Expression Of Porcine Parvovirus Recombinant Protein RVP2 And Immunogenicity Of PPI-JE Combined Vaccine

Japanese encephalitis（JE）is a central nervous systemic and acute infectious disease of swine herds infected with Japanese encephalitis virus（JEV）,which can cause miscarriage,stillbirth and mummified fetus in pregnant sows.Porcine parvovirus infection（PPI）is a reproductive dysfunctional infectious disease of pigs caused by Porcine parvovirus（PPV）,which causes miscarriages,stillbirths and mummies in pregnant sows.At present,vaccine immunization is an effective measure to prevent and control JE and PPI.The combined immunization of JE and PPI vaccines reduces the immunization procedures on the farm,reduces production costs,and reduces animal stress.Therefore,the prevention and control of swine diseases urgently need a combination of mμLtiple vaccines to provide protection for the healthy development of the pig industry.In this study,the JEV GD strain was propagated on Vero cells,and the conditions of virus infection time and dose were optimized to obtain a high-titer virus solution.The pathogenicity experiment was carried out in mice infected with 10^6.0 TID₅₀/m L of JEV GD strain virus.Twenty SPF-level Kunming mice were randomly divided into two groups,each with 10 mice.The mice in the experimental group were intraperitoneally injected with 100μL of JEV isolate virus solution,and the mice in the control group were intraperitoneally injected with 100μL saline.The results showed that on the 4th day of infection with the JEV GD strain,the mice in the experimental group were observed to be depressed,weakened,and secreted yellow-brown mucus at the eyelid.The mice were all morbid and died within 10 days.The mice in the control group had normal activities,and no brain tissue lesions were found on necropsy.Based on the VP2 gene of the pig PPV NJ strain（AY686602.1）included in Gen Bank,the bee venom signal peptide（Melittin）and His tag were introduced at the N and C ends,respectively,to synthesize the recombinant gene rVP2.The transfer plasmid vector p Fast Bac Dual is a dual-promoter vector.The recombinant rVP2 gene was cloned into the polyhedrin（PH）and p10 promoters of the transfer vector p Fast Bac Dual,respectively.The recombinant transfer plasmid was obtained after PCR,double digestion identification,and gene sequencing verification.The recombinant transfer plasmid p Fast Bac Dual-2rVP2 was transformed into DH10Bac competent cells carrying the baculovirus plasmid Bacmid,and the positive clones selected by blue and white spots were used to detect the recombination of its rVP2 gene and Bacmid using PCR technology.PCR results showed that there were specific bands at 2 000 bp and 2 200 bp,and the size was as expected;double digestion results showed that there were specific bands at 1 800 bp and 7 000 bp,and the size was as expected.The results showed that the target gene rVP2 and Bacmid were specifically recombined to obtain recombinant baculovirus plasmid r Bac-rVP2.Recombinant baculovirus plasmid r Bac-rVP2 was transfected into sf9 cells to obtain recombinant baculovirus Ac-rVP2.Recombinant baculovirus Ac-rVP2 was infected into sf9cells.Western blot and indirect immunofluorescence（Indirect immunofluorescence）were performed 72 hours after infection,IFA)to detect the expression of recombinant protein rVP2 in cells.Western blot results show that there is a specific band around 67 k Da,which is consistent with the molecular size of the recombinant protein rVP2;IFA results show that green fluorescence can be observed under fluorescent microscope in sf9 cells infected with recombinant baculovirus Ac-rVP2 and infected wild Baculovirus-type sf9 cells cannot observe fluorescence under a fluorescence microscope.High Five cells were infected with recombinant baculovirus Ac-rVP2 with M.O.I of0.1,1.0,and 10.0,and cell cultures were collected at 24 h,48 h,72 h,96 h,120 h,and 144h after infection.The cells were lysed by ultrasonication,the expression of recombinant protein rVP2 in the cell samples was detected by Western blot,and the protein was semi-quantitatively analyzed by Image J software.The results showed that the recombinant baculovirus Ac-rVP2 infected High Five cells between 24 h and 96 h.The content of recombinant protein rVP2 gradually increased;after 96 h,the content of recombinant protein rVP2 gradually decreased.When M.O.I.was 1.0,96 h after infection,the highest expression level of recombinant protein rVP2 in cell culture was detected,with a concentration of 380.5μg/m L.After inactivation of 10⁶TCID₅₀/m L JEV GD strain virus solution withβ-propiolacton e,emulsified with ISA 201VG adjuvant at a volume ratio of 1:1 to prepare JE inactivated vaccine;The agent is emulsified according to the volume ratio of 1:1 to prepare the PPV recombinant protein rVP2 subunit vaccine;the recombinant protein rVP2 is mixed with the inactivated JEV GD strain virus liquid in equal volumes,and then mixed with the ISA201VG adjuvant according to 1:1 Proportional emulsification to prepare PPI-JE（rVP2+GD strain）dual vaccine.The vaccine was immunized with guinea pigs to initially assess the immunogenicity of the vaccine.The experiments were divided into the following groups:Group 1 is JE inactivated vaccine,Group 2 is rVP2 subunit vaccine,Group 3 is PPI-JE（rVP2+GD strain）dual vaccine 4 is a commercial JE inactivated vaccine+PPI inactivated vaccine,and group 5 is a PBS control group.At the 14th day after the first immunization,booster immunization was performed at the same dose.Heart blood was collected from each group of guinea pigs 1 day before and 7 days,14 days,21 days,and 28days after the first immunization to detect specific antibody levels in guinea pig sera.The results showed that on the 14th day after the second immunization,except for the PBS control group,the JE inactivated vaccine group,the PPI-JE（rVP2+GD strain）combination vaccine group,and the commercial JE inactivated vaccine+PPI inactivated vaccine group all produced High level of JEV-specific antibodies,and no significant difference in antibody levels between groups（P>0.05）;rVP2 subunit vaccine group,PPI-JE（rVP2+GD strain）dual vaccine group,commercial JE inactivated vaccine+The PPI inactivated vaccine group all produced higher levels of PPV-specific antibodies,and there was no significant difference in antibody levels between the groups（P>0.05）.In summary,the synthetic gene rVP2 can efficiently express the recombinant protein rVP2 in High Five cells and has high antigenicity.The prepared PPI-JE（rVP2+GDstrain）dual vaccine can effectively induce guinea pigs to produce higher levels The PPV and JEV specific antibodies provide a reference for the development of a new PPI-JE dual vaccine.