重组SV40大T抗原慢病毒表达载体的构建

发布时间：2018-05-09 05:01

本文选题：SV大T抗原 + 慢病毒载体　；参考：《肿瘤基础与临床》2008年05期

【摘要】：目的构建含有重组SV40大T抗原基因的慢病毒表达载体pLentiGFP-Tag,转染真核细胞Eca-9706并检测SV40大T抗原基因在真核细胞内的表达。方法以PCR方法从含有SV40大T抗原基因的质粒上扩增出SV40大T抗原基因作为靶基因;将其连接至pGEM-TEasy载体上,并对靶基因进行DNA测序确认;再将靶基因与慢病毒表达载体pLentiGFP重组构建成为pLentiGFP-Tag;以脂质体介导转染真核细胞Eca-9706,观察慢病毒表达载体所携带的GFP基因在细胞内的表达,使用RT-PCR检测SV40大T抗原基因在细胞内的表达水平。结果成功克隆SV40大T抗原基因,经由DNA测序确认;成功筛选出含SV40大T抗原基因正插子的慢病毒表达载体pLentiGFP-Tag;转染真核细胞Eca-9706后48 h,观察到绿色荧光的广泛表达并检测出宿主细胞内高效表达的SV40大T抗原mRNA。结论成功构建含SV40大T抗原基因的慢病毒表达载体pLentiGFP-Tag,并观察到SV40大T抗原在真核细胞中的成功表达。
[Abstract]:Objective to construct a lentivirus expression vector pLentiGFP-Tagcontaining recombinant SV40 large T antigen gene, and to transfect Eca-9706 into eukaryotic cells and detect the expression of SV40 large T antigen gene in eukaryotic cells. Methods the large T antigen gene of SV40 was amplified from the plasmid containing large T antigen gene of SV40 by PCR and ligated to pGEM-TEasy vector, and the target gene was confirmed by DNA sequencing. The target gene and lentivirus expression vector pLentiGFP were recombined into pLentiGFP-Tag.Eca-9706 eukaryotic cells were transfected with liposome to observe the expression of GFP gene carried by lentivirus expression vector. The expression of large T antigen gene of SV40 was detected by RT-PCR. Results the large T antigen gene of SV40 was cloned successfully and confirmed by DNA sequencing. The lentivirus expression vector pLentiGFP-Tagcontaining the positive insertion of SV40 large T antigen gene was successfully screened, and the extensive expression of green fluorescence was observed at 48 h after transfection into eukaryotic cell Eca-9706, and the highly expressed SV40 large T antigen mRNA was detected in host cells. Conclusion the lentivirus expression vector pLentiGFP-Tagcontaining SV40 large T antigen gene was successfully constructed and the expression of SV40 large T antigen in eukaryotic cells was observed.
【作者单位】：郑州大学第一附属医院检验科;郑州大学基础医学院微生物学与免疫学教研室;
【分类号】：R392

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