Abstract： Objective To construct the recombinant pEGFP/IA-2 that can be used as the gene vaccine to prevent type 1 diabetes mellitus. Methods IA-2 cDNA and pEGFP were recombined with T4-ligase in BamHI and Hind Ⅲ. The recombinant pEGFP/IA-2 was transfected into RIN5F cell line and selected with G418 for eight weeks. The expression of pEGFP/IA-2 was detected by Western blot and identifed under the fluorescence microscope. The proliferation of spleen lymphocyte of NOD mice was assayed with MTS after being co-cultured with IA-2-overexpressing RIN5F cell line. ~Results The recombinant can be identified after cutting with BamHI and HindⅢ. The high expression of IA-2 was manifested by Western blot and fluorescence microscope. The proliferation of spleen lymphocyte was significantly increased after co-cultured with IA-2 overexpressing cell line when compared with control group (P<0.05). Conclusion The pEGFP/IA-2 is successfully constructed, which is the first step to prepare type 1 diabetes gene vaccine and has an important role in the prevention of type 1 diabetes and the study of biological function of IA-2.