A-type Seneca mutant virus strain and application thereof

Abstract

The invention relates to the technical field of molecular biology, in particular to an A-type Seneca mutant virus strain and application thereof. According to the invention, a Seneca virus CH-FJ-2017 strain A is subjected to continuous subculture for 80 generations in a CRL-2843 cell, and then a Seneca mutant virus strain CRL-FJ-P80 is obtained; compared with a CH-FJ-2017 parent virus strain, the Seneca mutant virus strain CRL-FJ-P80 disclosed by the invention has the advantages that the virus nucleic acid and virion replication levels are obviously increased, so that the Seneca mutant virus strain CRL-FJ-P80 disclosed by the invention has stronger replication and production capacity; the antigenicity of the Seneca mutant virus strain CRL-FJ-P80 is not changed, and the immunocompetence of the Seneca mutant virus strain CRL-FJ-P80 is enhanced; the strain can be used as an inactivated vaccine candidate strain, is used for preparing Seneca virus inactivated vaccines, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a type A Seneca mutant virus strain and application thereof. Background Art

[0002] Senecavirus type A (SVA), also known as Seneca Valley virus (SVV), is the only member of the Senecavirus genus of the Picornaviridae family, and currently has only one serotype. In 2002, Senecavirus was first discovered and identified in cell culture contaminants in Maryland, USA, and was subsequently named SVV-001. The complete genome sequence of SVV-001 was resolved in 2005. SVV-001 is known as NTX-010 in the medical field. Research data show that NTX-010 replicates specifically in tumor cells and exhibits high levels of oncolytic activity both 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 blisters and / or ulcerative lesions on the nose, oral mucosa, coronary 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 vesicular exanthema virus (VESV). Newborn piglets infected with Seneca virus within seven days will also be accompanied by various clinical signs, including weakness, salivation, skin congestion, neurological signs, diarrhea, high mortality, etc. The infection and spread of Seneca virus has caused huge economic losses to the pig farming 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 huge challenge to the prevention and control of Seneca virus.

[0005] The virus particles of Seneca virus are icosahedral in structure, without envelope, and have a diameter of about 30nm. The capsid of Seneca virus is composed of four structural proteins (VP1, VP2, VP3, and 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 viral protein (VPg) is covalently linked to the 5′ end of the viral RNA genome and encapsulated in the virus particle cavity. The genome of Seneca virus is a single-stranded positive-strand RNA with a total length of about 7.3kb, consisting 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 linker protein (VPg) sequence and the type IV internal ribosome entry site (IRES), and the downstream of the 3′UTR contains a poly (A) tail. There are poly(C) structures and IRES inside the 5′UTR of Seneca virus. Under the guidance of IRES, the ribosome translates the only ORF of Seneca virus into polyprotein. Then, under the action of specific proteases encoded by the virus and host proteases, the polyprotein is first cleaved into the leader protein L and P1, P2, and P3. Then P1 is further cleaved into four structural proteins: VP4, VP2, VP3, and VP1, while P2 is cleaved into three non-structural proteins: 2A, 2B, and 2C, and P3 is cleaved into four non-structural proteins: 3A, 3B, 3C, and 3D. Among them, the 3B protein (i.e., VPg protein) acts as a primer for the synthesis of viral RNA, and the 3D protein constitutes RNA-dependent RNA polymerase (RdRp), which participates in viral replication.

[0006] A porcine immune cell model that supports efficient replication of Seneca virus, namely 3D4 / 21, or CRL-2843, is a commercially available cell line, a type of immortalized porcine alveolar macrophage, a cell line susceptible to Seneca virus, and the virus induces an efficient natural immune response effect in the cells after infecting the cells. The present invention continuously subcultures Seneca virus CH-FJ-2017 (NCBI accession number is KY747510.1) in CRL-2843 cells, sets the virus infection amount of each subculture to MOI=0.1, co-cultures the cells and the virus for 36 hours, and then performs the next subculture, continuously and uninterruptedly subcultures, and temporarily names the subcultured virus CRL-FJ, and subcultures the virus for 80 generations in total; and performs virus whole genome sequencing analysis on CRL-FJ-P80, 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 obtains a type A Seneca mutant virus strain CRL-FJ-P80.

