CN117327664A - Thermostable coxsackievirus A group 6 virus strain and application thereof - Google Patents
Thermostable coxsackievirus A group 6 virus strain and application thereof Download PDFInfo
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Abstract
The invention particularly relates to a thermostable coxsackievirus A group 6 virus strain and application thereof. The heat-stable coxsackievirus A group 6 virus strain can withstand high temperature treatment below 55 ℃, and the protein amino acid sequence is shown as SEQ ID NO: 1-4, the virus strain is separated from a parent strain which is adapted to Vero cells through human rhabdomyoma cells (RD) and is subjected to passage, the strain is adapted to passage under the heat selection pressure of 52-55 ℃ and is obtained after limited dilution and purification, the virus strain has thermal stability, high yield and strong immunogenicity, the virus strain can be used as an inactivated vaccine candidate strain, and the sequence of the virus strain can be used for the subsequent research and development of enterovirus vaccines and the related research of enterovirus stability.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a thermostable coxsackie virus A group 6 virus 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-A6 belongs to enterovirus genus of picornaviridae, enterovirus particles are in icosahedral spherical symmetry, have diameters of about 27-30nm, and are single-stranded positive strand RNA with genome of about 7400 nt. CV-A6 viral structural proteins are composed of precursor protein P1 sheared into VP4, VP2, VP3 and VP1 by protease, VP1-VP3 is positioned on the surface of the virus particle, and VP4 is connected with virus RNA and positioned in the interior of the virus particle. 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
The invention takes CV-A6/CHN XY/2017 of 3391 sample of the patient suffering from hand-foot-mouth disease (CHN XY) of the Xianbei province of China, which is separated from 2017, as a mother strain, and the mother strain is adapted to passage under the hot selection pressure, and is obtained after dilution and purification, and is respectively named as a stable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]Simultaneously, the virus liquid which is subjected to the same passage times on Vero cells and is not subjected to heat treatment is directly subjected to three rounds of limiting dilution purification to obtain a plurality of virus liquidThe cloned strain is inoculated into Vero cells after being treated for 30 minutes at 52 ℃ to select out a strain which can not be proliferated and is named as a heat-unstable strain TS - [16]Compared with a heat-labile strain, the stability and the virus particle yield of the stable strain are obviously improved, the humoral immunogenicity is superior to or equivalent to that of the heat-labile strain, the heat-labile strain can be used as an inactivated vaccine candidate strain, and the sequence of the heat-labile strain can be used for the research and development of subsequent enterovirus VLP vaccines and related basic research and application of enterovirus stability.
One of the purposes of the invention is to protect a thermostable coxsackievirus A group 6 virus strain, the thermostable coxsackievirus A group 6 virus strain can withstand high temperature treatment below 55 ℃, and the protein amino acid sequence is shown as SEQ ID NO:1 to 4.
The 4 strains and the heat-labile strain are composed of structural proteins VP4, VP2, VP3 and VP1, and non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D.
Specifically, VP4 protein is the amino acid sequence of 1-69 th site of the sequence shown in SEQ ID NO 1-4, VP2 protein is the amino acid sequence of 70-325 th site of the sequence shown in SEQ ID NO 1-4, VP3 protein is the amino acid sequence of 326-565 th site of the sequence shown in SEQ ID NO 1-4, VP1 protein is the amino acid sequence of 566-870 th site of the sequence shown in SEQ ID NO 1-4, 2A protein is the amino acid sequence of 871-1029 th site of the sequence shown in SEQ ID NO 1-4, 2B protein is the amino acid sequence of 1021-1119 th site of the sequence shown in SEQ ID NO 1-4, 2C protein is the amino acid sequence of 1120-1448 th site of the sequence shown in SEQ ID NO 1-4, 3A protein is the amino acid sequence of 1449-1534 of the sequence shown in SEQ ID NO 1-4, 3B protein is the amino acid sequence of 5-1556 th site of the sequence shown in SEQ ID NO 1-4, 3C protein is the amino acid sequence of 1550-1740 th site of the sequence shown in SEQ ID NO 1-1534.
The second object of the present invention is to protect the nucleotide sequence encoding the above-mentioned thermostable coxsackievirus group a type 6 strain.
Further, the nucleotide sequence is shown in any one of SEQ ID NO. 6-9.
