稳定表达转录因子增强子结合蛋白α基因的白血病细胞株的构建及鉴定  

Construction and identification of leukemia cell strain for stable expression of CCAAT enhancer-binding protein α

作　　者：邵会媛[1] 栾材富[1] 刘杰[1] 张守信[1] 马新衡[1] 张贵丽[1] 孙成铭[1] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院检验中心,山东烟台264000

出　　处：《中国生物制品学杂志》2014年第5期636-638,641,共4页Chinese Journal of Biologicals

基　　金：山东省优秀中青年科学家科研奖励金(BS2011YY064);烟台市科技发展计划项目(2011203)

摘　　要：目的构建稳定表达转录因子增强子结合蛋白α(CCAAT-enhancer-binding proteinα,C/EBPα)基因的白血病髓性细胞系。方法利用DNA重组技术将C/EBPα基因插入到慢病毒表达载体PLVX-EGFP-3FLAG-Puro中,构建重组慢病毒质粒PLVX-C/EBPα-3FLAG-Puro,与慢病毒包装辅助质粒plp1、plp2、plp/VSVG(1∶1∶1)共转染293细胞,包装出慢病毒,实时荧光定量PCR法检测病毒滴度;收集病毒,分别以10、20 MOI感染K562细胞,G418筛选阳性克隆,利用有限稀释法筛选稳定转染的单克隆细胞株,并采用Western blot法检测C/EBPα蛋白的表达。结果重组慢病毒质粒PLVX-C/EBPα-3FLAG-Puro经菌落PCR及测序鉴定证明构建正确;包装后的慢病毒pSB957的病毒滴度为5×106 TU/ml;pSB957-K562细胞中C/EBPα蛋白的表达水平较高,且20和10 MOI pSB957感染的K562细胞中,C/EBPα蛋白的表达水平无明显差异。结论成功构建了pSB957-K562白血病细胞株,为进一步研究C/EBPα在白血病发生发展中的作用提供了良好的细胞模型。Objective To construct a myeloid leukemia cell strain for stable expression of CCAAT-enhancer-binding protein α（C / EBPα）. Methods C / EBPα gene was inserted into lentivirus expression vector PLVX-EGFP-3FLAG-Puro by recombinant DNA technology. The constructed recombinant plasmid PLVX-C / EBPα-3FLAG-Puro was co-transfected to 293 cells with helper plasmids plp1,plp2 and pol / VSVG（at a ratio of 1 ∶ 1 ∶ 1） for packaging. The obtained lentivirus was determined for titer by real-time fluorescent quantitative PCR,then harvested and infected to K562 cells at MOIs of 10 and 20 respectively. Positive clones were screened with G418,from which the monoclonal cell strains transfected stably were screened by limiting dilution method,and determined for expression of C / EBPα protein by Western blot. Results Colony PCR and sequencing proved that recombinant lentivirus expression vector PLVX-C / EBPα-3FLAG-Puro was constructed correctly. The titer of obtained lentivirus was 5 × 10^6TU / ml. The expression level of C / EBPα protein was high in pSB957-K562 cells,while showed no significant difference in the K562 cells infected with pSB957 at MOIs of 20 and 10. Conclusion The pSB957-K562 cell line was successfully constructed,which provided a good cell model for further study on the role of C / EBPα in genesis and progress of leukemia.

关 键 词：转录因子增强子结合蛋白α 慢病毒 基因表达 

分 类 号：R733.7[医药卫生—肿瘤] Q789[医药卫生—临床医学]