小鼠Aire逆转录病毒表达载体的构建及表达

摘要

目的:将小鼠Aire基因定向连入MigR1-GFP质粒,得到长期稳定的Aire逆转录病毒表达载体,收集逆转录病毒上清感染胸腺基质细胞,为研究Aire在胸腺细胞终末分化中的作用奠定基础.方法:以小鼠胸腺基质细胞的cDNA为模板扩增得到Aire的ORF片段;将该片段连接入MigR1-GFP质粒;将构建成功的Aire-MigR1-GFP质粒转染293T细胞,收集逆转录病毒上清;使用逆转录病毒上清感染胸腺基质细胞系MTEC9,流式细胞仪检测感染效率,Western blot确定Aire蛋白的表达.结果:(1)成功得到Aire的ORF片段.(2)构建的Aire逆转录病毒表达质粒能够成功表达Aire蛋白.(3)逆转录病毒上清成功感染胸腺基质细胞系MTEC9.结论:成功构建了小鼠Aire逆转录病毒表达载体,并实现了Aire蛋白在胸腺基质细胞系中的强制性表达.%Objective: To construct a recombinant retroviral vector bearing murine Aire gene and establish a thymic epithelial cell line for stable expression of Aire. Methods: Aire ORF was amplified by PCR using the cDNA of mouse thymic stromal cells freshly isolated as template, and subcloned into MigRl-GFP vector to obtail MigRl-Aire-GFP vector. After introducing the MigRl-Aire-GFP into 293T package cells, the cell culture supernatant was used to infect MTEC9. The efficiency of infection was determined by FACS. Western blot was used to detect the expression of Aire. Results:(1) The full-length Aire ORF was obtained. (2)The constructed MigRl-Aire-GFP vector successfully expressed Aire. (3)The retroviral supernatant successfully infected MTEC9. Conclusion: The recombinant retroviral vector bearing murine Aire gene is successfully constructed, and the expression of Aire in mouse stromal cell line is implemented.