Construction Of Recombinant Baculovirus With HA Gene Of H5N1 Avian Influenza Virus And Research On Its Immune Effect

The highly pathogenic avian influenza caused by influenza A H5N1 virus,is an acute and highly contagious disease.As the major protective antigen of avian influenza virus,hemagglutinin performs a vital role in its antigenicity and pathogenicity.Promoter is one of the major factors that affect the expression of recombinant baculovirus,and a stronger promoter can enhance the expression efficiency of exogenous genes.WSSV ie1 promoter and CMV promoter are both recognized as strong promoters due to their respective advantages of driving exogenous gene expression efficiently in mammalian cells and insect cells.In the research,HA gene derived from H5Nlsubstype avian influenza virus was cloned into the baculovirus transfer vector,which was driven by CMV promoter and WSSV ie1 promoter respectively and contained VSVG,woodchuck hepatitis virus post-transcriptional regulatory element(WPRE)and inverted terminal repeats(ITRs)regulatory elements.The recombinant Bacmid DNA was obtained via Bac-to-Bac system and then it was transfect into the Sf9 insect cells.The acquired baculoviruses were utilized to infect the chicken embryo fibroblast cells,and the expression time and expression levels of exogenous gene were compared by SDS-PAGE and Western blotting analysis.The recombinant baculoviruses carrying HA gene were directly used as vaccines to immunize trials chickens which could transferred the target gene into the poultry cells to effectively and stimulate the body to produce protective antibody and specific immune response.The differences of the expression time as well as the immune effects among different recombinant baculoviruses were compared and analysed in order to select more efficient promoter and determine the optimized baculovirus transfer vector.The study may lay a foundation for the development of poultry vaccine by using baculovirus.The specific primers according to the HA sequence reported in the GenBank were designed by Primier 5.0 software,and His-tag gene sequences were added into the 5'end of target gene.HA gene was amplified by polymerase chain reaction and was ligated to pMD18-T vector.The positive recombinant plasmids were named pT-HA via the restriction enzyme analysis and nucleotide sequence analysis.Then HA gene was cloned into the transfer vector pS,pS-ITRs,pS-con drived by CMV promoter and pAQ,pAQ-ITRs,pAQ-con drived by WSSV iel promoter.The recombinant expression vectors containing different transfer boxes and regulatory elements were named pS-HA,pS-ITRs-HA,pS-con-HA,pA-HA,pA-ITRs-HA,pA-con-HA respectively.The transfer vectors were transformed into the E.coli DH10 Bac competent cells in order to obtain the recombinant shuttle plasmid rBac-S-HA,rBac-S-ITRs-VP2,rBac-S-ITRs-HA,rBac-S-con-HA,rBac-A-HA,rBac-A-ITRs-HA and rBac-A-con-HA.By using the shuttle plasmids to transfect Sf9 insect cells,we obtained recombinant baculoviruses and then use it to infect the chicken embryo fibroblast cells.The target protein HA was successfully detected by SDS-PAGE and Western blotting.The protein expression levels of BV-S-HA and BV-S-ITRs-HA were respectively 2.43 times and 2.67 times than that of BV-S-con-HA.The protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 times and 2.69 times than that of BV-S-con-HA.The specific pathogen free chickens were respectively immuned with the six recombinant baculoviruses and commercialized vaccine,and the positive and negative control groups were set in the meanwhile.Then the immune serum were draw from the vein of chickens 14 days,28 days,42 days,56 days and 70 days post-immunization.The IgG antibody levels of each group were detected by enzyme linked immunosorbent assay and the chicken peripheral T lymphocyte proliferation effect was estimated by MTT.In the meantime,the IL-2,IL-4 and IFN-y cytokines were measured in order to compare and analyze the differences.The result revealed that IgG antibody levels induced by BV-A series recombinant baculovirus and BV-S series recombinant baculovirus were significantly higher than that of the commercialized vaccine group and the control group(P<0.05).Among the groups with same promoter,the IgG antibody levels induced by the baculovirus containing VSV-GED,WPRE and ITRs regulatory elements were significantly higher than that of the control.The lymphocyte proliferation results showed that the stimulation index of BV-A series with ie1 promoter can reach 1.349,which significantly higher than BV-S series with CMV promoter and vaccine group(P<0.05).The cytokine detection results demonstrated that cytokine levels of each group reached the maximum value two weeks post-immunization.The content of IL-2,IL-4 and IFN-y cytokines could come up to 99.13ng/L,86.42ng/L and 76.93ng/L,respectively.The results revealed that the immune effects induced by BV-A series recombinant baculoviruses with WSSV iel promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter.In this study,the avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses,which may provide useful references for the development of avian influenza vaccine and lay foundation for the prevention and control of avian influenza.