[0007] Compared with the parent strain CH-FJ-2017, the viral nucleic acid and virus particle replication levels of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application are significantly increased, indicating that the Seneca mutant virus strain CRL-FJ-P80 described in the present application has stronger replication and production capabilities; and the antigenicity of the Seneca mutant virus strain CRL-FJ-P80 has not changed, and the immune capacity is enhanced; it can be used as an inactivated vaccine candidate strain for the preparation of Seneca virus inactivated vaccines, and has broad application prospects. Summary of the invention

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

[0009] Specifically include the following:

[0010] In a first aspect, the present invention provides a type A Seneca mutant virus strain CRL-FJ-P80, wherein the type A Seneca mutant virus strain CRL-FJ-P80 is isolated after continuous subculture of the SVA CH-FJ-2017 strain in CRL-2843 cells for 80 generations.

[0011] Preferably, the type A Seneca mutant virus strain CRL-FJ-P80 inserts a base T between bases 108-109 of the whole genome of the SVA CH-FJ-2017 strain, and mutates 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 1591st base from G to A, the 1955th base from A to G, the 2242nd base from T to C, the 2345th base from G to A, the 3023rd base from T to A, and the 3527th base from G to A. The base mutated from C to T, the 3695th base mutated from T to A, the 4972th base mutated from C to T, the 5071st base mutated from C to T, the 5665th base mutated from A to G, the 5707th base mutated from A to G, the 5762nd base mutated from G to A, the 6095th base mutated from G to A, the 6451st base mutated from C to T, the 6510th base mutated from G to A, the 6588th base mutated from A to C, the 6913th base mutated from C to T, the 7023rd base mutated from T to C, and the 7031st base mutated from T to C.

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

[0013] In a second aspect, the present invention provides a use of the type A Seneca virus mutant strain CRL-FJ-P80 described in the first aspect as a candidate strain for a Seneca virus vaccine.

[0014] In a third aspect, the present invention provides a use of the type A Seneca virus mutant strain CRL-FJ-P80 described in the first aspect in the preparation of a drug for preventing and / or controlling related diseases caused by animal Seneca virus.

[0015] In a fourth aspect, the present invention provides a use of the type A Seneca virus mutant strain CRL-FJ-P80 described in the first aspect in the preparation of a type A Seneca virus vaccine.

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

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

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

[0019] Preferably, the adjuvant is one or more of a chemical immunoadjuvant, a microbial immunoadjuvant, or a biochemical immunoadjuvant. In the present application, the adjuvant includes (1) mineral oil adjuvants: ISA 206, ISA 201; (2) nucleic acid adjuvants: immunostimulatory sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units; (3) water-in-oil emulsions, such as SPT emulsions, MF59 emulsions, etc.; (4) cationic lipids containing quaternary ammonium salts, such as 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 invention are as follows: the present invention obtains a type A Seneca mutant virus strain CRL-FJ-P80 after continuously subculturing 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 virus particle replication levels of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application are significantly increased, indicating that the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application has stronger replication and production capabilities; and the antigenicity of the type A Seneca mutant virus strain CRL-FJ-P80 has not changed, and the immune ability is enhanced; it can be used as an inactivated vaccine candidate strain for the preparation of Seneca virus inactivated vaccines, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of continuous passage of type A Senecavirus in CRL-2843 cells described in the present application;

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

[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 was detected by fluorescent quantitative PCR;

[0024] Figure 4 The viral particle replication level of the type A Seneca mutant virus strain CRL-FJ-P80 in CRL-2843 cells was measured by TCID 50 Detection;

[0025] Figure 5 The viral particle replication level of the type A Seneca mutant virus strain CRL-FJ-P80 in IBRS-2 cells was measured by TCID 50 Detection;

