Biotinylated AcMNPV Displaying PEDV S1 Based On VSV-G Transmembrane Domain And Its Immunogenicity On Rat Model

Porcine epidemic diarrhea(PED)is one of the biggest threats to the global swine industry.Porcine epidemic diarrhea virus(PEDV)is the causative agent of porcine epidemic diarrhea.swines of all ages can be infected by this virus,and the main clinical symptoms are vomiting,small yellow intestine,and watery diarrhea.Among them,piglets in lactation are most threatened by PEDV,and the mortality rate is high up to over 90%.Since its first discovery in the 1970s,PED had caused huge economic losses to the swine industry in China and all over the world.However,the marketing PEDV vaccines are not effective enough to prevent the re-occurrence of Porcine epidemic diarrhea(PED).Therefore,it is urgent to develop the new effective and safe PEDV vaccine.Studies had shown that recombinant baculovirus displaying the antigenic proteins of the virus as a vaccine analog could stimulate the animal to produce a specific immune response effectively.However,it still remained some problems,such as low titer and inefficient purification.This project aimed to display protein S1 of PEDV on the surface of biotinylated recombinant baculovirus,and to purify the virus efficiently by combining the high affinity of the biotin-streptavidin system.Then,the SD rats in pregnancy were immunized with the virus or control reagents,and the immune activity was evaluated by detecting S1-specific IgG and milk sIgA produced by the rat.To prepare the desired recombinant baculovirus,we cloned the modified PEDV S1 gene,the biotin ligase gene(BirA),and the baculovirus gene gp64 fused with the biotin receptor peptide sequence(BAP).The recombinant baculovirus was constructed using the Bac-to-Bac system and linearized homologous recombination as well as non-homologous end joining.The expression and localization of S1 protein in cells or on virus surface were analyzed by Western blot,observation of direct fluorescence and indirect immunofluorescence,immunoelectron microscopy.The biotinylated recombinant baculovirus was purified by streptavidin magnetic beads,and the purification effect was analyzed by Western blot,silver staining.and qRT-PCR.Furthermore,purified recombinant baculovirus,wild-type PEDV,baculovirus Ac-WT,and saline were used as immunogens to immunize pregnant SD rats by subcutaneous injection.S1-specific serum IgG,S1-specific slgA and the total sIgA in milk were determined by ELISA for immunogenicity analysis.The study showed that the recombinant baculovirus modified by biotin could express PEDV S1 protein efficiently and displayed it on the surface of infected cells and viruses.After purification by magnetic beads,some structural proteins of baculovirus could be recognized by silver staining,which indicated the study improved the purity of virus significantly,and the result of purification could also be proved by qRT-PCR.ELISA analysis showed that the recombinant virus could significantly stimulate the high-level production of PEDV S1-specific IgG in the serum and slgA in the milk of the lactating rats.This finding indicated the baculovirus purified by magnetic beads displaying PEDV S1 can stimulate the lactation rats to produce an immune response,and improve the level of S1-specific slgA in the milk significantly,which lays an experimental foundation for further practical application of this baculovirus vaccine.