A type a seneca valley mutant virus strain and applications

Abstract

The present application relates to the technical field of molecular biology, and particularly relates to a type A Seneca mutant virus strain and application. The present application obtains a Seneca mutant virus strain CRL-FJ-P80 by continuously subculturing a type A Seneca virus CH-FJ-2017 strain in CRL-2843 cells for 80 generations. Compared with the CH-FJ-2017 parent strain, the virus nucleic acid and virion replication levels of the Seneca mutant virus strain CRL-FJ-P80 are both significantly increased, indicating that the Seneca mutant virus strain CRL-FJ-P80 has stronger replication and production capacity. Moreover, the antigenicity of the Seneca mutant virus strain CRL-FJ-P80 does not change, and the immune capacity is enhanced. The Seneca mutant virus strain CRL-FJ-P80 can be used as a candidate strain of inactivated vaccine, and is used for preparation of Seneca virus inactivated vaccine, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a type A Seneca mutant virus strain and application. BACKGROUND

[0002] Seneca Valley virus A (SVA), also known as Seneca Valley virus (SVV), is the only member of Senecavirus genus in the family Picornaviridae, and there is only one serotype. Seneca virus was first discovered and identified in cell culture contaminants in Maryland, USA in 2002, and was named SVV-001. The complete genome sequence of SVV-001 was resolved in 2005. SVV-001 is called NTX-010 in the medical field. Research data shows that NTX-010 specifically replicates in tumor cells and exhibits high levels of oncolytic activity in vitro and in vivo.

[0003] It is currently believed that pigs are the natural host of Seneca virus. The typical clinical signs of adult pigs infected with Seneca virus are vesicles and / or ulcerative lesions on the nasal, oral mucosa, coronary arterial band and hoof, which are difficult to distinguish from foot-and-mouth disease virus (FMDV), vesicular stomatitis virus (VSV), swine vesicular disease virus (SVDV) and swine vesicular exanthema virus (VESV). Newborn piglets infected with Seneca virus within seven days also exhibit various clinical signs, including weakness, drooling, skin hyperemia, neurological signs, diarrhea, high mortality and the like. The infection and transmission of Seneca virus has caused huge economic losses to the pig breeding industry.

[0004] As an RNA virus, the genome of Seneca virus is prone to mutation during natural transmission, forming new virus strains, which poses a great challenge to the prevention and control of Seneca virus.

[0005] The virus particle of Senecavirus is a icosahedral structure without envelope, and the diameter is about 30nm. The capsid of Senecavirus is composed of four structural proteins (VP1, VP2, VP3, VP4). Among them, VP1, VP2 and VP3 are spliced together to form the virus shell, and VP4 is located on the inside of the virus capsid. Among them, the virus protein (VPg) is covalently connected with the 5' end of the viral RNA genome and is encapsulated in the virus particle cavity. The genome of Senecavirus is a single-stranded positive strand RNA, about 7.3kb in length, composed of a 5' non-coding region (5'UTR), an open reading frame (ORF) encoding a polyprotein, and a 3' non-coding region (3'UTR). Among them, the 5'UTR contains the genome connecting protein (VPg) sequence and the type IV internal ribosome entry site (IRES), and the 3'UTR downstream contains a poly(A) tail. There is a poly(C) structure and IRES in the 5'UTR of Senecavirus, and under the guidance of IRES, the ribosome translates the only ORF of Senecavirus into a polyprotein, which is then cleaved into the leader protein L and P1, P2, P3 under the action of specific viral-encoded proteases and host proteases. Then P1 is further cleaved into VP4, VP2, VP3 and VP1 four structural proteins, and P2 is cleaved into 2A, 2B and 2C three non-structural proteins, and P3 is cleaved into 3A, 3B, 3C and 3D four non-structural proteins. Among them, the 3B protein (i.e. VPg protein) acts as a primer for viral RNA synthesis, and the 3D protein constitutes an RNA-dependent RNA polymerase (RdRp) and participates in viral replication.

