Silkworm Baculovirus Surface Display Of SARS-CoV N.RBD Protein And Immunogenicity Of The Displayed Protein

Severe Acute Respiratory Syndrome, also known as infectious atypical pneumonia,hereinafter referred to as SARS, is a kind of new respiratory infectious disease caused bySARS-CoV.Using Bombyx mori baculovirus surface display technique to simultaneously display themajor structural protein N (Nucleocapsid protein) and the Receptor Binding Domain (RBD) of Sprotein (Spike protein), the recombinant baculovirus vBmgp64-N-RBD was purified bylow-speed, high-speed and ultra-centrifugation and was inactived to get the antiserum throughinoculating the BALB/c, then further analyzed its immunogenicity, in order to lay a certainfoundation for SARS bivalent genetic engineering subunit vaccine. At the same time, constructedand dealt with the recombinant baculovirus vBmgp64-N and vBmgp64-RBD the same way asthe vBmgp64-N-RBD, then studied the immunogenicity of three recombinant baculovirus bycomparing the three kinds of antiserum. First,by E.coli BL21expression system, expressed the Nprotein and RBD protein respectively, purified protein of interest by Ni+affinity chromatography,and gained highly purified N protein and RBD protein respectively, then inoculated NewZealand male rabbit with these proteins to get polyclonal antibody. After purification, antibodieswere measured their titers by indirect ELISA method respectively, the titer of the anti-N serumwas1：25600, the titer of the anti-RBD serum was1∶12800. Using Bac-to-Bac system to gainrecombinant baculovirus vBmgp64-N, vBmgp64-RBD, vBmgp64-N-RBD, analyzed bySDS-PAGE, Western Blotting and immunofluorescene microscopy, the results showed that thecorresponding target protein of three recombinant baculovirus were successfully located on thecytoplasmic membrance of BmN cell.In this article, we inoculated BALB/c mice with the threepurified recombinant baculovirus particles after inactivation and preliminary studiedimmunogenicity of target protein. Three types of recombinant baculovirus were injected intomice to obtain the corresponding antiserum,the titer of which was even1∶6400. Neutralizationassays showed the neutralization titers of antiserums coming from vBmgp64-N, vBmgp64-RBD,vBmgp64-N-RBD inoculated BALB/c mice were1∶16,1∶48,1∶50respectively. These resultsconfirmed N, RBD protein had good antigenicity and antibody had higher titer.This study attemped to develop a new way for SARS vaccine and achieved good tentativeresults, which laid a foundation for subsequent vaccine research and development, and had amethodological innovation of SARS vaccine. In this article, surface display technology on BmNcell is used to develop new vaccines for the first time. Although the SARS is being silent for many years, making it become a kind of technical reserves or strategic reserves is of greatsignificance.