Porcine Japanese encephalitis virus and porcine parvovirus VP2 protein bivalent vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of vaccine preparation, in particular to a porcine Japanese encephalitis virus and porcine parvovirus VP2 protein bivalent vaccine as well as a preparation method and application thereof. According to the invention, the JEV-PPV VP2 combined vaccine is prepared by combining the JEV whole-virus inactivated vaccine with the VLP assembled by the expressed PPV VP2 protein, so that the organism can be induced to generate good cellular immunity and humoral immunity, the organism is protected from being damaged by PPV and JEV, and a certain basis and direction are provided for research on the aspect of combined use of vaccines in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and particularly relates to a bivalent vaccine of Japanese encephalitis virus of pigs and VP2 protein of porcine parvovirus, and a preparation method and application thereof. Background Art

[0002] Infection of swine herds with Japanese encephalitis virus (JEV) and porcine parvovirus (PPV) can both cause reproductive disorders in sows, resulting in abortion, stillbirth, and infertility. Moreover, the two are extremely likely to be co-infected clinically, bringing huge challenges and significant economic losses to the prevention and control of large-scale pig farms. At present, the prevention and control of Japanese encephalitis virus disease and porcine parvovirus disease at home and abroad mainly rely on vaccination.

[0003] Most of the JEV vaccines approved for use on the market at present are vaccines developed against genotype GIII [Yun S I, Lee YM. Japanese encephalitis: the virus and vaccines[J]. Human Vaccines&Immunotherapeutics, 2014, 10(2):263 - 279]. Due to the evolution of JEV, the dominant position of genotype GIII has gradually been replaced by genotype GI. Therefore, the vaccines developed based on genotype GIII can no longer completely and effectively control the prevalence of JEV [Fan YC, Chen YY, Chen JM, et al. Effectiveness of live-attenuated genotype iii japanese encephalitis viral vaccine against circulating genotype i viruses in swine[J]. Viruses-Basel, 2022, 14(1)].

[0004] In addition, the currently mainly approved PPV vaccine on the market is an inactivated vaccine. However, the inactivated PPV vaccine has obvious defects, such as high cost, large immunization dose, long immunization program, and adverse reactions in pigs after immunization, including fever, anorexia, listlessness, and swelling. Compared with inactivated vaccines and attenuated vaccines, genetically engineered subunit vaccines have the advantages of weak toxicity, small immunization dose, simple immunization program, and no adverse reactions in immunized pigs. As the main component of PPV, the VP2 protein can be assembled into VLPs alone. It contains most of the B-cell epitopes, which play a key role in eliciting neutralizing antibodies [Hua T, Zhang D, Tang B, et al. The immunogenicity of the virus-like particles derived from the VP2 protein of porcine parvovirus [J]. Veterinary Microbiology, 2020, 248: 108795]. Therefore, the VP2 protein is usually considered as the target antigen of genetically engineered subunit vaccines. Ling et al. successfully constructed VLPs through the baculovirus-insect cell system, which had excellent immunogenicity, could induce humoral and cellular immune responses, and could protect against PPV challenge [Ling Z, Zhang H, Chen Y, et al. A Subunit Vaccine Based on the VP2 Protein of Porcine Parvovirus 1 Induces a Strong Protective Effect in Pregnant Gilts [J]. Vaccines, 2023, 11(11)]. Antonis et al. successfully expressed PPV VLPs through the BEVS system. The results of animal experiments showed that high levels of neutralizing antibodies could be detected in the sera of guinea pigs when the single immunization dose was 0.2 μg, and piglets could be protected from PPV challenge when the single immunization dose for pregnant gilts was 0.7 μg. The results indicated that PPV VLPs could prevent PPV from spreading through the placenta [Antonis AF, Bruschke C J, Rueda P, et al. A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure [J]. Vaccine, 2006, 24(26): 5481 - 5490].

[0005] The baculovirus expression vector system (BEVS) has been proven to be a system suitable for producing a diverse set of proteins, with advantages including high safety, ease of operation, and suitability for serum-free culture. It also has good post-translational modification functions, and the recombinant proteins prepared have good immunogenicity. Currently, it has been widely used in the preparation of commercial vaccines and therapeutic drugs [Hitchman R B, Possee R D, Crombie A T, et al. Genetic modification of a baculovirus vector for increased expression in insect cells [J]. Cell Biology & Toxicology, 2010, 26(1): 57].

[0006] Since the co-infection of JEV and PPV is difficult to eradicate, there is only an inactivated single vaccine on the market so far, and there is no commercial combined vaccine for JEV and PPV, which results in too frequent, time-consuming and laborious immunizations in pig farms. Therefore, there is an urgent need to develop an effective combined vaccine for multiple preventions with a single injection to simultaneously prevent JEV and PPV in the prevention and control work of pig farms. Summary of the Invention

[0007] In order to overcome the deficiencies and disadvantages of the prior art, the primary object of the present invention is to provide a combined vaccine of Japanese encephalitis virus of pigs and VP2 protein of porcine parvovirus. This vaccine mixes the inactivated vaccine strain of Japanese encephalitis virus of pigs with the VLP particles of VP2 protein of porcine parvovirus expressed by recombinant baculovirus to jointly prepare a combined vaccine of Japanese encephalitis virus of pigs and VP2 protein of porcine parvovirus, which can simultaneously prevent Japanese encephalitis virus disease of pigs and porcine parvovirus disease.

[0008] Another object of the present invention is to provide a preparation method of the above-mentioned combined vaccine of Japanese encephalitis virus of pigs and VP2 protein of porcine parvovirus.

[0009] Another object of the present invention is to provide the application of the above-mentioned combined vaccine of Japanese encephalitis virus of pigs and VP2 protein of porcine parvovirus.

[0010] The object of the present invention is achieved by the following technical solutions:

[0011] A combined vaccine of Japanese encephalitis virus of pigs and VP2 protein of porcine parvovirus, comprising inactivated Japanese encephalitis virus of pigs, PPV-VP2 recombinant protein and adjuvant;

[0012] The amino acid sequence of the said PPV-VP2 recombinant protein is as shown in SEQ ID No: 1;

[0013] The nucleotide sequence encoding the PPV-VP2 recombinant protein is shown in SEQ ID No: 2;

[0014] In the JE virus and porcine parvovirus VP2 protein bivalent vaccine, the content of inactivated JE virus is preferably 10 5.5 -10 6.0 TCID 50 / mL, and the content of PPV-VP2 recombinant protein is preferably 45 - 55 μg / mL;

[0015] In the JE virus and porcine parvovirus VP2 protein bivalent vaccine, the volume ratio of the aqueous phase to the oil phase is preferably (1:1) - (1:1.5);

[0016] The JE virus is preferably the GI type JEV GD strain;

[0017] The inactivated JE virus is prepared by the following method:

[0018] Inoculate the JE virus strain into Vero cells, harvest the virus solution after culturing for 48 - 96 h; add formaldehyde with a final volume percentage of 0.1 - 0.3%, and inactivate at 37°C for 24 - 48 h to obtain the inactivated JE virus;

[0019] The M.O.I. of the JE virus strain is preferably 0.1;

[0020] The culture time is preferably 60 h;

[0021] The final concentration of formaldehyde is preferably 0.1%;

[0022] The inactivation time is preferably 36 h;

[0023] The preparation method of the PPV-VP2 recombinant protein includes the following steps:

