重组人脂联素融合蛋白在大肠埃希菌BL21中的表达

Expression,purification characterization of recombination human adiponectin in Escherichia coli BL21

目的在大肠埃希菌BL21中对构建的重组人脂联素质粒pET-32a(+)/AP进行诱导表达,并对产物进行纯化、鉴定。方法将pET-32a(+)质粒和重组pET-32a(+)/AP质粒转化到大肠埃希菌BL21中进行诱导表达,建立表达体系,筛选高表达的克隆子,用不同诱导时间和不同异丙基-β-D-硫代半乳糖苷诱导浓度确定最佳诱导条件,用Ni^(2+)金属层析柱进行蛋白亲和层析,对纯化蛋白进行浓缩,蛋白质印迹鉴定所诱导的蛋白。结果得到分子量为43 kD的新生蛋白,经蛋白质印迹鉴定为重组脂联素融合蛋白。结论将重组pET-32a(+)/AP质粒转化到大肠埃希菌BL21中,建立表达体系,且获得高效表达。

Objective To construct the recombinant adiponectin plasmid, induce the pET-32a（＋） /AP expression in Escherichia coli BL21, and purify as well as identify its products. Methods The recombinant pET-32a/AP and pET-32a（＋） were transformed into Escherichia coli BL21, made to induce and express. The expression system was built and the high expressed clone was selected. The best induction condition was confirmed by using different induction time and different isopropyl thiogalactoside induction concentration. The recombinant adiponectin was proceeded much more induction and expression, and the albumen was taken affinity chromatograph through the Ni2＋ chromatographic column, and the purified albumen was concentrated and finally induced albumen was identified by westen-blotting. Results The westen-blotting identified that the marker 43 kD was the recombinant adiponectin fusion alumen. Conclusion The recombinant adiponectin fusion albumen is successfully induced and purified, which provides a good basis for further investigation of its physiological mechanism.