转录因子Bach2的构建及表达

Construction and expression of the transcrption factor Bach2

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 lentivirus shuttle plasmids which can express human Bach2 gene, so as to explore its mechanism in the immune diseases. Methods The peripheral blood mononuclear cells（PBMC） were isolated from the healthy individuals and the total RNA was extracted and reverse transcripted to eDNA, then, Bach2 CDS was amplified and inserted to the pLVX- IRES- ZsGreenl. Calcium phosphate co -transfection method was used to package the lentivirus, which infected human HEK293T cells. Blank plasmid infected with HEK293T cells were used as control. Expression of Bach2 gene in blank plasmid and over expression plasmid in HEK293T cells was analyzed by Western blot. Results PCR results showed that Bach2 was successfully inserted into the pLVX - IRES - ZsGreenl plasmid, real - time PCR and Western blot confirmed that Bach2 gene was highly expressed in HEK293T cells. Conclusion Lentivirns recombinant plasmid highly expressing Bach2 gene was suecessfully constructed and expressed in HEK293T cells.