Cloning And Expression Of The Capripoxvirus P32 Gene In Pichia Pastoris

Capripoxvirus(CPV) can cause the sheeppox, goatpox and lumpy skin disease. Capripox(sheeppox and goatpox) is classified as group A diseases by Office International des Epizooties(OIE) and group first disease in china. The sequence of P32 gene is conserved to CPV genome, is specific to CPV, and contains a major antigentic determinant.The P32 gene was amplified by PCR, and was inserted into pMD18-T vector resulting in recombinant plasmid pMD18-P32. The recombinant plasmid was identified by PCR, and digestion by restriction endonuclease EcoRâ… and Notâ… ; the results showed that P32 gene was obtained. Compared the sequence with the AF124517,AY159333 in Genbank, the homologies were 98.2%,99.4%, respectively.Then the P32 gene was subcloned into the Pichia pastoris expression vector pPIC9K, resulting in recombinant plasmid pPIC9K-P32. The plasmid was transformed into Pichia pastoris GS115 by electroporation. The multi-copy recombinant P.pastoris strains were selected out by G418 and induced by methanol. The expression product was analyzed by SDS-PAGE and Western-blot. The results indicated that the P32 protein was expressed in supernatant, and could react with the positive serums of CPV.