类法尼醇X受体α基因的原核表达及多克隆抗体制备

Prokaryotic expression and antibody preparation of farnesoid X receptor α

根据NCBI上公布的序列设计引物,从Hep G2细胞系中克隆了FXRα的开放阅读框(ORF),构建原核表达载体p ET-28a-FXRα,转化E.coli BL21(DE3),经1.0 mmol·L-1IPTG诱导表达,SDS-PAGE检测,成功诱导FXRα蛋白;以镍柱亲和层析纯化获得FXRα蛋白,免疫家兔,制备多克隆抗体。Western Blot分析表明:制备的多克隆抗体能够特异识别重组蛋白,为深入研究此受体的功能及新药发现奠定了基础。

The open reading frame（ ORF） sequence for FXRα was amplified by RT-PCR form Hep G2 cell line. The sequence was sub-cloned into vector p ET-28 a for expression in E. coli BL21（ DE3）.The expression of recombinant fusion FXRα with 6 × His-tag was induced by isopropylthio-β-D-galactoside（ IPTG）. The expression of the target protein was detected by SDS-PAGE and then purified by Ni NTA chelating agarose. SDS-PAGE analysis showed that FXRα protein was success fully expressed in E. coli BL21（ DE3）. A rabbit polyclonal antibody against recombinant FXRα was obtained by immunization rabbit with the purified FXRα protein. It was showed that the anti-sera could bind to the expressed protein specifically by Western blot analysis. This work provides a foundation for further study on receptor function and new drug development.