleotidesMutations, wherein missense mutations are 7, synonymous mutations are 4, and the 7 missense mutant nucleotides and corresponding amino acid positions of the coding region are respectively: VP2-C1533G/L194V, VP-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 of the coding region are respectively: VP2-C1532T; VP3-A1910G;2A-C3377T;3D-T7205G.
CV-A10-3108-TS + The coding region IV generates 11 nucleotide mutations in total, wherein the missense mutations are 6, the synonymous mutations are 5, and the coding region 6 missense mutant nucleotides and corresponding amino acid positions are respectively: VP2-C1533G/L194V, VP-A2557G/E40G, VP-A2745G/T103A, 2B-A3951G/I57V, 2A-T3754C/L141S, 3A-a5198G/I44M; the nucleotide positions of 5 synonymous mutations in the coding region are: VP2-C1532T; VP3-A1910G;2A-C3377T;2C-T4838C;3D-T7205G.
The invention also aims at protecting the culture method of the heat-resistant coxsackievirus A10 type thermostable strain, and culturing the coxsackievirus A10 type strain by using Vero cells as hosts.
The invention also aims to protect the heat-resistant coxsackievirus A group 10 thermostable strain and application of the coding gene in researching the pathogenic mechanism of coxsackievirus.
The invention also aims at protecting the heat-resistant coxsackievirus A group 10 heat-stable strain and the application of the coding gene in preparing vaccines for hand-foot-mouth disease.
The fifth purpose of the invention is to protect the heat-resistant coxsackievirus A group 10 thermostable strain and the application of the coding gene in testing the protection effect of coxsackievirus vaccine.
The invention has at least the following beneficial technical effects:
(1) According to the invention, four thermostable strains are obtained by a cell culture virus technology;
(2) Through sequence determination and comparison of the whole genome of the virus strain, nucleotide and amino acid sites which influence proliferation of CV-A10 strain after 52 ℃ treatment are found, and the mutation sites are different from the related sites of poliovirus heat stability reported in the prior literature, namely, the mutation sites are not positioned in a VP1 hydrophobic pocket, and VP1 amino acid outside the hydrophobic pocket is not positioned in the heat stability sites of other enteroviruses reported in the literature;
(3) Further culturing the thermally unstable strain and the thermally stable strain in a cell factory in a large quantity, purifying, and comparing and analyzing the virus titer, the particle yield and the humoral immunity, wherein the titer of the thermally stable strain is not lower than that of the thermally unstable strain, and the virus particle yield and the humoral immunity are superior to and not inferior to those of the thermally unstable strain;
(4) The quantitative analysis of the particles after the virus is cultured and purified by Vero cells shows that after 2 amino acids in VP1 sequence are changed, the yield of the particles after the virus is cultured by the virus cells is higher than that of a parent strain (a heat-labile strain), and the characteristics are opposite to the reduction of the yield of the virus after the amino acids at a plurality of sites of a polio heat-stable strain are changed, so that the virus is favorable for being used as the high yield requirement of an inactivated vaccine strain;
(5) The obtained thermostable strain can be used as an inactivated vaccine candidate strain, can also be applied to constructing VLP vaccine based on the discovered thermostable site, and can also provide theory and application foundation for other enterovirus yield, particle stability and immunogenicity related site research.
Drawings
FIG. 1 is a comparison of the growth status of the original strain and the screened thermostable strain on Vero cells;
FIG. 2 is a comparison of growth curves of original strain and selected thermostable strain on Vero cells;
FIG. 3 shows a comparison of growth status of the original strain and the selected thermostable strain after 30min of treatment at 52℃after inoculation with Vero cells;
FIG. 4 is a comparison of the heat resistance of the original strain and the screened thermostable strain;
FIG. 5 shows the centrifugation results of the original strain and the selected thermostable strain virus particles in separation and purification;
FIG. 6 shows the results of purification and identification of original strain and selected thermostable strain viruses;
FIG. 7 is a comparison of the heat resistance of virus particles of the original strain and the screened thermostable strain;
FIG. 8 shows the results of comparison of humoral immunogenicity of the original strain and the selected thermostable strain.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention. In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
The reagents used in the examples of the present invention are all commercially available, without specific explanation. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as molecular cloning, as described in Sambrook et al: conditions described in the laboratory Manual (New York: coldSpring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
EXAMPLE 1 selection of thermostable strains
(1) Virus sample CV-A10-3108-TS - (sample No. 3108 separated from the patient suffering from hand-foot-and-mouth disease of Xiangyang in Hubei province of 2017) is treated at 52 ℃ for 30min, inoculated with Vero cells, cultured in a 37 ℃ incubator until lesions appear on the cells, and stored at-20 ℃;
(2) Repeatedly freezing and thawing for three times to obtain virus liquid, centrifuging 6000g for 5min, removing cell debris precipitate, and collecting supernatant;
(3) The virus liquid collection was repeated (1) to (2), and the heat treatment was continued for 5 passages.
