Cloning, Expression,Purification Of Recombinant Human GAd And Its Biological Activity Detection

Objective: In order to construct the prokaryotic expression plasmid pBV220-gAd,to purify and detect the activity of target protein gAd. Method: We separated lymphocytes from healthy volunteers blood and then extracted genome DNA.According to the DNA sequence of adiponectin acquired from genbank specific primers for PCR were designed by using the software Oligo 6.44.The purified PCR product was connected with T vector and then transformed E.coli JM109.We screened and identified positive plasmid named as pMD18T-Ad.Then pMD18T-Ad was used as template and amplified with up stream primer containing EcoR â… site and down stream primer containing Pst â… site.We subcloned this PCR product into pBV220 and transformed E coli DH5α.The positive bacteria was induced at 42℃.The inclusion body containing target protein gAd was separarted and washed.The preliminary purified gAd was disolved in 8 mol/L urea and renatured by gradient dilution.The renatured gAd was concentrated by ultrafiltration .We injected this gAd into rabbit to investigate its bioactivity of decreasing the level of plasma glucose and free fatty acid.Also we constructed expression plasmid of gAd fusion protein with His Tag by adding encoding sequence of six consecutive histidine to down stream specific primer.The interesting fusion protein was identified by Western-blotting with His Tag antibody and purified by NTA-Ni²⁺ resin. Result: The pBV220-gAd expression plasmid was constructed successfully.The target protein gAd can express as inclusion body in E coli DH5αand JM109,but the expressin quantity in DH5αis more than in JM109.In DH5αthe gAd protein accounted for 20% of whole bacteria protein.Compared to control group,renatured gAd can decrease the level of glucose and free fatty acid.Meanwhile we constructed gAd fusion protein expression system and after NTA-Ni2+ resin purification SDS-PAGE analysis showed a single band in the target protein. Conclusion: We have successfully constructed the prokaryotic expression plasmid pBV220.The target protein gAd can significantly decrease glucose and free fatty acid.At the same time gAd fusion protein with high purity was obtained.The results of our study have provided the reliable experimental methods for expression and purification in large scale as well as the foundation for prevention and treatment of obesity,diabetes mellitus,atherosclerosis.