微小牛蜱Bm86基因的原核表达与表达条件的优化  

Prokaryotic Expression of Bm86 Gene of Boophilus microplus and Optimization of the Expression Condition

作　　者：马米玲[1] 关贵全[1] 李有全[1] 刘爱红[1] 任巧云[1] 牛庆丽[1] 殷宏[1] 罗建勋[1] 

机构地区：[1]家畜疫病病原生物国家重点实验室,甘肃省动物寄生虫病重点实验室,中国农业科学院兰州兽医研究所,兰州730046

出　　处：《中国寄生虫学与寄生虫病杂志》2009年第6期531-533,共3页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家科技支撑计划项目(No.2007BAD40B06);国家高技术研究发展计划(863计划)(No.2006AA10A207);科技部自然资源平台项目(No.2005DKA21100)~~

摘　　要：根据微小牛蜱Bm86基因序列设计引物,在重组质粒pMD18-T-Bm86中克隆,并将其定向亚克隆入原核表达载体pGEX-4T-1,转化至大肠埃希菌BL21(DE3)株,用不同浓度异丙基-β-D-硫代半乳糖苷(IPTG)在不同时间进行诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测结果表明,37℃条件下经1mmol/LIPTG诱导8h后,目的重组蛋白表达量最大,表达相对分子质量(Mr)约为94000的包涵体蛋白,与预期大小一致,目的蛋白约占蛋白总量的29%。蛋白质印迹(Western blotting)分析表明,该重组蛋白可被兔抗微小牛蜱全蜱阳性血清所识别。A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates,and subcloned into the prokaryotic plasmid pGEX-4T-1.The recombined plasmid was transformed into E.coli BL21（DE3） and followed by expression of the protein induced by different concentration of IPTG for different time.SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST（Mr 94 000） after being induced with IPTG. High level expression of Bm86-GST was found at 1 mmol/L IPTG condition after incubation for 8 h at 37 ℃, and the expression level of the recom-binant Bm86-GST reached up to 29% of total E.coli proteins. Western-blotting analysis showed that the recombinant Bm86- GST was recognized by the rabbit anti-B, microplus positive serum.

关 键 词：微小牛蜱 Bm86基因 表达 

分 类 号：R384.4[医药卫生—医学寄生虫学]