葡萄糖调控的胰岛素原真核表达载体的构建

Construction of eukaryotic glucose-regulated insulin expression vector

【目的】构建胰岛素原真核表达载体,在肝细胞内实现葡萄糖调控下的成熟胰岛素的表达。【方法】(1)RT-PCR方法从胎儿胰腺获取野生型人胰岛素原cDNA;(2)重叠延伸PCR法在人胰岛素原cDNA上制造两个点突变,该胰岛素原cDNA片段命名为胰岛素/furin(INS/furin);(3)将INS/furin亚克隆至含葡萄糖反应元件(GlRE)的p(GlRE)3BP-1Luc的多克隆位点上。【结果】HindⅢ和EcoRV酶切及测序均证实载体p(GlRE)3BP-11×furin构建成功。【结论】含葡萄糖反应元件的点突变胰岛素原基因真核表达载体p(GlRE)3BP-11×furin有望成为糖尿病基因治疗理想的候选载体之一。

ObjectiveA eukaryotic expression vector was constructed in order to realize glucose-regulated mature insulin gene express in hepatocytes.MethodsNative human promisulin cDNA was obtained from fetus pancreas by RT-PCR. Using site-directed mutagenesis(overlap extension PCR), furin consensus cleavage sequence was introduced into proinsulin cDNA, and the new sequence was named INS/furin,which allowed for the recognition and processed of furin and efficient proteolytic maturation of proinsulin to insulin within most of the cells containing furin. Subquently, INS/furin was subcloned into the multiple clone sites of plasmid p(GlRE)_3BP-1Luc,which containing glucose reaction element GlRE in its promotor.ResultsDouble digestion with Hind and EcoRV, as well as sequencing verified the vector containing target gene in specific sites.ConclusionThe recombinant mammalian expression vector p(GlRE)_3BP-11furin, containing glucose reaction element and site-mutated proinsulin,is a novel ideal candidate vector for diabetic gene therapy.