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

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.