BAD慢病毒载体的构建及其对A549细胞增殖的影响

目的探讨促凋亡基因BAD(Bcl-2-associated death protein)重组慢病毒表达载体构建及其对肺癌A549细胞株增殖的影响。方法应用PCR法从含有目的基因的质粒pAV-MCMV-BAD-GFP中扩增BAD基因片段,克隆至慢病毒载体(pLVX-IRES-ZsGreen 1),应用PCR、酶切和测序鉴定正确后,经病毒包装,感染A549细胞,经流式细胞仪筛选稳定表达细胞株,Western blot鉴定重组慢病毒感染的A549细胞BAD的表达。采用CCK-8试剂盒定点检测细胞的光密度(OD)值,分析BAD蛋白过表达对A549细胞增殖能力的影响。结果酶切鉴定和基因测序证实长度为507bp的BAD基因成功克隆至慢病毒表达载体pLVX-IRES-ZsGreen1;重组慢病毒通过感染A549细胞株和流式细胞仪筛选出单克隆细胞株BAD-A549,Western blot检测结果显示,细胞培养BADA549细胞可稳定过表达BAD蛋白。体外增殖结果显示,细胞培养120~144h,BAD组的OD值均较对照组低(P<0.05)。结论成功构建了BAD重组慢病毒载体,获得稳定表达BAD的A549细胞株。BAD过表达能有效抑制A549细胞的增殖速度。

Construction of BAD Lentivirus Vector and Its Effect on Proliferation in A549 Cell Lines

Objective To construct the recombinant lentivirus expressing vector BAD (Bcl-2-associated death protein) gene and to study its effect on A549 cell proliferation. Methods The BAD gene was amplified from plasmid pAV-MCMV-BAD-GFP by PCR. The purified BAD gene fragment was inserted into a lentivirus vector (pLVX-IRES-ZsGreen 1), and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. A549 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with flow cytometry. The expression of BAD in A549 cell lines stably transduction with a lentivirus was examined using Western blot. The effect of BAD overexpression on proliferation of A549 cells was evaluated by using CCK-8 kit. Results Restriction enzyme digestion and DNA sequencing showed that the full-length BAD gene (507 bp) had been successfully subcloned into the lentiviral vector to result in the recombinant vector pLVX-IRES-ZsGreen 1. Monoclonal cell lines BAD-A549 was produced after transfection with the recombinant lentivirus and selected with flow cytometry. Stable expression of BAD protein was verified by Western blot. In vitro, the OD value in BAD group was significantly lower than that of control groups from 120-144 h (P<0.05). Conclusion A549 cell lines stably transduced with a lentivirus expressing the BAD gene had been successfully generated. In vitro, BAD overexpression significantly inhibited A549 cells proliferation.