CDNA Cloning Of Complete Adiponectin Gene Coding Sequence From Chinese And Its MRNA Expression In Subcutaneous And Omental Adipose Tissues Of Obese Patients

Objective To provide the experimental basis for further researching on adiponectin function, human adiponectin gene was cloned. Methods The greater omental fat pat of Chinese was disrupted and homogenized, Total RNA was finally extracted by Qiaquick Rneasy Mini Kit. Adiponectin cDNA was amplified by a reverse transcriptase (RT-PCR ) by using the total RNA as a template. The PCR product was fractionated by electrophoresis in agarose gels and purified by Qiaquick Gel Extraction Kit, Then it was subcloned into plasmid pMD18-T to generate the recombinant plasmid pMD18T-adiponectin. The blue-white blot screened out positive clones, which was confirmed to contain the amplified target gene segments by digest of restrictive endonuclease and nucleotide sequencing. Results The RT-PCR product was consistent with theoretic value 745bp. The recombinant pMD18T-adiponectin plasmid was digested into two fragments by EcoR I and Sph I . They are consistent with theoretic values 800bp and 2637bp. The sequencing results for amplified target gene showed that the sequence of adiponectin from omental fat pat of Chinese is similar to that of human adiponectin gene in Genbank., only at the site of 37 amino acid where Ala(GCA) substitutes Thr(ACA). This proved that has successfully constructed the recombinant plasmid containing adiponectin gene. Conclusions Complete adiponectin cDNA was successfully cloned from Chinese, which laid a foundation for constructing eukaryotic expression and further studing on adiponectin function.