Construction And Immunogenicity Of Recombinant Baculovirus Expressing Bovine Viral Diarrhea Virus E0 Protein

Bovine viral diarrhea(BVD)is an infectious disease of cattle caused by the bovine viral diarrhea virus(BVDV)that has been widespread worldwide.Although BVD was classified as the third class of animal blight in our country,it caused huge economic losses to cattle industry cannot be ignored.In addition to causing acute infection in cattle,BVDV can also cross the placental barrier early in cow pregnancy and lead to the birth of persistent infection(PI)calves.Persistent infected animals tend to transmit BVDV more efficiently than acutely infected animals,and PI animals excrete large amounts of virus throughout their lives,so PI cattle are considered to be the primary reservoir of BVDV.Control schemes used in many countries around the world rely primarily on the early detection and culling of PI animals.E0 protein is one of the membrane proteins of BVDV.This protein has been widely used in the study of BVDV genetic engineering subunit vaccines.The aim of this study was to develop subunit vaccine using insect cells to express BVDV E0 protein.Firstly,specific primers were designed to amplify the BVDV E0 gene,and the E0 gene fragment was linked to the insect expression vector plasmid p Fast Bac HTB by homologous recombination method.The pfastbach Tb-E0 was transformed into the DH10 Bac receptor state,and the target gene was transposited to the shuttle vector Bacmid,which was screened by blue and white spots.The recombinant rod r Bacmid-E0 was obtained.Recombinant baculovirus r Bv-E0 entered Sf9 insect cells with transfection reagent to obtain cytopathic effect of recombinant baculovirus RBV-E0.The recombinant virus was passed through the passage to obtain recombinant disease venom with high expression level of P4.The recombinant virus was bound to nickel column and purified E0 protein was obtained under the elution of imidazole.The recombinant baculovirus rBV-E0 was mixed with Freudel’s adjuvant to form an oil-in-water recombinant subunit vaccine.The immunogenicity of the vaccine was evaluated by indirect ELISA,neutralization assay,flow cytometry and double antibody sandwich ELISA.The recombinant vaccine can stimulate the production of specific antibodies,and the neutralizing antibodies can neutralize BVDV,and increase the content of CD4+ and CD8+ T lymphocyte subsets and the secretion level of Th1 cytokine.