重组人源性脂联素球状结构的表达、纯化与活性检测

Expression,Purification of Human gAd in E.coli and Identification of Its Biological Activity

目的:利用基因工程方法构建人源性脂联素球状结构(gAd)基因的高效原核表达体系,并对重组蛋白进行诱导表达、纯化、鉴定及活性检测。方法:从正常人脂肪组织里面提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒pET-22b(+)-gAd,重组质粒转化大肠杆菌BL21(DE3)感受态细胞。经诱导剂诱导后目的蛋白以包涵体形式产生,采用强碱促溶包涵体并用丙酮沉淀蛋白的方法进行复性和纯化,得到高纯度的人源性gAd。运用SDS-PAGE、Western blotting对重组蛋白进行鉴定,通过对蛋白激酶(AMPK)的磷酸化水平和对小鼠的心肌缺血再灌注损伤的保护作用来检测纯化蛋白的生物学活性。结果:成功构建了原核表达载体pET-22b(+)-gAd,实现了人源性gAd在原核细胞中的表达,并对形成的包涵体变性、复性和纯化,纯化出的蛋白经过SDS-PAGE和Western分析证实为gAd;通过对AMPK的磷酸化水平的检测和对小鼠的心肌缺血再灌注损伤的保护作用证明纯化出的gAd具有高生物学活性。结论:成功构建、表达和纯化了无标签、高生物学活性的人源性脂联素球状结构(gAd),为其进一步的理论研究、生产开发奠定了基础。

Objective： To construct the prokaryotic expression vector of human gAd gene and obtain the no- tagged recombinant human gAd protein. Methods： Total RNA was extracted from human adipose tissue. The gAd cDNA was obtained with RT-PCR technique and subcloned into a prokaryotic exprssion vector pET-22b（ ＋ ） to generate pET-22b （ ＋ ）-gAd. The expression of no-tagged recombinant human gAd protein was induced by IPTG in E. coli BL21 （DE3）. The gAd protein produced as inclusion bodies at an elevated level. The inclusion bodies were solubilized by alkaline shock, and then refolded and purified after acetone precipitation. SDS-PAGE and Western blot were used for identification of the purified gAd. The abilities of gAd to induce the phosphorylation of AMPK in HUVECs and to protect hearts after myocardial ischemia/reperfusion in mice were used for its biological activity assay. Results： The human gad coding sequence was correctly cloned into pET-22b （ ＋ ） vector. After expression and purification, the recombinant human gAd significantly induced the phosphorylation of AMPK in HUVECs and protected hearts from myocardial isehemia/reperfusion injury. Conclusion： The no-tagged recombinant human gAd was successfully expressed and purified in prokaryotic expression system with high biological activity.