The 4 thermostable viral strains TS + [1]、TS + [4]、TS + [9]、TS + [12]With a parent strain or a heat-labile virus strain TS - [16]In contrast, the 4 thermostable strain coding regions produce a total of 23 nucleotide mutations, 9 of which are missense mutations and 14 of which are synonymous mutations. The 9 missense mutation nucleotides and the corresponding amino acid positions of the coding region are respectively as follows: 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 of 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 invention also aims to protect the culture method of the thermostable coxsackievirus A group 6 virus strain, which specifically comprises the following steps: the coxsackievirus A6 strain was cultured using Vero cells as hosts.
The invention also aims to protect the application of the thermostable coxsackievirus A group 6 virus strain and the nucleotide sequence in researching the pathogenic mechanism of the coxsackievirus.
The invention also aims to protect the application of the heat-stable coxsackievirus A group 6 virus strain and the nucleotide sequence in preparing vaccines for hand-foot-mouth disease.
The invention also aims to protect the application of the thermostable coxsackievirus A group 6 virus strain and the nucleotide sequence in testing the protection efficacy of the coxsackievirus vaccine.
The invention obtains 4 strains CV-A6 thermostable strain and 1 strain thermostable strain from wild strains CV-A6/CHN XY/2017 through screening and separation by cell culture virus technology, and compared with the prior art, the invention has the beneficial effects that:
(1) The thermostable strain and the thermostable strain are subjected to cell factory mass culture and then purification, virus titer, particle yield and humoral immunogenicity after live-virus immunization of rats are compared, the titer of the 4 thermostable strains and the humoral immunogenicity after live-virus immunization of rats are superior or equivalent to those of the thermostable strain, and the stability and the virus particle yield are superior to those of the thermostable strain, and the 4 thermostable strains can meet the high yield requirement as inactivated vaccine strains, prolong the shelf life of the vaccine and reduce the cold chain transportation cost of the vaccine;
(2) The nucleotide and amino acid mutation sites which affect the continuous proliferation of CV-A6 viruses after the hot-pressing gradient treatment at 52-55 ℃ are found by carrying out the whole genome sequence measurement on the thermostable strain and the thermostable strain, which are different from the enterovirus thermostability related sites reported in the prior literature, the gene nucleotide and amino acid sites related to the thermostability can be applied to the construction of other enteroviruses except CV-A6, the construction of thermostable strains, and the construction of CV-A6 and other enterovirus thermostable virus-like particle candidate vaccines according to the change of structural protein gene sequence sites, and can also provide theory and application basis for the research of other enterovirus yield, particle stability and immunogenicity related sites.
Drawings
FIG. 1 shows the growth state of wild strains and heat-labile strains after being treated at 52-56 ℃ for 30min and inoculated with Vero cells;
FIG. 2 is a comparison of the growth status of thermostable and thermostable strains on Vero cells;
FIG. 3 is a comparison of the heat resistance of thermostable and thermostable strains;
FIG. 4 is a comparison of the humoral immunogenicity of a thermostable strain and a thermostable strain;
FIG. 5 shows the results of purification and identification of thermostable and thermostable strains.
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: screening of thermostable high-yielding strains
(1) Virus sample CV-A6/CHN XY/2017 (sample 3391 of hand-foot-and-mouth disease patient (CHN XY) isolated from Xiangyang City, hubei province, china) was treated at 52deg.C for 30min and immediately stored at 4deg.C;
(2) After the treated sample is dissolved, the Vero cells are inoculated, and the sample is placed at 37 ℃ and 5 percent CO 2 Culturing in incubator until the cells have pathological changes, and storing at-20deg.C;
(3) Repeatedly freezing and thawing for three times to obtain virus liquid, centrifuging 6000g for 5min, removing cell debris precipitate, and collecting supernatant;
(4) Repeating steps (1) - (3) on the harvested virus liquid, continuously carrying out heat treatment for 5 times, and gradually increasing the heat treatment temperature to 1-56 ℃;
(5) And selecting a batch of virus harvest liquid which can withstand the highest temperature treatment (in this example, the fourth batch, namely, the screening temperature reaches 55 ℃), and sub-packaging and freezing for use as a thermally stable high-yield quasi-population stock.