[0024] (1) Gene synthesis of the nucleotide sequence encoding the PPV-VP2 recombinant protein, introduce restriction enzyme sites at both ends, and connect the nucleotide sequence encoding the PPV-VP2 recombinant protein to the transfer plasmid through the restriction enzyme sites to obtain a recombinant transfer plasmid;

[0025] (2) Transform the recombinant transfer plasmid prepared in step (1) into DH10Multibac competent cells to obtain the recombinant baculovirus plasmid rBac-VP2;

[0026] (3) Transfect the recombinant baculovirus plasmid rBac-VP2 prepared in step (2) into sf9 cells to obtain the Ac-VP2 recombinant baculovirus;

[0027] (4) Transfect the Ac-VP2 recombinant baculovirus prepared in step (3) into High Five cells and culture them. Collect the cell culture suspension, lyse the cells by sonication, and centrifuge to collect the supernatant. Purify the supernatant by sucrose density gradient centrifugation to obtain the PPV-VP2 recombinant protein;

[0028] The transfer plasmid described in step (1) is preferably pFastBac TM Daul vector;

[0029] The M.O.I. of the transfection described in step (4) is preferably 1 - 10;

[0030] The culture time described in step (4) is preferably 48 - 120 h;

[0031] The conditions of the sucrose density gradient centrifugation described in step (4) are preferably: the sucrose concentration (mass percentage) gradient is successively 70%, 50%, 30%, and centrifuge at 30000 r / min at 4°C for 6 h;

[0032] The adjuvant is preferably ISA201 adjuvant;

[0033] The preparation method of the Japanese encephalitis virus of pigs and porcine parvovirus VP2 protein bivalent vaccine comprises the following steps:

[0034] Mix the inactivated Japanese encephalitis virus of pigs and the PPV-VP2 recombinant protein in equal volume to obtain an aqueous phase; mix the aqueous phase and the adjuvant in equal volume and emulsify to obtain the Japanese encephalitis virus of pigs and porcine parvovirus VP2 protein bivalent vaccine;

[0035] The application of the Japanese encephalitis virus of pigs and porcine parvovirus VP2 protein bivalent vaccine in the preparation of a product for preventing and treating at least one of the Japanese encephalitis virus of pigs and porcine parvovirus;

[0036] The present invention has the following advantages and effects compared with the prior art:

[0037] (2) The JEV strain used in the present invention is of genotype GI, which is the current prevalent strain of JEV and has a better protective effect; at the same time, the cultured virus is inactivated before use, which has the advantage of high safety compared with the attenuated JEV vaccine.

[0038] (2) The present invention selects a baculovirus expression system with high safety, simple operation, and good immunogenicity of recombinant protein to express the VP2 protein of PPV. The expressed VP2 protein can self-assemble into virus-like particles (VLP).

[0039] (3) At present, there is no combined vaccine for JEV and PPV on the market. The present invention combines inactivated JEV whole virus with VLP assembled from PPV VP2 protein expressed by baculovirus expression system to prepare a combined vaccine for Japanese encephalitis virus of swine and porcine parvovirus VP2 protein (JEV-PPV VP2). This vaccine can induce good cellular immunity and humoral immunity in the body, protecting the body from the invasion of PPV and JEV. Description of the Drawings

[0040] Figure 1 It is a growth curve result diagram of JEV GD strain. Among them, a: the morphology of vero cells inoculated with JEV GD strain; b: TCID of JEV GD strain harvested at different time points 50 / mL.

[0041] Figure 2 It is a PCR identification result diagram of recombinant baculovirus plasmid rBac-VP2. Among them, M: 10000 DNA Marker, 1: baculovirus blank group, 2, 3: recombinant baculovirus plasmid rBac-VP2.

[0042] Figure 3 It is a microscopic observation diagram after transfection of recombinant baculovirus plasmid into sf9 insect cells. Among them, a: sf9 cells infected with recombinant baculovirus, b: sf9 cell blank control group.

[0043] Figure 4 It is a Western blot identification result diagram of P3 generation Ac-VP2 recombinant baculovirus. Among them, 1, 2, 3: High-Five cell samples after infection with Ac-VP2 recombinant baculovirus, 4: negative control (wild baculovirus infecting sf9 cells).

[0044] Figure 5 It is an indirect immunofluorescence detection result diagram of P3 generation Ac-VP2 recombinant baculovirus. Among them, a: negative control group (wild baculovirus infecting sf9 cells), b: Ac-VP2 recombinant baculovirus.

[0045] Figure 6 It is a Western blot result diagram of recombinant protein expression at different culture times after P3 generation recombinant baculovirus Ac-VP2 was inoculated into High-Five cells at different M.O.I.. Among them, 1, 4, 7, 10: cell samples infected with M.O.I. = 1, 2, 5, 8, 11: cell samples infected with M.O.I. = 5, 3, 6, 9, 12: cell samples infected with M.O.I. = 10.

[0046] Figure 7 It is a transmission electron microscopy result diagram of PPV-VP2 recombinant protein after sucrose density gradient purification.

[0047] Figure 8 It is a graph showing the results of the JEV-specific antibody levels in mouse serum. Among them, ns, P > 0.05; *, P < 0.05, **, P < 0.01; ****, P < 0.0001.

[0048] Figure 9 It is a graph showing the detection results of JEV neutralizing antibodies in mouse serum. Among them, the P value was calculated by two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0049] Figure 10 It is a graph showing the detection results of the secretion levels of cytokines IL-2 and IL-4 in mouse serum. Among them, the P value was calculated by two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0050] Figure 11 It is a graph showing the results of the mouse lymphocyte proliferation experiment. Among them, the P value was calculated by two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0051] Figure 12 It is a graph showing the detection results of JEV virus loads in the brain tissue and blood of mice after virus challenge.

[0052] Figure 13 It is a graph showing the survival rate results of mice after JEV virus challenge.

[0053] Figure 14 It is a graph showing the detection results of PPV-specific antibody levels in guinea pig serum. Among them, the P value was calculated by two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0054] Figure 15 It is a graph showing the detection results of PPV neutralizing antibodies in guinea pig serum. Among them, the P value was calculated by two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0055] Figure 16 It is a graph showing the detection results of the hemagglutination inhibition (HI) antibody titers of PPV in guinea pig serum. Among them, the P value was calculated by two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0056] Figure 17 It is a graph showing the detection results of the secretion levels of cytokines IL-2 and IL-4 in guinea pig serum.

[0057] Figure 18 It is a graph of the results of the guinea pig lymphocyte proliferation experiment. Among them, the P value was calculated using two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Specific implementation manners

[0058] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0059] The sf9 cells and High Five cells were purchased from Beijing Sino Biological Inc.; the Vero cells and the JEV GD strain (CH / GD2018 / 2018 strain) (Sun Y, Ding H, Zhao F, et al. Genomic Characteristics and E Protein Bioinformatics Analysis of JEV Isolates from South China from 2011 to 2018. Vaccines (Basel). 2022; 10(8): 1303) were preserved in the Laboratory of Veterinary Microbiology and Immunology, South China Agricultural University; the inactivated JEV vaccine (SA14-14-2 strain) and the inactivated PPV vaccine (WH-1 strain) were purchased from Wuhan Keqian Biologic Co., Ltd.; the experimental mice and guinea pigs were purchased from the Experimental Animal Center of Southern Medical University; the ISA 201VG adjuvant was purchased from Seppic, France.

