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目的通过逆转录病毒介导HBV抗原特异性细胞毒性T细胞(CTL)的T细胞受体(TCR)转基因表达,初步观察其结合活性。方法从HLA-A2阳性急性乙肝患者外周血中诱导、分选、克隆和扩增HBV抗原特异性CTL;提取细胞RNA,用RT-PCR、5’-RACE和OVER-LAP PCR等方法获取TCR的α和β链编码基因;构建TCR重组逆转录病毒,介导特异性TCR分别在人Jurkat T细胞和HLA-A2阳性健康人CD8 T淋巴细胞上表达。结果从1例HLA-A2阳性急性乙肝患者样本中分别获得了2组TCR Vα、Vβ配对,分别命名为α21β13、α15β13,包装的重组逆转录病毒滴度为(1.5～5.0)×105IU/mL,用针对目标TCR的特异性Vβ链抗体(抗Vβ13 TCR-PE)和HLA-A2限制性表位特异性五聚体(pentamer)进行免疫荧光染色,重组TCR在T细胞表面获得表达:其中在Jurkat细胞上转入的Vβ13链表达细胞占1.06%～2.25%,在HLA-A2阳性健康人T细胞上Vβ13阳性细胞和pentamer阳性细胞分别占到1.03%～2.06%和1.05%～1.12%,在HLA-A2阴性健康人T细胞上Vβ13阳性细胞和pentamer阳性细胞均低于0.05%。结论通过逆转录病毒介导可以使HBV特异性CTL TCR获得转基因表达,具有结合HLA-A2限制性表位的活性。
Objective To observe the T-cell receptor (TCR) transgene expression of retrovirus-mediated HBV antigen-specific cytotoxic T lymphocytes (CTL) and to observe its binding activity. Methods HBV antigen-specific CTLs were induced, sorted, cloned and amplified from the peripheral blood of patients with HLA-A2 positive acute hepatitis B. The RNAs were extracted and the expression of TCR was obtained by RT-PCR, 5’-RACE and OVER-LAP PCR α and β chain coding genes. TCR recombinant retrovirus was constructed and specific TCRs were expressed on human Jurkat T cells and HLA-A2 positive healthy human CD8 T lymphocytes, respectively. Results Two samples of TCR Vα and Vβ were obtained from one patient with HLA-A2 positive acute hepatitis B, and named as α21β13 and α15β13 respectively. The packaged recombinant retrovirus titer was (1.5-5.0) × 105IU / mL, Immunofluorescence staining was performed with specific Vβ chain antibodies against the target TCR (anti-Vβ13 TCR-PE) and HLA-A2 restricted epitope-specific pentamers, where the recombinant TCR was expressed on T cell surfaces: where Jurkat Vβ13 chain transfected cells accounted for 1.06% ~ 2.25%, Vβ13 positive cells and pentamer positive cells accounted for 1.03% ~ 2.06% and 1.05% ~ 1.12% of HLA-A2 positive healthy human T cells, Vβ13-positive cells and pentamer-positive cells on -A2-negative healthy human T cells were less than 0.05%. Conclusion HBV-specific CTL TCR can be obtained transgene expression by retroviral vector and bind to HLA-A2-restricted epitopes.