新型抗2-型糖尿病基因重组RMBAY的克隆、表达及生产环节优化  

Cloning and Expression of the New Gene Recombinant RMBAY Against Type-2 Diabetes and Its Production Optimization

作　　者：马义[1] 余榕捷[1] 曾乐[1] 谢珊珊[1] 洪岸[1] 

机构地区：[1]暨南大学生物工程研究所,广州510632

出　　处：《中国生物工程杂志》2009年第4期17-21,共5页China Biotechnology

基　　金：国家"十一五""863"计划(2006AA02Z125)资助项目

摘　　要：利用基因重组方法构建RMBAY的原核表达载体pKY-BAY,并研究其生产的优化条件。选用大肠杆菌偏爱密码子,用PCR方法合成全长RMBAY多肽基因,并定向插入到高效表达载体pKYB-mcs中,用大肠杆菌ER2566进行表达,融合蛋白经Chitin-Beads柱纯化后,结合在柱上的融合蛋白用β-巯基乙醇诱导蛋白内含肽的N端自动切割,释放目的肽,目的肽由质谱鉴定。实验结果表明:利用载体pKY-B在大肠杆菌ER2566中,RMBAY能够实现高效表达;在优化的生产条件下,RMBAY的产量可达到6.7mg/L发酵产物,纯度大于98%,质谱鉴定RMBAY的分子量为3.887kDa.与理论值相符合。Constructing prokaryotic expression vector pKY-RMBAY by gene recombination and research its optimizing productive conditions. By PCR technology synthesizing the gene of the RMBAY with preference codon of E. coli and the RMBAY gene was inserted into high efficiency expression vector pKYB-MCS. Expressed fusion proteins in E. coli ER2566 were purified with Chitin-Beads column. Fusion proteins binding on Chitin-Beads was cut on N-terminus of intein due to the induction of β-mercaptoethanol and the target peptide RMBAY was released. The RMBAY was identified by mass spectrum. Experiment results showed RMBAY can be high efficiently expressed in E. coli ER2566, with optimizing productive conditions the yield of the RMBAY may be 6.7mg/L fermentation product and its purity is greater than 98%. The molecular weight of RMBAY is 3. 887 kDa by mass spectrum and that accords with its theory value.

关 键 词：基因重组 VPAC2受体激动剂 生产环节优化 2-型糖尿病 电喷雾质谱 

分 类 号：Q785[生物学—分子生物学] Q786