文档介绍：利用重组酵母菌生产人胰岛素
摘要：利用重组酵母菌生***胰岛素为以下流程：获得目的基因T构建重组质粒
一构建基因工程菌一工程菌发酵一产物分离纯化一产品检验。其中前三个流程为
产人胰岛素酵母工程菌的构建，根据人胰岛素的氨基酸序列和酵母偏爱的密码子，采用基因半合成技术合***胰岛素基因。将合成的胰岛素基因克隆到pPIC9
质粒，构建了毕赤（Pichia）酵母重组表达载体pPINS319,用BglII线性化后用电转化法导入毕赤酵母GSII5菌株，经过营养筛选和PCR复筛，得到含人胰岛素基因的毕赤酵母工程菌株。后三个流程为胰岛素的大规模发酵生产，通过补料 -分批
发酵获得发酵液，分离纯化后获得人胰岛素。纯化后的重组人胰岛素产品经 SDS-PAGE检测，氨基酸组成分析及小鼠惊厥实验证明制得产品为有生物活性的人胰岛素。
关键词：重组酵母；人胰岛素；发酵；纯化；鉴定
Using recombinant yeast to produce human insulin
HuShe ng 1142043040
Abstract： The proceed ings of produce huma n in suli n by recomb inant yeast show as follow:get purpose gene^con struct the recomb inant plasmid -con struct the gen etic engineering bacteria — fermentationof engineering bacterium — separationand purificati on of the product — iden tificati on of the product. The front three processes is buildi ng engin eeri ng bacteria of huma n in sulin yeasts . In sulin gene was syn thesized accord ing to the amino acid seque nee of huma n in suli n and yeast prefere ntial cod ons. The in suli n gene was cloned in to pPIC9 vector an dexpressi on vector pPINS319 was con structed. The expressi on vector was digested with BgllI and the n used to tran sform Pichia Pastoris GSll5 by electroporation. The expression analysis showed that the insulin gene w s able to expressed efficiently in Pichia Pastoris. The posterior three processes is large-scale producti on by ferme ntatio fed -