【摘要】 目的 :构建带信号肽人胰岛素原突变体的真核表达载体并在COS - 7细胞表达出成熟的人胰岛素。方法 :采用PCR体外定点突变技术 (PCR -SDM) ,将普通细胞内furin蛋白酶的识别和切割位点Arg -X -Lys -Arg -样序列 ,引入人胰岛素原C肽两端 ,同时加上信号肽、Kozak序列和限制性内切酶的酶切位点并克隆至载体pcDNA3.1(+)中 ,构建成人胰岛素原突变体的真核表达载体 ,经测序验证后转染COS - 7细胞 ,RT -PCR、Western印迹、放射免疫法检测其在COS - 7细胞中的表达。结果 :DNA测序结果表明在预期位点突变符合要求 ,经RT -PCR、Western印迹、放射免疫鉴定 ,构建的人胰岛素原突变体的真核表达载体转染的COS - 7细胞可表达、分泌人成熟胰岛素。结论 :构建了人胰岛素原突变体的真核表达载体 ,并成功地在COS - 7细胞中获得人胰岛素的分泌表达更多还原

【Abstract】 Objective:To construct a mutant of human proinsulin with signal peptide code,so that it may be expressed in eukaryotic system to produce mature human insulin in COS-7 cells.Methods:Human proinsulin gene was mutated by the polymerase chain reaction (PCR) for the site-directed mutagenesis method.Two furin recognized sites of Arg-X-Lys-Arg-motifs were introduced into human proinsulin.Signal peptide,Kozak sequence and restriction enzyme cutting site were added and cloned into pcDNA3.1(+) vector and verified by sequencing analysis.Eukaryotic expression vector was constructed and transfected into COS-7 cells.Insulin were evaluated respectively with RT-PCR,radioimmunoassay and Western blot analysis.Results:The sequencing analysis showed that the mutation sites were correct,and the mutant of human proinsulin was constructed successfully.Human insulin mRNA was highly expressed in transfected COS-7 cells,and secretive recombinant human insulin was detected in the culture supernatant of transfected COS-7 cells by Western blotting.Conclusion:A mutant human proinsulin eukaryotic expression vector is obtained and secretive recombinant human insulin is successfully expressed in COS-7 cells.更多还原