用于IDDM基因治疗的rAAV2-GAD-Ig的研制  

Development of rAAV2-GAD-Ig for IDDM gene therapy

作　　者：王仁喜[1] 王建安[1] 宋伦[1] 韩根成[1] 沈倍奋[1] 吴小兵 黎燕[1] 

机构地区：[1]军事医学科学院基础医学研究所分子免疫室,北京100850 [2]军事医学科学院国家"863"计划病毒载体研发基地,北京100850

出　　处：《细胞与分子免疫学杂志》2005年第1期24-28,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助(No. 2001AA217061 );国家重点基础研究发展规划(973)资助(No.2001CB510005)

摘　　要：　目的: 构建重组真核表达载体pSNAV- GAD -Ig[GAD- Ig融合基因是由小鼠GAD(glutamicaciddecarboxylase)基因插入到小鼠IgG3重链(Ig)基因的N端而成的 ], 制备含GAD- Ig融合基因cDNA的重组缺陷型腺相关病毒 -2 (rAAV2- GAD -Ig), 并探讨rAAV2所介导的GAD- Ig是否能诱导I型糖尿病(IDDM)动物模型 NOD(nonobesediabetic)小鼠产生特异性免疫耐受。方法: 从含GAD- Ig融合基因的中介载体BSSK(BSSK- GAD- Ig)中, 用PCR方法扩增目的基因片段, 并克隆入真核表达载体pSNAV中, 构建重组真核表达载体pSNAV- GAD Ig。以脂质体法将pSNAV GAD -Ig转染到rAAV2包装细胞BHK -21中, 产生rAAV2 -GAD- Ig。采用R-T PCR、Westernblot和ELISA法, 检测rAAV2- GAD -Ig感染的BHK- 21细胞中目的基因的表达。应用GAD抗原体外诱导特异性T细胞的二次应答, 来检测rAAV2所介导的GAD -Ig是否能诱导NOD小鼠产生特异性免疫耐受。结果: GAD- Ig目的基因已整合到靶细胞的基因组中, 并能以分泌形式表达目的蛋白GAD- Ig。将rAAV2 GAD- Ig肌注后, NOD小鼠能产生特异性免疫耐受。结论: 获得了可用于NOD小鼠治疗研究的rAAV2 GAD- Ig。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 TM2000 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. 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.

关 键 词：谷氨酸脱羧酶(GAD) GAD-Ig pSNAV-GAD-Ig rAAV2一GAD—Ig 特异性免瘦耐受 

分 类 号：Q78[生物学—分子生物学]