Secretive Expression of White Spot Syndrome Virus Envelope Proteins VP28 and VP26 in Pichia pastoris Induced by Constitutive Promoter

Abstract:

WSSV has been a globally recognized highly harmful pathogen in shrimp farming industry that causes tremendous economic loss. The envelope proteins of WSSV, VP28 and VP26, play important roles in interacting with host cells, initiating virus infection and mediating virus intrusion. In this study, we used pGAPZαA as the expression vector and X-33 Pichia pastoris as the host cell to express VP28 and VP26 in a secretive manner. The coding sequences of VP28 and VP26 (GenBank: AF332093.3) were amplified from WSSV using PCR, and the sequences of EcoR I (GAATTC) and Xba I (TCTAGA) were added to the 5¢ and 3¢ ends of the target genes. The purified PCR products were then cloned into the EcoR I/Xba I sites of the pGAPZαA vector. Sequencing analysis verified whether the target genes were correctly inserted into the reading frame. The construct was linearized by Bln I (Avr Ⅱ) and then was integrated into P. pastoris X-33 through electroporation while being screened by Zeocin. The expressed proteins were identified with SDS-PAGE. The VP28 and VP26 recombinant proteins could not be detected by coomassie brilliant blue R250 staining, however, the bands of the fusion proteins appeared after silver staining. The sizes of VP28 and VP26 fusion proteins were about 32 kDa. These results suggest that the P. pastoris system was effective in expressing WSSV envelope proteins VP28 and VP26, although the expression level was not sufficient. Nonetheless, our study still established a novel tool for the study of subunit vaccine, and provided basic information for the large scale vaccine production.