[0006] A pig immune cell model supporting efficient replication of Senecavirus, namely 3D4 / 21, or CRL-2843, is a commercial passaged cell line, which is a kind of immortalized porcine alveolar macrophage, a kind of susceptible cell line of Senecavirus, and can induce efficient natural immune response effect in cells after viral infection. The Senecavirus CH-FJ-2017 (NCBI accession number KY747510.1) is continuously passaged and cultured in CRL-2843 cells, and the virus infection amount of each passage is set to MOI=0.1, the cells and the virus are co-cultured for 36 hours, and then the next passage is carried out, and the virus is continuously passaged without interruption, and the virus after passage is temporarily named as CRL-FJ, and the virus is passaged and cultured for 80 generations; and the CRL-FJ-P80 is subjected to viral whole genome sequencing analysis, and compared with the CH-FJ-2017 parent strain, the genome of CRL-FJ-P80 has many nucleic acid base mutations and corresponding amino acid mutations, and an A type Senecavirus mutant strain CRL-FJ-P80 is obtained.

[0007] Compared with the CH-FJ-2017 parent strain, the viral nucleic acid and virion replication of the A-type Seneca mutant virus strain CRL-FJ-P80 described in the application are obviously increased, indicating that the Seneca mutant virus strain CRL-FJ-P80 described in the application has stronger replication and production capacity; moreover, the antigenicity of the Seneca mutant virus strain CRL-FJ-P80 is not changed, and the immune capacity is enhanced; and the Seneca mutant virus strain CRL-FJ-P80 can be used as an inactivated vaccine candidate strain for the preparation of Seneca virus inactivated vaccine, and has a broad application prospect. SUMMARY

[0008] The A-type Seneca mutant virus strain CRL-FJ-P80 with high virus titer, high antigen production capacity and good production performance is screened and obtained, and can be used as an excellent vaccine candidate strain for the preparation of Seneca virus inactivated vaccine, and has a broad application prospect.

[0009] Specifically includes the following contents:

[0010] In a first aspect, the application provides an A-type Seneca mutant virus strain CRL-FJ-P80, which is obtained by isolating the SVA CH-FJ-2017 strain after being cultured in CRL-2843 cells for 80 generations of continuous passage.

[0011] Preferably, the A-type Seneca mutant virus strain CRL-FJ-P80 is obtained by inserting a base T between 108-109 bases of the whole genome of the SVA CH-FJ-2017 strain, and mutating the 265th base from G to A, the 875th base from C to A, the 1434th base from A to G, the 1447th base from A to G, the 1570th base from C to T, the 1575th base from A to G, the 1591th base from G to A, the 1955th base from A to G, the 2242th base from T to C, the 2345th base from G to A, the 3023th base from T to A, the 3527th base from C to T, the 3695th base from T to A, the 4972th base from C to T, the 5071th base from C to T, the 5665th base from A to G, the 5707th base from A to G, the 5762th base from G to A, the 6095th base from G to A, the 6451th base from C to T, the 6510th base from G to A, the 6588th base from A to C, the 6913th base from C to T, the 7023th base from T to C, and the 7031th base from T to C.

[0012] Preferably, the gene sequence of the A Seneca mutant virus strain CRL-FJ-P80 is shown as SEQ ID NO. 1.

[0013] In a second aspect, the present application provides an application of the A Seneca mutant virus strain CRL-FJ-P80 of the first aspect as described above as a Seneca virus vaccine candidate strain.

[0014] In a third aspect, the present application provides an application of the A Seneca mutant virus strain CRL-FJ-P80 of the first aspect as described above in the preparation of a medicine for preventing and / or controlling the relevant diseases caused by Seneca virus in animals.

[0015] In a fourth aspect, the present application provides an application of the A Seneca mutant virus strain CRL-FJ-P80 of the first aspect as described above in the preparation of a Seneca virus vaccine.

[0016] Preferably, the Seneca virus vaccine is an inactivated vaccine.

