人胰岛素原乳腺表达载体的构建  

Construction of a Expression Vector Carrying Human Insulin Gene in Mammary Gland

作　　者：森布尔[1] 郭旭东[1] 旭日干[1] 

机构地区：[1]内蒙古大学实验动物研究中心

出　　处：《内蒙古大学学报（自然科学版）》1997年第1期99-106,共8页Journal of Inner Mongolia University：Natural Science Edition

基　　金：国家自然科学基金

摘　　要：通过ＰＣＲ方法扩增了人胰岛素（ＨＩ）基因１．３４ｋｂ的ＤＮＡ片段并克隆于ｐＵＣ１８的ＳｍａＩ位点内．经ＤＮＡ序列分析表明，该片段为ＨＩ基因的氨基酸编码区及３′端调控区序列．同时，应用ＰＣＲ方法对牛α－乳白蛋白（ＢαＬＡ）基因进行了扩增，得到了０．８４ｋｂＤＮＡ扩增片段．将其克隆于去除了ＥｃｏＲＩ位点的ｐＵＣ１８ＳｍａＩ位点内，经内切酶分析和ＤＮＡ测序证明该片段是ＢαＬＡ基因５′调控区序列．ＥｃｏＲＩ酶切ＨＩ基因重组质粒，回收１．３４ｋｂＤＮＡ片段，并亚克隆于ＢαＬＡ重组质粒插入片段的３′端ＥｃｏＲＩ位点内．筛选出ＨＩ基因片段正确连接的克隆，经内切酶分析证实成功地构建了人胰岛素原乳腺表达载体．The 1.34 kb DNA fragment of human insulin (HI) gene was amplified by using PCR method,and cloned into plasmid pUC18.The insert was proved to be the coding sequence and 3′ flanking region of human insulin gene by DNA sequencing.Meanwhile the 0.84 kb DNA fragment of bovine α lactalbumin (BαLA) gene was obtained by PCR amplification from bovine chromosome DNA,and cloned into plasmid pUC18 after eliminating Eco RI site from the polycloning sites.The insert was demonstrated to be the 5′ flanking region of BαLA gene by digesting with restriction endonucleases and DNA sequencing.The 1.34 kb insert was isolated from the HI recombinant plasmid by digesting with Eco RI,and subcloned into Eco RI site of 3′ end of the 0.84 kb insert in the BαLA recombinant plasmid.The orientation of the subcloned HI insert was identified by restriction endonucleases.The result confirmed that the expression vector of human insulin gene in mammary gland was constructed successfully.

关 键 词：乳腺表达载体 胰岛素 糖尿病 基因工程 

分 类 号：TQ467.32[化学工程—制药化工] R394.8[医药卫生—医学遗传学]