细胞内病原体抗性基因1的原核表达  

Prokaryotic expression of intracellular pathogen resistance 1 gene

作　　者：佘茜[1,2,3,4] 徐蕾 何永林[1,2,3] 靳志栋[1,2,3] 张丹[1,2,3] 杨春[1,2,3] 

机构地区：[1]重庆医科大学病原生物学教研室,重庆400016 [2]重庆医科大学神经科学研究中心,重庆400016 [3]重庆市神经生物学重点实验室,重庆400016 [4]第二军医大学长征医院检验科,上海200003

出　　处：《中国生物制品学杂志》2012年第4期426-428,436,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30771922)

摘　　要：目的构建细胞内病原体抗性基因1(Intracellular pathogen resistance 1,Ipr1)的原核表达质粒,并在大肠杆菌中进行表达。方法以pMD19-T simple-Ipr1质粒为模板,采用PCR法扩增Ipr1基因,克隆至表达载体pET-32a(+)中,构建重组原核表达质粒pET-32a(+)-Ipr1,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE和Western blot鉴定。结果重组表达质粒经双酶切及测序鉴定构建正确;表达的重组Ipr1蛋白相对分子质量约为70 000,以1.5 mmol/L IPTG诱导4 h,目的蛋白的表达量最高,约占菌体总蛋白的16%,重组Ipr1蛋白可与His标签单克隆抗体特异性结合。结论成功构建了Ipr1基因重组原核表达质粒,并在大肠杆菌中表达了重组Ipr1蛋白,为进一步探讨Ipr1蛋白的功能及Ipr1重组BCG的构建奠定了基础。Objective To construct a prokaryotic expression for intracellular pathogen resistance 1 （Iprl） gene and express in E. coll. Methods Iprl gene was amplified by PCR using plasmid pMD19-T simple-Iprl as a template and cloned into vector pET- 32a （＋）. The constructed recombinant plasmid pET-32a （＋）-Iprl was transformed to E. coli BL21 （DE3） and induced with IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a （＋）-Iprl was constructed correctly. The relative molecular mass of expressed recombinant protein was about 70 000. The expression level reached the maximum after induction with 1.5 mmol/L IPTG for 4 h, which was about 16% of total somatic protein. Recombinant Iprl protein showed specific binding to monoclonal antibody with His tag. Conclusion The prokaryotic expression vector for Iprl gene was successfully constructed, which laid a foundation of further study on function of Iprl protein and construction of recombinant BCG with Iprl.

关 键 词：结核病 细胞内病原体抗性基因1 原核细胞 基因表达 

分 类 号：R37[医药卫生—病原生物学] Q782[医药卫生—基础医学]