1型糖尿病自身抗原胰岛素瘤相关蛋白2真核表达系统的构建和鉴定  

The construction and identification of the gene vaccination encoding insulinoma-associated protein 2 (IA-2)

作　　者：刘煜  朱虎 [2] 曾丽玲 [2] 陈家伟  杨金奎 [3] 钟耀华 [2] 王若宁 [4] 陈小章  

机构地区：[1]210029,南京医科大学第一附属医院内分泌科 [2]香港中文大学上皮细胞研究中心 [3]北京同仁医院内分泌科 [4]南京医科大学生化与分子生物学研究室

出　　处：《中华糖尿病杂志（1006-6187）》2005年第3期230-233,共4页

基　　金：国家自然科学基金资助项目(30300167)

摘　　要：目的构建能高效表达自身抗原胰岛素瘤相关蛋白2(IA2)的真核表达载体作为预防发生1型糖尿病(T1DM)的基因疫苗。方法利用基因工程技术构建重组pEGFP/IA2真核表达系统,并且转染、筛选稳定高表达IA2的RIN5F胰岛细胞株,用免疫印迹法并在荧光显微镜下鉴定其表达,利用共培养体系检测IA2刺激淋巴细胞增殖率。结果双酶切结果表明成功构建了重组pEGFP/IA2真核表达系统,免疫印迹法证实可在RIN5F细胞内高表达IA2。并且该表达的IA2可显著促进NOD小鼠淋巴细胞增殖反应(P<0.05)。结论成功构建的IA2真核表达系统对于T1DM基因疫苗预防策略和对于IA2生理功能的研究都具有重要的意义。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.

关 键 词：1型糖尿病 自身抗原 胰岛素瘤相关蛋白2 真核表达系统 IA-2 疫莳 

分 类 号：R587.1[医药卫生—内分泌]