Effect Of PPAR-γ 2mRNA Expression In 3T3-L1 Cells With The Human Recombinant Adiponectin By TNF-α, Insulin Or Dexamethasone

Objective To provide the experimental basis for further investigating on adiponectin (ADPN) function, its eukaryotic recombinant was constructed and expression in 3T3-L1 preadipocytes. Methods The recombinant plasmid pMD18-T/ADPN and eukaryotic expression vector pcDNA3.1+were digested by two restrictive endonuclease and ADPN and linear pcDNA3.1+were obtained. Then, they were ligated and translated into JM109. At last, the recombinant pcDNA3.1+/ADPN was obtained and was identified by digest of restrictive endonuclease and nucleotide sequencing. The 3T3-L1 preadipocytes were transfected using SuperFect Transfection Reagent(QIAGEN). The positive clones screened out by G418, the positive clones were cloned again.total mRNA was extracted from the 3T3-L1 preadipocytes,the quality of the total RNA was analyzed by electrophoresis in agarose gel.The concentration and the purity of total RNA were estimated based on absorbance at 260nm and 280nm. Specific primers were designed from the published human adiponectin gene sequence,the adiponectin cDNA was synthesized by a reverse transcription by using the total RNA as a template,then complete adiponectin was synthesized by PCR by the cDNA as a template. Results 800bp fragment and 5.4kb fragment were consistent with theoretic values after eukaryotic recombinant was digested by HindⅢ and EcoR I and was identified by the nucleotide sequence scaning.The total RNA showed 5S,18S and 28S bands in the agarose gel. A ration of A260 and A280 is 1.9,these results showed that the quality of the total RNA was good.the concentration of the total RNA was 1 μ g/ml.PCR product was showed only one band in front of 250bp,which was consistent with theoretic value...