【摘要】 目的构建重组原核表达质粒,对类鼻疽伯克霍尔德菌分子伴侣Hfq蛋白进行高效表达和纯化。方法 PCR扩增类鼻疽伯克霍尔德菌hfq基因并克隆至pMD19-T克隆载体中,测序验证后,用NdeⅠ和XhoⅠ双酶切pM D19T-hfq与pET30a(+),将目的基因片段插入含His标签序列的原核表达载体PET30a(+)中,构建重组表达质粒pET30a(+)-hfq,并转化至大肠埃希菌BL21(DE3),IPTG诱导目的基因表达,采用SDS-PAGE与Western blot对表达蛋白进行鉴定,通过Ni 2+螯合柱对目的蛋白进行纯化。结果 PCR扩增出正确的hfq基因片段,大小为237bp,与预期值相符。亚克隆质粒与原核表达质粒载体连接后进行双酶切鉴定,重组质粒pET30a(+)-hfq构建正确。重组质粒转化DE3后经IPTG诱导5h,从细菌裂解液中检测到Hfq融合蛋白,分子质量单位(Mr)约为9.4×103。通过Ni 2+螯合柱纯化,获得单一SDS-PAGE条带的目的蛋白。结论 hfq基因原核表达质粒构建成功,Hfq在大肠埃希菌中成功表达,得到Ni 2+柱层析获得的高纯度的sRNA伴侣蛋白Hfq,为进一步筛选与该蛋白有相互作用的sRNA及sRNA功能研究奠定了基础。更多还原

【Abstract】 Objectives To express and purify the sRNA chaperone protein Hfq of Burkholderia pseudomallei in a prokaryotic expression system. Methods The recombinant vector pET30 a(+)-hfq carrying the hfq gene was constructed,and it was verified as correct with PCR and restriction enzyme digestion analysis.The vector pET30 a(+)-hfq was transfected into Escherichia coli BL21(DE3).Expression of recombinant protein was induced with IPTG.The expressed product was detected with SDS-PAGE,identified with a Western blot assay,and then further purified using an Ni 2+chelate column. Results An Hfq gene fragment was amplified.Agarose gel electrophoresis indicated that the fragment was237 bp in size,which was consistent with expectations.T-A subclones were successfully constructed.the recombinant plasmid pET30 a(+)-hfq was verified as correct with double enzyme digestion and sequencing.The expressed protein was detected with SDS-PAGE and identified with a Western blot assay using mouse anti-His mAb.Results indicated that the protein had a molecular weight of about 9.4×103.The fusion protein mainly existed in T-cell filtrate.The target protein was purified via Ni 2+-chelating chromatography. Conclusion A recombinant prokaryotic expression plasmid was successfully constructed.Hfq was successfully expressed in E.coli.and sRNA chaperone protein Hfq was purified using Ni 2+column chromatography.Overall,these results have laid the foundation for further study of the function of sRNA and further screening of sRNA interaction with the protein.更多还原