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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 a highly efficient prokaryotic expression system of human adiponectin globular structure (gAd) gene by genetic engineering and to induce, purify, identify and assay the recombinant protein. Methods: Total RNA was extracted from normal human adipose tissue and reverse transcribed into cDNA. The recombinant plasmid pET-22b (+) - gAd was ligated into pET-22b (+ Escherichia coli BL21 (DE3) competent cells. After induced by the inducer, the target protein is produced in the form of inclusion body, renaturation is carried out by using strong alkali to solubilize the inclusion body and the protein is precipitated by acetone to be renatured and purified to obtain high-purity human-derived gAd. The recombinant protein was identified by SDS-PAGE and Western blotting, and the biological activity of the purified protein was tested by the phosphorylation of protein kinase (AMPK) and the protective effect on myocardial ischemia-reperfusion injury in mice. Results: The prokaryotic expression vector pET-22b (+) - gAd was successfully constructed. The expression of human gAd in prokaryotic cells was achieved. The inclusion bodies were denatured, renatured and purified. The purified proteins were analyzed by SDS- PAGE and Western analysis proved to be gAd. The purified gAd was proved to have high biological activity by detecting the phosphorylation level of AMPK and the protective effect on myocardial ischemia-reperfusion injury in mice. CONCLUSION: The successful construction, expression and purification of unlabeled and highly biologically active human adiponectin globular structure (gAd) have laid the foundation for its further theoretical research and production development.