J亚群禽白血病病毒gp85基因在毕赤酵母中的分泌表达

Secretory Expression of Avian Leukosis Virus Subgroup J gp85 Gene in Pichia pastoris

为获得J亚群禽白血病病毒gp85蛋白,将gp85基因克隆至毕赤酵母表达载体pPIC9K,构建了pPIC9K-gp85表达载体。将鉴定正确的表达载体经SalⅠ酶切线性化后电转化毕赤酵母宿主菌GS115,经MD及G418抗性平板筛选后挑取阳性克隆进行PCR鉴定,获得重组酵母菌株GS115/pPIC9K-gp85。对阳性重组菌进行诱导表达,表达产物经SDS-PAGE、Western blotting和Dot-ELISA分析,结果表明,gp85基因在毕赤酵母中获得分泌表达,表达产物与J亚群禽白血病病毒多克隆抗体有良好的反应活性,分泌表达量达40mg/L。本研究为J亚群禽白血病病毒诊断方法的建立及gp85蛋白的深入研究奠定了基础。

To express recombinant gp85 protein of avian leukosis virus subgroup J（ALV-J） in P.pastoris,the gp85 gene was amplified and cloned into the pPIC9K vector.The recombinant plasmid pPIC9K-gp85 was identified,linearized with Sal Ⅰ and transformed into P.pastoris strain GS115 by electroporation.Transformants were selected on MD plates then on YPD plates containing G418 and confirmed by PCR.SDS-PAGE assay demonstrated that gp85 protein was expressed and secreted into the culture medium.The expression product amounted to 40 mg/L of culture under optimized condition.The recombinant gp85 protein showed good antigenicity in Western blotting and Dot-ELISA assays.This study laid the foundation for further study of the gp85 protein and the development of the diagnostic methods for ALV-J.