Construction Of Recombinant Baculovirus Expressing F And HN Genes Of Newcastle Disease Virus And Research On Its Immune Effect

Newcastle disease is fatal infectious disease in poultry,and it was caused by the paramyxovirus family of Newcastle disease virus(NDV).The breakout of Newcastle disease is urgent and highly pathogenic,which cause serious damage to the poultry in our country.F protein,one of the major protective antigens of NDV,can induce the body to produce neutralizing antibody,and it plays an important role in the immune responses.In addition,HN protein is another protective antigen of NDV and it also plays a role in the protection of the body.However,the immune protection of F and HN protein is generally different in the different expression systems.Therefore,the appropriate antigen gene and expression system are critical to the immune response.In this study,the Lasota F and HN gene were inserted into the optimized baculovirus with CMV promoter and VSV-GED,WPRE,ITRs regulatory elements,respectively.The baculovirus without modification was used as the control.The recombinant bacmid DNA was obtained via Bac-to-Bac system and then it was transfected into Sf9 insect cells in order to acquire recombinant baculovirus.The chicken embryo fibroblast cells were infected with recombinant baculovirus.The different expression levels of antigen genes were compared by SDS-PAGE and Western blotting analysis;meanwhile,the recombinant baculovirus was used as vaccine to directly immune the poultry.The expression and immune effect of recombinant baculoviruses with different regulatory elements and antigen genes were compared and analyzed.The results showed that the expression of F and HN protein were successfully detected by SDS-PAGE and Western blotting.The protein expression levels of recombinant baculovirus with WPRE and VSV-GED were higher than that of recombinant baculovirus without any regulatory element.The protein expression levels of BV-LM-F and BV-LM-ITRs-F were respectively 3.45 times and 3.62 times than that of BV-LM(-)-F;The protein expression levels of BV-LM-HN and BV-LM-ITRs-HN were respectively 3.99 times and 4.60 times than that of BV-LM(-)-HN.Indirect immunofluorescence test results showed that the VSV-GED was successfully expressed in SJ9 insect cells in a surface display way,which could strengthen the transduction efficiency.The 14 days-old SPF chickens were immuned with six recombinant baculoviruses and the commercial vaccine,respectively.At the same time,the empty vector control group and PBS control group were set.The immune experiment results showed that the serum antibody level of F and HN series recombinant baculovirus vaccine were significantly higher than that of PBS control group and empty vector group(P<0.05).In the serum neutralization test,antibody titer of F series reached up to 1:1387.47,and there was no significant difference with the F and HN co-immune series(1:1272.59).However,significant difference was found between HN series(1:764.56)and the control group(1:9.37).The detection of IgG antibody level showed that the order of IgG antibody level as follows:Lasota vaccine group>F series vaccine group?F+HN vaccine group>HN series vaccine group>the control group.In addition,the IgG antibody level of pLM and pLM-ITRs vaccine group were higher than that of pLM(-)vaccine group.This results indicated that WPRE and VSV-GED regulatory elements could increase the expression level of antigen gene.The ELISA results of IL-2,IL-4 and IFN-y showed that the recombinant baculovirus vaccines could effectively stimulate the humoral and cellular immune response.The concentration of IL-2,IL-4 and IFN-y reached up to 71.26 ng/mL,131.93 ng/mL and 69.65 ng/mL,respectively.In addition,ITRs element could effectively prolong the expression time of cytokines in vivo.The results of chicken attack test showed that the F series,HN series and commercial vaccine all stimulated the certain protection for chickens.The average protection ratio of F series vaccine was 95.8%and the average protection ratio of HN series vaccine was 75.0%.The difference was significant compared with protection ratio of the control group.This study may lay the foundation for the research of screening more effective NDV antigen gene and expression vector.At the same time,it also provides a novel method for developing a new type of Newcastle disease vaccine.