【摘要】 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。更多还原

【Abstract】 Aim To construct and identify an eukaryotic expression plasmid of human proinsulin and EGFP gene driven by rat insulin 1 promoter. Method A rat insulin 1 promoter (RIP) fragment,spanning nucleotides -412 to +43(from transcription start site 4006,rat insulin 1,GenBank J00747)was isolated using PCR method and inserted into the pIRES2-EGFP plasmid replacing original CMV promoter. The human proinsulin gene was isolated from human whole blood genome DNA using overlap extension PCR and cloned into the multiple cloning site of pIRES2-EGFP plasmid. Results The rat insulin 1 promoter (RIP) fragment and the full length cDNA sequence of human proinsulin was obtained. Digestion with restriction endonuclease and sequencing of the recombinant plasmids both confirmed the results. Conclusions The human proinsulin and EGFP-expressing plasmid vectors were constructed under the rat insulin 1 promoter.更多还原