Construction Of Baculovirus Expressing The Main Protective Antigens Of IBDV And The Study Of Its Immune Effect

Infectious bursal disease(IBD)is an acute,thermal resistance,highly contagious disease which is cause by the infectious bursal diseases virus(IBDV).VP2 protein is the main protective antigen of IBDV,it is connects with the induction of virus neutralizing antibodies,the variation of antigens,virulence and cell apoptosis.The polyprotein VP2/4/3 can be exactly cut into a natural configuration of VP2 protein.Therefore,choosing the appropriate target gene and expression system is crucial.This research using baculovirus vector to express VP2 gene and VP2/4/3 gene,we obtain the recombinant Bacmid DNA via Bac-to-Bac system and then transfect to Sf9 insect cells to acquire recombinant baculovirus.The optimized baculovirus were constructed by adding VSV-GED,woodchuck hepatitis virus post-transcriptional regulatory element(WPRE)and inverted terminal repeats(ITRs)regulatory elements.Using the optimized baculovirus and baculovirus without modification respectively as gene transfer vectors,we compare the expression time and expression level of exogenous gene in avian cells by SDS-PAGE and Western blotting analysis approach.At the same time,using the recombinant baculovirus as vaccine to immune the poultry directly and transfer exogenous antigen genes efficiently to the poultry cells can stimulate the body to produce specific protective antibodies and strong cellular immune responses.Ultimately,we determined the main protective antigen gene,the expression effect and immune effect of recombinant baculovirus with different regulatory elements were compared and analyzed.the study will lay a foundation for the development of poultry vaccine by using baculovirus.We used Primier 5 to design specific primers according to IBDV-VP2 sequence and IBDV-VP2/4/3 sequence in GenBank and added His-tag gene sequences to the 5'end of the target gene.By polymerase chain reaction(PCR),the His-VP2 gene and His-VP2/4/3 gene were amplified and ligated to pMD18-T vector respectively.It turned out that the positive recombinant plasmids which contained His-VP2 and His-VP2/4/3 fusion gene were named pT-VP2 and pT-VP2/4/3 respectively via the restriction enzyme analysised and nucleotide sequenced.Then His-VP2 gene and His-VP2/4/3 gene were respectively cloned to the transfer vector pS,pS-ITRs and pS-con.By combined enzymes identifications,it is proved that obtained recombinant expression vectors containing the different transfer boxes and regulatory elements named pS-VP2,pS-ITRs-VP2,pS-con-VP2,pS-VP2/4/3,pS-ITRs-VP2/4/3 and pS-con-VP2/4/3 respectively.The transfered vectors were transformed into the E.coli DH10 Bac competent cells in order to obtain the recombinant shuttle plasmid rBac-S-VP2,rBac-S-ITRs-VP2,rBac-S-con-VP2,rBac-S-VP2/4/3,rBac-S-ITRs-VP2/4/3.And then transfected them into SJ9 insect cells,to obtain recombinant baculovirus PI stocks,continued to amplify P1 stock up to P3 and then calculated the virus titer by plaque analysis.As a result the Virus titer was 1.5×108 pfu/mL.The six recombinant baculovirus were transfected chicken primary cell respectively,the His-VP2 protein and His-VP2/4/3 protein were successfully detected by SDS-PAGE and Western blotting.The protein expression quantity of BV-S-VP2 and BV-S-ITRs-VP2 was respectively 2.5 times and 2.4 times of BV-S-con-VP2.The protein expression quantity of BV-S-VP2/4/3 and BV-S-ITRs-VP2/4/3 was respectively 2.4 times and 2.2 times of BV-S-con-VP2/4/3.With Serum neutralization test,the antibody titer of VP2/4/3 series was up to 1:2885.42,which was significantly higher than the vaccine group 1:1326.68 and the attacking poison control group 1:8.32,the VP2/4/3 series ELISA test result showed that the antibody levels in 42 days can reach 1.225,higher than 1.125 of the vaccine group and 0.249 of attacking poison control group.At the same time,the 14 day-old SPF chicken were immuned with six recombinant baculovirus respectively.Then we drawed blood from the vein under chicken wing by 7,14 days after the first immunization and the second immunization.Through the separatation of the serum,serum neutralization test and the ELISA kits,the immunogenicity of different antigen proteins were compared and analysed.The results showed that serum antibody level of the recombinant baculovirus contains VP2/4/3 gene was significantly higher than the blank group and empty vector group(P<0.05).Poison attack test results showed that the VP2 series and VP2/4/3 protection rate were 95.8%and 100%respectively,higher than protective vaccine group 87.5%,with significant difference in the control group 50%.This study will lay the theoretical and practical foundation for the further development of poultry vaccines,and provide new ideas and useful references for developing the non-replicating live vaccine against other viral diseases.