西农萨能羊脂肪分化相关蛋白(ADRP)基因的原核表达、纯化及多克隆抗体制备

Prokaryotic Expression, Purification and Preparation of Polyclonal Antibody for Adipose Differentiation-Related Protein(ADRP) of Xi′nong Saanen Goat

在大肠杆菌中表达西农萨能羊脂肪分化相关蛋白(ADRP),并制备其多克隆抗体。以实验室保存的pMD19-T-ADRP质粒为模板,扩增ADRP的CDS区,将其克隆到pET32a(+)载体,阳性克隆转化到大肠杆菌菌株E.coli BL21(DE3)中诱导表达,优化融合蛋白表达条件,使用Ni-NTA亲和层析柱纯化融合蛋白,纯化的ADRP蛋白经复性后免疫兔子制备多克隆抗体,ELISA和Western blot检测血清效价和特异性。结果表明:成功构建pET32a(+)-ADRP原核表达载体并诱导其表达,获得最佳表达条件,即37℃、0.5 mmol/L IPTG诱导6 h;ELISA检测显示抗体效价达1∶64 000;经Western blot鉴定,制备的多克隆抗体能特异性结合原核表达以及乳腺上皮细胞表达的ADRP蛋白。本实验成功表达、纯化了ADRP融合蛋白,获得具高特异性、高效价的兔抗ADRP多克隆抗体。

To express ADRP protein of Xinong Saanen goat and prepare its polyclonal antibody,the ADRP gene was amplified by PCR using the pMD19-T-ADRP as a template and was then cloned into prokaryotic expression vector pET32a （＋）.We transformed the prokaryotic expression vector pET32a （＋）-ADRP into E.coli BL21 （DE3） competent cells to induce and optimize its expression.ADRP protein was purified by Ni-NTA column,refolded by gradient urea dialysis and then inoculated to rabbit to prepare polyclonal antibody.ELISA and Western blot analysis was used to determine the titter and specificity of antibody.We successfully constructed pET32a （＋）-ADRP vector and optimized the environmental factors of its expression （37℃,0.5 mmol/L IPTG and 6 h）.The titer of antibody was over 1∶64 000,measured by ELISA.Western blot showed that the antibody could specifically identify ADRP protein expressed in prokaryotic cells and mammary epithelial cells.These results indicated that recombinant ADRP protein was expressed and purified.The polyclonal antibody with high affinity and specificity was generated successfully.