[0017] In a fifth aspect, the present application provides an inactivated Seneca virus vaccine, which comprises the inactivated A Seneca mutant virus strain CRL-FJ-P80 of the first aspect. In the present application, the "inactivated A Seneca mutant virus strain CRL-FJ-P80" refers to the A Seneca virus which is not infectious but still maintains its immunogenicity and can cause an immune response in animals. The inactivated A Seneca virus can be prepared by methods well known in the art, for example, the A Seneca virus can be inactivated by using diethylene imine.

[0018] Preferably, the inactivated Seneca virus vaccine further comprises a pharmaceutically acceptable adjuvant or carrier. The Seneca virus vaccine of the present application is in the form of water-in-oil, water-in-oil-in-water, oil-in-water or freeze-dried.

[0019] Preferably, the adjuvant is one or several of chemical immunological adjuvants, microbial immunological adjuvants, biochemical immunological adjuvants. In the present application, the adjuvant includes (1) mineral oil adjuvants: ISA 206, ISA 201; (2) nucleic acid adjuvants: immunostimulatory sequences (ISS), for example, oligodeoxyribonucleotide sequences with one or more non-methylated CpG units; (3) oil-in-water emulsions, for example, SPT emulsion, MF59 emulsion, etc.; (4) cationic lipids containing quaternary ammonium salts, for example, DDA; (5) cytokines; (6) aluminum adjuvants: aluminum hydroxide or aluminum phosphate; (7) plant adjuvants: saponins or (8) any combination or mixture thereof.

[0020] The beneficial effects of the present application are: the present application obtains a type A Seneca mutant virus strain CRL-FJ-P80 after culturing the Seneca virus CH-FJ-2017 strain in CRL-2843 cells for 80 generations; compared with the CH-FJ-2017 parent strain, the viral nucleic acid and virion replication levels of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application are obviously increased, indicating that the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application has stronger replication and production capacity; moreover, the antigenicity of the type A Seneca mutant virus strain CRL-FJ-P80 does not change, and the immune capacity is enhanced; the type A Seneca mutant virus strain CRL-FJ-P80 can be used as a candidate strain of inactivated vaccine, for the preparation of Seneca virus inactivated vaccine, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The type A Seneca virus described in the present application is schematically shown in the continuous passage in CRL-2843 cells;

[0022] Figure 2 The reverse genetic plasmid (r-SVA FJ-cDNA) of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application is schematically shown;

[0023] Figure 3 The viral nucleic acid replication level of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application in CRL-2843 cells is detected by fluorescence quantitative PCR;

[0024] Figure 4 The virion replication level of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application in CRL-2843 cells is detected by TCID 50 ;

[0025] Figure 5 The virion replication level of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application in IBRS-2 cells is detected by TCID 50 ;

[0026] Figure 6 The virion replication level of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application in CRL-2843 cells is detected by Western Blot;

[0027] Figure 7 The inactivated virus immunization mouse experiment of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application is schematically shown;

[0028] Figure 8The antibody detection results of the A-type Seneca Valley mutant virus strain CRL-FJ-P80 inactivated virus after immunizing mice are detected by ELISA (Enzyme-linked Immunosorbent Assay). DETAILED DESCRIPTION

[0029] The above scheme is further described in combination with specific examples. It should be understood that these examples are used to illustrate the present application and are not intended to limit the scope of the present application. The implementation conditions used in the examples can be further adjusted according to the conditions of specific manufacturers, and the implementation conditions not mentioned are usually the conditions in conventional experiments.

[0030] Explanation and interpretation of related terms in the present application:

[0031] The experiments described in the following examples are obtained with biological safety permission:

[0032] According to the requirements of the biological safety level 3 laboratory (BSL-3), the Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, through the biological safety committee of Lanzhou Veterinary Research Institute, the experimental animal ethics committee, the biological safety committee of Chinese Academy of Agricultural Sciences, is reported, permitted, recorded and meets the requirements of national biological safety level.

[0033] The experimental methods used in the following examples are conventional conditions unless otherwise specified.

