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目的构建能够靶向作用于大鼠胰岛细胞的绿色荧光蛋白及人类胰岛素原表达载体,为糖尿病的特异性基因治疗奠定基础。方法采用PCR法从SD大鼠全血基因组中扩增大鼠胰岛素I启动子(RIP)片段,将其克隆至pIRES2-EGFP质粒载体的启动子位点,用重叠延伸PCR法从人类全血基因组DNA扩增获得人类胰岛素原基因片段,插入pIRES2-EGFP质粒载体的多克隆位点,构建大鼠胰岛素I启动子驱动的人类胰岛素原及绿色荧光蛋白表达载体。结果构建的RIP-人类胰岛素原及绿色荧光蛋白表达载体经酶切及测序结果均正确。结论构建了受大鼠胰岛素I启动子驱动的人类胰岛素原及EGFP表达载体,为开展糖尿病的特异性基因治疗奠定了基础。
Objective To construct green fluorescent protein and human proinsulin expression vector targeting rat pancreatic islet cells and to lay the foundation for the gene therapy of diabetes. Methods The rat insulin I promoter (RIP) fragment was amplified by PCR from whole blood of SD rats and cloned into the promoter region of pIRES2-EGFP plasmid vector. The human proinsulin gene fragment was amplified by DNA and inserted into the multiple cloning site of the pIRES2-EGFP plasmid vector to construct the rat insulin-I promoter-driven human proinsulin and green fluorescent protein expression vector. Results The constructed RIP-human proinsulin and green fluorescent protein expression vector were correctly digested and sequenced. Conclusion The constructed human proinsulin and EGFP expression vector driven by rat insulin I promoter laid the foundation for the gene therapy of diabetes.