脂联素及其球状区在大肠杆菌中的可溶性表达

Cloning and Soluble Expession of Adiponectin and Gadiponectin in E.coli

目的:克隆表达有生物学活性的脂联素及其球状区蛋白,并制备抗体。方法:以pQE30-adiponectin质粒为模板,PCR扩增脂联素及其球状区蛋白基因片段,插入pGEX-4T-2载体,转化大肠杆菌BL21后获得表达,用GSTrap柱亲和纯化可溶性表达的蛋白。用纯化的蛋白免疫家兔制备多抗,Westernblot鉴定抗体与人血清中脂联素的反应性。结果:PCR扩增脂联素基因片段长约710bp,脂联素球状区基因片段长约430bp。表达的GST-脂联素融合蛋白表观Mr约51000,GST-脂联素球状区融合蛋白表观Mr约42000,纯化后纯度高于90%。免疫产生的抗体与人血清中的脂联素能特异性结合。结论:表达获得的脂联素蛋白和制备的抗体为脂联素的检测及对其功能的研究奠定了基础。

Objectives： To clone and express adiponectin and gadiponectin with biological activities in E, coli expression system and prepare anti-adiponectin and anti-gadiponectin polyclonal antibodies. Methods： The adiponectin and gadiponectin-coding DNAs were amplified from pQE30-adiponectin plasmid by PCR. Then the genes were subcloned into pGEX-4T-2 vector to construct recombinant expression plasmids and transformed into competent host E.coli BL21 to induce and express fusion proteins. The soluble proteins were purified by GSTrap affinity chromatography. The specificity of the polyclonal antibody prepared was examined by Western blot. Results- 700 bp adiponectin and 400 bp gadiponectin DNA fragments were amplified from pQE30-adiponectin plasmids by PCR. The molecular weight of GST-adiponectin and GST-gadiponectin recombinant fusion proteins were about 51 kD and 42 kD, respectively. After purification, the purity was over 90%. The specificity of the polyclonal antibodies prepared with serum adiponectin were confirmed by Western blot. Conclusion： The expressed proteins and antibodies prepared may be used for detection and functional analysis of adiponectin.