[0034] Example 1 Isolation and identification of A-type Seneca Valley mutant virus strain CRL-FJ-P80

[0035] Cell culture and virus continuous passage: as shown in Figure 1 CRL-2843 cells (3D4 / 21 cells) were cultured in a T25 cell culture flask, and the cell culture box was set to 37℃, 5% CO2. When the cell adhesion density reached about 70%, the parent strain CH-FJ-2017 (NCBI accession number KY747510.1) was inoculated into the cells with MOI = 0.1, and was placed in the incubator for 1h. The supernatant culture in the cell bottle was discarded, and the cells were washed with PBS for 3 times. New complete culture medium (RPMI1640 medium + 10% FBS + 1% P / S, Gibco) was added, and then the cells were placed in the incubator for 36h. The CPE of the cells was observed, the supernatant culture was collected, and the virus CRL-FJ-P1 was obtained by centrifugation at 1800rpm for 3min. The virus was continuously passed and stopped at CRL-FJ-P80, and was stored at -80℃.

[0036] During the process of cell culture and SVA virus continuous passage, only SVA virus was in the culture system, so the obtained CRL-FJ-P80 was the SVA virus progeny.

[0037] The virus whole genome of CRL-FJ-P80 was sequenced (Shanghai Taopu Biotechnology Co., Ltd.), and compared with SVA CH-FJ-2017 (CRL-FJ-P0). The sequencing results showed that the gene sequence of CRL-FJ-P80 was highly matched with SVA CH-FJ-2017, further indicating that a mutant strain of type A Seneca virus was successfully isolated and obtained, and the mutation site was as previously described. The whole genome sequencing results of CRL-FJ-P80 are shown in SEQ ID NO. 1.

[0038] Example 2 Reverse genetic construction and virus rescue of type A Seneca mutant virus strain CRL-FJ-P80

[0039] A perfect reverse genetic system of foot-and-mouth disease virus and Seneca virus has been established in our laboratory, which can be used to modify and rescue RNA viruses conveniently and quickly. As shown in Figure 2 The core structural elements of the SVA reverse genetic plasmid used in the present patent include: complete cDNA clone (r-SVA FJ-cDNA) of SVA CH-FJ-2017 strain (NCBI accession number KY747510.1), but the original sequence bases of r-SVA FJ-cDNA are mutated to be consistent with CRL-FJ-P80, and there is no termination codon at the 3' end of r-SVA FJ-cDNA. The upstream and downstream of r-SVA FJ-cDNA respectively have T7 promoter sequence and rabbit globin terminator sequence, and a CMV enhancer sequence and a chicken actin promoter sequence are inserted upstream of the T7 promoter, and a monkey vacuole virus PolyA (SV40 poly(A) signal) sequence is inserted downstream of the rabbit globin terminator.

[0040] IBRS-2 cells were cultured in a cell 6-well plate, and the cell incubator was set to 37℃, 5% CO2. When the cell adherent density reached about 70%, the CRL-FJ-P80 reverse genetic plasmid (r-SVAFJ-cDNA) was transfected into the cells using jetPRIME transfection reagent (Polyplus) at a dose of 1 ug / well. After 6h of transfection, the cells were rinsed once with PBS and replaced with new complete culture medium (DMEM + 10% FBS + 1% P / S, Gibco) for continuous culture for 48h. The cell culture supernatant was collected and centrifuged at 1800rpm for 3min, and the supernatant was retained. The supernatant was used to infect IBRS-2 cells cultured in a 6-well plate. After 48h of culture, obvious CPE appeared. The cell culture supernatant was collected and centrifuged at 1800rpm for 3min, and the supernatant was retained. The supernatant was used to infect cells again, and obvious CPE also appeared, indicating that the CRL-FJ-P80 mutant virus was successfully rescued.

