微小牛蜱磷酸丙糖异构酶基因片段的克隆与原核表达  

Cloning and Expression of Triosephosphate Isomerase Gene of Boophilus microplus

作　　者：梁怡琳[1] 高攀[1] 柯泽楷[1] 罗新萍[1] 刘志刚[1] 

机构地区：[1]深圳大学过敏反应与免疫学研究所,深圳518060

出　　处：《中国寄生虫学与寄生虫病杂志》2012年第4期331-332,共2页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.39660073);广东省科技重点专项(No.2003A3080502);深圳大学校创新科研团队基金(No.200904)~~

摘　　要：为克隆和表达微小牛蜱磷酸丙糖异构酶(triosephosphate isomerase,Tim)编码基因,提取微小牛蜱成虫总RNA,采用RT-PCR方法扩增目的基因,将其连入pMD-18T载体,经酶切和测序鉴定后,将正确的目的基因与pET-28a表达载体连接,并转入大肠埃希菌(E.coli)Rosetta(DE3)中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定分析。结果发现,克隆所得的微小牛蜱成虫tim基因片段长度为750bp(登录号为JX112888),其中开放阅读框为750 bp,编码249个氨基酸,与微小牛蜱胚胎中克隆出的tim基因序列的同源性为99%。SDS-PAGE结果显示,重组蛋白Tim相对分子质量(Mr)约为27 000。Total RNA was extracted from adult Boophilus microplus. RT-PCR was used to amplify the gene and the fragment was subcloned into the expression vector pET-28a. The cloned gene was expressed in E. coli Rosseta （DE3）, induced by IPTG, and identified by SDS-PAGE. The results showed that the triosephosphate isomerase （tim） gene of B. microplus has 750bp and encodes 249 amino acids （GenBank No. JX112888）. The cloned tim gene shares 99% homology with that in tick embryos. The relative molecular weight （Mr） of the expressed recombinant protein is about 27 000.

关 键 词：微小牛蜱 磷酸丙糖异构酶 克隆 表达 

分 类 号：R384.41[医药卫生—医学寄生虫学]