[0060] Example 1 Cultivation and condition optimization of JEV GD strain

[0061] I. Test method

[0062] (1) The isolated GI type JEV GD strain was cultured and proliferated on Vero cells. The specific method was as follows: The Vero cells (Guangdong Yongshun Biopharmaceutical Co., Ltd.) grown to 80% density were discarded the culture medium, washed 3 times with PBS, and then inoculated with the virus solution according to M.O.I = 0.1. After incubating in a 37°C cell culture incubator for 1 h, the liquid was discarded, washed 3 times with PBS, and then added with DMEM maintenance medium containing 2% (volume fraction) FBS, and the cytopathic effect was observed.

[0063] (2) Explore the optimal virus culture conditions: Inoculate the JEV GD strain at an M.O.I. of 0.1 into Vero cells grown to 80% density, and harvest the virus solution at 12h, 24h, 36h, 48h, 60h, 72h, 84h, and 96h after virus inoculation for the determination of virus TCID 50 to obtain the growth curve of the JEV GD strain.

[0064] (3) Explore the optimal virus inactivation conditions: Obtain the virus solution according to the optimal virus culture conditions in step (2), and prepare JEV GD strain virus solutions with final formaldehyde concentrations (volume percentages) of 0.05%, 0.1%, 0.2%, and 0.3%. Take 5 mL of each and place them in an incubator at 37°C for inactivation for 12h, 24h, 36h, and 48h. At the same time, set up a negative control group. After the inactivated virus solution is blindly passaged three times on cells, observe the cytopathic effect under a microscope. If no cytopathic effect appears in the cells, it proves that the virus has been inactivated.

[0065] Table 1 Optimization of the inactivation conditions of the JEV GD strain

[0066]

[0067]

[0068] Note: + Cytopathic effect appears in the cells, - No cytopathic effect appears in the cells.

[0069] II. Test results

[0070] Under the microscope, it was observed that the volume of Vero cells inoculated with the JEV GD strain became larger, showed filamentation, rupture, and detachment and death ( Figure 1 a). The titer of the virus solution harvested 60h after inoculation of the JEV GD strain at an M.O.I. of 0.1 was the highest, and the virus titer was 10 8 TCID50 / mL( Figure 1 b). The optimal formaldehyde inactivation concentration for the JEV GD strain was 0.1%, and the inactivation time was 36h.

[0071] Example 2

[0072] I. Test method

[0073] 1. Construction of the recombinant pFBD-VP2 transfer vector

[0074] Based on the complete gene sequence of porcine PPV NADL-2 strain (KF913351.1) deposited in GenBank, a His tag was added before the stop codon of its VP2 gene sequence. The gene sequence of the VP2 recombinant protein was codon-optimized for insect cells (the specific sequence is shown below), and BamH I and Hind III restriction enzyme sites were introduced at both ends, and then it was sent to Suzhou Hongxun Biotechnology Co., Ltd. for gene synthesis, and was ligated into pFastBac TM Dual vector (Thermo Fisher, USA) by double digestion and ligation. The constructed recombinant transfer plasmid was named pFBD-VP2.

[0075] Nucleotide sequence of the gene encoding the VP2 recombinant protein:

[0076]

[0077] Amino acid sequence of VP2 recombinant protein:

[0078] MSENVEQHNPINAGTELSATGNESGGGGGGGGGRGAGGVGVSTGTFNNQTEFQYLGEGLVRITAHASRLIHLNMPEHETYKRIHVLNSESGVAGQMVQDDAHTQMVTPWSLIDANAWGVWFNPADWQLISNNMTEINLVSFEQEIFNVVLKTITESATSPPTKIYNNDLTASLMVALDTNNTLPYTPAAPRSETLGFYPWLPTKPTQYRYYLSCIRNLNPPTYTGQSQQITDSIQTGLHSDIMFYTIENAVPIHLLRTGDEFSTGIYHFDTKPLKLTHSWQTNRSLGLPPKLLTEPTTEGDQHPGTLPAANTRKGYHQTINNSYTEATAIRPAQVGYNTPYMNFEYSNGGPFLTPIVPTADTQYNDDEPNGAIRFTMDYQHGHLTTSSQELERYTFNPQSKCGRAPKQQFNQQAPLNLENTNNGTLLPSDPIGGKSNMHFMNTLNTYGPLTALNNTAPVFPNGQIWDKELDTDLKPRLHVTAPFVCKNNPPGQLFVKIAPNLTDDFNADSPQQPRIITYSNFWWKGTLTFTAKMRSSNMWNPIQQHTTTAENIGNYIPTNIGGIRMFPEYSQLIPRKLYHHHHHH

[0079] 2. Generation and identification of recombinant Bacmid

[0080] (1) Transform the recombinant transfer vector pFBD-VP2 into DH10Multibac competent cells. The specific steps are as follows:

[0081] ① Take out 100 μL of DH10Multibac competent cells and 5 μL of the recombinant transfer vector pFBD-VP2 in a laminar flow hood, gently mix them in an EP tube, and insert it on ice for 30 min; after ice bath, take it out and heat shock it in a 42 °C metal bath for 60 s, and finally quickly place it on ice for 3 min;

[0082] ② Add 850 μL of SOC medium to the EP tube and culture it in a shaker at 220 r / min for 6 h;

[0083] ③ After culturing, the bacterial liquid is diluted by a factor of 10 -1~10 -3 Gradient dilution. Pipette 150 μL and slowly spread it on the LB solid medium containing Amp, Kan, Gen, IPTG and X-Gal. Invert it and place it in a bacterial incubator at 37 °C for 48 h, and observe the blue and white colonies on the medium.

[0084] (2) Pick white colonies and inoculate them into the LB liquid medium containing resistance. Culture them in a shaker at 37 °C and 220 r / min for 32 h. Use the Plasmid Mini Kit I (D6943-02) of Omega Bio-Tek, Inc. in the United States to extract the baculovirus plasmid according to the following steps:

[0085] ① Add the bacterial liquid into a 2 mL EP tube, centrifuge at 14000 r / min at room temperature for 1 min, and discard the supernatant until all the bacterial liquid is centrifuged.

[0086] ② Add 250 μL of Solution I to the precipitate, resuspend it, and place it at room temperature for 8 min to fully lyse the RNA.

[0087] ③ Add 250 μL of Solution II to the tube, slowly resuspend it, and after placing it at room temperature for 5 min, the suspension becomes transparent.

[0088] ④ Add 350 μL of Solution III to the tube, shake the EP tube while adding, and after adding, invert the EP tube several times. After placing it at room temperature for 12 min, white flocculent precipitates can be observed.

[0089] ⑤ Centrifuge the EP tube at 4 °C and 14000 r / min for 15 min. Transfer the supernatant to an EP tube containing 500 μL of pre-cooled isopropanol, mix well, and place it in an ice bath for 10 min.

[0090] ⑥ After centrifuging the EP tube at 12000 r / min for 20 min, discard the supernatant. Add 500 μL of ethanol solution with a volume fraction of 70% to the tube, resuspend the precipitate, centrifuge at 12000 r / min for 20 min, and discard the supernatant.

