[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].

ABSTRACT

OBJECTIVE: To express the fusion protein of glutathione S-transferase (GST) and peroxisome proliferator-activated receptor-gamma coactivator-1 (PPARgammaC1) in E. coli. and prepare the polyclonal antibody against PPARgammaC1. METHODS: The coding sequence of PPARgammaC1 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-PPARgammaC1 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-PPARgammaC1. RESULTS: Restriction endonuclease digestion analysis demonstrated that the PPARgammaC1 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 PPARgammaC1 in Western blot analysis. CONCLUSION: The successfully prepared polyclonal antibody against PPARgammaC1 peptide provides a useful reagent for PPARgammaC1 detection.