title: [Development of rAAV2-GAD-Ig for IDDM gene therapy].
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  rAAV2-GAD-Ig was obtained, which can be used in IDDM gene therapy in NOD mice.
authors.affiliation: Laboratory of Molecular Immunology, Basic Medical Institute, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Ren-Xi
authors.initials: RX
authors.lastname: Wang
authors.forename: Jian-An
authors.initials: JA
authors.lastname: Wang
authors.forename: Lun
authors.initials: L
authors.lastname: Song
authors.forename: Gen-Cheng
authors.initials: GC
authors.lastname: Han
authors.forename: Bei-Fen
authors.initials: BF
authors.lastname: Shen
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Wu
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D049109:Cell Proliferation; D004274:DNA, Recombinant / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000276:immunology / Q000628:therapy*; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000276:immunology; D007108:Immune Tolerance; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D051379:Mice; D016688:Mice, Inbred NOD; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011506:Proteins; D012333:RNA, Messenger; D005968:Glutamate Decarboxylase