微小牛蜱Bm86基因的真核表达  

Eukaryotic expression of Bm86 gene of Boophilus microplus in Pichia pastoris

作　　者：李文卉[1] 罗建勋[1] 殷宏[1] 关贵全[1] 马米玲[1] 刘志杰[1] 刘爱红[1] 党志胜[1] 高金亮[1] 任巧云[1] 

机构地区：[1]中国农业科学院兰州兽医研究所甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科技》2005年第9期718-722,共5页Chinese Journal of Veterinary Science and Technology

基　　金：国家高技术研究发展计划(863)项目(2003AA241110);国家自然科学基金与JSPS国际合作项目(30270992)

摘　　要：采用RT-PCR技术从微小牛蜱饥饿幼蜱破解物中扩增到Bm86基因,将其与巴斯德毕赤酵母分泌型表达载体pPIC9K重组构建了重组表达载体pPIC9K-Bm86,测序正确后将其用SacⅠ内切酶线性化后采用电穿孔法转化巴斯德毕赤酵母菌GS115,经G418抗性筛选高拷贝重组菌株后用甲醇诱导表达,SDS-PAGE和Western-blotting分析结果表明,诱导表达的培养上清液中表达出具有反应活性的68 ku重组Bm86蛋白,目的蛋白约占培养上清液中蛋白总量的32%以上,诱导96 h目的蛋白的表达量为0.36 mg/mL。A Bm86 gene was cloned from larvae of Boophilus microplus, then inserted into a secretory expression vector pPICgK, and the expression vector pPICgK-Bm86 was constructed successfully. The recombinant was linearized with Sac Ⅰ and electroporated into the yeast cell GS115. 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. Results showed that Bm86 gene was expressed successfully at 48th hour. The concentration of expressed Bm86 protein was 0.36 mg/mL and its molecular weight is approximately 68 ku. The expressed Bm86 protein reached up to 32% of total proteins in culture supernatant and could be specifically recognized by the positive serum of rabbit infected by B. microplus.

关 键 词：微小牛蜱 Bm86基因 毕赤酵母 真核表达 

分 类 号：S852.746[农业科学—基础兽医学] Q786[农业科学—兽医学]