论文部分内容阅读
目的:构建TAP1基因慢病毒过表达载体及检测其过表达效率,为制备前列腺癌的免疫细胞疫苗提供基础。方法:将pDONR223-TAP1质粒和pLenti6.3-IRES-EGFP构建形成pLenti6.3-TAP1-IRES-EGFP表达质粒,采用慢病毒包装试剂盒包装产生慢病毒;感染293T细胞后RT-PCR检测目的基因TAP1的表达;采用荧光FACS计算病毒活性滴度。RT-PCR检测转染PC-3细胞后TAP1表达。结果:PCR和测序证实pLenti6.3-TAP1-IRES-EGFP慢病毒质粒构建成功;包装纯化后收集的病毒液滴度经流式细胞仪检测滴度为2.87×107 TU/ml;转染后的PC-3后,TAP1转染组TAP1的表达量显著高于对照组。结论:成功构建人TAP1基因慢病毒过表达载体,可以上调PC-3细胞TAP1的表达。
OBJECTIVE: To construct a lentivirus overexpression vector for TAP1 gene and to detect its over-expression efficiency, and to provide a basis for preparation of immune cell vaccine for prostate cancer. Methods: The pLONR223-TAP1 plasmid and pLenti6.3-IRES-EGFP were constructed into pLenti6.3-TAP1-IRES-EGFP expression plasmid and packaged by lentivirus packaging kit to generate lentivirus. 293T cells were infected with 293T cells for detection of the target gene TAP1 expression; using fluorescent FACS calculate viral activity titers. The expression of TAP1 in transfected PC-3 cells was detected by RT-PCR. Results: The plasmid pLenti6.3-TAP1-IRES-EGFP was successfully constructed by PCR and sequencing. The titer of the virus collected after purification was 2.87 × 107 TU / ml by flow cytometry. After transfection After PC-3, the expression level of TAP1 in TAP1 transfection group was significantly higher than that in control group. Conclusion: The successful construction of human TAP1 lentivirus overexpression vector can up-regulate the expression of TAP1 in PC-3 cells.