GST-PPARγC1原核表达载体的构建、表达及抗GST-PPARγC1多克隆抗体的制备

Prokaryotic expression vector construction, expression and polyclonal antibody preparation of the fusion protein of glutathione S-transferase and peroxisome proliferator-activated receptor-gamma coactivator-1

目的在大肠杆菌中表达过氧化物酶体增殖物激活受体γ共激活因子-1(PPARγC1)与谷胱甘肽-S转移酶(GST)的融合蛋白,并制备抗PPARγC1的多克隆抗体。方法从肝癌细胞系HepG2提取总RNA,用RT-PCR扩增出PPARγC1cDNA的编码序列,克隆入表达载体pGEX-4T-1中,重组载体在酶切鉴定后,在大肠杆菌中经IPTG诱导表达获得GST-PPARγC1蛋白,SDS-PAGE分析表达产物。通过亲和层吸法纯化表达的GST-PPARγC1融合蛋白,并以此为抗原制备多克隆抗体。Westernblotting检测重组抗原的免疫活性。结果经测序、酶切鉴定证明,PPARγC1基因已正确插入到pGEX-4T-1中,经IPTG诱导后,表达出相对分子量为44000的融合蛋白。Westernblotting鉴定所制备的多克隆抗体可以与PPARγC1特异性结合。结论PPARγC1片段在大肠杆菌中的成功表达及制备得到的多克隆抗体,为检测PPARγC1及其在其他组织中的表达提供了一种检测途径。

Objective To express the fusion protein of glutathione S-transferase (GST) and peroxisome proliferator-activated receptor-gamma coactivator-1 (PPARγC1) in E. coli. and prepare the polyclonal antibody against PPARγC1. Methods The coding sequence of PPARγC1 gene was amplified by reverse transcriptase-PCR (RT-PCR) from the total RNA of Hep G2 cells and inserted into pGEX-4T-1 vector. The recombinant vector was identified by restriction endonuclease digestion analysis and the fusion protein GST-PPARγC1 was expressed in E. coli. via IPTG induction. The expressed fusion protein was purified by glutathione-agarose affinity chromatography and used to immunize the egg-laying hens for preparing the polyclonal antibody against GST-PPARγC1. Results Restriction endonuclease digestion analysis demonstrated that the PPARγC1 gene had been correctly inserted into pGEX-4T-1 vector, and the expressed fusion protein had a relative molecular mass of approximately 39 000 as shown by SDS-PAGE. The polyclonal antibody obtained from the egg yolk immunoglobulins was found to specifically bind to purified PPARγC1 in Western blot analysis. Conclusion The successfully prepared polyclonal antibody against PPARγC1 peptide provides a useful reagent for PPARγC1 detection.