[0091] ⑦ Open the lid of the EP tube and let it stand at room temperature until the precipitate becomes transparent. Add 30 μL of Elution Buffer to dissolve it to obtain the recombinant baculovirus plasmid. Measure the concentration of the recombinant baculovirus plasmid with a UV spectrophotometer and store it at -20 °C.

[0092] (3) Since the nucleotide sequence of the recombinant baculovirus plasmid is relatively long, it is difficult to identify whether the foreign gene fragment is successfully inserted by double digestion. The foreign gene is transposed to the Tn7 transposition site of Bacmid through Tn7R / L. Therefore, the universal primers M13-F and M13-R are used to identify the recombinant baculovirus plasmid. The nucleic acid sequences of M13-F and M13-R are as follows:

[0093] M13-F: 5'-CCCAGTCACGACGACGTTGTAAAACG-3';

[0094] M13-R: 5'-AGCGGATAACAATTTCACACAGG-3'.

[0095] II. Test Results

[0096] The recombinant baculovirus plasmid was amplified by PCR using M13-F / R to verify whether the target gene was successfully inserted into the baculovirus plasmid. The Bacmid PCR amplification product without the inserted target gene was the mini-attTn7 element, with a size of 296 bp, while the amplification product of the recombinant baculovirus plasmid was the Tn7R, Tn7L elements and the target gene, with a size of target gene + 2560 + 296 bp, that is, the PCR amplification product size of the recombinant baculovirus plasmid rBac-VP2 was 4696 bp. The electrophoresis results Figure 2 showed that the specific band was around 4500 bp, which was consistent with the expected amplification product size, indicating that the recombinant baculovirus plasmid was successfully constructed.

[0097] Example 3

[0098] I. Test Method

[0099] 1. Packaging of Ac-VP2 Recombinant Baculovirus

[0100] The steps of mediating the transfection of sf9 cells with the recombinant baculovirus plasmid prepared in Example 2 using the cationic liposome cellfectin II method are as follows:

[0101] (1) Prepare a six-well cell culture plate. First, add sf9 cells at a density of 8×10 5 cells / well, and then add 2 mL of Grace's incomplete medium to each well. Incubate in a 27°C incubator for 30 min to allow the cells to adhere fully.

[0102] (2) Take an EP tube, add 100 μL of Grace's Insect Medium (Thermo Fisher, USA) and 2 μg of the recombinant baculovirus plasmid prepared in Example 2 and mix well; take another EP tube, add 100 μL of Grace's Insect Medium and 8 μL of Cellfectin II Reagent transfection reagent (Thermo Fisher, USA) and mix well. Incubate both at room temperature for 15 min.

[0103] (3) Mix the liquids in the two EP tubes in (2) and incubate at room temperature for 30 min.

[0104] (4) Add the transfection mixture drop by drop into the corresponding cell wells and incubate in an incubator at 27°C for 5 h.

[0105] (5) Aspirate the supernatant in the cell wells, supplement with 2 mL of SIM SF medium, and statically culture in an incubator at 27°C.

[0106] (6) Compared with the control group cells without recombinant baculovirus plasmid, the experimental group cells generally showed phenomena such as increased cell nucleus and cell diameter after transfection for 72 h, and even stopped growing, detached, and floated. Collect the supernatant of the experimental group and add it to an EP tube. After centrifugation at 1000 r / min for 6 min, the supernatant is the P1 generation recombinant baculovirus, named Ac-VP2 recombinant baculovirus. Add FBS to a final concentration of 2% (volume percentage) and store at -80°C for later use.

[0107] 2. Amplification culture of Ac-VP2 recombinant baculovirus

[0108] The virus titer of the P1 generation recombinant baculovirus is relatively low (about 1×10 7 pfu / mL). A large amount of high-titer recombinant baculovirus can be subcultured using suspension-cultured sf9 cells. The specific steps are as follows:

[0109] (1) Add 10 mL of sf9 cells with a density of 1×10 6 cells / mL to a sterile 250 mL conical flask.

[0110] (2) According to the following formula, add the corresponding amount of P1 generation recombinant baculovirus to the conical flask with M.O.I. = 0.1, and place it on a shaker at 27°C and 140 r / min for 96 h.

[0111]

[0112] (3) Add 50 mL of sf9 cells with a density of 1×10 6 cells / mL to a sterile 500 mL conical flask. Add the supernatant after centrifugation of the cell solution in (2) to it with M.O.I. = 0.1, which is the P2 generation Ac-VP2 recombinant baculovirus.

[0113] (4) Place it on a shaker at 27°C and 140 r / min for 96 h. The supernatant after centrifugation of the cell solution is the P3 generation Ac-VP2 recombinant baculovirus.

[0114] 3. Titer determination of recombinant baculovirus Ac-VP2

[0115] (1) Construction of standard plasmid pMD18-T-Ac

[0116] ①Using baculovirus DNA as a template, primers AcMK107-F and AcMK107-R for amplifying a partial sequence (AcMK107) of baculovirus DNA were designed using Snapgene software; using Beacon Designer software, with the fragment amplified by the above primers as a template, a pair of detection primers QF and QR for SYBR Green I fluorescence quantification were designed within the fragment, and the primer sequences are shown in Table 2.

[0117] Table 2 Primer sequences

[0118]

[0119] ②Using wild-type baculovirus (AcMNPV) DNA as a template, AcMK107-F and AcMK107-R as primers, the target gene was amplified according to the reaction system shown in Table 3. After agarose gel electrophoresis with a mass fraction of 1%, the target fragment was recovered and purified and ligated to the pMD18-T vector by a conventional method. The ligation product was transformed into DH5α Escherichia coli competent cells, and the colonies were identified by PCR using vector universal primers. Then, the identified positive colonies were inoculated into LB liquid medium with Amp resistance for culture; the recombinant plasmid was extracted and identified by PCR using M13 universal primers. The plasmid with correct identification was sequenced and named pMD18-T-Ac, and stored at -20 °C.

[0120] Table 3 PCR reaction program

[0121]

[0122] (2) Measure the concentration of the extracted pMD18-T-Ac standard quality plasmid using a micro ultraviolet spectrophotometer, and calculate the standard plasmid DNA copy number according to the following formula.

[0123]

[0124] (3) Dilute the above standard quality plasmid concentration to 10 8 、10 7 、10 6 、10 5 、10 4Using the plasmid copies / μL as a template, three replicates were performed for each concentration. A standard curve was constructed based on the copy number and Cq value of the standard plasmid. The DNA of the P3 generation of Ac-VP2 recombinant baculovirus obtained in Step 2 was extracted according to the operation instructions of the OMEGA Viral DNA Kit (Omega Bio-Tek, USA). Using this DNA as a template, three replicates were performed for each concentration, and a fluorescence quantitative PCR reaction was carried out using primers QF (5’-AGATGCGACACAGATGGA3’) and QR (5’-TCAACAAGAATGGACCGAAT-3’). Additionally, Elution Buffer was used as a negative control. Among them, the fluorescence quantitative PCR reaction system was as follows: SYBR qPCR SuperMix Plus 10.0 μL, QF 0.4 μL, QR 0.4 μL, standard plasmid / viral DNA 2.0 μL, ddH2O 7.2 μL; The reaction conditions were: 95 °C for 1 min; 95 °C for 10 s, 60 °C for 15 s, for a total of 35 cycles; Fluorescence signals were collected at 60 °C.

