CAJ | 학술논문

目的：应用分子克隆技术，构建CYP2E1基因过表达载体，转染L02肝细胞，建立CYP2E1过表达细胞株并进行鉴定，为深入研究环境和职业毒物的毒作用机制提供模型细胞。方法：根据GenBank提供的基因cDNA序列设计引物，PCR扩增该基因并将其克隆到慢病毒过表达载体pLVX-acGFP1-C1中，将已经构建的慢病毒载体对293FT细胞进行转染，收集病毒上清，感染正常L02肝细胞。用嘌呤霉素进行筛选得到转染CYP2E1基因的L02细胞，通过基因测序、荧光定量PCR和Western blot对细胞株进行鉴定。结果：测序证明重组慢病毒过表达载体CYP2E1基因序列与GenBank提供的CYP2E1序列一致，荧光定量PCR检测转染CYP2E1基因的L02细胞比正常肝细胞CYP2E1 mRNA表达提高273倍，Western blot实验显示转染CYP2E1基因的L02细胞CYP2E1蛋白表达水平比正常肝细胞提高3.2倍。结论：成功构建了CYP2E1基因过表达细胞株，可应用于环境毒物和职业毒物的分子机制研究。CYP2E1
목적：응용분자극륭기술，구건CYP2E1기인과표체재체，전염L02간세포，건립CYP2E1과표체세포주병진행감정，위심입연구배경화직업독물적독작용궤제제공모형세포。방법：근거GenBank제공적기인cDNA서렬설계인물，PCR확증해기인병장기극륭도만병독과표체재체pLVX-acGFP1-C1중，장이경구건적만병독재체대293FT세포진행전염，수집병독상청，감염정상L02간세포。용표령매소진행사선득도전염CYP2E1기인적L02세포，통과기인측서、형광정량PCR화Western blot대세포주진행감정。결과：측서증명중조만병독과표체재체CYP2E1기인서렬여GenBank제공적CYP2E1서렬일치，형광정량PCR검측전염CYP2E1기인적L02세포비정상간세포CYP2E1 mRNA표체제고273배，Western blot실험현시전염CYP2E1기인적L02세포CYP2E1단백표체수평비정상간세포제고3.2배。결론：성공구건료CYP2E1기인과표체세포주，가응용우배경독물화직업독물적분자궤제연구。CYP2E1
OBJECTIVE:To construct the lentivirus vector with CYP2E1 gene overexpression,lentivirus was packaged,then transduced into normal human liver cells (L02 cells),finally the CYP2E1 overexpressing cells (CYP2E1 OE cells) were constructed and identified. METHODS:Primers were designed according to cDNA sequence of CYP2E1 gene from GenBank. The gene was amplified using PCR and ligated into the lentiviral vector pLVX-acGFP-C1. 293FT cells were transfected with the recombinant vector,viral supernatant was collected,L02 liver cells were then transfected. After puromycin screening,L02 cells with CYP2E1 gene transfection were constructed. Finally,gene sequencing,real-time fluorescent quantitative PCR and western blot assays were performed for identification. RESULTS:The sequence contained in the recombinant vector was exactly the same as CYP2E1 gene from GenBank. CYP2E1 gene expression level in L02 cells with CYP2E1 gene transfection was 273 times higher than that of normal L02 cells. CYP2E1 protein level in L02 cells with CYP2E1 gene transfection was 3.2 times higher than that of normal L02 cells. CONCLUSION:CYP2E1-overexpressing cell strain was successfully constructed by using lentiviral vector,this cell strain expressed CYP2E1 with high efficiency,and could become a useful tool for metabolism research of environmental or occupational pollutants.