简化的人纤溶酶原饼环区5基因的克隆及其在大肠杆菌中的融合表达  

Cloning and Fusion Expression of Predigested Human Plasminogen Kringle-5 in Escherichia coli

作　　者：王健琪[1] 翟朝阳[1] 孙剑[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物化学与分子生物学实验室,成都610041

出　　处：《四川大学学报（自然科学版）》2006年第2期435-440,共6页Journal of Sichuan University(Natural Science Edition)

摘　　要：从正常人肝脏组织中提取总RNA,合理设计引物,利用RT-PCR直接得到简化的hPK-5基因.将该基因克隆至原核表达质粒pGEX-1λT,将此重组载体转化大肠杆菌JM109,经PCR、酶切及测序鉴定阳性克隆.用IPTG诱导阳性克隆表达融和蛋白,SDS-PAGE观察表达产物.再通过Western-Blotting鉴定.简化的hPK-5基因的RT-PCR产物为264bp.通过酶切、测序等方法鉴定简化的hPK-5基因正确克隆至原核表达质粒pGEX-1λT中.重组质粒pGEX-1λT/predhPK-5在大肠杆菌中成功地表达出了GST/predhPK-5融合蛋白,并通过免疫学测定,相对分子质量约为3.6×104,与理论值3.58×104相符.目的蛋白表达量占菌体总蛋白的23.6%.The predhPK-5 gene was cloned from hominal liver tissue with RT-PCR, and then the predhPK-5 gene was cloned into prokaryotic expression plasmid pGEX-1λT to construct the co-expression plasrnid pGEX-1λT/predhPK-5. The pGEX-1λT/predhPK-5 was transformed into E. coli JM109. The positive clone was i- dentified by PCR,restriction enzyme analysis and sequence analysis. The fusion protein GST/predhPK-5 was expressed by IPTG induced and identified by Western-Blotting. The size of RT-PCR product of predhPK-5 is 264bp. The evidences of PCR, enzyme digestion and sequence analysis confirmed that predhPK-5 gene has been correctly recombinant with pGEX-1λT. The recombinant expression plasrnid pGEX-1λT/predhPK-5 successfully expressed GST/predhPK-5 fusion protein in E. coli JM109, and the Mr of the fusion protein is about 3.6 × 10^4, which is according with the Mr forecasted by Expert Protein Analysis System on interuet. And the fusion protein expression quantity occupies 23.6 % according to the whole expression protein.

关 键 词：简化的hPK-5 Kimgle环 基因重组 融和表达 

分 类 号：Q78[生物学—分子生物学]