IRF4逆转录病毒表达载体的构建及鉴定

Construction and Identification of Retroviral Expression Vector Harboring Murine IRF4

目的将小鼠IRF4基因定向克隆入MSCV-IRES-GFP质粒,得到长期稳定表达IRF4的逆转录病毒载体.方法以含IRF4 cDNA质粒作为模板,PCR扩增所需目的片段;将该片段连接入MSCV-IRES-GFP质粒;以磷酸钙法转染293T细胞,收集病毒上清感染GP+E86细胞后,流式细胞术和蛋白印迹技术检测IRF4的表达.结果经琼脂糖凝胶电泳鉴定,目的片段成功连接到逆转录病毒MSCV载体上.逆转录病毒上清成功感染GP+E86细胞系.结论成功构建了小鼠IRF4逆转录病毒表达载体,并实现了IRF4在GP+E86细胞系中的外源性表达.

Objective To construct a recombinant retroviral vector bearing murine IRF4 cDNA and establish a GP＋E86 cell line for stable expression of IRF4. Methods IRF4 cDNA fragment was amplified from a plasmid containing IRF40RF by PCR, and cloned into MSCV-IRES-GFP retroviral vector. The recombinant vector was transfected into 293T. The virus from the supernatant was harvested and used to infect GP＋E86 cell line. Expression of IRF4 in infected cells was analyzed by fluorescence activated cell sorter and confirmed by Western blotting. Results IRF4 cDNA was successfully cloned into MSCV-IRES-GFP retroviral vector. The resulting retrovirus successfully infected GP＋E86 cell line. Conclusion The recombinant retroviral vector bearing murine IRF4 cDNA has been successfully constructed, and the expression of exogenous IRF4 gene in mouse GP＋E86 cell line has been achieved.