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目的构建高效表达人Bach2基因的慢病毒重组质粒,并探索其在免疫疾病中的机制。方法取健康人外周血分离单个核细胞并提取总RNA逆转录为c DNA,扩增Bach2基因CDS序列,插入p LVX-IRES-Zs Green1慢病毒质粒。磷酸钙共转染法包装慢病毒,感染人HEK293T细胞。空白质粒感染HEK293T细胞作为对照。免疫印迹分析空白质粒和过表达质粒HEK293T细胞中Bach2基因表达。结果 PCR结果显示,Bach2成功插进p LVX-IRES-Zs Green1质粒中,real-time PCR以及Western blot证实,Bach2基因成功在HEK293T细胞中高效表达。结论成功构建高效表达Bach2基因的慢病毒穿梭质粒,并成功在HEK293T细胞表达。
Objective To construct a recombinant lentiviral vector expressing human Bach2 gene efficiently and to explore its mechanism in immune diseases. Methods Peripheral blood mononuclear cells were isolated from healthy volunteers and the total RNA was reverse transcribed into c DNA. The CDS sequence of Bach2 gene was amplified and inserted into p LVX-IRES-Zs Green1 lentiviral plasmid. The lentivirus was packaged by calcium phosphate cotransfection and infected human HEK293T cells. Blank plasmids were infected with HEK293T cells as a control. Bach2 gene expression in blank plasmids and overexpression plasmid HEK293T cells was analyzed by immunoblotting. Results The PCR results showed that Bach2 was successfully inserted into p LVX-IRES-Zs Green1 plasmid. Real-time PCR and Western blot confirmed that Bach2 gene was successfully expressed in HEK293T cells. Conclusion The lentiviral shuttle plasmid with high expression of Bach2 gene was successfully constructed and successfully expressed in HEK293T cells.