论文部分内容阅读
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 further study the invasion activity of IpaC in Shigella dysenteriae, IpaC recombinant fusion protein must be obtained first. Methods The IpaC gene was subcloned into the expression vector pET-24α (+) and transformed into E. coli BL21 (DE3) expressing host. The expression of IpaC was induced by IPTG, and then the recombinant protein was purified. Results The optimal conditions for the recombinant protein expression were 0.50mmol / L IPTG and induced at 30 ℃ for 6h to obtain recombinant IpaC with a molecular weight of 33kDa. Western blotting confirmed the specificity of the recombinant protein. The IpaC recombinant protein is expressed mainly in the form of inclusion bodies in host bacteria, and purified to obtain a single target protein. Conclusion The prokaryotic expression system of IpaC was successfully constructed and the expression and optimization of the protein was achieved. Preliminary establishment of IpaC recombinant protein purification program. Which laid the foundation for further study on the invasiveness of IpaC.