Secretion Expression of the Gene Encoding Classical Swine Fever Virus E2 A/D Antigenic Domain in Pichia pastoris and Identification of the Protein

Based on the fact that the envelope glycoprotein E2 which can protect swine from virulent attack of Classical swine fever virus(CSFV) has two structural antigenic domains B/C and A/D, a pair of specific primers was designed to amplify the gene fragment encoding A/D antigenic domain of E2 protein. The 373 bp PCR product was directionally cloned into Pichia pastoris secretory expression vector pPICZαC under the control of the AOX1 promoter and α-factor secretion signal sequence. After being linearized with restriction endonuclease Dra I, the recombinant plasmid was transformed into Pichia pastoris by electroporation. Five transformants with high copies were acquired when selected under ZeocinTM and were induced with methanol. SDS-PAGE indicated that the supernatant of the induced P. pastoris culture contained the recombinant protein E2 (175.8ug/mL). Western-blot analysis proved that the recombinant protein had good reactimmunity against positive CSFV serum. N-glycosylation analysis of expressed products showed that the recombinant protein was glycosylated in the process of secretion. Our research provided a basis to develop sub-unit vaccine and diagnostic antigen against CSFV.