EXAMPLE 2 plaque purification of thermostable strains
(1) Digesting the prepared Vero cells with 5X 10 5 Cell density of individual/mL was seeded into 6-well cell culture plates;
(2) Discarding the culture medium when the cells grow to a compact monolayer, and washing with 0.01M PBS for 2 times;
(3) The virus solution obtained from the fifth generation of heat treatment is subjected to continuous dilution of the virus 10 times after being treated for 30min at 52 ℃ again, and the total dilution is 6 gradients;
(4) Adding the virus liquid into a 6-hole plate, adsorbing for 2 hours at 37 ℃, and discarding the virus liquid;
(5) Mixing 2% low-melting agarose with 2 XDMEM maintaining solution at equal volume of 1:1, adding into each well of 6-well plate, 2ml each well, and placing into incubator after solidification;
(6) After 2-3 days, plaques were picked at the lowest dilution, as observed;
(7) Repeating plaque purification for three times to obtain a clone strain;
(8) The clone obtained by three rounds of plaque purification is continuously transmitted on Vero cells for three generations in a blind way with low MOI to obtain a thermostable strain virus harvest.
Example 3 Virus titre assay
(1) Vero cells were grown at 1X 10 5 Cell density per mL was plated into 96-well plates, every 100 μl;
(2) Carrying out continuous 10-time gradient dilution on the harvested virus liquid by using DMEM maintenance liquid, and diluting 8 gradients in total;
(3) After cells in the 96-well plate are attached, diluted virus liquid is added, each gradient is provided with 8 compound wells, 100 mu l of each well is added, and the mixture is placed in a 37 ℃ incubator for culture;
(4) Observing pathological changes in the 96-well plate after 5 days, and calculating the virus titer according to a Reed-mesh formula;
(5) The titration was repeated 3-6 times to determine the virus titer.
Example 4 comparison of thermostable strains with wild-type growth Capacity
(1)CV-A10-3108-TS + The strain (thermostable strain purified in example 2, respectively designated 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 - Strains were grown at the same MOI (moi=5) in 6-well plates confluent with Vero cells, incubated in an incubator at 37 ℃, and lesions were observed as shown in fig. 1.
(2) Titrating the treated virus liquid according to the virus titer determination method in example 3;
(3) 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 is inoculated into Vero cells according to the same inoculum size, viruses are respectively harvested at different time and CCID is used for 50 The titer of the strains on Vero cells is detected by the method, a growth curve is drawn, the growth capacities of the two strains are compared and analyzed, and the result is shown in figure 2, and the growth capacities of the thermostable strain and the thermostable strain are not different.
Example 5 comparison of thermostable strain and wild-type strain
(1)CV-A10-3108-TS + Strain and CV-A10-3018-TS - The plants were treated at 37℃at 42℃at 45℃at 48℃at 50℃at 52℃and at 54℃for 30min, respectively, and immediately stored at 4 ℃; then inoculating into Vero cells, observing pathological state, and after inoculating virus for 4 days, CV-A10-3108-TS + Strain I, CV-A10-3018-TS + Strain II, CV-A10-3108-TS + Strain III and CV-A10-3108-TS + Complete lesions of strain IV, while CV-A10-3018-TS - No significant lesions were observed after 4 days of inoculation, see fig. 3 for specific results.
