山羊痘病毒P32基因真核表达载体的构建及表达  

Construction and expression of recombinant antigen P32 gene of goat poxvirus

作　　者：谢芳[1,2] 张强[1] 颜新敏[1] 吴国华[1] 李健[1] 朱海霞[1] 郭爱疆[1] 韩若婵[1] 施程洪[1] 鞠厚斌[1] 朱彩珠[1] 岳城[2] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,甘肃兰州730046 [2]新疆农业大学动物医学院,新疆乌鲁木齐830052

出　　处：《中国兽医学报》2009年第8期982-985,共4页Chinese Journal of Veterinary Science

基　　金：国家基础平台建设资助项目(2005DKA21205-3)

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31000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。The structural protein P32 gene of the goat poxvirus in the recombinant plasmid pMD18-P32 was subcloned into the Pichia pastoris expression vector pPICgK. The resultant recombinant plasmid pPICgK-P32 was transformed into P. pastoris GSl15 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 aim protein was expressed in P. pastoris, and had an immunological activity, the protein could be recognized by anti-serum against goat poxvirus.

关 键 词：山羊痘病毒 P32基因 毕赤酵母 诱导表达 

分 类 号：S852.65[农业科学—基础兽医学]