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

[0027] Figure 7 Schematic diagram of the experiment of immunizing mice with inactivated virus of type A Seneca mutant virus strain CRL-FJ-P80 described in the present application;

[0028] Figure 8The antibody detection results after mice were immunized with the inactivated virus of the type A Seneca mutant virus strain CRL-FJ-P80 described in the present application were detected by ELISA (Enzyme-linked Immunosorbent Assay). DETAILED DESCRIPTION

[0029] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not limited to the scope of the present invention. The implementation conditions adopted in the examples can be further adjusted according to the conditions of the specific manufacturer, and the unspecified implementation conditions are usually the conditions in conventional experiments.

[0030] Description and explanation of relevant terms in the present invention:

[0031] The experiments described in the following examples were performed with biosafety approval:

[0032] In accordance with the relevant requirements of Biosafety Level 3 Laboratory (BSL-3), the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences has been reported, approved, and filed step by step by the Biosafety Committee of the Lanzhou Veterinary Research Institute, the Experimental Animal Ethics Committee, and the Biosafety Committee of the Chinese Academy of Agricultural Sciences, and is in compliance with the national biosafety level requirements.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all based on conventional conditions.

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

[0035] Cell culture and virus serial passage: Figure 1 As shown, CRL-2843 cells (3D4 / 21 cells) were cultured in T25 cell culture flasks, and the cell culture incubator was set at 37°C and 5% CO 2 When the cell adhesion density reached about 70%, the parent strain CH-FJ-2017 (NCBI accession number is KY747510.1) was inoculated into the cells with MOI = 0.1, and the cells were placed in an incubator for incubation for 1 hour. The cells were taken out and the supernatant culture in the cell bottle was discarded. The cells were rinsed with PBS 3 times, and new complete culture medium (RPMI1640 medium + 10% FBS + 1% P / S, Gibco) was added. Then the cells were returned to the incubator and cultured for 36 hours. The cells were taken out and obvious cell CPE was observed. The supernatant culture was collected and centrifuged at 1800rpm for 3 minutes. The supernatant was retained to obtain the virus CRL-FJ-P1. The cells were continuously passaged in this way until CRL-FJ-P80 was stopped, and the virus was stored at -80°C.

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

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

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

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

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

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

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

[0043] CRL-2843 cells (3D4 / 21 cells) were cultured in 12-well plates in a cell culture incubator set at 37°C and 5% CO 2 When the cell adhesion density reached about 70%, CRL-FJ-P0 (SVA CH-FJ-2017) and CRL-FJ-P80 were inoculated into the cells at MOI = 0.1, and incubated in an incubator for 1 h. The supernatant culture in the cell bottle was removed and discarded, and the cells were rinsed 3 times with PBS, and then new complete culture medium (RPMI 1640 medium + 10% FBS + 1% P / S, Gibco) was added. Then the cells were returned to the incubator and cultured for 24 h. The relative replication level of viral nucleic acid in the culture was detected by fluorescent quantitative PCR.

[0044] Relative quantitative PCR detection is as follows:

[0045] (1) Extraction of total RNA from cell culture: The specific operation is strictly in accordance with the commercial kit ( Cell / Tissue Total RNA Isolation Kit V2, Novozymes, RC112-01) instructions.

[0046] (2) RNA was reverse transcribed into cDNA: 10ul of the total reaction system contained 2ul of 5× PrimeScript RT MasterMix (TaKaRa, RR036A-1) and 8ul of total RNA extract (RNA concentration was 62.5ng / ul), and the reaction was carried out in a PCR thermal cycler: 25°C for 30 seconds, 37°C for 15 minutes, 85°C for 5 seconds, and 4°C for 5 minutes.

