受血糖调控的定点突变胰岛素原真核表达载体的构建

Construction of mammalian glucose-stimulated insulin expression vector by combining promoter of glucose 6-phosphatase with modified proinsulin cDNA

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 a mammalian expression vector, bearing proinsulin cDNA ( modified for processing by furin) driven by glucose-6-phosphatase promoter to be used for gene therapy of diabetes. Methods Using site-directed mutagenesis( splicing by overlap extension) , furin consensus cleavage sequences were introduced into proinsulin cDNA, and the new sequence was named INS/furin, which allowed for the recognition and procedure of furin and efficient proteolytic maturation of proinsulin to insulin within most of the cells containing furin. Subsequently , INS/furin was subcloned into the multiple clone sites B of the mammalian expression vector pIRES after substitution of CMV promotor in pIRES with glucose-6-phosphatase promoter. Results Two combinations of double enzyme digestion, SgfⅠ and SacⅠ、XbaⅠ and NotⅠ, as well as sequencing verified the vectors containing targeted gene in specific sites. Conclusion The recombined mammalian expression vector, containing both G6p promoter and site-mutated proinsulin, is a novel ideal candidate vector for diabetic gene therapy.