CAJ | 학술논문

目的构建能高效表达自身抗原胰岛素瘤相关蛋白2(IA-2)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/IA-2真核表达系统,并且转染、筛选稳定高表达IA-2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IA-2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/IA-2真核表达系统,免疫印迹法证实可在RIN5F细胞内高表达IA-2.并且该表达的IA-2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IA-2真核表达系统对于T1DM基因疫苗预防策略和对于IA-2生理功能的研究都具有重要的意义.
목적구건능고효표체자신항원이도소류상관단백2(IA-2)적진핵표체재체작위예방발생1형당뇨병(T1DM)적기인역묘. 방법이용기인공정기술구건중조pEGFP/IA-2진핵표체계통,병차전염、사선은정고표체IA-2적RIN5F이도세포주,용면역인적법병재형광현미경하감정기표체,이용공배양체계검측IA-2자격림파세포증식솔. 결과쌍매절결과표명성공구건료중조pEGFP/IA-2진핵표체계통,면역인적법증실가재RIN5F세포내고표체IA-2.병차해표체적IA-2가현저촉진NOD소서림파세포증식반응(P<0.05). 결론성공구건적IA-2진핵표체계통대우T1DM기인역묘예방책략화대우IA-2생리공능적연구도구유중요적의의.
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.