人胰岛素A链与B链基因的化学合成、克隆与表达  

Chemical synthesis,cloning and expression of human insulin A chain and B chain gene separately in E. coli

作　　者：袁汉英[1] 闵永洁 王启松[2] M.Malhotra 

机构地区：[1]复旦大学遗传学研究所,上海200433 [2]复旦大学 [3]UNIDO.InternationalCentreforGeneticEngineeringandBoltechnology

出　　处：《复旦学报（自然科学版）》1997年第5期489-494,共6页Journal of Fudan University：Natural Science

基　　金：联合国遗传工程和生物技术中心研究项目

摘　　要：采用大肠杆菌密码子，用化学法分别合成人胰岛素21个氨基酸的A链．30个氨基酸的B链，并在基因的5’端引入甲硫氨酸密码子．尾部加入终止密码，两端加入限制性内切酶．A链为83个核苷酸，B链为110个核苷酸，经纯化后的寡聚核苷酸进行酶促连接反应后，分别克隆了人胰岛素A链、B链基因．然后选用pL启动子，构建了以牛生长激素前134个氨基酸的大片段基因，分别与人胰岛素A链、B链基因的融合基因的表达质粒pLWYM－A、PLWYM－B转化大场杆菌BL21．表达受42℃温度调控．表达的融合蛋白按电泳扫描计算，可占细胞总蛋白的60％～80％．表达产物Westernblot呈阳性反应．Two genes in E. coli encoding for human insulin A and B chains have been chemically synthesized. This synthetic DNA duplex consists of structural genes encoding insulin A and B chain, a methionine codon at their 5' end a stop codun at 3' end and several convenient restriction sites at both ends. These genes were separately cloned into vector pBS KS(+),and recombinant colonies identified by hybridization restriction enzyme digestion and sequence analysis. A and B chain genes were cloned and fused separately with a bGH 1 ~ 134amino acids gene. The hybrid genes: WYM-A and WYM-B were inserted into a pL promoter expression vector and expressed in E. coil at 42℃. A high Products yield of 60 ％-80％ of the total cell protein was obtained,and ed7ression confirmed by Western blot analysis.

关 键 词：人胰岛素 B链基因 基因表达 A链基因 

分 类 号：Q578[生物学—生物化学] Q753