[0047] (3) Relative quantitative PCR: The total reaction system of 25ul contained 12.5ul TB Green Premix Ex Taq II (TaKaRa, RR820), 1ul PCR Forward Primer (10uM), 1ul PCR Reverse Primer (10uM), 2ul cDNA template, and 8.5ul sterile pure water. The following PCR program was performed in the QuantStudio real-time fluorescence quantitative PCR system:

[0048] Stage 1: Pre-denaturation

[0049] 95°C, 30 seconds

[0050] 1Cycle

[0051] Stage 2: PCR reaction

[0052] 95℃, 15 seconds

[0053] 60℃, 30 seconds

[0054] 40Cycles

[0055] Stage 3: Melting curve analysis

[0056] 95℃, 15 seconds

[0057] 60℃, 1min

[0058] 95℃, 0.1 sec

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

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

[0061] IBRS-2 cells (SVA susceptible cells) were cultured in a 96-well cell plate in a cell culture incubator set at 37°C and 5% CO2. When the cell adhesion density reached about 70%, the collected culture supernatant was diluted 10-fold in a continuous gradient, and then the diluted samples were added to a 96-well plate at 50ul / well. Eight replicates were made for each diluted sample, and then the cells were returned to the incubator for continued culture. After 24 hours of culture, the CPE of the cells in the cell 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 6-well cell plate, and the cell culture incubator was set at 37°C and 5% CO2. When the cell adhesion 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. The cells were placed in an incubator and incubated for 1 h. The supernatant culture in the cell bottle was removed and discarded. The cells were rinsed 3 times with PBS, and new complete culture medium (RPMI 1640 medium + 10% FBS + 1% P / S, Gibco) was added. After culturing for 24 h, the cells were collected and 1X working solution prepared with 5X Protein Loading Buffer (Reducing) (Apexbio, K1164-10) was used to dissolve the cells and prepare protein samples.

[0064] Western blot detection samples: a one-step PAGE gel rapid preparation kit (Yazyme, PG212) was used to prepare a 15-well, 10% concentration PAGE gel, 10ul / well of sample was loaded, a protein marker (ThermoFisher Scientific, 26616) was used to indicate the position of the electrophoresis sample, and the electrophoresis was performed at 90V constant voltage throughout the process; a PVDF membrane (Merck-Millipore, ISEQ00010) was used for transfer, and a constant current of 200mA was used for transfer throughout the process; the mouse SVA-VP2 antibody homemade in this laboratory was used for antigen-antibody antisense incubation at 4°C for 10h, and HRP-goat anti-mouse IgG (Biolong, BF03001) was used for primary and secondary antibody reaction incubation at room temperature for 35 minutes; an ultrasensitive ECL chemiluminescence kit (New Saimei, P10300) was used for sample luminescence.

[0065] 4. Results

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

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

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

[0069] Example 4 Inactivated vaccine preparation and immune effect evaluation

[0070] 1. Preparation of inactivated vaccines

[0071] Virus inactivation: SVA virus heat inactivation: 56°C, 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: Use adjuvant and antigen in a volume ratio of 1:1. Use a three-way syringe for emulsification. Lubricate with adjuvant first, then draw in sample and adjuvant separately and connect them. Pay attention to eliminating bubbles during the process. Push and pull repeatedly to mix. Add a cup of clean water. If the emulsified droplets condense on the water surface without dispersing, it means the emulsification is successful.

[0074] 2. Immunization of mice by back injection

[0075] Use a 1ml syringe, which is more accurate, and connect it to the emulsifying tee to absorb the sample and avoid bubbles.

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

[0077] Subcutaneous injection at the back, 200 μl / mouse per immunization, avoid liquid leakage, injection at two sites, 100 ul / site.

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

[0079] After the first vaccination, there will be a second vaccination 14 days later, and a third vaccination 14 days later, and blood will be collected after another 14 days.

[0080] 3. Blood collection

[0081] Orbital blood collection: The amount of blood collected from each mouse is about 500ul, and the mouse is killed afterwards.

[0082] The collected blood was processed by placing it at 37°C for 1 hour, centrifuging it at 4°C, 8000rpm, for 5min, and keeping the supernatant, i.e. serum, which was frozen at -80°C for later use.

