【摘要】 从重组克隆载体pMD18-P32中扩增出山羊痘病毒P32基因,与毕赤酵母分泌性表达载体pPIC9K相连接,构建重组表达载体pPIC9K-P32。重组质粒pPIC9K-P32用SalⅠ线性化后,与毕赤酵母(Pichia pastoris)GS115混合后电转化,使重组表达载体与酵母染色体发生同源重组。采用G418抗性梯度筛选法得到高拷贝重组菌株,甲醇诱导目的基因表达。SDS-PAGE和Western-blotting分析结果表明,用酵母成功表达出了31 kD的重组蛋白,该蛋白具有生物学活性,能被山羊痘阳性血清识别。更多还原

【Abstract】 The structural protein P32 gene of the goat pox virus in the recombinant plasmid pMD18-P32 was subcloned into the Pichia pastoris expression vector pPIC9K.The resultant recombinant plasmid pPIC9K-P32 was transformed into P.pastoris GS115 by electroporation.The multi-copy recombinant P.pastoris strains were screened by G418 and induced by methanol.The expressed product was analyzed by SDS-PAGE and Western-blotting tests.The results indicated that the recombinant protein was expressed in P.pastoris,and had biological activity,the protein could be recognized by anti-serum against goat pox virus.更多还原