人胰岛素原基因突变体的构建

目的　完成人胰岛素原基因的定点突变 ,使之能在普通细胞中被Furin蛋白酶识别去除C肽分泌成熟胰岛素。方法　采用PCR体外定点突变技术 (PCRsite directedmutagenesis ,PCR SDM) ,设计 4对引物 ,引入 5个Furin识别的突变位点 ,通过重叠延伸法PCR扩增 ,使人胰岛素原编码基因B10密码子由CAC突变为GAC ;C3密码子由GAA突变为AAA ;C4密码子由GCT突变为CGT ;C3 2密码子由CTT突变为CGT ,将扩增片段克隆入T载体并测序。结果　DNA测序结果表明在预期位点突变符合要求。结论　应用PCR诱导突变技术 ,在体外能准确、简便有效的诱导胰岛素原基因突变 ,使体外应用非内分泌细胞构建高效分泌成熟胰岛素的细胞克隆成为可能。

Construction of a mutant human proinsulin gene

Objective To construct a mutant of human proinsulin gene for the purpose of removal of C peptide in non-endocrine cells by Furin proteolytic enzyme to excrete mature insulin. Methods We used polymerase chain reaction (PCR) for the site-directed mutagenesis. Four sets of primers were designed according to the gene sequence of human proinsulin and mismatches were introduced into F02 for histidine (CAC) to aspartic acid (GAC) substitution at site B10, F03/R03 for glutamic acid (GAA) to lysine (AAC) substitution at site C3, alanine (GCT) to arginine (CGT) substitution at site C4, and F04/R02 for leucine (CTT) to arginine (CGT) substitution at site C32. Mutagenesis was performed in a two-step PCR and the amplified fragments from the second PCR containing the mutation sites were subcloned into the T vector and verified by sequencing analysis. Results The sequencing analysis showed that the mutation sites were correct, and the mutant of human proinsulin was constructed successfully. Conclusion A mutant human proinsulin, successfully constructed by PCR for the site-directed mutagenesis, is potential to construct a cell clone to efficiently excret mature insulin with non-endocrine cells in vitro.