Wu Jing, Wang Lin, Liu Yan, Ye Haiyan, Ding Ning, Liu Yongming, Xu Dongping
Guilin Medical University, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 May;29(5):453-7.
To express T lymphocyte receptors (TCRs) on hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) mediated by retrovirus and investigate their binding affinity.
Peripheral blood mononuclear cells were isolated from acute hepatitis B patients with HLA-A2⁺; phenotype, and after induced, HBV-specific CTLs were sorted out followed by cloning and proliferating. Cell RNAs were extracted. The sequences of TCR's α and β chains were obtained by means of RT-PCR, 5'RACE and OVER-LAP PCR, for constructing TCR retrovirus vectors. Through retrovirus-mediated transduction, HBV-specific TCRs were expressed on Jurkat cells and CD8⁺; T cells from HLA-A⁺; healthy subjects.
Two paired TCR Vα and Vβ, respectively named α21β13 and α15β13, were obtained from one patient with acute hepatitis B and HLA-A2⁺;. The titers of packaged recombinant retroviruses were 1.5×10;-5.0×10⁵; IU/mL. Immunofluorescence staining by anti-Vβ13 TCR-PE targeting the specific TCR and HLA-A2 restricted epitope-specific pentamer showed a positive expression of reconstructed TCR on T cells. The positive cells accounted for 1.06%-2.25% for Vβ13 on Jurkat cells, 1.03%-2.06% for Vβ13 chain and 1.05%-1.12% for the epitope-specific pentamer on T cells from healthy HLA-A2⁺; subjects respectively. By contrast, only less than 0.05% cells from healthy HLA-A2⁻ ;subjects were positive for either Vβ13 or the pentamer.
TCRs on HBV-specific CTLs could be expressed by TCR gene transfer mediated by retrovirus, and they were proved with binding affinity to HLA-A2-restricted epitope.
通过逆转录病毒介导表达乙型肝炎病毒（HBV）特异性细胞毒性T淋巴细胞（CTL）上的T淋巴细胞受体（TCR），并研究其结合亲和力。
从HLA-A2⁺急性乙型肝炎患者中分离外周血单个核细胞，经诱导后筛选出HBV特异性CTL，进行克隆和增殖。提取细胞RNA。通过RT-PCR、5'RACE和重叠PCR获得TCR的α和β链序列，构建TCR逆转录病毒载体。通过逆转录病毒介导的转导，在Jurkat细胞和来自HLA-A⁺健康受试者的CD8⁺T细胞上表达HBV特异性TCR。
从1例HLA-A2⁺急性乙型肝炎患者中获得了两对TCR Vα和Vβ，分别命名为α21β13和α15β13。包装的重组逆转录病毒滴度为1.5×10⁵-5.0×10⁵IU/mL。用抗Vβ13 TCR-PE针对特异性TCR和HLA-A2限制性表位特异性五聚体进行免疫荧光染色，结果显示重建的TCR在T细胞上呈阳性表达。Jurkat细胞上Vβ13的阳性细胞占1.06%-2.25%，来自健康HLA-A2⁺受试者的T细胞上Vβ13链的阳性细胞占1.03%-2.06%，表位特异性五聚体的阳性细胞占1.05%-1.12%。相比之下，来自健康HLA-A2⁻受试者的细胞中，Vβ13或五聚体阳性的细胞均不到0.
