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目的:构建Jurkat细胞株独特型TCR Vα1-pIRES-TCR Vβ8表达载体,转染后了解其体外表达情况。方法:根据聚合酶链反应-基因扫描(PCR-Genescan)检测Jurkat细胞株TCR Vα及Vβ亚家族表达情况,分别将其所表达的单克隆性的TCR Vα1及TCR Vβ8亚家族基因片段克隆至质粒pIRES的多克隆位点A(MCS A)和多克隆位点B(MCS B)中。通过限制性酶切分析、序列分析、RT-PCR、间接免疫荧光、流式细胞术(FCM)手段鉴定重组表达载体的正确性以及转染A549和Molt4细胞后基因及蛋白的表达情况。结果:构建了2组TCR Vα1-pIRES-TCR Vβ8表达载体,该表达载体转染A549和Molt4细胞后,在mRNA和蛋白水平检测到了TCR Vα1和TCR Vβ8的表达。结论:成功构建了2种独特型Vα1-pIRES-TCR Vβ8真核表达载体,为利用特异性TCR基因修饰T细胞的研究提供方法学依据。
OBJECTIVE: To construct a unique TCR Vα1-pIRES-TCR Vβ8 expression vector for Jurkat cell line and to investigate its expression in vitro. Methods: The expression of TCR Vα and Vβ subfamilies of Jurkat cells were detected by PCR-Genescan. The cloned TCR Vα1 and TCR Vβ8 subfamilies were cloned into plasmid Multiple cloning site A (MCS A) and multiple cloning site B (MCS B) of pIRES. The correctness of the recombinant expression vector and the expression of the gene and protein after transfected A549 and Molt4 cells were identified by restriction analysis, sequence analysis, RT-PCR, indirect immunofluorescence and flow cytometry (FCM). Results: Two TCRVα1-pIRES-TCR Vβ8 expression vectors were constructed. The expression of TCR Vα1 and TCR Vβ8 were detected at mRNA and protein levels after transfection into A549 and Molt4 cells. CONCLUSION: Two unique Vα1-pIRES-TCR Vβ8 eukaryotic expression vectors were successfully constructed, which provided the methodological basis for the study of T cells modified by specific TCR gene.