Wherein, the CV-A6/CHN XY/2017 wild type strain still showed significant lesions after 30min treatment at 55deg.C, and no macroscopic lesions were observed until the temperature was raised to 56deg.C, see FIG. 1. Selecting virus supernatant still having lesions after 55 ℃ treatment, and carrying out limited dilution purification to obtain a thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]. Then, directly limiting dilution is carried out on CV-A6/CHN XY/2017 wild-type strains to obtain a plurality of clone strains, and the clone strains are respectively treated at 52 DEG CInoculating into Vero cells after 30min, and collecting clone strain incapable of making Vero cells obviously pathological changes as heat-labile strain TS - [16]。
Example 2: limiting dilution purification of thermostable strains
(1) After digestion of Vero cells with good growth status, the cells were grown in the appropriate ratio (5X 10 5 individual/mL) was inoculated into 96-well cell culture plates, placed at 37℃and 5% CO 2 The cells are kept in an incubator for later use.
(2) Adding cell maintenance liquid DMEM (DMEM) and 900 mu L of each well into a clean 24-well plate cell culture plate, taking 100 mu L of virus sample into a first well, repeatedly blowing and mixing uniformly, sucking 100 mu L into the next well, sequentially carrying out continuous ten-time gradient dilution, replacing gun heads for each dilution, sequentially adding diluted virus liquid into a 96-well plate according to the dilution gradient from high to low, setting 8 compound wells (100 mu L/well) for each dilution, and setting 8 compound wells (100 mu L/well), at 37 ℃ and 5% CO 2 The results of the incubation for 7 days and the observation under a microscope are shown in FIG. 2.
(3) Harvesting of the virus: and (3) microscopic examination of cytopathic effect (Cytopathic effect, CPE) of each well, picking clone strains from a compound well corresponding to the last CPE positive dilution, and transferring 200 mu L of virus harvest liquid in a suction well into a 1.5mL centrifuge tube. Taking 100 mu L of harvest liquid to carry out ten times dilution, repeating the steps for three times to carry out continuous limiting dilution, and finally obtaining the purified virus clone TS + [1]、TS + [4]、TS + [9]、TS + [12]And TS - [16]。
(4) Expansion culture of virus clone: the virus clone obtained after three times of continuous purification is inoculated into Vero cells for expansion culture according to low MOI.
Example 3: virus titer assay
(1) RD cells grown to a dense monolayer state are digested and then treated with 1X 10 5 Cell density per mL was plated into 96-well plates, 100 μl per well;
(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 the cells in the 96-well plate are attached, diluted virus liquid is added, each gradient is 8 compound wells, each well is 100 mu L, and the disease is curedMixing the venom with cells, and placing at 37deg.C and 5% CO 2 Culturing in an incubator;
(4) Observing pathological changes in the 96-well plate after 7 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: thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]And thermostable strain TS - [16]Comparison of Heat treatment resistance
(1) The purified clone CV-A6 TS obtained in example 2 was subjected to + [1]、TS + [4]、TS + [9]、TS + [12]Plants and TS - [16]The plants were treated at 37℃at 42℃at 45℃at 48℃at 50℃at 52℃at 54℃at 56℃at 58℃and at 60℃for 30min, respectively, and immediately stored at 4 ℃;
(2) The treated strains were seeded at the same MOI (calculated as titer measured before heat treatment) into 6-well plates filled with Vero cells at 37℃with 5% CO 2 Culturing in an incubator, observing the pathological conditions, and finding out a thermally unstable strain TS - [16]Most of the viruses lose infectivity after treatment at 52 ℃, but thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]The high titer can be maintained after the treatment at 56 ℃, and the heat stability is obviously better than that of the heat unstable strain;
(3) Titrating the treated virus liquid according to the virus titer determination method in example 3;
(6) A heat-resistant treatment curve was drawn, and the heat-resistant treatment capacity of each strain was analyzed by comparison, and the results are shown in FIG. 3.
Example 5: thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]And heat labile strain TS- [16 ]]Is determined by genome-wide sequencing
(1) Viral RNA extraction: CV-A6 viral RNA was extracted and purified using QIAGEN viral RNA extraction and purification kit QIAamp Viral RNA Mini Kit (cat# 52906);
(2) Reverse transcription synthesis of cDNA: adding the reagents shown in table 1 into a PCR tube, uniformly mixing, placing into a PCR instrument, reacting for 5min at 65 ℃, and rapidly quenching on ice; adding the reagents shown in Table 2 into the reaction solution, uniformly mixing, and placing the mixture in a PCR instrument under the reaction condition of 30 ℃ for 15min;42 ℃ for 60min;95℃for 15min.
