Construction Of Recombinant Baculovirus Virus Expressing The Major Immunogenic Genes Of Swine O Serotype Foot-and-mouth Disease Viruse And Study On Its Biological Characteristics

Foot and Mouth Disease(FMD)is caused by foot-and-mouth disease virus(FMDV)which can infect animals especially cloven-hoofed animals,acute,fever,and contagious enzootic disease.FMDV have a lot of serum type,strong infectivity,fast spreading,high infection rate,big epidemic range,wide host spectrum of known characteristics,FMDV can reproduce infection and serious harm to the global aquaculture.So it is considered to be one of the most serious diseases of Class A by The United Nations Food and Agriculture Organization(FAO)and World Organization for Animal Health(OIE).Foot-and-mouth disease was included in the major animal disease of the global eradication program.Currently,Foot-and-mouth disease vaccine include inactivated vaccine,subunit vaccine(protein or polypeptide),DNA vaccines and live carrier vaccine virus etc.The inactivated vaccine play an important role in the prevention and control of foot-and-mouth disease in our country,but it has not completely inactivated,and the existence of biological security.The P1 protein is the major immune protein of FMDV,The P1 protein is structural protein of FMDV and can be cleaved into VP0、VP1、VP3 structural protein of the viral capsid formation under the action of protease.Therefore,P1 and VP0、VP1、VP3 gene is the major gene that vaccine is studied.The main content of our research can be described as follows:1.Construction of recombinant Baculovirus virus rBac-VP130、rBac-P111、rBac-P11-3C expressing of the major immunogenic genes of swine O serotype Foot-and-mouthThe main antigen genes of FMDV VP0、VP 1、VP3;Three copies of theGMPl gene and GMP1 and 3C gene were inserted into the baculovirus expression vector pFast BacTMDual.This can obtain transfer plasmid vector of pFBD-VP130,pFBD-Pl 111、pFBD-P11-3C.The transfer plasmid vector was transformed into DH10Bac competent cells.We got recombinant bacmid rBacmid-VP130,rBacmid-P111,rBacmid-P11-3C through blue/white selection.These three recombinant bacmids were transfected into sf9 cells to get three recombinant baculovirus rBac-VP130,rBac-P111,rBac-P11-3C.The indirect immunofluorescence technique(IFA)and Western Blotting analysis showed that the exogenous genes were effectively expressed in Sf9 and HF cells.The recombinant baculovirus rBac-VP130,rBac-P11-3C infected sf9 cells,the cells were collected.After the cells were repeated freezing and thawing,used of sucrose density gradient centrifugation,after collected protein that is correct by detection Virus-like particles(VLPs)was observed under electron microscopy.We observed about 26nm in diameterThe VLPs of shape and size were similar with FMDV particles.This can show FMDV capsid protein VPO、VP1、VP3 can form virus-like particles in vitro.The 3C protein can cleavage P1 protein in vitro that form virus-like particles.2.The experimental on immune efficacy study of the recombinant baculovirus rBac-VP130、rBac-P111、rBac-P11-3CTaking recombinant baculovirus rBac-VP130、rBac-P111 and rBac-P11-3C infected HF cells respectively,collecting cells after 72h and emulsified by freund’s complete adjuvant,then immuned Balb/c mice in a dose of 1 x 106 cells.FMDV neutralizing test results showed that the recombinant baculovirus rBac-VP130、rBac-Pllland rBac-P11-3C can induce mice to produce antibodies against FMDV,degree of neutralization antibody are 1:18、1:24 and 1:16 respectively.ELISA test results showed that ELISA antibody levels of the rBac-VP 130、rBac-P111、rBac-P11-3C immune group and commercialization vaccine group are on the rise,the antibody levels reached the highest levels in 42 days after the first vaccination,antibody levels of rBac-P11-3C was the highest of the three groups.but this three groups was not significant difference(P>0.05).In addition,Real-Time PCR technique was detected immune interferon(IFN)mRNA expression in spleen cells of the mice.The results have showed that the three groups of recombinant baculovirus can induce immune response in immune mice.PCR,we also detected cytokine,like IFN-alpha,IFN-beta and from spleens of immunized mice.The results show that this three subunit vaccine were very good immunization effect,There well be a new candidate vaccines of FMDV.