Recombinant Human Peroxisome Proliferator-activated Receptor γ- Glucagon-like Peptide-1 Cloning, Expression And Purification

Background:Peroxisome proliferator activated receptor γ(PPARγ) belong to the members of the family of PPARs in the nuclear receptor superfamily, of ligand dependent transcription factors. PPARγ ligands including natural ligand and forming ligands. Synthetic ligands (thiazolidinediones, TZD s) can be selectively combined with PPAR gamma. TZDs drugs can increase the utilization of glucose in skeletal muscle, and reduce the liver synthesis of glucose in the body. Research has shown that PPAR gamma function damage can lead to severe insulin resistance.Glucogon like peptide-1is islets alpha cells and intestinal mucosa L cells secrete a hormone that contains30amino acids, the glucagon after processing, its N-terminal glucagon formation,C-terminal formation of GLP-1and GLP-2.Objective:The srudy was to construst people peroxidase proliferation of activated receptor γ-glucogon like peptide-1of prokaryotic expression vector(pET32a-PPARγ-GLP-1). Get a lot of fusion protein by prokaryotic expression system. In order to further test its biological activity provides research foundation.Methods:This study extracting total RNA from human adipose and amplified PPAR Y gene sequence by RT-PCR method. The human genome DNA was extracted from human peripheral blood nucleated cells and amplified GLP-1gene sequence by PCR. Through gene recombination technology to construct pET32a-PPARγ-GLP-1and by double enzyme digestion and sequencing identification of positive clones. pET32a-PPARγ-GLP-1into Rosseta (DE3) cells and expressed by IPTG induction.Expression product by10%SDS-PAGE and Western boltting identification. Fusion protein by the nickel ion chromatography purification.Result:1.Successful construct pET32a ppar-gamma gamma glp-1expression vector, by double enzyme digestion and sequencing identification.2.10%SDS-PAGE results show that the purpose gene in E. coli Rosseta (DE3) expressed in the form of inclusion body, relative molecular weight (Mr)73kda.3. The induction condition optimization, at37℃and inducer IPTG concentration tendency for1L, induced10h, can get a lot of PPARγ-GLP-1fusion protein.4. The Ni-NTA purification with high fusion protein.5. Western blotting show thar target protein can be anti-his tag identification, confirmed that successfully target protein expression. Conclusion:This study confirmed that ppar-gamma gamma can successfully construct pET32a-PPARγ-GLP-1recombinant plasmid.Target gene expressed in E. coli Rosseta (DE3) prokaryotic expression system efficiently by inclusion body form. This research method have been established for PPARγ-GLP-1fusion proteins. The research basis for drug research and development provides a good study.