南方医科大学学报

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (05): 558-561.

• • 上一篇    下一篇

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

张燕1, 薛耀明1, 郭爱林2, 张文敏2   

  1. 1. 南方医科大学南方医院内分泌科, 广东, 广州, 510515;
    2. 中山医科大学病理教研室, 广东, 广州, 510089
  • 出版日期:2005-05-20 发布日期:2005-05-20
  • 基金资助:
    收稿日期:2004-11-15。
    基金项目:国家自然科学基金(10703-H52)
    作者简介:张燕(1978- ),女,第一军医大学在读硕士研究生,电话:020-61645855,E-mail:xinchristina@163.com

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

ZHANG Yan1, XUE Yao-ming1, GUO Ai-lin2, ZHANG Wen-min2   

  1. 1. 南方医科大学南方医院内分泌科, 广东, 广州, 510515;
    2. 中山医科大学病理教研室, 广东, 广州, 510089
  • Online:2005-05-20 Published:2005-05-20

PDF

1063

摘要： 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PPAR γC1的多克隆抗体.方法 从肝癌细胞系Hep G2提取总RNA,用RT-PCR扩增出PPAR γ C1 cDNA的编码序列,克隆人表达载体pGEX-T-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-肿ARγC1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-T-1中,经IPTG诱导后,表达出相对分子量为000的融合蛋白.Western blotting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.

Abstract: 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.

中图分类号: