Construction of glutamic acid decarboxylase-65 (GAD-65) baculovirus expression plasmid and its expression in insect cells

Objective To construct the recombinant glutamic acid decarboxylase-65(GAD65)baculovirus expression plasmid and express GAD65 in Sf9 cell line.Methods The GAD65 cDNA was cloned into a pFASTBACHTb donor plasmid,and then the recombinant plasmid was transformed into DH10BAC competent cells.The transformant containing the recombinant bacmid was selected and named as Bacmid/GAD65.The recombinant baculoviruses were obtained after the transfection of Bacmid/GAD65 into Sf9 lines.The techniques of IFA,SDS-PAGE and Western-blot were used to detect and identify the products expressed in Sf9 lines.Results The recombinant baculavirus was obtained.The recombinant GAD65 protein was expressed in Sf9 insect cells and detected by IFA and Western-blot using monoclonal antibody CS1-4.The MW of the recombinant GAD65 protein was 65kDa,and could be detected in the supernatant of Sf9 cell culture.Conclusion Recombinant GAD65 could be expressed in Sf9 lines using the baculovirus expression system,and thus provided the basic material for studying of the function of GAD65 and its application in immunotherapy.