[0041] Example 3 Replication and titer determination of the A-type Seneca Valley mutant virus strain CRL-FJ-P80

[0042] 1. Replication level of the A-type Seneca Valley mutant virus strain CRL-FJ-P80

[0043] CRL-2843 cells (3D4 / 21 cells) were cultured in a cell 12-well plate, and the cell incubator was set to 37℃, 5% CO2. When the cell adherent density reached about 70%, CRL-FJ-P0 (SVA CH-FJ-2017) and CRL-FJ-P80 were inoculated into the cells at an MOI of 0.1, and incubated in the incubator for 1h. The supernatant culture in the cell bottle was discarded, the cells were rinsed with PBS for 3 times, and then new complete culture medium (RPMI 1640 medium + 10% FBS + 1% P / S, Gibco) was added. The cells were then placed back in the incubator for culture for 24h. The relative replication level of viral nucleic acid in the culture was detected by fluorescent quantitative PCR.

[0044] The relative quantitative PCR detection was as follows:

[0045] (1) Extraction of total RNA in cell culture: The specific operation was strictly performed according to the instructions of the commercial kit (Cell / Tissue Total RNA Isolation Kit V2, Norgen, RC112-01). Cell / Tissue Total RNA Isolation Kit V2, Norgen, RC112-01).

[0046] (2) RNA reverse transcription into cDNA: 10ul reaction system contains 2ul 5x PrimeScript RT MasterMix (TaKaRa, RR036A-1) and 8ul total RNA extract (RNA concentration is 62.5ng / ul), reaction in PCR thermal cycler: 25℃ reaction for 30 seconds, 37℃ reaction for 15 minutes, 85℃ reaction for 5 seconds, 4℃ reaction for 5 minutes.

[0047] (3) Relative quantitative PCR: 25ul reaction system contains 12.5ul TB Green Premix Ex Taq II (TaKaRa, RR820), 1ul PCR Forward Primer (10uM), 1ul PCR Reverse Primer (10uM), 2ul cDNA template, 8.5ul sterile water, in QuantStudio real-time fluorescent quantitative PCR system, the following PCR program is carried out:

[0048] Stage 1: pre-denaturation

[0049] 95℃, 30 seconds

[0050] 1 Cycle

[0051] Stage 2: PCR reaction

[0052] 95℃, 15 seconds

[0053] 60℃, 30 seconds

[0054] 40 Cycles

[0055] Stage 3: melting curve analysis

[0056] 95℃, 15 seconds

[0057] 60℃, 1 minute

[0058] 95℃, 0.1 seconds

[0059] 2. Virus titer determination of A-type Seneca Valley mutant virus strain CRL-FJ-P80

[0060] CRL-2843 cells and IBRS-2 cells were cultured in a cell 12-well plate, and the cell incubator was set to 37°C and 5% CO2. When the cell adherent density reached about 70%, CRL-FJ-P0 (SVA CH-FJ-2017) and CRL-FJ-P80 were inoculated into the cells at an MOI of 0.1, and were placed in the incubator for 1 h of incubation. Then, the cells were taken out, the supernatant culture in the cell bottle was discarded, the cells were washed with PBS for 3 times, and new complete culture solution was added. At this time, the culture supernatant at 0 h was collected, and the culture was continued. The culture supernatant was collected at 12 h, 24 h, 36 h, and 48 h, respectively. All the culture supernatants were centrifuged at 1800 rpm for 3 min, and the supernatant was reserved.

[0061] IBRS-2 cells (SVA susceptible cells) were cultured in a cell 96-well plate, and the cell incubator was set to 37°C and 5% CO2. When the cell adherent density reached about 70%, the collected culture supernatant was centrifuged and diluted by 10 times in a gradient. Then, the diluted sample was added to the 96-well plate at 50ul / well, and 8 repeats were made for each diluted sample. Then, the plate was placed back into the incubator for further culture. At 24 h of culture, the CPE of the cells in the plate was observed and counted, and both positive and negative wells were counted.

