天花粉蛋白突变体TCS_(KR173-174CG)的构建、表达与纯化  

Construction and Expression of Trichosanthin Mutant Gene TCS _(KR173-174CG) and Purification of Expressed Product

作　　者：安群星[1] 穆士杰[1] 张献清[1] 陈蕤[1] 夏爱军[1] 张清平[1] 陈晨[1] 雷迎峰[2] 黎志东[2] 徐志凯[2] 

机构地区：[1]第四军医大学西京医院输血科,西安710033 [2]第四军医大学微生物学教研室,西安710032

出　　处：《中国生物制品学杂志》2006年第6期557-559,563,共4页Chinese Journal of Biologicals

基　　金：陕西省科技计划项目(2003K10G9).

摘　　要：目的构建天花粉蛋白(TCS)突变体基因,并进行表达及纯化。方法应用计算机预测TCS分子上可能的抗原决定簇,并进行定点突变。以栝楼基因组DNA为模板,经PCR扩增突变基因,与pRSET-A表达载体连接,转化大肠杆菌BL21(DE3),经IPTG诱导表达,并对表达产物进行Ni-NTA层析纯化。结果目的蛋白在大肠杆菌中获得高效可溶性表达,表达产物经纯化后,得到均一的TCS突变体蛋白。结论已成功构建了TCS突变体基因,并获得高效表达。Objective To construct and express a trichosanthin（TCS） gene mutant and purify the expressed product. Methods Predict the potential antigenic determinant on TCS molecule by computer modeling and induce site-directed mutation. Amplify gene mutant TCSKR173-174CG by PCR using the genomic DNA of Trichosanthes kirilowii as a template and insert into expression vector pRSET-A,then transform to E. coll BL21 （DE3） for expression under induction of IPTG. Purify the expressed product by Ni-NTA affinity column chromatography. Results The target protein in a soluble form was successfully expressed in E. coll. Homogenous TCS mutant protein was obtained after purification of expressed product. Conclusion TCS mutant gene TCSKR173-174CG was successfully constructed and expressed.

关 键 词：天花粉蛋白 定点突变 原核表达 

分 类 号：R512.63[医药卫生—内科学]