微小牛蜱磷酸丙糖异构酶基因片段的克隆与原核表达

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。

Cloning and Expression of Triosephosphate Isomerase Gene of Boophilus microplus

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 750 bp 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.