微小牛蜱Bm86基因的克隆与原核表达  

Cloning and expression of Bm86 gene of Boophilus microplus in E.coli

作　　者：樊瑞泉[1] 罗建勋[1] 杨孝朴[2] 殷宏[1] 高金亮[1] 关贵全[1] 刘志杰[1] 党志胜[1] 马米玲[1] 任巧云[1] 刘爱红[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甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《甘肃农业大学学报》2007年第1期15-19,共5页Journal of Gansu Agricultural University

基　　金：国家高新技术研究发展计划(863)项目(2006AA10A207);国家自然科学基金资助项目(30270992)

摘　　要：根据已发表的Bm86基因序列,设计表达型引物,利用RT-PCR技术,从微小牛蜱饥饿幼蜱的研磨物中扩增Bm86基因,将PCR产物连入pGEM-T Easy载体,构建重组克隆载体pGEM-T easy-Bm86.测序分析表明:克隆的微小牛蜱Bm86基因序列与GenBank上登录的Bm86基因的核苷酸和氨基酸序列的同源性分别为97%和95.6%.然后对重组克隆载体pGEM-T easy-Bm86进行双酶切,获得带有粘性末端的Bm86基因片段,并将此片段定向亚克隆入原核表达载体pGEX-4T-1,构建重组原核表达载体pGEX-4T-1-Bm86,将其转化到BL21宿主菌中,用IPTG进行诱导表达.SDS-PAGE检测表明表达产物为分子量为88 Ku的融合蛋白,目的蛋白约占蛋白总量的39%,表达量约为1.08 mg/mL.Western blot分析表明此表达产物能被兔抗微小牛蜱阳性血清所识别.Bin86 gene was amplified by RT--PCR from unfed larvae of Boophilus microplus and cloned into pGEM--T Easy vector. Sequen quence of the cloned Bin86 gene shar respectively. fragment dig Recombinant expression cing result showed tha ed97 % and 95.6% h pGEX-- 4T-- 1-- Bm86 t the nucleotide sequence and amino acid se- omologies with the data published in GenBank plasmid was constructed by sub-cloning Bin86 ested from pGEM--T easy--Bm86 plasmid into l nearized pGEX--4T-- 1 vector, pGEX--4T --1--Bm86 plasmid was expressed in E. coli induced by IPTG. SDS--PAGE showed that a fusion protein with a molecular weight of 88 Ku was expressed. The concentration of expressed Bm86 protein was about 1.08 mg/mL. The recombinant fusion protein was about 39 % of total protein of the host and it could be detected by rabbit anli--B, microplus positive serum in the Western Blot.

关 键 词：微小牛蜱 Bm86基因 克隆 表达 

分 类 号：Q786[生物学—分子生物学]