Cloning and Prokaryotic Expression of Rat GAD65 Gene

Objective To construct prokaryotic expression vector of PGEX-6P-3/GAD65, and obtain bulk of purified GAD65 protein. Method GAD65 cDNA was amplified from brain tissue of rat with RT-PCR, cloned into PGEM-Teasy and sequenced. GAD65 gene was inserted into PGEX-6P-3 through pre-set enzyme digestion sites of BamHI and EcoRI in primers to construct recombinant gene, then the plasmid was transfected into E.coli BL21 and the transformants were identified by restriction enzyme digestion method. The recombinant protein was purified with sepharose 4B affinity column and the fusion protein was cleaved by PrescissionProtesa enzyme, and then identified by SDS-PAGE and Western-blot analysis. Result The length of amplified GAD65 fragment was 1758 base pairs, and the sequence of the gene is the same as the reported sequence. The expression plasmid, PGEX-6P-3 /GAD65, was constructed. Recombinant GAD65 fusion protein was expressed in E.coli with a M.W. of 91kDa. The fusion protein was puirifed and cleaved to generate the GAD65 with a M.W.of 65kDa. Results of Western-blot indicate the recombination protein can be idio-conjugated with GAD65 monoclonal antibody. Conclusion The GAD65 gene was cloned and recombinant GAD65 was produced and purified from E.coli. It is an important reagent for the studies of the biological function of GAD65.