| 魏丹芸,张洪,彭锐,等.ABCC2基因过表达细胞株的建立及鉴定[J].安徽医药,2016,20(3):433-436. |
| ABCC2基因过表达细胞株的建立及鉴定 |
| Construction and identification of the recombinant cell line over-expressing ABCC2 gene |
| 投稿时间：2015-10-09 |
| DOI： |
| 中文关键词: ABCC2基因 过表达 慢病毒载体 HEK293细胞 |
| 英文关键词: ABCC2 gene over-expression lentiviral vector HEK293 cell |
| 基金项目: |
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| 摘要点击次数: 3091 |
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| 中文摘要: |
| 目的 构建携带ABCC2基因的慢病毒载体,转染HEK293细胞,筛选出稳定过表达ABCC2基因的细胞株并进行鉴定,为体外实验确定与多药耐药相关蛋白2(MRP2)外排转运相关的底物药物及其转运机制提供细胞模型。方法 根据GenBank提供的ABCC2基因cDNA序列设计引物,PCR扩增该基因并将其连接至慢病毒载体PZE-LV105,包装病毒并感染HEK293细胞。用嘌呤霉素进行筛选得到过表达ABCC2基因的稳转细胞株,通过基因测序、实时荧光定量PCR(RTQ-PCR)和蛋白免疫印迹(Western blot)对稳转细胞进行鉴定。结果 测序结果证明重组慢病毒过表达载体所携带的ABCC2基因序列与GenBank提供的ABCC2序列一致；RTQ-PCR分析结果显示转染了ABCC2基因的HEK293细胞中MRP2 的mRNA相对表达比正常HEK293细胞和空白载体对照HEK293细胞高出约320倍；蛋白免疫印迹试验显示,转染了ABCC2基因的HEK293细胞中MRP2蛋白表达水平比正常HEK293细胞和空白载体对照HEK293细胞高出约150倍。结论 该实验成功构建了稳定过表达ABCC2基因的细胞株,该细胞株可用于初步筛选确定MRP2的特异性外排转运底物及其转运机制。 |
| 英文摘要: |
| Objective To construct the lentiviral vecter carrying human ABCC2 gene,then to screen out the stable cell line over-expressing ABCC2 after package and transfection to HEK293 cells,thus to provide cell model to determine substrate drugs of multidrug resistance protein 2(MRP2) in vitro.Methods The primers were designed according to the cDNA sequence of ABCC2 gene from GenBank. The gene were amplified by PCR and connected to lentiviral vector PEZ-LV105. The recombined lentiviral vector was packaged and transfected to HEK293 cells. After puromycin screening,HEK293 cells over-expressing human ABCC2 gene were selected and cloned. Finally,gene sequencing,real-time quantitative PCR(RTQ-PCR)and Western blot assays were performed for identification.Results RTQ-PCR showed that the ABCC2 mRNA in HEK293 cells transfected with exogenous ABCC2 gene was about 320 times higher than that of normal HEK293 cells and blank carrier HEK293 cells. MRP2 protein level in HEK293 cells with ABCC2 gene transfection was nearly 150 times higher than that of normal HEK293 cells and blank carrier HEK293 cells according to the Western blot.Conclusions Stable recombinant cell line over-expressing ABCC2 was successfully generated. The cell line could be useful in the confirmation of substrate drugs of multidrug resistance protein 2(MRP2)and the transport mechanism in vitro. |
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