(2) Titrating the treated virus liquid according to the virus titer determination method in example 3;
(3) CV-A10-3018-TS - 、CV-A10-3108-TS + Ⅰ、CV-A10-3108-TS + Ⅱ、CV-A10-3108-TS + III and CV-A10-3108-TS + The strain IV is treated for 30min at 37-54 ℃, immediately cooled at 4 ℃, and then treated by CCID 50 The titer of the strain on Vero cells was measured by the method, a heat-resistant treatment curve was drawn, and the heat-resistant treatment capacities of the two strains were compared and analyzed, and the results are shown in FIG. 4, in which the heat-labile strain (CV-A10-3018-TS - ) The vast majority of viral inactivation lost infectivity upon treatment at 48℃but the thermostable strain (CV-A10-3108-TS + Ⅰ、CV-A10-3108-TS + Ⅱ、CV-A10-3108-TS + III and CV-A10-3108-TS + IV) can still keep higher titer after being treated at 50 ℃, and the thermal stability is obviously better than that of a thermally unstable strain.
Example 6 comparison of thermostable strains with Heat resistance of wild-type Virus particles
(1) The obtained virus liquid is subjected to ultrafiltration concentration, sucrose bedding centrifugation and CsCl density gradient centrifugation, the result is shown in figure 5, 3 opalescence bands appear in a centrifuge tube, the result is shown in figure 6 after CsCl is removed by centrifugation, and the virus particles such as EP, FP and AP of the virus are obtained through purification.
(2) Buffer (2mM HEPES,200mM NaCl,1 ×sybr green II, ph=8) was prepared;
(3) Taking 1 microgram of purified FP in a qPCR tube, and supplementing the buffer solution to 50 mu L;
(4) Drawing a melting curve by using a qPCR instrument; the temperature program is 25 to 95 ℃, the temperature rising speed is 30 s/DEG C, a heat-resistant curve is drawn, and the heat-resistant capability of virus particles is compared and analyzed, so that as shown in figure 7, the RNA release temperature of a heat-unstable strain is lower than that of the heat-stable strain, and the heat stability of the heat-stable strain is obviously better than that of the heat-unstable strain.
EXAMPLE 7 Whole genome sequencing of thermostable and unstable strains
(1) Viral RNA extraction: extracting and purifying viral RNA by using an artificial column type viral RNA extraction and purification kit (product number: B518667);
(2) Reverse transcription synthesis of cDNA: RNA reverse transcription kit PrimeScript Using Takara TM II 1st Strand cDNASynthesis Kit (cat# 6210A) cDNA was synthesized by reverse transcription;
(3) And (3) PCR amplification: PCR amplification was performed using 2X Phanta Flash Master Mix (Dye Plus) (product number: P520) manufactured by Norpran Corp., 98℃for 30sec to 98℃for 10sec,61℃for 5sec, and 72℃for 10sec/kb at a cycle number of 30-35, wherein the total sequence of the primers is as shown in Table 1 below:
TABLE 1 primers for full sequence determination
Note that: f, forward primer; r, reverse primer.
(4) Agarose gel electrophoresis: after the PCR reaction was completed, the product was subjected to 1% agarose gel electrophoresis to identify a PCR amplified band. After the PCR products are identified correctly, the amplified products are sent to a biological engineering service company for sequencing, and all fragments are spliced to obtain a complete genome sequence.
Sequencing results showed CV-A10-3018-TS - The whole genome sequence and the polyprotein amino acid sequence of the strain are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 1, and CV-A10-3108-TS + The whole genome sequence and the polyprotein amino acid sequence of the strain I are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 3, and CV-A10-3018-TS + The whole genome sequence and the polyprotein amino acid sequence of the II strain are respectively shown as SEQ ID NO. 6 and SEQ ID NO. 5, and CV-A10-3108-TS + The whole genome sequence and the polyprotein amino acid sequence of the III strain are respectively shown as SEQ ID NO. 8 and SEQ ID NO. 7, and CV-A10-3108-TS + The whole genome sequence and the polyprotein amino acid sequence of the IV strain are respectively shown as SEQ ID NO. 10 and SEQ ID NO. 9.
