Molecular Cloning Of Human Peroxisome Proliferator Activated Receptor γ2 And Identifying Of Its Stable Expressing Cell Strain

Objective To provide the basis for investigating human PPARγ2 gene by molecular cloning complete PPARγ2 cDNA from omentum fat pat of Chinese and constructing its eukaryotic expression vector.Methods The greater omental fat pat of Chinese was disrupted and homogenized, total RNA was finally extracted by Qiaquick Rneasy Mini Kit. Human PPARγ2 cDNA was amplified by using RT-PCR and then subcloed into pcDNA3.1 empty vector with using the restrictive endonucleases Kpn I and Xho I . The blue-white blot was used to screen out positive clones, which was further confirmed to contain the amplified target gene segment by digestion of the restrictive endonucleases and gene sequencing.Results The RT-PCR product was consistent with theoretic value 1518 bp. The recombinant vector was digested into two fragments by Kpn I and Xho I , were consistent with theoretic values 1518bp(hPPARγ2) and 5400bp(empty vector pcDNA3.1). The sequencing results for amplified target gene showed that the sequence of human PPARγ2 from omental fat pat of Chinese was the same as that of human PPARγ2 gene in Genbank.Conclusion Complete encoding hPPARγ2 cDNA of hPPARγ2 was successfully cloned from omentum fat pat of Chinese and its eukaryotic expression vector pcDNA3.1(+)/hPPARγ2 was successfully constructed.