Bcl-2修饰因子过表达及敲减A549稳转细胞株的构建

Construction of A549 stable cell lines with Bcl-2 modifying factor overexpression and knockdown

目的构建Bcl-2修饰因子(BMF)过表达及BMF敲减A549稳转细胞株。方法本研究为实验研究。根据BMF基因序列设计合成特异性引物并扩增目的基因,将其定向连接至经限制性内切酶BstBⅠ/SpeⅠ酶切后的GL159载体上,构建GL159-BMF重组质粒,经过筛选和测序鉴定后的阳性克隆转染293T细胞,包装慢病毒并测定病毒滴度。构建BMF-shRNA,连接到AgeⅠ和EcoRⅠ双酶切后的pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE载体上,经鉴定后转染293T细胞,包装慢病毒、测定病毒滴度,最终转染至A549细胞内。采用RT-PCR技术检测A549稳转细胞株BMF mRNA的表达情况。结果重组质粒GL159-BMF及BMF-shRNA在慢病毒的介导下转染至A549细胞内且稳定表达。RT-PCR检测结果表明:BMF过表达组的BMF mRNA表达量显著高于其对照组,差异倍数为986.23±35.47(P<0.001),BMF敲减组的BMF mRNA表达量显著低于其对照组,差异倍数为0.18±0.06(P<0.001)。结论成功构建BMF过表达及敲减A549细胞株,为后续BMF在慢性阻塞性肺疾病细胞凋亡机制中的研究奠定了基础。

Objective To construct A549 stable cell lines with Bcl-2 modifying factor(BMF)overexpression and BMF knockdown.Methods This was an experimental study.According to the BMF gene sequence,specific primers were designed and synthesized,the target gene was amplified,and the target primers were connected to GL159 vector digested by restriction endonuclide BstBⅠ/SpeⅠ.The GL159-BMF recombinant plasmid was constructed.After screening and sequencing,the positive clones were transfected into 293T cells,lentivirus was packaged,and virus titer was determined.BMF-shRNA was constructed and connected to pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE vector with enzymedigested by Age I and EcoR I.After identification,it was transfected into 293T cells,then lentivirus was packaged,virus titer was determined,and finally it was transfected into A549 cells.The expression of BMF mRNA in A549 cells was detected by the real-time fluorescent quantitative PCR.Results The recombinant plasmids GL159-BMF and BMF shRNA were transfected into A549 by the mediation of the lentivirus and stably expressed.RT-PCR results showed that the expression of BMF mRNA in the BMF overexpression group was significantly higher than that in the control group(Fold change 986.23±35.47,P<0.001).The expression of BMF mRNA in BMF knockdown group was significantly lower than that in the control group(Fold change 0.18±0.06,P<0.001).Conclusions A549 cell lines with BMF-overexpression and BMF-knockdown were successfully established,which can lay the foundation for the subsequent study of BMF in the mechanism of cell apoptosis in chronic obstructive pulmonary disease.