NY-ESO-1抗原T细胞受体基因慢病毒载体的构建及鉴定

Construction and expression in Hela cell of NY-ESO-1 antigen T cell receptor lentiviral vector

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 establish an NY-ESO-1（New York esophageal squamous cell carcinoma 1） T Cell receptor（TCR） recombinant lentiviral vector.Methods Specific NY-ESO-1 TCR primers were designed,and NY-ESO-1 TCR gene was amplified by PCR（polymerase chain reaction） with the template of NY-ESO-1 TCR cDNA,which we had cloned before in our lab.The PCR products were digested by EcoR I and Not I,and directly cloned into pCL20c-MSCV-GFP vector named pCL20c-MSCV-ESOTCR and confirmed by digestion,PCR and sequencing.pCL20c-MSCV-ESOTCR and other three package plasmids pCL20c-HIV-gp、pCAG4-RTR2 and CAG-VSV-G were co-infected into T293 cell.Hela cells were infected with above collected supernatants and ESOTCR gene and protein were detected.Results NY-ESO-1 TCR lentiviral vector was successfully constructed by PCR and sequenceing and packaged.Recombinant lentiviral ESOTCR gene was detected by PCR and protein observed under the flurescence microscope.Conclusion We successfully established a pCL20c-MSCV-ESOTCR lentiviral vector which could express NY-ESO-1 TCR gene and protein.