Expression of E2 Gene from Classical Swine Fever HL-LY Isolate in Pichia Pastoris

摘要

A cDNA of classical swine fever virus (CSFV) HL-LY isolate E2 gene was amplified by RT-PCR with the virus total RNA as template. The amplified cDNA fragment was cloned into vector pMD-18 T and sequenced. The full length E2 gene was inserted downstream of the Pichia pastoris vector pPIC9 a-mating factor secretion signal. Then the recombinant (pPIC9-E2) was lineared and introduced into the host genome of Pichia pastoris GS115 by electrotransformation. The His~+ Mut~+ transformations screened out on minimal medium (MM) and MD plates was cultured in MM supplemented with methanol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficient expression and secretion of E2 protein into culture fluid. The yield of secreted E2 protein was estimated to be 0. 34g/l with the optimal expression conditions being explored. This E2 protein was detected on Western-blot with anti-CSFV serum which showed E2 protein has specifically natural antigenivity.