【摘要】 目的　构建融合基因sOVA linker β2m ,探讨通过内源性途径递呈的肽在细胞表面形成MHCⅠ类分子复合物的情况。方法　构建了真核表达载体pcDNA3/sOVA linker β2m及不含有 β2m基因的对照载体 ,以研究 β2m是否能在此过程中起到辅助的作用。结果　RT PCR与原位杂交结果显示在各转导细胞中均有融合基因的正确表达 ,且二者的表达水平差异无显著性 (P >0 .0 5 )。进一步对细胞内、细胞表面H 2Kb OVA复合物的分布进行分析 ,表明各转导细胞中均有正确的蛋白质分子翻译合成 ,并能表达在细胞表面 ,但融合有 β2m基因的EL4 /eb2m细胞的表达量远远高于没有融合 β2m基因的转导细胞EL4 /OVA。结论　β2m基因与特异性抗原肽的融合 ,易于形成稳定的MHCⅠ类分子复合物 ,提高抗原呈递效率。更多还原

【Abstract】 Objective To construct sOVA linker β2m fusion gene, and explore antigen presentation by endogenous pathway. Methods Eukaryotic expression vectors pcDNA3/sOVA linker β2m and pcDNA3/sOVA linker were constructed and two transfectants (EL4/eb2m and EL4/OVA) were obtained after G418 selection. RT PCR and in situ hybridization were used to evaluate the expression of the fused gene. Laser scanning confocal microscopy and FACS can were used to detect the distribution of H 2K b OVA complex. Results The results of RT PCR and in situ hybridization showed that both cells had correct expression of fusion genes, and no significant difference was founded ( P >0.05). But much more H 2K b OVA molecules were detected on the cell surface of EL4/eb2m than EL4/OVA. Conclusion It suggested that human β2m with a tethered H 2K b restricted epitope (OVA), was able to form CTL target structures endogenously through expression in transfected murine cell lines. This result provides a technical approach for the in vivo induction of epitope specific CTL responses. [更多还原