人tumstatin基因的克隆、表达和抗体制备

Cloning and Expression of Human Tumstatin in Escherichia coli and Preparation of Its Polyclonal Antiserum

应用PCR技术从人胎肺cDNA文库中扩增tumstatin基因编码序列,克隆至pET_3c载体构建非融合表达的重组质粒pET_3c_tum,并在大肠杆菌E.coliBL21(DE3)中获得高效表达。表达蛋白经SDS_聚丙烯酰胺凝胶电泳(SDS_PAGE)分离、切胶回收后免疫新西兰大白兔,获得ELISA效价高达1∶1000的特异性兔抗人tumstatin抗血清。

Human tumstatin cDNA was amplified from embryo lung cDNA library by PCR technique and cloned into expression vector pET-3c resulting in recombinant plasmid pET-3c-tum which was subsequently transformed into Escherichia coli BL21 （DE3）. Tumstatin was expressed by 0.5 mmol/L IPTG induction at 37 ℃ for 3 hours as a 27 000 protein as detected by SDS-PAGE and accounted for more than 30% of total bacterial protein as estimated by densitometry. Tumstatin was recovered and purified from the separated target band on SDS-PAGE gels and used to immunize New Zealand rabbits and a polyclonal antiserum against human tumstatin with the titer of 1 ： 1 000 as detected by ELISA was obtatined.