[0125] (4) After substituting the Cq values of each sample well into the standard curve, the copy number of the recombinant baculovirus DNA was calculated, and the virus titer was calculated according to the following formula:

[0126]

[0127] II. Test Results

[0128] The recombinant baculovirus plasmid was transfected into sf9 insect cells in the logarithmic growth phase. After 72 h of transfection, it could be observed under an inverted microscope that the cell volume increased significantly, the cell nucleus swelled, the cell morphology was irregular, the refractive index decreased, some cells floated, and in severe cases, cell rupture, detachment, and death occurred; while normal cells adhered well, the cell shape was round, the cell body was translucent, the outline was clear, and the cell dish was completely confluent ( Figure 3 ).

[0129] The recombinant baculovirus was amplified continuously for two generations to obtain 50 mL of the P3 generation virus. Using the constructed standard plasmid pMD18-T-Ac, the titer of the recombinant baculovirus was determined by absolute fluorescence quantitative PCR. The results showed that the copy number of the recombinant baculovirus Ac-VP2 was 1.5×10 8 copies / μL, and the calculated virus titer was 1.2×10 9 pfu / mL.

[0130] Example 4

[0131] I. Test Methods

[0132] 1. Identification of recombinant protein PPV-VP2

[0133] (1) Identification of recombinant protein by Western Blot

[0134] ① Extraction and denaturation of recombinant protein: Take 200 μL of the P3 cell solution cultured for 96 h in step 2(4) of Example 3 and place it in a 1.5 mL EP tube. Add 50 μL of 5×loading buffer and mix well. Boil it in a metal bath at 100 °C for 10 min. Store the sample at -20 °C;

[0135] ② Gel preparation: Prepare a 12.5% (volume percentage) SDS-PAGE gel according to the instructions of the PAGE gel rapid preparation kit of Yaenzyme Biotechnology Co., Ltd.;

[0136] ③ SDS-PAGE gel electrophoresis: After assembling the SDS-PAGE gel, place it in the electrophoresis tank, pour in 1×SDS-PAGE electrophoresis buffer, and add the denatured recombinant protein to the wells of the gel, 10 μL per well; Assemble the electrophoresis tank and transfer it. Run at a voltage of 80 V for 30 min and 120 V for 60 min;

[0137] ④ Blotting: Prepare 1×SDS-PAGE electrotransfer buffer in advance and pre-cool it on ice; Immerse the PVDF membrane in methanol for 1 min and then transfer it to the pre-cooled electrotransfer buffer; After electrophoresis, take out the gel and place it in order from the negative electrode to the positive electrode: sponge - 3 layers of filter paper - SDS-PAGE gel - PVDF membrane - 3 layers of filter paper - sponge. Pay attention not to generate bubbles during this process; Place the fixed blotting device in the electrotransfer tank, pour in the electrotransfer solution, and transfer the membrane at a constant current of 200 mA (determine the transfer time according to the protein size, 1 KDa / min);

[0138] ⑤ Blocking: After washing the taken-out PVDF membrane three times with PBST, place it in 5% (prepared by dissolving 5 g of skim milk powder in water to a final volume of 100 ml) skim milk powder and block it at room temperature at 50 r / min for 1 h;

[0139] ⑥ Primary antibody incubation: Wash the blocked PVDF membrane three times with PBST for 10 min each time. Use the His-tag antibody (mouse source) (Beijing Sino Biological Inc.) diluted at a ratio of 1:1000 as the primary antibody to incubate the recombinant protein;

[0140] ⑦ Secondary antibody incubation: Discard the primary antibody, wash it three times with PBST for 10 min each time. Use the goat anti-mouse IgG-HRP antibody (1:1000 dilution) (Shanghai Beyotime Biotechnology Co., Ltd.) as the secondary antibody and incubate it in an incubator at 37 °C for 1 h;

[0141] ⑧ Color development: Discard the secondary antibody, wash three times with PBST for 10 minutes each time; mix solution A and solution B of the ECL chemiluminescent solution (Shanghai Yaen Biotechnology Co., Ltd.) at a volume ratio of 1:1, immerse the PVDF membrane in the chemiluminescent solution, and detect by exposure in the Fine-do X6 chemiluminescent imaging system.

[0142] (2) Indirect immunofluorescence detection of recombinant protein: Infect healthy sf9 cells with the P3 generation of Ac-VP2 recombinant baculovirus at a dose of M.O.I. = 1. After culturing in a constant temperature incubator at 27 °C for 72 h, perform indirect immunofluorescence detection according to the conventional method, using the His monoclonal antibody (Beijing Sino Biological Inc.) as the primary antibody and FITC-labeled goat anti-mouse IgG (Shanghai Beyotime Biotechnology Co., Ltd.) as the secondary antibody. Observe under a fluorescence microscope, and set sf9 cells not infected with the recombinant baculovirus as the negative control group.

[0143] II. Test results

[0144] The P3 generation of cell samples after infection with the recombinant virus was identified by Western blot to detect the reactivity of the VP2 recombinant protein to specific antibodies, and Western blot identification was performed using the murine His-tag antibody as the primary antibody. The results showed that a specific band appeared at approximately 65 kDa in the cell samples infected with the Ac-VP2 recombinant virus, and no specific band was observed in the protein samples of sf9 cells infected with the wild baculovirus, proving that the recombinant virus was successfully constructed and could correctly express the target protein (see Figure 4 ).

[0145] Collect the P3 generation of Ac-VP2 recombinant baculovirus, infect adherent sf9 cells in the logarithmic growth phase at a dose of M.O.I. = 1, set the non-infected recombinant baculovirus as the negative control group, place it in a constant temperature incubator at 27 °C for 72 h, and perform indirect immunofluorescence detection using the murine His-tag monoclonal antibody as the primary antibody. The results are as Figure 5 shown. Bright green fluorescence appeared in the sf9 cells infected with Ac-VP2, while specific fluorescence was not detected in the sf9 cells of the negative control group.

[0146] Example 5

[0147] I. Test methods

[0148] 1. Optimization of recombinant protein expression conditions

[0149] In order to improve the expression efficiency of the recombinant protein, the present invention uses High Five suspension cells with high protein expression to express the PPV-VP2 protein. Explore the optimal expression conditions of the recombinant target protein from two aspects: different harvest times and different virus inoculation doses. The specific operation method is as follows:

[0150] (1) Adjust the suspension of High Five cells with a concentration of 1×10 6 cells / mL, add High Five-SFM medium, and culture in a shaker at 27°C and 140 r / min.

[0151] (2) When the cell concentration is 2×10 6 cells / mL, inoculate the Ac-VP2 recombinant baculovirus into the cells at an M.O.I. of 1.0, 5.0, and 10.0 respectively. Collect the cell culture suspension at 48 h, 72 h, 96 h, and 120 h after virus inoculation.

[0152] (3) After collecting 200 μL of cell samples from each group, add 50 μL of 5×loading buffer and mix well, then boil in a metal bath at 100°C for 10 min. Detect the expression level of the recombinant protein in the protein samples by Western Blot, and analyze the gray value through ImageJ software to determine the change trend of protein expression.

[0153] 2. Electron microscopy observation of recombinant virus-like particles

[0154] According to the optimal conditions explored above, ultrasonically lyse the harvested High Five cells, centrifuge to harvest the supernatant, and purify it by centrifugation at 30,000 r / min for 6 h at 4°C with a sucrose concentration gradient of 70%, 50%, and 30%. Add 10 μL of the purified sample to a carbon-coated grid, negatively stain the sample with 2% (mass fraction) phosphotungstic acid (purchased commercially) for 1 min, then dry the grid for 6 h, and observe the VLP assembly under a TEM transmission electron microscope.