Wherein, the nucleotide at 1-746 in the whole genome sequence is 5' UTR, the nucleotide at 747-953 encodes VP4 protein, the nucleotide at 954-1718 encodes VP2 protein, the nucleotide at 1719-2438 encodes VP3 protein, the nucleotide at 2439-3332 encodes VP1 protein, the nucleotide at 3333-3782 encodes 2A protein, the nucleotide at 3783-4079 encodes 2B protein, the nucleotide at 4080-5066 encodes 2C protein, the nucleotide at 5067-5324 encodes 3A protein, the nucleotide at 5325-5390 encodes 3B protein, the nucleotide at 5391-5939 encodes 3C protein, the nucleotide at 5940-7328 encodes 3D protein, the nucleotide at 7329-7439 is 3' UTR, the polynucleotide with undetermined length after 3' UTR;
VP4 protein is the 1st to 69 th amino acid sequence, VP2 protein is the 70 th to 324 th amino acid sequence, VP3 protein is the 325 th to 564 th amino acid sequence, VP1 protein is the 565 th to 862 th amino acid sequence, 2A protein is the 863 th to 1012 th amino acid sequence, 2B protein is the 1013 th to 1111 th amino acid sequence, 2C protein is the 1112 th to 1440 th amino acid sequence, 3A protein is the 1441 th to 1526 th amino acid sequence, 3B protein is the 1527 th to 1548 th amino acid sequence, 3C protein is the 1549 th to 1731 th amino acid sequence, and 3D protein is the 1732 th to 2193 th amino acid sequence.
Example 8: critical site analysis for differences in virulence stability, yield and immunogenicity
CV-A10-3108-TS + Strain and CV-A10-3018-TS - The whole genome sequences of the strains were aligned using snapge software.
CV-A10-3018-TS - The 69 th amino acid of the VP4 protein is asparagine (N); the 194 th amino acid of VP2 protein is leucine (L); the 40 th amino acid of VP1 protein is glutamic acid (E); the 103 th amino acid of VP1 protein is threonine (T); the 85 th amino acid of the 2A protein is glutamic acid (E); leucine (L) at amino acid 141 of the 2A protein; the 57 th amino acid of the 2B protein is isoleucine (I); valine (V) at amino acid 232 of the 2C protein; the 44 th amino acid of the 3A protein is isoleucine (I).
CV-A10-3108-TS + Serine (S) is the 69 th amino acid of the VP4 protein of the I strain; valine (V) at 194 th amino acid of VP2 protein; the 40 th amino acid of VP1 protein is glycine (G); valine (V) at amino acid 57 of the 2B protein; the 44 th amino acid of the 3A protein is methionine (M).
CV-A10-3018-TS + The 194 th amino acid of the VP2 protein of the II strain is valine (V); the 40 th amino acid of VP1 protein is glycine (G); valine (V) at amino acid 57 of the 2B protein; the 44 th amino acid of the 3A protein is methionine (M).
CV-A10-3108-TS + The 194 th amino acid of the VP2 protein of the III strain is valine (V); the 40 th amino acid of VP1 protein is glycine (G); the 85 th amino acid of the 2A protein is glycine (G); serine (S) at amino acid 141 of the 2A protein; valine (V) at amino acid 57 of the 2B protein; the 232 th amino acid of the 2C protein is isoleucine (I); the 44 th amino acid of the 3A protein is methionine (M).
The 194 th amino acid of VP2 protein of CV-A10-3108-TS+IV strain is valine (V); the 40 th amino acid of VP1 protein is glycine (G); the 103 th amino acid of VP1 protein is alanine (A); serine (S) at amino acid 141 of the 2A protein; valine (V) at amino acid 57 of the 2B protein; the 44 th amino acid of the 3A protein is methionine (M).
And heat-labile strain CV-A10-3018-TS - The plant is compared with: CV-A10-3108-TS + The coding region I generates 9 nucleotide mutations in total, wherein the missense mutations are 5, the synonymous mutations are 4, and the coding region 5 missense mutant nucleotides and corresponding amino acid positions are respectively: VP4-A952G/N69S, VP2-C1533G/L194V, VP-A2557G/E40G, 2B-A3951G/I57V, 3A-A5198G/I44M; the nucleotide positions of the 4 synonymous mutations of the coding region are respectively: VP2-C1532T; VP3-A1910G;2A-C3377T;3D-T7205G.
CV-A10-3108-TS + The coding region II generates 9 nucleotide mutations in total, wherein the missense mutations are 4, the synonymous mutations are 5, and the coding region 4 missense mutant nucleotides and corresponding amino acid positions are respectively: VP2-C1533G/L194V, VP-A2557G/E40G, 2B-A3951G/I57V, 3A-A5198G/I44M; the nucleotide positions of 5 synonymous mutations in the coding region are: VP2-C1532T; VP3-A1910G;2A-C3377T;2C-T4148C;3D-T7205G.