TABLE 1 first reaction System for synthesizing cDNA by reverse transcription
| Name of the name | Volume (mu L) |
| RNA | 5.0 |
| RNase Free ddH 2 O | 3.0 |
| dNTPs Mixture(10mM each) | 1.0 |
| Random 6mers primers(50μM) | 1.0 |
TABLE 2 second step reaction System for cDNA Synthesis by reverse transcription
| Name of the name | Volume (mu L) |
| Reaction solution 1 | 10.0 |
| RNase Free ddH 2 O | 4.5 |
| 5×PrimerⅡBuffer | 4.0 |
| Primer ScriptⅡRTase(200U/μL) | 1.0 |
| RNase Inhibitor(40U/μL) | 0.5 |
(3) And (3) PCR amplification: CV-A6 full sequence determination primers are shown in Table 3, and PCR amplification is carried out by using KOD-Plus-Neo (product number: KOD-401) manufactured by Toyobo Co., ltd under the reaction condition of 98℃for 10min;98℃for 30sec;55 ℃,30sec;68 ℃ (extension speed 30 sec/kb); the cycle number is 30-35.
TABLE 3 Whole genome sequencing primers
(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 show that 4 CV-A6 thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]The amino acid sequences of the polyprotein are respectively shown as SEQ ID NO. 1-4, and the whole genome sequences are respectively shown as SEQ ID NO:6 to 9, the heat-labile strain TS - [16]The amino acid sequence of the polyprotein is shown as SEQ ID NO. 5, and the whole gene is shown in the specificationThe group sequence is shown as SEQ ID NO. 10. The coding region consists of non-coding region 5' -UTR, 3' -UTR and structural proteins VP4, VP2, VP3, VP1 and non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D, wherein the length of genome is 7430 nucleotide residues, the nucleotide positions of the non-coding region 5' UTR and the 3' UTR in the genome are respectively 1-724 and 7331-7430, the length of the 3' UTR is not determined, the length of the encoded polyprotein is 2201 amino acid residues, and the amino acid sequences and positions of each structural protein and the non-structural protein of the virus are respectively:
VP4 protein is the 1 st-69 th amino acid sequence shown in SEQ ID NO 1-4;
VP2 protein is the 70 th-325 th amino acid sequence shown in SEQ ID NO. 1-4;
VP3 protein is the 326 th to 565 th amino acid sequence shown in SEQ ID NO 1-4;
VP1 protein is 566-870 amino acid sequence shown in SEQ ID NO 1-4;
the 2A protein is the 871-1020 amino acid sequence shown in SEQ ID NO 1-4;
the 2B protein is the 1021 th-1119 th amino acid sequence shown in SEQ ID NO 1-4;
the 2C protein is the 1120-1448 amino acid sequence shown in SEQ ID NO. 1-4;
the 3A protein is the 1449-1534 amino acid sequence shown in SEQ ID NO 1-4;
the 3B protein is the 1535 th-1556 th amino acid sequence shown in SEQ ID NO. 1-4;
the 3C protein is the 1557 th to 1739 th amino acid sequence shown in SEQ ID NO. 1-4;
the 3D protein is the 1740-2201 th amino acid sequence shown in SEQ ID NO. 1-4.
Example 6: critical site analysis leading to differences in stability and yield of individual strains
The 4 CV-A6 thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]And heat labile strain TS- [16 ]]Sequence alignment was performed using snapge software.
Heat labile strain TS - [16]The 41 st amino acid of VP2 protein is tyrosine (Y); VP2 eggAmino acid 143 is glycine (G); the 4 th amino acid of VP3 protein is threonine (T); serine (S) is the 10 th amino acid of VP3 protein; the 54 th amino acid of VP1 protein is alanine (A); the 135 th amino acid of VP1 protein is asparagine (N); valine (V) at amino acid 80 of the 2C protein; serine (S) at amino acid 56 of the 3A protein; the 445 th amino acid of the 3D protein is alanine (A).
