γ-PGA合成酶基因在大肠杆菌中的克隆和表达  

Cloning and Expression of Poly-glutamic Acid Synthase Gene in Escherichia coli

作　　者：乔广军[1] 汪晨[2] 周志蕙[2] 张凯[2] 蔡恒[2] 

机构地区：[1]南京绿叶思科药业有限公司,江苏南京210061 [2]南京工业大学,江苏南京211816

出　　处：《食品与发酵科技》2013年第1期8-12,共5页Food and Fermentation Science & Technology

基　　金：江苏省自然科学基金(BK2009363);国家重点基础研究发展计划(973计划)资助项目(2011CB707405)

摘　　要：研究了γ-PGA合成酶基因pgsBCA在大肠杆菌中的克隆和表达,以pET28a(+)为载体,构建表达载体pET28a(+)-pgsBCA,导入宿主Escherichia coli Rosetta中,诱导使之表达。将发酵液离心去除菌体,得到上清液用旋转蒸发仪浓缩后,采用SDS-PAGE电泳检测重组菌E.coli Rosetta/pET28a-pgsBCA产生的γ-PGA分子量在200-300kDa之间,将产物水解,采用薄层层析法鉴定产物由单一的谷氨酸组成,表明γ-PGA合成酶基因pgsBCA在大肠杆菌中成功表达。Studied poly-glutamic acid synthase gene pgsBCA cloned and expressed in the the E.coli, pET28a （＋） was selected as the carrier to construct the expression vector pET28a （＋）-pgsBCA and to be imported into host E. coli Rosetta, and induced it to express. Dealing with fermentation broth, centrifuged to remove bacteria body and obtained supernatant, using SDS-PAGE electrophores to detect the PGA molacular weight between 200-300kDa pro- duced by recombinant bacteria, hydrolysised the product, using the thin-layer chromatography identification, we found that the product was composed by a single glutamic acid, which showed that-PGA synthase gene pgsBCA was successfully expressed in E.coli.

关 键 词：大肠杆菌 聚谷氨酸合成酶 克隆 表达 

分 类 号：TQ925[轻工技术与工程—发酵工程]