CV-A10-3108-TS + The coding region III generates 11 nucleotide mutations in total, wherein the missense mutations are 7, the synonymous mutations are 4, and the coding region 7 missense mutant nucleotides and corresponding amino acid positions are respectively: VP2-C1533G/L194V, VP-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 of the coding region are respectively: VP2-C1532T; VP3-A1910G;2A-C3377T;3D-T7205G.
CV-A10-3108-TS + The coding region IV generates 11 nucleotide mutations in total, wherein the missense mutations are 6, the synonymous mutations are 5, and the coding region 6 missense mutant nucleotides and corresponding amino acid positions are respectively: VP2-C1533G/L194V, VP-A2557G/E40G, VP-A2745G/T103A, 2B-A3951G/I57V, 2A-T3754C/L141S, 3A-a5198G/I44M; the nucleotide positions of 5 synonymous mutations in the coding region are: VP2-C1532T; VP3-A1910G;2A-C3377T;2C-T4838C;3D-T7205G.
Nucleotide variations that caused the above amino acid changes and variations of nonsense nucleotides in the genome are shown in Table 2. All nucleotide and amino acid variations listed may be related to the ability of the two strains to grow in a heat-treated environment, viral particle yield, and viral immunogenicity.
TABLE 2 Critical sites affecting the proliferation of strains in Vero cells after 52℃treatment
Example 9: viral particle purification, antigen preparation
(1) Inoculating CV-A10 strain into a cell factory with the same MOI when Vero cells of ten-layer cell factory grow to a compact single-layer state, and culturing in a 37 ℃ incubator;
(2) After more than 90% of cells are diseased, virus particles are harvested after ultrafiltration concentration, sucrose bedding centrifugation and CsCl density gradient centrifugation are carried out, and centrifugal filtration and desalination are carried out;
(3) The BCA method detects the protein concentration of the purified virus particles and calculates the total protein amount of the harvested virus. The results of the harvested EP, FP, AP antigen content are shown in table 3:
TABLE 3 comparison of antigen yields of thermostable and thermostable strains
The viral particle yield was 2 and 10 layers of the yield after purification of the viral supernatant harvest from the cell factory culture.
CV-A10-3018-TS - The single ten-layer cell factory yield of strain virus particles was 0.29mg;
CV-A10-3108-TS + the single ten-layer cell factory yield of the virus particles of strain I was 0.18mg;
CV-A10-3018-TS + the single ten-layer cell factory yield of strain II virus particles was 0.25mg;
CV-A10-3108-TS + single ten-layer cell factory yield of strain III virus particles was 0.26mg;
CV-A10-3108-TS + the amount of antigen harvested from a single ten-layer cell factory of strain IV was 0.29mg.
(4) 4% formaldehyde is added to EP and FP particle suspensions of the strains respectively for inactivation, the inactivation is carried out at 37 ℃ for 48 hours, and the 10kd ultrafiltration concentration tube is used for centrifugation to remove formaldehyde.
Example 10: immunization of animals
(1) BCA method to determine the concentration of formaldehyde inactivated virus particles, EP harvested after purification: FP: AP yield ratio the inactivated EP, FP, AP were mixed with chromatography salt buffer and aluminium adjuvant to make up three antigen dose groups of 0.1. Mu.g, 0.5. Mu.g and 2.5. Mu.g;
(2) Female Wistar rats weighing 180-200g were given 8 per group, each injected intraperitoneally, 500 μl per dose, and an aluminum adjuvant control group was set. Primary immunization on day 0, booster immunization on day 14, and blood collection on day 28.
Example 11: serum neutralizing antibody titer detection
(1) Serum samples from 1: 16-ratio was diluted with a diluent (e.g., MEM maintaining solution), and added to column A of 96-well plates at 100. Mu.l/well, 2 multiplex wells were set, and 50. Mu.l of diluent was added to each of columns B to H. Serum samples from column A were serially diluted 2-fold to column H, and 50 μl was discarded after dilution of the last column.