[0155] II. Test results

[0156] Inoculate the P3 generation recombinant baculovirus Ac-VP2 into High-Five cells at an M.O.I. of 1.0, 5.0, and 10.0, culture in a constant temperature shaker at 27°C and 140 r / min, collect cell samples at 48 h, 72 h, 96 h, and 120 h after inoculation respectively, detect the expression of the recombinant protein under different conditions by Western blot, and draw a gray value-time line graph. The results are as Figure 6 shown. The expression level of the recombinant protein changes significantly with time. Considering the time cost and production cost in the actual production process, use an M.O.I. of 5.0 as the infection dose, and harvest the PPV-VP2 recombinant protein sample 96 h after infection.

[0157] It can be found by transmission electron microscopy observation of the PPV-VP2 recombinant protein purified by sucrose gradient that the recombinant protein self-assembled into polygonal hollow particles with a diameter of about 25 nm, indicating that the recombinant protein can self-assemble into virus-like particles in vitro ( Figure 7)。

[0158] Example 6

[0159] I. Test method

[0160] To evaluate the immunogenicity of the vaccine, the immune effect of the vaccine was evaluated on mice and guinea pigs in this example.

[0161] 1. Vaccine preparation

[0162] (1) The inactivated JEV GD strain virus solution in Example 1 was diluted and emulsified with ISA 201 adjuvant according to the volume ratio of aqueous phase to oil phase = 1:1 to obtain the inactivated single JEV vaccine, where the JEV virus content was 10 6.0 TCID 50 / mL;

[0163] (2) The concentration of the PPV-VP2 recombinant protein prepared in Example 5 was adjusted to 100 μg / mL and mixed with the inactivated JEV GD strain virus solution in equal volume, and then emulsified with ISA 201VG adjuvant according to the volume ratio of aqueous phase to oil phase = 1:1 to obtain the JEV-PPV VP2 combined vaccine, where the JEV virus content was 10 6.0 TCID 50 / mL and the PPV-VP2 recombinant protein content was 50 μg / mL.

[0164] 2. Immune and challenge protection tests on mice

[0165] Healthy 4-week-old Kunming mice were randomly divided into 5 groups. After one week of adaptive feeding, the mice were immunized by subcutaneous injection. The booster immunization was carried out on the 14th day after the first immunization. The specific immune grouping is shown in Table 4 below.

[0166] Table 4 Immune grouping of mice

[0167]

[0168] (1) The JEV-specific antibody levels in the sera of each vaccine group on the 14th, 21st, 28th, and 35th days were measured using the Porcine Japanese Encephalitis Virus Antibody ELISA Kit (EY-elisa-0203) from Shanghai Yiyan Biological Co., Ltd.

[0169] (2) The JEV neutralizing antibody titers of the sera of each vaccine group on the 14th, 21st, 28th, and 35th days were measured using the fixed virus-diluted serum method. The specific operation is as follows:

[0170] ① Vero cells were cultured in a 96-well cell culture dish until the cell density reached 80 - 100%;

[0171] ②Collect the sera of each vaccine group and inactivate them in a 56 °C water bath for 30 min;

[0172] ③Dilute the JEV GD strain virus solution to approximately contain 10 2.5 TCID 50 in every 150 μL, and then adjust the final dilution of the serum to 2 1 ~2 10 in a 2-fold serial dilution manner with the above inactivated serum. Place the mixture in a CO2 incubator and incubate for 1 h;

[0173] ④Discard the liquid in the cell plate wells, and respectively pipette 50 μL of the serum-virus mixture into a 96-well plate. Make 4 replicates for each serum dilution, and statically culture for 5 d to observe the results.

[0174] (3) Use the mouse IL-2 ELISA kit (MM-0701M1) and IL-4 ELISA kit (MM-0165M2) from Jiangsu Enzyme Immunoassay Co., Ltd. to measure the cytokine levels in the sera on day 35.

[0175] (4) On day 42, sacrifice 3 mice in each group, take the spleens for isolating splenocytes, and measure the lymphocyte proliferation response. The specific method is as follows:

[0176] ①Sacrifice the mice / guinea pigs by cervical dislocation and isolate the spleens;

[0177] ②Put the mouse / guinea pig spleen into a disposable bacteriological culture dish, repeatedly wash the spleen with a 2 mL PBS syringe, and use a 200-mesh cell sieve to collect the liquid;

[0178] ③Add 5 mL of red blood cell lysate to the liquid in step ②, pipette and mix well; let it stand for 5 min to fully lyse the red blood cells, then centrifuge at 2000×g at room temperature for 10 min, and discard the red blood cells in the supernatant;

[0179] ④Repeat step ③, add 5 mL of PBS to the centrifuge tube to wash the cells, centrifuge at 2000×g for 10 min, discard the supernatant, collect the precipitate, which is the splenocytes. Resuspend the splenocytes with 1640 complete medium containing 10% FBS, adjust the cell density to 5×10 6 cell / mL, and add the lymphocytes to a 96-well cell culture plate, 100 μL per well;

[0180] ⑤For the splenocytes of each mouse, set up a positive control (treated with ConA, using 100 μL of concanavalin A with a final concentration of 10 μg / mL as the stimulator), a negative control (treated with 100 μL of 1640 complete medium), and a virus stimulation group (100 μL of 1×10 6 TCID 50The JEV GD strain virus solution at [specific concentration] / mL was used as the stimulant, and three replicates were made for each well.

[0181] ⑥ The above-treated 96-well plate was placed in an incubator at 37 °C with 5% CO2 for 48 h. Then, 10 μL of CCK-8 solution was added to each well, and the plate was further incubated in the incubator at 37 °C with 5% CO2 for 2 h. The OD value of the cell culture suspension was measured. 450 The stimulation index (SI, the ratio of the lymphocyte proliferation response in the experimental group to that in the negative control group, which reflects the response degree of lymphocytes to a specific stimulant) was calculated according to the following formula:

[0182] Stimulation index SI = OD 450 Mean value (virus stimulation group or positive control group) / OD 450 Mean value (negative control group)

[0183] (5) The remaining mice 42 days after immunization were each intracranially challenged with the JEV GD strain (10 8 TCID 50 / mL) 50 μL. After observing for 14 days, they were sacrificed. The heart, liver, spleen, lung, kidney, brain, and blood were collected. After extracting RNA and reverse-transcribing it into cDNA, a fluorescence quantitative PCR reaction was performed using the primers JEV-NS1-F (5’-CAGCCTCACAAAACGGCAAG-3’) and JEV-NS1-R (5’-GTACATGGCAGTGATAGCCAG-3’). Among them, the fluorescence quantitative PCR reaction system was as follows: SYBR qPCR SuperMix Plus 10.0 μL, primer JEV-NS1-F 0.4 μL, primer JEV-NS1-R 0.4 μL, viral DNA 2.0 μL, ddH2O 7.2 μL; The reaction conditions were: 95 °C for 1 min; 95 °C for 10 s, 60 °C for 15 s, for a total of 35 cycles; Fluorescence signals were collected at 60 °C to detect the JEV virus load in each organ.

