人CD271基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Human CD271 Gene

作　　者：徐艳红[1] 石琳[1] 张涛[1] 冯春玲[1] 刘红芹 屈浩 黄丽华[1] 朱卫彬 张宇光[1] 

机构地区：[1]中国医学科学院北京协和医学院天津血液学研究所,天津300020 [2]协和干细胞基因工程有限公司,天津300384

出　　处：《中国生物制品学杂志》2008年第10期858-860,868,共4页Chinese Journal of Biologicals

摘　　要：目的克隆人CD271基因,并在大肠杆菌中表达。方法采用RT-PCR技术从人REH细胞系中扩增CD271基因,将其克隆入pET22b表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行纯化及Western blot鉴定。结果重组表达质粒pET22b/CD271经双酶切鉴定,可见1197bp的目的基因条带。表达产物经SDS-PAGE分析,在相对分子质量约75000处可见表达条带。IPTG浓度为0.5mmol/L时,目的蛋白表达量最高。纯化后蛋白纯度为82.5%。Western blot检测表明纯化后的蛋白具有良好的反应原性。结论已成功克隆了人CD271基因,并在大肠杆菌中获得表达。Objective To clone human CD271 gene and express in E. coli. Methods Amplify CD271 gene from human REH cell line by RT-PCR and clone into expression vector pET22b. Transform the constructed recombinant plasmid pET22b / CD271 to E. coli BL21（DE3） for expression under induction of IPTG. Purify the expressed product by affinity chromatography and identify by Western blot. Results The restriction analysis of recombinant plasmid pET22b / CD271 showed a target gene fragment at length of 1197 bp. SDS-PAGE showed that the relative molecular mass of expressed protein was about 75 000. The expression level of target protein reached a peak value under induction of 0. 5 mmol / L IPTG. The purified target protein reached a purity of 82. 5% and showed good reactogenicity as proved by Western blot. Conclusion Human CD271 gene was successfully cloned and expressed in E. coli.

关 键 词：人CD271基因 克隆 原核表达 

分 类 号：Q785[生物学—分子生物学] Q786