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目的 构建受血糖调控的含葡萄糖-6-磷酸酶(G6p)启动子的定点突变胰岛素原基因真核表达载体。方法 应用基因突变技术(重叠延伸拼接法)改变胰岛素原C肽两端的氨基酸序列,使之能被存在于大多数细胞中的furin酶所识别并剪切为成熟的胰岛素,新序列命名为INS/furin;应用基因重组技术将G6p启动子序列替代真核表达载体pIRES中的CMV启动子序列,并将INS/furin亚克隆至pIRES的多克隆位点B。结果 Sgf Ⅰ和Sac Ⅰ、Xba Ⅰ和Not Ⅰ两种组合的双酶切及特定引物测序均证实载体构建成功。结论 带有G6p启动子的含定点突变胰岛素原基因的真核表达载体有望成为糖尿病基因治疗理想的候选载体。
Objective To construct eukaryotic expression vector of site-directed mutated proinsulin gene with glucose-6-phosphatase (G6p) promoter regulated by blood glucose. Methods The amino acid sequence at both ends of proinsulin C peptide was changed by gene mutation technique (overlap extension splicing method), which was recognized and cut into mature insulin by furin enzyme existing in most cells. The new sequence was named as INS / furin. The G6p promoter sequence was substituted for the CMV promoter sequence in the eukaryotic expression vector pIRES using gene recombination technique, and INS / furin was subcloned into the multi-cloning site B of pIRES. Results The double digestion of Sgf Ⅰ and Sac Ⅰ, Xba Ⅰ and Not Ⅰ and the sequencing of specific primers all confirmed that the vector was successfully constructed. Conclusion The eukaryotic expression vector containing the site-directed mutated proinsulin gene with the G6p promoter is expected to be an ideal candidate vector for gene therapy of diabetes.