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《Journal of First Military Medical University》 2005-05
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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 1Department of Endocrinology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 2Department of Pathology, Sen Yet-sen University, Guangzhou 510089, China  
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.
【Fund】: 国家自然科学基金(10703-H52)~~
【CateGory Index】: R392
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