[0062] 3. Antigen content determination

[0063] CRL-2843 cells were cultured in a cell 6-well plate, and the cell incubator was set to 37°C and 5% CO2. When the cell adherent density reached about 70%, CRL-FJ-P0 (SVA CH-FJ-2017), CRL-FJ-P20, CRL-FJ-P40, CRL-FJ-P60, and CRL-FJ-P80 were inoculated into the cells at an MOI of 0.1, and were placed in the incubator for 1 h of incubation. Then, the cells were taken out, the supernatant culture in the cell bottle was discarded, the cells were washed with PBS for 3 times, and new complete culture solution (RPMI 1640 medium + 10% FBS + 1% P / S, Gibco) was added. At 24 h of culture, the cells were collected, and the cells were dissolved to prepare protein samples using 1X working solution prepared by 5X Protein Loading Buffer (Reducing) (Apexbio, K1164-10).

[0064] Western blot analysis of samples: 15-well, 10% concentration PAGE gels were prepared using a one-step PAGE gel rapid preparation kit (Yamei, PG212). 10 μL of sample was loaded per well. A protein marker (ThermoFisher Scientific, 26616) was used to indicate the sample position during electrophoresis, and electrophoresis was performed at a constant voltage of 90 V throughout. Transfer was performed using a PVDF membrane (Merck-Millipore, ISEQ00010) at a constant current of 200 mA throughout the transfer process. Antigen-antibody antisense incubation was performed using a self-made mouse SVA-VP2 antibody at 4°C for 10 h, followed by primary and secondary antibody incubation with HRP-goat anti-mouse IgG (Bio-Lon, BF03001) at room temperature for 35 minutes. Sample luminescence was detected using a high-sensitivity ECL chemiluminescence kit (Xinsemei, P10300).

[0065] 4. Results

[0066] like Figure 3 As shown, in the co-culture of CRL-2843 cells and the virus, the nucleic acid replication level of the type A Seneca mutant virus strain CRL-FJ-P80 described in this application was significantly increased compared to the CH-FJ-2017(P0) strain.

[0067] like Figures 4-5 As shown, in co-culture of CRL-2843 cells and virus, and in co-culture of IBRS-2 cells and virus, the viral titer of the type A Seneca mutant virus strain CRL-FJ-P80 described in this application was significantly enhanced compared to the CH-FJ-2017(P0) strain.

[0068] like Figure 6 As shown, in the co-culture of CRL-2843 cells and the virus, compared with the CH-FJ-2017(P0), CRL-FJ-P20, CRL-FJ-P40, and CRL-FJ-P60 strains, the CRL-FJ-P80 strain of the type A Seneca mutant virus described in this application had the highest VP2 antigen accumulation, which also reflects that its viral particle replication is significantly enhanced.

[0069] Example 4: Preparation and Evaluation of Inactivated Vaccine Immunization Efficacy

[0070] 1. Preparation of inactivated vaccines

[0071] Virus inactivation: SVA virus heat inactivation: 56℃, 30 minutes.

[0072] Freund's adjuvant: first immunization: complete adjuvant (Sigma-Aldrich, F5881-10X10ML); second immunization: incomplete adjuvant (Sigma-Aldrich, F5506-10X10ML); third immunization: incomplete adjuvant.

[0073] Emulsification: adjuvant and antigen are used at a volume ratio of 1:1, a three-way needle tube is used for emulsification, the adjuvant is first lubricated, then the sample and the adjuvant are respectively sucked, connected, and air bubbles are prevented during the process. Mix well by repeatedly pushing and pulling, and then add a cup of water. The emulsion droplets do not disperse when they coagulate on the water surface, indicating that the emulsification is successful.

[0074] 2. Mouse back injection

[0075] Use a 1ml syringe for more accurate connection to the emulsification three-way tube to avoid air bubbles.

[0076] Mice: BALB / c, five weeks old, randomly divided into groups, 8 mice per group.

[0077] Subcutaneous injection on the back, 200ul per mouse per injection, avoid liquid leakage, inject in two places, 100ul per place.

[0078] Before injection, wipe the mouse back with alcohol cotton, and observe the mouse state for about 30 minutes after injection.

[0079] The second immunization is 14 days after the first immunization, the third immunization is 14 days after the second immunization, and blood sampling is 14 days after the third immunization.

[0080] 3. Blood sampling

[0081] Orbital blood sampling: each mouse is sampled at about 500ul, and the mouse is sacrificed after the sampling.

[0082] Blood sampling: 37℃ for 1 hour, 4℃ centrifugation, 8000rpm, 5min, keep the supernatant, i.e. serum, and store at -80℃ for standby.

[0083] 4. Elisa detection

[0084] (1) Coating Elisa plate: blank plate, viral antigen protein (SVA-VP2), coating solution, blocking solution;

[0085] The coating amount of antigen protein is 100ng / well, the antigen protein is diluted with coating solution and added to the blank plate, 50ul / well, 4℃ for 12 hours, and the plate is washed 4 times and dried.

[0086] Add blocking solution, 50ul / well, 4℃ for 12 hours, discard the solution, do not wash the plate, and dry.

[0087] The coated plate is immediately used, otherwise it needs to be stored at 4℃ in a plastic bag.

[0088] (2) Samples to be tested: multi-channel pipette, sample diluent, secondary antibody, substrate, and terminator;

[0089] First, dilute the serum to 1:100 in a 1.5ml EP tube, and then dilute the sample to 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000 in a dilution plate; the dilution ratio depends on the specific situation.

[0090] Add the diluted sample to the coated ELISA plate, 50 μl / well, and perform 3 replicates for each diluted sample. Incubate at 37°C for 30 minutes, discard the liquid, wash the plate 4 times, and pat dry.

[0091] Add HRP-goat anti-mouse IgG (Bio-Lon, BF03001) at a concentration of 1:10000, 50 μl / well, incubate at 37°C for 30 minutes, discard the liquid, wash the plate 4 times, and pat dry.

[0092] Protect from light, add substrate, 50 μl / well, and let stand at 37°C for 10 minutes.

[0093] Add the stop agent, 50 μl / well, and immediately run the instrument for testing.

[0094] 5. Results

[0095] like Figure 8 As shown, under the same cell count, same inoculation amount, same amplification time, same immunization protocol, and same detection method, compared with the CH-FJ-2017(P0) strain, the type A Seneca virus mutant strain CRL-FJ-P80 described in this application can lead to a relatively higher level of specific antibodies detected in the serum of immunized BALB / c mice. This indicates that CRL-FJ-P80 can enhance the immune capacity of immunized BALB / c mice, and CRL-FJ-P80 can be used as an amplification seed candidate for Seneca virus inactivated vaccine.

[0096] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make other improvements without departing from the concept of the present invention, and these improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A Seneca Valley Virus strain CRL-FJ-P80 of type A, characterized in that, The gene sequence of the A Seneca mutant virus strain CRL-FJ-P80 is shown as SEQ ID NO.

1.

2. The use of the A Seneca mutant virus strain CRL-FJ-P80 of claim 1 in the preparation of a medicine for preventing the related diseases caused by the A Seneca virus in animals.

3. The use of the A Seneca mutant virus strain CRL-FJ-P80 of claim 1 in the preparation of an A Seneca virus vaccine.

4. The use according to claim 3, wherein the compound is ###0002### The A Seneca virus vaccine is an inactivated vaccine.

5. An inactivated vaccine of a Senecavirus type A, characterized in that, The A Seneca virus vaccine comprises the inactivated A Seneca mutant virus strain CRL-FJ-P80 of claim 1.

6. The Seneca Valley virus inactivated vaccine of claim 5, wherein the virus is a type A Seneca Valley virus. The A Seneca virus inactivated vaccine further comprises a pharmaceutically acceptable adjuvant or carrier.

7. The Seneca Valley virus inactivated vaccine of claim 6, wherein the Seneca Valley virus is a type A Seneca Valley virus. The adjuvant is one or several of a chemical immunoadjuvant, a microbial immunoadjuvant, and a biochemical immunoadjuvant.

Images