[0184] 3. Immunization test of guinea pigs

[0185] Healthy guinea pigs weighing about 400 g were randomly divided into 4 groups: PPV commercial vaccine, JEV-PPV VP2 combined vaccine, PBS + ISA201 adjuvant, and PBS group. After one week of adaptive feeding, the guinea pigs were immunized by subcutaneous injection. Booster immunization was carried out on the 14th day after the first immunization. The specific immunization grouping is shown in Table 5 below.

[0186] Table 5 Guinea pig immunization grouping

[0187]

[0188] (1) The specific antibody levels of PPV in the peripheral sera of guinea pigs on the 14th, 21st, 28th, and 35th days after the first immunization were determined using the Porcine Parvovirus Antibody Detection Kit (E310601) from Shenzhen Finder Biotechnology Co., Ltd.

[0189] (2) Referring to the above-mentioned immunization and challenge protection test of mice, the neutralizing antibody titers of PPV in the sera of each vaccine group on the 14th, 21st, 28th, and 35th days were determined by the fixed virus - diluted serum method.

[0190] (3) Hemagglutination inhibition test of the peripheral sera of guinea pigs on the 14th, 21st, 28th, and 35th days after the first immunization: The sera of each vaccine group in the guinea pig immunization experiment on the 14th, 21st, 28th, and 35th days were collected, and the hemagglutination inhibition antibody titers were determined by the fixed virus - diluted serum method on a 96 - well V - shaped microplate. The specific method is as follows:

[0191] ① First, add 25 μL of PBS to wells 1 - 10, add 4 units of the virus solution of PPV GD strain (Sun Xiu. Genome sequence analysis of porcine parvovirus and preparation and immunological evaluation of inactivated vaccine [D]. South China Agricultural University [2025 - 04 - 01]. DOI: CNKI: CDMD: 2.1016.923203.) to well 11, and add PPV - positive antiviral serum (According to the conventional method, the serum collected after immunizing guinea pigs with the PPV commercial vaccine WH - 1 strain and detected as positive by the antibody detection kit, HI titer 1:256) to well 12;

[0192] ② Add 25 μL of the serum to be tested to well 1, mix well and then pipette 25 μL and add it to well 2, and repeat the operation successively until well 10;

[0193] ③ After adding the serum to be tested, add 25 μL of 4 - unit virus solution of PPV GD strain (the same as step ① above) to each well, shake gently and evenly, and then incubate in a 37°C incubator for 30 min;

[0194] ④ After taking out from the incubator, add 25 μL of 0.6% guinea pig red blood cell suspension (from guinea pigs without any treatment, prepared by collecting blood according to the conventional method) to each well, shake gently and evenly, and then incubate in a 37°C incubator for 30 min;

[0195] ⑤ Determine the result: The highest dilution multiple of the serum that can inhibit 100% red blood cell agglutination is the hemagglutination inhibition antibody titer of the serum to be tested.

[0196] (4) The cytokine levels in the sera on the 35th day were determined using the IL - 2 ELISA Kit (MM - 0894O2) and IL - 4 ELISA Kit (MM - 0332O2) for guinea pigs from Jiangsu Enzyme Immunoassay Industry Co., Ltd.

[0197] (5) At 42 d after the grouping, three guinea pigs were sacrificed in each group, and the spleen was taken for the isolation of splenic lymphocytes. Referring to the above-mentioned immunization and challenge protection test of mice, the lymphocyte proliferation reaction was measured. The positive control and negative control were the same as those in the mouse experiment. The virus stimulation group used 100 μL of 1×10 6 TCID 50 / mL of the PPV GD strain virus solution as the stimulator.

[0198] II. Test Results

[0199] 1. Immunization and challenge protection test of mice

[0200] (1) Detection of the level of JEV-specific antibodies in mouse serum

[0201] The results of the specific antibody detection are as Figure 8 shown. The level of JEV-specific antibodies in the sera of the vaccinated mice increased continuously over time and reached the peak at 35 d after the first immunization. The levels of specific antibodies in the JEV inactivated single vaccine group and the JEV-PPV VP2 combined vaccine group were significantly higher than those in the PBS-ISA201 group and the PBS group (P<0.01), but the levels of the two were similar. This indicates that JEV-PPV VP2 can better induce the body to produce specific antibodies against JEV.

[0202] (2) Detection of the level of JEV neutralizing antibodies in mouse serum

[0203] The JEV neutralizing antibody level in mouse serum was measured by virus neutralization experiment. The results are as Figure 9 shown. The peak was reached at 35 d after the first immunization in each vaccine group. The average neutralizing antibody titer of the JEV-PPV VP2 group (1:181) was significantly higher than that of the PBS-ISA201 group and the PBS group (P<0.01). The results indicate that the JEV-PPV VP2 group can effectively induce the secretion of neutralizing antibodies.

[0204] (3) Detection of the levels of IL-2 and IL-4 cytokines in mouse serum

[0205] To evaluate the immune efficacy of the vaccine, an Elisa kit was used to detect the levels of IL-2 and IL-4 in the peripheral serum of mice. Compared with the PBS group, each vaccine group could stimulate the body to produce higher levels of IL- and IL-4.

[0206] The detection results of IL-2 are as Figure 10 shown. The JEV-PPV VP2 group was comparable to the JEV inactivated single vaccine group; there was a significant difference between the JEV commercial vaccine group and the JEV inactivated single vaccine group (P<0.05). This indicates that the prepared vaccines can all induce the secretion of the Th1-type cytokine IL-2 and improve the cellular immune function of the body.

[0207] The detection results of IL-4 are as follows Figure 10 shown. The level of IL-4 secretion in the JEV commercial vaccine group was the highest; the secretion levels of the JEV-PPV VP2 group and the inactivated JEV single vaccine group were comparable. This indicates that all vaccine groups can induce the secretion of the Th2 cytokine IL-4 well and improve the humoral immune function of the body.

[0208] (4) Mouse lymphocyte proliferation assay

[0209] To verify whether the prepared vaccines can induce good cellular immunity, mice in each group were sacrificed 42 days after the first immunization, and spleen lymphocytes were isolated for culture for subsequent lymphocyte proliferation assays. The results are as follows Figure 11 shown. All vaccine groups could effectively induce lymphocyte proliferation, with stimulation indices greater than 1, significantly higher than those of the PBS-ISA201 group and the PBS group (P < 0.01). The stimulation index of the JEV-PPV VP2 combined vaccine group was 1.8. The results indicate that the JEV-PPV VP2 combined vaccine can stimulate lymphocyte proliferation.

[0210] (5) Detection of survival rate of mice after challenge and JEV viral load in various brain tissues and blood

[0211] To verify the protective effect of the combined vaccine against challenge, immunized mice were challenged with JEV GD (10 8 TCID 50 / mL), and 50 μL was injected intracranially into each mouse. The results are as follows Figure 13 shown. One mouse in the JEV-PPV VP2 vaccine group died after challenge, and all the remaining mice in the other vaccine groups survived after immunization and challenge. All the mice in the PBS-ISA201 and PBS groups died. During the challenge experiment of the JEV-PPV VP2 group, one mouse died on the day of challenge. We speculate that the cause of death of the mouse may be due to stress during the vaccination process.

