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目的构建NY-ESO-1抗原T细胞受体基因(TCellReceptor,TCR)的慢病毒载体并获得重组慢病毒。方法设计特异性NY-ESO-1TCR的引物,用聚合酶链反应(PCR)对我们前期已克隆的NY-ESO-1TCRcDNA进行体外扩增,双酶切后定向插入到pCL20c-MSCV-GFP质粒,构建NY-ESO-1抗原特异性TCR基因的重组慢病毒载体pCL20c-MSCV-ESOTCR,并经酶切、PCR及测序鉴定。用pCL20c-MSCV-ESOTCR﹑pCL20c-HIV-gp、pCAG4-RTR2和CAG-VSV-G共转染T293包装细胞,获得上清,上清浓缩后感染Hela细胞,观察ESOTCR基因及蛋白水平的表达。结果经PCR及测序证实,成功构建重组慢病毒载体pCL20c-MSCV-ESOTCR,包装产毒后感染Hela细胞,RT-PCR法及荧光显微镜均能检测及观察到基因及蛋白水平的表达。结论成功构建了表达NY-ESO-1抗原T细胞受体基因(TCellReceptor,TCR)的重组慢病毒载体。
Objective To construct lentiviral vector of NY-ESO-1 antigen T cell receptor gene (TCR) and obtain recombinant lentivirus. Methods NY-ESO-1 TCR primers were designed and the NY-ESO-1 TCR cDNAs cloned in our previous study were amplified by polymerase chain reaction (PCR). After double digestion, we inserted the plasmid into pCL20c-MSCV-GFP plasmid, Recombinant lentiviral vector pCL20c-MSCV-ESOTCR for NY-ESO-1 antigen-specific TCR gene was constructed and identified by restriction enzyme digestion, PCR and sequencing. The T293 packaging cells were cotransfected with pCL20c-MSCV-ESOTCR, pCL20c-HIV-gp, pCAG4-RTR2 and CAG-VSV-G to obtain the supernatant. The supernatant was concentrated and then infected into Hela cells to observe the expression of ESOTCR gene and protein. Results The recombinant lentiviral vector pCL20c-MSCV-ESOTCR was successfully constructed and confirmed by PCR and sequencing. Hela cells were infected and infected by the packaging. The expression of both gene and protein was detected by RT-PCR and fluorescence microscopy. Conclusion Recombinant lentiviral vector expressing NY-ESO-1 antigen T cell receptor gene (TCR) was successfully constructed.