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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 a eukaryotic expression vector that can efficiently express the autoantigen insulinoma related protein 2 (IA2) as a gene vaccine for the prevention of type 1 diabetes mellitus (T1DM). Methods Recombinant pEGFP / IA2 eukaryotic expression system was constructed by gene engineering and transfected into RIN5F islet cells stably expressing IA2. The expression of IAA was detected by immunoblotting and fluorescent microscopy. The co-culture system was used to detect IA2 stimulus Lymphocyte proliferation rate. Results The results of double enzyme digestion showed that the eukaryotic expression system of recombinant pEGFP / IA2 was constructed successfully. Western blotting confirmed that IA2 was highly expressed in RIN5F cells. And the expression of IA2 could significantly promote lymphocyte proliferation in NOD mice (P <0.05). Conclusion The successful construction of IA2 eukaryotic expression system is of great significance for the prevention of T1DM gene vaccine and the study of IA2 physiological function.