[0212] To verify the effect of each immunized group in reducing the viral load after challenge, the remaining mice were sacrificed 14 days after challenge, and brain and blood were collected as samples, and RNA was extracted for detection of the viral load. The results are as follows Figure 12 shown. The viral loads in the brain tissues and blood of all vaccine groups were significantly lower than those of the PBS-ISA201 and PBS groups (P < 0.05); the viral loads of the inactivated JEV single vaccine group and the JEV-PPV VP2 combined vaccine group were slightly higher than those of the JEV commercial vaccine group, and there was no difference between the groups.

[0213] 2. Immunization test in guinea pigs

[0214] (1) Detection of PPV-specific antibody levels in guinea pig sera

[0215] To further evaluate the immune efficacy of different vaccines, blood samples were collected on days 14, 21, 28, and 35 after the first immunization to isolate serum, and the specific antibody levels of PPV in the serum were detected using an ELISA specific antibody detection kit. The results are as Figure 14 shown. The specific antibody levels of PPV in each vaccine group gradually increased after the first immunization and reached the peak on day 35. At this time, both the JEV-PPV VP2 group and the commercial vaccine group were much higher than the PBS-ISA201 group and the PBS group (P<0.001).

[0216] (2) Detection of PPV neutralizing antibody levels in guinea pig serum

[0217] To detect the PPV neutralizing antibody levels in guinea pig serum, blood samples were collected on days 14, 21, 28, and 35 after the first immunization to isolate serum for virus neutralization antibody experiments. The results are as Figure 15 shown. The PPV neutralizing antibody levels in each vaccine group were relatively low on day 14 after the first immunization, and there were no significant differences among the vaccine groups. The PPV neutralizing antibody titers in both the JEV-PPV VP2 combined vaccine group and the commercial vaccine group reached the peak on day 35 after the first immunization, greater than 7log2.

[0218] (3) Detection of the hemagglutination inhibition (HI) antibody titers of PPV in guinea pig serum

[0219] To detect the HI titers in guinea pig serum, blood samples were collected on days 14, 21, 28, and 35 after the first immunization to isolate serum, and the HI titers were detected by hemagglutination inhibition experiments. The results are as Figure 16 shown. The HI titers in both the JEV-PPV VP2 combined vaccine group and the commercial vaccine group reached the peak on day 21 after the first immunization. The HI titers in each vaccine group were above 6log2 and then gradually decreased.

[0220] (4) Detection of IL-2 and IL-4 cytokine levels in guinea pig serum

[0221] To evaluate the immune efficacy of the vaccine, an Elisa kit was used to detect the levels of IL-2 and IL-4 in the peripheral serum of guinea pigs. The detection results of IL-2 are as Figure 17 shown. Each vaccine group could induce the secretion of a relatively high level of IL-2; the results showed that the prepared vaccines could all induce the secretion of the Th1-type cytokine IL-2 well and improve the cellular immune function of the body.

[0222] The detection results of IL-4 are as Figure 17 shown. The secretion level was the highest in the PPV commercial vaccine group. The IL-4 secretion levels in both the JEV-PPV VP2 group and the commercial vaccine group were much higher than those in the PBS-ISA 201 and PBS groups. The results showed that the prepared vaccines could induce the secretion of the Th2-type cytokine IL-4 well.

[0223] (5) Guinea pig lymphocyte proliferation assay

[0224] To verify whether the prepared vaccine could induce good cellular immunity, guinea pigs in each group were sacrificed on the 42nd day after the first immunization, and the spleen lymphocytes of guinea pigs were isolated for culture, which were used for subsequent lymphocyte proliferation assays. The results were as Figure 18 shown. The lymphocyte stimulation indices of the JEV-PPVVP2 group and the commercial vaccine group were significantly higher than those of the PBS-ISA 201 group and the PBS group (P<0.001), and there was no significant difference between the two. The JEV-PPV VP2 bivalent vaccine group had the best stimulation effect, which was higher than that of the commercial PPV vaccine group.

[0225] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine, characterized in that It contains inactivated Japanese encephalitis virus of swine, PPV-VP2 recombinant protein and adjuvant; The amino acid sequence of the said PPV-VP2 recombinant protein is as shown in SEQ ID No: 1; In the described Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine, the content of inactivated Japanese encephalitis virus of swine is 10 5.5 -10 6.0 TCID 50 / m, and the content of PPV-VP2 recombinant protein is 45 - 55 μg / mL; In the Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine, the volume ratio of the aqueous phase to the oil phase is (1:1)-(1:1.5).

2. The Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to claim 1, characterized in that: The nucleotide sequence encoding the said PPV-VP2 recombinant protein is as shown in SEQ ID No:

2.

3. The Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to claim 1, characterized in that: The said Japanese encephalitis virus of swine is GI type JEV GD strain.

4. The Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to claim 1, characterized in that: The said inactivated Japanese encephalitis virus of swine is prepared by the following method: Inoculate the Japanese encephalitis virus strain of swine into Vero cells, harvest the virus solution after culturing for 48 - 96h; add formaldehyde with a final volume percentage of 0.1 - 0.3%, inactivate at 37°C for 24 - 48h to obtain inactivated Japanese encephalitis virus of swine; The M.O.I. of the said Japanese encephalitis virus strain of swine is 0.1; The said culturing time is 60h; The final concentration of the said formaldehyde is 0.1%; The said inactivating time is 36h.

5. The Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to claim 1, characterized in that: The preparation method of the said PPV-VP2 recombinant protein comprises the following steps: (1) Gene synthesis of the nucleotide sequence encoding the said PPV-VP2 recombinant protein, introduce restriction enzyme sites at both ends, and connect the nucleotide sequence encoding the said PPV-VP2 recombinant protein to the transfer plasmid through the restriction enzyme sites to obtain a recombinant transfer plasmid; (2) Transform the recombinant transfer plasmid prepared in step (1) into DH10Multibac competent cells to obtain a recombinant baculovirus plasmid rBac-VP2; (3) Transfect the recombinant baculovirus plasmid rBac-VP2 prepared in step (2) into sf9 cells to obtain an Ac-VP2 recombinant baculovirus; (4) Transfect the Ac-VP2 recombinant baculovirus prepared in step (3) into High Five cells and culture, collect the cell culture suspension, ultrasonically lyse the cells, centrifuge to collect the supernatant; purify the supernatant by sucrose density gradient centrifugation to obtain the PPV-VP2 recombinant protein.

6. The Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to claim 1, characterized in that: The transfer plasmid described in step (1) is pFastBac TM Daul vector.

7. The Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to claim 1, characterized in that: The M.O.I. of the transfection in step (4) is 1 - 10; The culturing time in step (4) is 48 - 120h; The conditions for sucrose density gradient centrifugation described in step (4) are as follows: the sucrose density gradient is successively 70%, 50%, 30%, centrifuging at 30000 r / min at 4°C for 6 h.

8. The Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to claim 1, characterized in that: The adjuvant is ISA201 adjuvant.

9. The preparation method of the Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to any one of claims 1-8, characterized in that It comprises the following steps: Mixing the inactivated Japanese encephalitis virus of swine and PPV-VP2 recombinant protein in equal volumes to obtain an aqueous phase; mixing the aqueous phase and the adjuvant in equal volumes and emulsifying to obtain the Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine.

10. Use of the Japanese encephalitis virus of swine and porcine parvovirus VP2 protein bivalent vaccine according to any one of claims 1-8 in the preparation of a product for preventing and treating at least one of Japanese encephalitis virus of swine and porcine parvovirus.

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