Cloning Of Resistin Gene CDNA From Chinese And Expression Of Resistin Gene In Adipose Tissus Of Obese Individuals

Objective to obtain cDNA cloning of resistin gene plasmid vector of Chinese , which laid a foundation for study on resistin gene. Methods Total mRNA was extracted from omentum adipose tissue of Chinese , 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 resistin gene sequence (GI:14211896),the resistin cDNA was synthesized by a reverse transcripase by using the total RNA as a template. Complete resistin cDNA was synthesized by RT-PCR method . An aliquot of the PCR prduct was analyzed by electrophoresis in agarose gel and verified by using BamHI digestion . The PCR product was isolated from agarose gel and purified by QIAquick Gel Extraction Kit . The purified PCR product was ligated into pMDIST vector and the recombinant pMD18T-resistin plasmid was transformed into E.coli JM109. After the blue-white blot screened out positive clones, positive clones were cloned again, the recombinant pMD18-T-RETN plasmid was extracted by QIAprep Spin Miniprep Kit and verified by using EcoRI and MndIII digestion and by an analysis of the nucleotide sequenced later. Results The total RNA showed nice 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 g/ 1. The RT-PCR product was showed only one band between 250bp and 500bp,which was consistent with theoretic value 363bp; It was digested into two fragments by BamHI, they consistent with theoretic value 239bp and 124bp. the recombinant pMD18-T-RETN plasmid was digested into two fragments by EcoRI and Mndlll, they consistent with theoretic value 2635bp and 420bp, which indicated the PCR products had inserted pMDIST vectors. The nucleotide sequence analysis was almost consistent with the resistin data ofgenebank ,only TCC turned into TTC at the 23rd amino acid site. Conclusion Theresistin gene cDNA was successfully cloned.