Screening,Expression,Purification And Immunogenicity Identification Of Several African Swine Fever Antigens

African swine fever is an acute infectious disease caused by African swine fever virus infecting pigs,which has caused serious harm to the breeding industry in our country.The large number of proteins encoded by African swine fever virus with unclear immunogenicity has hindered the development of genetically engineered vaccines.In order to obtain an effective genetically engineered vaccine,it is necessary to screen and identify the immunogenicity of African swine fever antigens.Insect baculovirus expression system is a very widely used eukaryotic expression system,which can modify proteins to make them highly consistent with natural proteins in terms of structure and immunogenicity.In order to maximize the expression capacity of the baculovirus expression system,accurate virus titers need to be obtained to optimize the multiplicity of infection.In this experiment,the endpoint dilution method was used to measure the virus titer.First,the Hi Bi T tag and the fluorescent protein GFP were fused and cloned into the vector to construct a new vector p UCDM-p10-GFPHi Bi T.The recombinant vector was then transfected into insect cells,and the virus titer was determined after virus amplification.The luminescence intensity of Hi Bi T and the fluorescence results of GFP were recorded,and the successful expression of GFP protein was identified by Western-blot.After analysis,it was found that Hi Bi T tag can replace GFP protein as the reporter gene of baculovirus.Subsequent validation in different recombinant baculoviruses showed that the Hi Bi T tag could be used for baculovirus titer determination.Then,the expression of three antigen proteins of CD2 v,P30 and CD2 v transmembrane transport region which on the envelope of African swine fever virus was studied.The above three proteins were cloned into the vector p UCDM-p10-GFPHi Bi T,and then transfected into insect Sf9 cells for recombinant protein expression.The recombinant protein was successfully obtained by Western-blot identification and affinity column chromatography,and finally injected into mice for immunogenicity identification.The results showed that the highest specific antibody levels induced by the three antigen proteins were 2.85,3.05,and 3.02,respectively,which proved that the three proteins were successfully expressed in the baculovirus system and had good immunogenicity.This study explored the feasibility of using the Hi Bi T tag to measure the baculovirus titer,and constructed a new expression vector p UCDM-p10-GFP-Hi Bi T.And screened and expressed three antigenic proteins of African swine fever virus CD2 v,P30 and CD2 v transmembrane transport region and identified immunogenicity.It has made useful explorations for the development of safe and efficient African swine fever virus subunit vaccines.