微小牛蜱Bm91基因的原核表达  

Prokaryotic Expression of Bm91 Gene of Boophilus microplus

作　　者：马米玲[1] 刘军龙[1] 关贵全[1] 刘志杰[1] 郝雪峰[1] 王玙[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]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,农业部草食动物疫病重点实验室,甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《动物医学进展》2010年第12期6-9,共4页Progress In Veterinary Medicine

基　　金：国家支撑计划项目(2007BAD40B06);国家863项目(2006AA10A207);国家自然资源平台项目(2005DKA21100)

摘　　要：根据GenBank收录的微小牛蜱Bm91基因序列设计一对表达引物,以微小牛蜱幼蜱总RNA反转录合成的cDNA为模板,扩增获得了Bm91基因,其长度为1 908 bp,包含一个大小为1 833 bp的完整开放阅读框,编码611个氨基酸,该蛋白预期分子质量为83 ku。将Bm91基因片段亚克隆到原核表达载体pET-30a,构建重组原核表达载体pET-30a-Bm91,转化BL21宿主菌,经IPTG诱导,发现在0.8 mmol/LIPTG,诱导时间2 h,温度37℃条件下,目的重组蛋白表达量最大,约占蛋白总量的20%。SDS-PAGE检测表明,表达产物为分子质量为83 ku的融合蛋白,与预期大小一致;Western blot分析表明,表达产物能被兔抗微小牛蜱阳性血清所识别。One pair of primers were designed based on the Bm91 gene of Boophilus microplus published in GenBank.Bm91 gene was amplified from total RNA of larva B.microplus.The sequence result showed that Bm91 gene has 1 908 nucleotide residue encoding 611 amino acid,the deduced protein is 83 ku.The gene was subcloned into pET-30a vector and expressed in E.coli BL21 induced by IPTG.SDS-PAGE analysis showed the recombinant protein was highest expressed in 37 ℃,2 h,0.8 mmol/L IPTG and about 83 ku,and Bm91 expression level could reach 20% of total E.coli proteins.Western-blot analysis suggested the recombinant protein couble be recognized by rabbit anti B.microplus serum.

关 键 词：微小牛蜱 Bm91基因 原核表达 

分 类 号：Q786[生物学—分子生物学]