人FGF21原核表达载体的构建及重组蛋白表达

Preparation of Prokaryotic Expression Construct of Human FGF21 cDNA and Its Recombinant Protein Expression

构建人FGF21(fibroblast growth factor,FGF)cDNA的原核表达载体并诱导其重组蛋白表达。提取人肝脏总RNA后,经RT-PCR扩增获得目的片段,构建其T载体进行保存。再构建重组原核表达载体pET-28a(+)-h FGF21,重组质粒转化至大肠杆菌菌株BL21(DE3)中,在IPTG诱导下得到可溶性表达,采用亲和层析法纯化表达产物后,进行Western blot鉴定。成功构建重组质粒pET-28(+)-hFGF21,对其进行可溶性表达后成功纯化出his-hFGF21,经Western blot鉴定该融合蛋白可与FGF21抗体特异性结合。成功构建pET-28(+)-hFGF21,并可溶性表达his-hFGF21蛋白。

Preparation of prokaryotic expression constructs of human FGF21（fibroblast growth factor,FGF）cDNA and induction of recombinant hFGF21 protein expression. Total RNA was extracted from human liver, and the target cDNA fragment was obtained using RTPCR. The amplified cDNA fragment was cloned into pMD19-T for preservation. Then the expression construct p ET-28a（＋）-hFGF21 was successfully constructed and expressed with IPTG induction, and the his-hFGF21 protein was purified with histide-selective nickel affinity gel and identified by Western blot. Western blot analysis showed that the fusion protein had specific binding with a FGF21 antibody.