Aim: To develop rAAV2 containing cDNA of GAD-Ig fusion gene [GAD-Ig fusion gene is constructed by inserting glutamic acid decarboxylase(GAD) into N-terminal of murine IgG3 heavy chain(Ig)], and to study whether rAAV2 mediated GAD-Ig fusion gene expression could induce specific tolerance in IDDM animal model-nonobese diabetic (NOD) mice.
Methods: GAD-Ig cDNA was amplified by PCR from the plasmid BSSK-GAD-Ig and inserted into an eukaryotic expression plasmid pSNAV to construct a recombinant expression plasmid pSNAV/GAD-Ig. Plasmid pSNAV-GAD-Ig was then transfected into the rAAV2 packaging cell-BHK-21 cells using LipofectAMINE TM 2000 to produce rAAV-GAD-Ig. Target gene expression in BHK-21 cells infected by rAAV-GAD-Ig was confirmed by RT-PCR, Western blot and ELISA respectively. Then rAAV-GAD-Ig was injected into intramuscularly NOD mice. Antigen-specific tolerance in NOD mice was detected by specific lymphocytic proliferation reaction to GAD antigen.
Results: GAD-Ig gene was inserted into the genome of target cells. Target cells transfected by rAAV2-GAD-Ig could secret GAD-Ig. rAAV2-GAD-Ig induced specific tolerance in NOD mice.
Conclusion: rAAV2-GAD-Ig was obtained, which can be used in IDDM gene therapy in NOD mice.