Construction and expression of eukaryotic recombinant with human complete adiponectin cDNA

Objective To provide experimental basis for further investigating on adiponectin (ADPN) function,whose eukaryotic recombinant was constructed and expression in 3T3-L1 preadipocytes. Methods The recombinant plasmid pMD18-T/hADPN and eukaryotic expression vector pcDNA3.1+ were digested by two restrictive endonuclease and hADPN and linear pcDNA3.1+ were obtained,then,they were linked and translated into JM109,and 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,and the positive clones were cloned again. The adiponectin cDNA was synthesized by a reverse transcription by using the total RNA as a template,and then complete adiponectin was synthesized by PCR by the cDNA as a template. Results 800 bp fragment and 5.4 kb fragment were consistant with theoretic values after eukaryotic recombinant was digested by HindⅢ and EcoRⅠ. Conclusion The eukaryotic expressing recombinant with complete adiponectin cDNA was constructed.