微小牛蜱Bm91基因的克隆及其真核表达载体的构建  

Cloning of Bm91 gene of Boophilus microplus and construction of eukaryotic expression vector with Bm91 gene

作　　者：王玙[1,2] 曾巧英[1] 罗建勋[2] 马米玲[2] 关贵全[2] 殷宏[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]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

出　　处：《甘肃农业大学学报》2009年第1期21-26,共6页Journal of Gansu Agricultural University

基　　金：国家863项目;国家自然资源平台项目;国家支撑计划

摘　　要：参照GenBank中收录的微小牛蜱Bm91基因的核苷酸序列,设计了1对特异性引物,从我国微小牛蜱半饱血雌性成蜱肠道和唾液腺研磨物中提取总RNA,利用M-MLV RTase cDNA Synthesis Kit合成双链cDNA,并采用PCR技术扩增获得的Bm91基因,将其克隆到pGEM-T Easy载体中并进行PCR、酶切和测序分析鉴定.结果表明:所克隆的Bm91基因序列与GenBank上收录的Bm91基因序列的同源性达97.1%,而且该片段含有完整的开放阅读框;将该基因从克隆载体上用EcoRⅠ单酶切消化并亚克隆到巴斯德毕赤酵母分泌型表达载体pP1C9K的EcoRⅠ酶切位点,再次进行PCR、酶切和测序分析鉴定,结果表明成功构建并获得了微小牛蜱Bm91基因的重组真核表达载体pP1C9K-Bm91.The total RNA was extracted from the salivary glands and the intestine of semiengorged adult female Boophilus microplus and double eDNA was synthesized using M-MLV RTase eDNA Synthesis Kit. According to the nucleotide sequences of Bin91 published in GenBank, a pair of primers was designed and a 1 908 bp Bin91 gene was amplified by PCR. The target gene was subcloned into pGEM-T Easy vector and tested by PCR, enzyme digestion and sequencing. The sequencing showed that the cloned Bin91 gene shared 97.1 % homology with the date published in GenBank and this fragment contained the complete open reading frame of Bin91 gene. Then the gene was cleaved from pGEM-T Easy vector by EcoR I and subcloned into pPIC9K secretory expression vector of Pichia pastoris. The recombinant eukaryotic expression vector of designated pPIC9K-Bmgl was constructed successfully.

关 键 词：微小牛蜱 Bm91基因 真核表达载体 

分 类 号：S852.746[农业科学—基础兽医学]