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目的 :克隆人Acrp30基因 ,并在大肠杆菌中表达。方法 :提取人脂肪组织总RNA ,经RT_PCR扩增目的cDNA片段 ,进行核苷酸序列分析 ;将该基因克隆入原核表达载体pQE_30 ,在大肠杆菌M15中表达 ,用Ni2 + 固相化的螯合SepharoseFastFlow亲和层析纯化重组蛋白 ;并用Western印迹检测所制备多克隆抗体的特异性。结果 :从人脂肪组织总RNA中扩增得到 736bp的目的cDNA ,其序列与已报道的序列完全一致 ,并在大肠杆菌中成功获得高水平表达 ,蛋白相对分子质量大约为 30× 10 3,其表达量占菌体总蛋白的 15 %左右。重组蛋白经Ni2 + 固相化的螯合SepharoseFastFlow亲和层析纯化 ,纯度 >90 %。Western印迹试验证实所制备多克隆抗体具有较高特异性。结论 :本研究为进一步探讨Acrp30在肥胖症和糖尿病患者中的表达情况以及Acrp30的作用机制奠定基础
Objective: To clone human Acrp30 gene and express in E. coli. Methods: The total RNA of human adipose tissue was extracted and the cDNA fragment of interest was amplified by RT-PCR. The gene was cloned into prokaryotic expression vector pQE_30 and expressed in E. coli M15. SepharoseFastFlow affinity chromatography purification of recombinant protein; and Western blot analysis of the specificity of the prepared polyclonal antibody. Results: The target cDNA of 736bp was amplified from total RNA of human adipose tissue. The sequence of the cDNA was exactly the same as that reported. The recombinant protein was successfully expressed in E. coli. The relative molecular mass of the cDNA was about 30 × 10 3 The amount of expression accounted for about 15% of total bacterial protein. Recombinant protein purified by Ni2 + immobilized SepharoseFastFlow affinity chromatography, purity> 90%. Western blot test confirmed that the prepared polyclonal antibody has high specificity. Conclusion: This study is to further investigate the expression of Acrp30 in obesity and diabetic patients and the mechanism of Acrp30