CV-A6 thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]Compared with TS- [16 ]]Together comprising the mutations: the 41 st amino acid of VP2 protein is histidine (H); the 143 th amino acid of VP2 protein is alanine (A); the 4 th amino acid of VP3 protein is alanine (A); the 10 th amino acid of VP3 protein is threonine (T); valine (V) at amino acid 54 of VP1 protein; the 135 th amino acid of VP1 protein is threonine (T); the 80 th amino acid of the 2C protein is (I); the 56 th amino acid of the 3A protein is asparagine (N); valine (V) at amino acid 445 of the 3D protein.
Nucleotide variations that caused the above amino acid changes and variations of nonsense nucleotides in the genome are shown in Table 4. All of the nucleotide and amino acid variations listed may be related to the ability of each strain to grow, viral particle yield and immunogenicity of the virus in a heat treatment environment.
TABLE 4 Critical sites affecting the thermal stability of strains
Example 7: antigen content quantification of virus harvest
(1) Preparing an ELISA plate: the purified CV-A6 rabbit polyclonal antibody was diluted to 2. Mu.g/mL with a carbonate buffer having a pH of 9.6 and 0.05mol/L, and the ELISA plate was coated at 100. Mu.L/well at 4℃overnight. After washing the plates, adding a PBST buffer solution containing 1% bovine serum albumin, incubating at 37 ℃ for 2 hours, discarding a sealing solution, and taking dry water and placing at 4 ℃ for storage for later use.
(2) Sandwich ELISA: the virus harvest and CV-A6 antigen standard to be tested are diluted by the PBST buffer solution containing 1% bovine serum albumin from the ratio of 1:4 in a doubling ratio, respectivelyAdd to ELISA plate, 100. Mu.L/well, incubate at 37℃for 1h, wash plate and pat dry. mu.L/well of murine anti-CV-A6 IgG-HRP conjugate diluted to working concentration was added, incubated at 37℃for 1h, plate washed and patted dry. Adding substrate A, B solution mixed in a ratio of 1:1, developing, adding 50 mu L/hole, incubating at 37 ℃ in dark place for 15min, and adding 2mol/LH 2 SO 4 The reaction was stopped at 50. Mu.L/well. And reading absorbance values of each hole of the ELISA plate at 450nm and 630nm, performing linear fitting by using the antigen content of the standard substance and the absorbance value (A450-A630) corresponding to the antigen content, and calculating the antigen content of the sample to be detected according to a standard curve equation.
Example 8: immunization of animals
(1) After the antigen content of each strain virus harvest liquid is measured by ELISA method, setting the antigen quantity of 500U as the dose of live virus immunity;
(2) 175-200g female SPF grade Wistar rats were given 8 per group, each dose was given 500U antigen by intraperitoneal injection, and a DMEM maintenance solution control group was set. Primary immunization on day 0, booster immunization on day 14, and blood collection on day 21.
Example 9: serum neutralizing antibody titer detection
(1) The serum sample is diluted by a diluent (such as DMEM maintaining solution) according to the proportion of 1:8, a 96-well plate A column is added, 100 mu L/well is arranged, 2 compound wells are arranged, 50 mu L of diluent is added to each well of B-H columns, the serum sample in the A column is serially diluted to H columns by 2 times, and 50 mu L of diluent is discarded after the last column is diluted.
(2) Diluting the neutralized seed to 100CCID 50 50. Mu.L of the sample is sucked up, 50. Mu.L of the sample is vertically suspended and dripped into the diluted serum sample, and the 96-well plate is placed in a culture box at 37 ℃ for neutralization for 2 hours. Diluting the diluted neutralizing poison to 10CCID in a 10-fold gradient 50 /50μL、1CCID 50 /50μl、0.1CCID 50 mu.L of maintenance solution and 50. Mu.L of virus solution (total of 4 dilutions, 100, 10, 1, 0.1CCID, respectively) were added per well 50 50. Mu.L) of 8 wells per dilution were placed as a drip-back plate at 4 ℃.
(3) After neutralization, digested RD cells were cultured at 1X 10 5 Density of/ml was spread into neutralization plate and drip plate, 37℃and 5% CO 2 Culturing for 5-7 days to judge the neutralization result. The result is shown in FIG. 4, where each point in the graph representsTable one Wistar rat, DMEM control group titre below 8, was assigned a plot and analyzed by single factor analysis of variance (ANOVA) using the Sidak multiplex comparison test, 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；***，P<0.001 and P<0.0001. As can be seen from FIG. 4, CV-A6 thermostable strain TS 7 days after the booster immunization + [1]、TS + [4]、TS + [9]、TS + [12]Serum neutralizing antibody titer of live-poison immune Wistar rat is superior to or equivalent to that of heat-unstable strain TS- [16 ]]Serum neutralization titers after immunization with antigen amounts.