[0083] 4. Elisa test

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

[0085] The coating amount of antigen protein is 100 ng / well. Dilute the antigen protein with coating solution and add it to the blank plate, 50 ul / well. Let it stand at 4°C for 12 hours, wash the plate 4 times, and pat dry.

[0086] Add blocking solution, 50 μl / well, incubate at 4°C for 12 hours, discard the solution, do not wash the plate, and pat dry.

[0087] The coated plates should be used immediately, otherwise they should be sealed with plastic and stored at 4°C.

[0088] (2) Detection samples: sample gun, 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 samples to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 in a dilution plate; the dilution ratio depends on the specific situation.

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

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

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

[0093] Add the stop substance, 50 μl / well, and immediately test on the instrument.

[0094] 5. Results

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

[0096] The above-described embodiments are only embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the present invention. It should be noted that, for a person of ordinary skill in the art, other improvements may be made without departing from the concept of the present invention, and these improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A type A Seneca virus mutant strain CRL-FJ-P80, characterized in that: The type A Seneca mutant virus strain CRL-FJ-P80 was isolated after the parent strain CH-FJ-2017 was continuously subcultured in CRL-2843 cells for 80 generations.

2. The type A Seneca virus mutant strain CRL-FJ-P80 according to claim 1, characterized in that The type A Seneca mutant virus strain CRL-FJ-P80 is a strain in which a base T is inserted between bases 108-109 of the whole genome of the parent strain CH-FJ-2017, and the base 265 is mutated from G to A, the base 875 is mutated from C to A, the base 1434 is mutated from A to G, the base 1447 is mutated from A to G, the base 1570 is mutated from C to T, the base 1575 is mutated from A to G, the base 1591 is mutated from G to A, the base 1955 is mutated from A to G, the base 2242 is mutated from T to C, the base 2345 is mutated from G to A, and the base 3023 is mutated from T to C. The base at position mutates from T to A, the base at position 3527 mutates from C to T, the base at position 3695 mutates from T to A, the base at position 4972 mutates from C to T, the base at position 5071 mutates from C to T, the base at position 5665 mutates from A to G, the base at position 5707 mutates from A to G, the base at position 5762 mutates from G to A, the base at position 6095 mutates from G to A, the base at position 6451 mutates from C to T, the base at position 6510 mutates from G to A, the base at position 6588 mutates from A to C, the base at position 6913 mutates from C to T, the base at position 7023 mutates from T to C, and the base at position 7031 mutates from T to C.

3. The type A Seneca virus mutant strain CRL-FJ-P80 according to claim 2, characterized in that The gene sequence of the type A Seneca mutant virus strain CRL-FJ-P80 is shown in SEQ ID NO.

1.

4. Use of the type A Seneca virus mutant strain CRL-FJ-P80 as claimed in any one of claims 1 to 3 as a candidate strain for a Seneca virus vaccine.

5. Use of the type A Seneca virus mutant strain CRL-FJ-P80 according to any one of claims 1 to 3 in the preparation of a medicament for preventing and / or controlling diseases related to animal Seneca virus.

6. Use of the type A Seneca virus mutant strain CRL-FJ-P80 according to any one of claims 1 to 3 in the preparation of a type A Seneca virus vaccine.

7. The use according to claim 6, characterized in that The type A Seneca virus vaccine is an inactivated vaccine.

8. An inactivated vaccine of Seneca virus type A, characterized in that: The type A Seneca virus vaccine comprises the inactivated type A Seneca virus mutant strain CRL-FJ-P80 described in any one of claims 1 to 3.

9. The inactivated Seneca virus type A vaccine according to claim 8, characterized in that The inactivated Seneca virus type A vaccine further comprises a pharmaceutically acceptable adjuvant or carrier.

10. The inactivated Seneca virus type A vaccine according to claim 9, characterized in that The adjuvant is one or more of a chemical immune adjuvant, a microbial immune adjuvant, and a biochemical immune adjuvant.

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