大肠杆菌O157:H7的sRNA伴侣蛋白Ufq的基因克隆、表达及纯化

摘要

Objective: To express and purify small RNA(sRNA) chaperone Hfq of EHEC O157:H7 in Escherichia coli. Methods: The hfq gene ceding Hfq protein was amplified by PCR from EHEC O157:H7 genome and inserted into plasmid pET28a(+) containing 6×His Tag sequence to construct a recombinant vector pET28a(+)-hfq. The recombinant vector was transformed into host strain E.coli BL21(DE3). The fusion Hfq was expressed by IPTG induction and detected by SDS-PAGE and Western blot. The fusion protein was purified by Ni2+-chelating chromatography. Results: The expression vector was digested by restriction enzyme, and the consequent electrophoresis showed the correct length DNA fragment, therefore, the plasmid pET28a(+)-hfq was correctly constructed. The 17 kD fusion protein expressed in E.coli BL21(DE3) was examined by SDS-PAGE. Finally, the purified fusion protein was successfully obtained from supernatant. Conclusion: The sRNA chaperone Hfq of EHEC O157:H7 was obtained, which will lay the foundation for screening the interacting sRNA and further research on sRNA's function.%目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157:H7的sRNA伴侣蛋白Hfq.方法:利用PCR方法从EHEC O157:H7基因组中扩增出基因hfq,并插入含6xHis标签序列的原核表达载体pET28a(+)的多克隆位点中,构建重组表达质粒pET28a(+)-hfq,以重组质粒转化大肠杆菌BL21(DE3),诱导目的基因表达,采用Western印迹法进行鉴定,通过Ni2+螯合柱纯化获得目的蛋白.结果:质粒酶切鉴定结果表明pET28a(+)-hfq重组质粒构建成功;对目的蛋白进行了融合表达,SDS-PAGE显示相对分子质量约17x103的蛋白特异表达条带;对表达产物进行亲和纯化,从上清中获得了Hfq的融合蛋白.结论:得到了sRNA伴侣蛋白Hfq,为进一步筛选与该蛋白有相互作用的sRNA,研究sRNA的功能奠定了基础.