Example 10: viral particle purification, antigen preparation
(1) When Vero cells of ten-layer cell factory grow to compact single-layer state, inoculating each CV-A6 strain into cell factory at the same MOI, placing at 37deg.C and 5% CO 2 Culturing in an incubator.
(2) After more than 90% of cells are diseased, virus particles are harvested after virus is harvested by ultrafiltration concentration, sucrose bedding centrifugation, csCl and other density gradient centrifugation, centrifugal filtration and desalination are carried out, the result is shown in figure 5A, 3 latex bands appear in a centrifuge tube, extracted bands are identified by SDS-PAGE after cesium chloride is removed by centrifugation, and the result is shown in figure 5B, and three types of CV-A6EP, FP and AP virus particles are obtained after purification.
(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 content of EP, FP and AP antigens obtained from each strain are shown in Table 5, and CV-A6 thermostable strain TS + [1]、TS + [4]、TS + [9]、TS + [12]The total amount of antigen harvested from a single ten-layer cell factory varies from 0.33mg to 0.76mg, with an average yield of 0.57mg; CV-A6 Heat labile strain TS - [16]The amount of antigen harvested in a single ten-layer cell factory was only 0.39mg.
TABLE 5 comparison of antigen yields for thermostable and thermostable strains
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 (8)
1. The heat-stable coxsackievirus A group 6 virus strain is characterized in that the heat-stable coxsackievirus A group 6 virus strain can withstand high-temperature treatment below 55 ℃, and the protein amino acid sequence is shown as SEQ ID NO:1 to 4.
2. The thermostable coxsackievirus group a type 6 strain according to claim 1, wherein the strain consists of structural proteins VP4, VP2, VP3, VP1 and non-structural proteins 2A, 2B, 2C and 3A, 3B, 3C, 3D, wherein
VP4 protein is the amino acid sequence shown in positions 1-69 of the sequence shown in SEQ ID NO. 1-4, VP2 protein is the amino acid sequence shown in positions 70-325 of the sequence shown in SEQ ID NO. 1-4, VP3 protein is the amino acid sequence shown in positions 326-565 of the sequence shown in SEQ ID NO. 1-4, VP1 protein is the amino acid sequence shown in positions 566-870 of the sequence shown in SEQ ID NO. 1-4, 2A protein is the amino acid sequence shown in positions 871-1020 of the sequence shown in SEQ ID NO. 1-4, 2B protein is the amino acid sequence shown in positions 1021-1119 of the sequence shown in SEQ ID NO. 1-4, 2C protein is the amino acid sequence shown in positions 1120-1448 of the sequence shown in SEQ ID NO. 1-4, 3A protein is the amino acid sequence shown in positions 1535-1556 of the sequence shown in SEQ ID NO. 1-4, and 3C protein is the amino acid sequence shown in positions 1730-1740 of the sequence shown in SEQ ID NO. 1-1444.
3. A nucleotide sequence encoding the thermostable coxsackievirus group a type 6 strain of claim 1 or 2.
4. A nucleotide sequence according to claim 3, wherein the nucleotide sequence is as set forth in any one of SEQ ID NOs 6 to 9.
5. The method for culturing a thermostable coxsackievirus a group 6 strain according to claim 1 or 2, characterized in that the coxsackievirus A6 strain is cultured using Vero cells as a host.
6. Use of the thermostable coxsackievirus group a6 strain of claim 1 or 2, the nucleotide sequence of claim 3 or 4 for studying the pathogenic mechanism of coxsackievirus.
7. Use of the thermostable coxsackievirus a group 6 strain of claim 1 or 2, the nucleotide sequence of claim 3 or 4, for the preparation of a vaccine against hand-foot-and-mouth disease.
8. Use of a thermostable coxsackievirus group a6 strain according to claim 1 or 2, a nucleotide sequence according to claim 3 or 4 for testing the protective efficacy of a coxsackievirus vaccine.
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