痢疾志贺菌侵袭素IpaC的原核表达及纯化复性  

Prokaryotic expression,purification and renaturation of invasin IpaC fromShigella dysenteriae

作　　者：姚晓坤[1,2] 伦永志[1] 成军[3] 刘顺爱[3] 韩铭[3] 孙杰[1] 

机构地区：[1]莆田学院药学与医学技术学院医学检验系,福建莆田351100 [2]浙江医药高等专科学校药学系,浙江宁波315100 [3]首都医科大学附属北京地坛医院传染病研究所,北京100015

出　　处：《中国微生态学杂志》2017年第4期373-376,共4页Chinese Journal of Microecology

基　　金：首都医科大学新发突发传染病研究北京市重点实验室开放研究项目资助(DTKF201505)

摘　　要：目的为进一步研究痢疾志贺菌侵袭素IpaC蛋白的侵袭活性,必须先获得IpaC重组融合蛋白。方法将IpaC基因亚克隆至表达载体pET-24α(+)中,转化至E.coli BL21(DE3)表达宿主菌中,采用IPTG优化诱导表达,然后纯化复性重组蛋白。结果重组蛋白最佳表达条件为0.50mmol/L IPTG、30℃诱导6h,获得分子量约为33kDa的IpaC重组蛋白。Western blotting证实了重组蛋白的特异性。IpaC重组蛋白主要以包涵体形式在宿主菌中表达,通过纯化后得到单一的目的蛋白。结论成功构建IpaC原核表达体系,并获得了蛋白的高效表达及优化。初步建立了IpaC重组蛋白的纯化方案。为进一步研究IpaC的侵袭性作用奠定了基础。Objective To obtain the recombinant fusion protein IpaC for further study of its invasiveness. Methods The invasive gene was amplified into the prokaryotic expression vector pET-24a （q-）, then the recombinant plasmid was transformed into the competent cell of E. coli BL21 （DE3） . The prokaryotic ex- pression of recombinant IpaC protein was induced by using IPTG, then the protein was purified and rena- tured. Results The optimal expression condition was induced by 0.50 mmol/L IPTG at 30℃ for 6 hours. The molecular weight of the IpaC protein was 33 kDa. Western blotting confirmed the specificity of the re- combinant protein. It was expressed in the form of inclusion bodies in the host bacteria. Finally, a single target protein was obtained. Conclusion The expression system of IpaC was successfully constructed and the expression of recombinant IpaC protein was optimized. A purification protocol of recombinant IpaC pro- tein was established.

关 键 词：侵袭素 IPAC 原核表达 蛋白纯化 

分 类 号：R378.2[医药卫生—病原生物学]