Prokaryotic Expression, Purification Of Human GAd And Its Biological Activity Detection

Objective To construct the prokaryotic expression plasmid pBV220-gAd, to expression,purify and detect the bioactivity of the target protein gAd.Methods We extracted genome DNA from healthy volunteers blood. The gAd gene was amplified by standard polymerase chain reaction (PCR) The amplified cDNA fragment was ligated into the EcoR I and Pst I sites of the pBV220 vector. The gAd plasmid was named pBV220/gAd and then co-transfected into JM109 cells. After it was expressed in the host cells, E.coli. JM109, SDS-PAGE analysis suggested that the bacteria containing the recombinant plasmid produced a protein as induced by temperature. The recombinant gAd was expressed in inclusion body, consisting of 28% of the total bacterial proteins. Western blot indicated that the protein could react with His-tag antibody. The protein purified by his-tag column. Western blot indicated that the purified protein could react with His-tag antibody. The purified protein was approved that it had the same active as wild one by diabetic rats test.Result PBV220-gAd plasmid expression vector was constructed and the correct sequence was identified by double digestion. Experimental results show that E.coli with the recombinant plasmid pBV220/gAd induced by the temperature-controlled can express the target protein accounted for 28% of the total bacterial proteins. Immunological identification results showed that the heterologous recombinant protein had His-tag antigen activity. gAd purified by TALON Metal Affinity Resin and tested by SDS-PAGE showed a single band. Immunological identification results showed that the purified protein had His-tag antigen activity. Animal experiments confirmed that recombinant gAd protein had the biological function to reduce blood glucose in diabetic rats.Conclusion The gAd gene was successfully cloned and expressed in E.coli and the research may provide a theoretical basis for developing a new antidiabetes drug.