(2) Diluting the neutralized seed to 100CCID 50 Mu.l of the sample is pipetted into a 50. Mu.l vertical suspension into the diluted serum sample. The 96-well plate was placed in an incubator at 37℃and neutralized for 2 hours. Diluting the diluted neutralizing poison to 10CCID in a 10-fold gradient 50 /50μl、1CCID 50 /50μl、0.1CCID 50 50 μl. Mu.l of maintenance solution and 50. Mu.l of virus solution (total of 4 dilutions, 100, 10, 1, 0.1CCID, respectively) were added to each well 50 50 μl), 8 wells per dilution, were placed as a drip-back plate at 4deg.C.
(3) After neutralization, digested RD cells were cultured at 1X 10 5 The density of the mixture/ml was spread into a neutralization plate and a drip-back plate, and the neutralization result was judged for 5 to 7 days. In particular CV-A10-3018-TS + Strain II was used as neutralizing virus, and the isolated serum was evaluated for humoral immunogenicity, CV-A10-3018-TS + Strain II 2.5 μg dose group showed CV-A10-3018-TS + The neutralizing antibody titer of strain II is higher than CV-A10-3018-TS - For specific results, see FIG. 7, where each dot represents a Wistar rat, the titer of the aluminum adjuvant vaccinated group was below 8, and the assignment was plotted. Multiplex comparison assay using SidakThe experiments were subjected to one-way analysis of variance (ANOVA), statistically significant as follows: ns, not significant (P is greater than or equal to 0.05); * P is more than or equal to 0.01<0.05；**，P<0.01. As can be seen from FIG. 8, CV-A10-3018-TS was initiated 14 days after the immunization + The high dose group neutralizing antibody titer of strain II is higher than CV-A10-3018-TS - A strain.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A thermostable strain of coxsackievirus a group 10, characterized in that the thermostable strain of coxsackievirus a group 10 is resistant to high temperature treatment at 52 ℃ or less, and has the following site mutation in any one of (1) to (4) compared with a wild-type strain:
(1) I44M in VP4 protein N69S, VP2 protein L194V, VP1 protein E40G, 2B protein I57V, 3A protein;
(2) VP2 protein L194V, VP1 protein E40G, 2B protein I57V, 3A protein I44M;
(3) L194V, VP in VP2 protein, E40G in 2A protein, E85G in 2B protein, I57V in 2A protein, L141S in 2A protein, V232I in 2C protein, I44M in 3A protein;
(4) L194V, VP in VP2 protein, T103A in E40G, VP1 protein, I57V in 2B protein, L141S in 2A protein, I44M in 3A protein;
the protein sequence of the wild strain is shown as SEQ ID NO. 1, wherein VP4 protein is the amino acid sequence shown as 1st to 69 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP2 protein is the amino acid sequence shown as 70 th to 324 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP3 protein is the amino acid sequence shown as 325 th to 564 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP1 protein is the amino acid sequence shown as 565 th to 862 th positions of the sequence shown as 1st position of the SEQ ID NO. 2A protein is the amino acid sequence shown as 863 th to 1012 th positions of the sequence shown as 1st position of the SEQ ID NO. 2B protein is the amino acid sequence shown as 1013 th to 1111 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, VP 2C protein is the amino acid sequence shown as 1441 th to 1526 th positions of the sequence shown as 1st position of the SEQ ID NO. 1, and 3B protein is the amino acid sequence shown as 1527 th to 1548 positions of the sequence shown as 1st position of the SEQ ID NO. 1732 nd sequence shown as 3C protein.
2. A gene encoding the thermostable coxsackievirus a group 10 strain of claim 1.
3. The coding gene according to claim 2, wherein the nucleotide sequence of the coding gene is 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 the heat-resistant coxsackievirus a group 10 according to claim 1, wherein the coxsackievirus a10 strain is cultured using Vero cells as a host.
5. Use of the thermostable coxsackievirus a group 10 strain of claim 1, the coding gene of claim 2 or 3 for studying the pathogenic mechanism of coxsackievirus.
6. Use of the thermostable coxsackievirus a group 10 strain of claim 1, the encoding gene of claim 2 or 3 in preparing vaccine against hand-foot-and-mouth disease.
7. Use of the thermostable coxsackievirus a group 10 strain of claim 1, the coding gene of claim 2 or 3 for testing the protective efficacy of coxsackievirus vaccine.
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