转基因表达HBV抗原特异性细胞毒性T淋巴细胞受体  

Transgenic expression of T cell receptors on HBV-specific cytotoxic T lymphocytes

作　　者：吴静[1,2] 王琳[2] 刘妍[2] 叶海燕 丁宁[2] 刘永明[1] 徐东平[1,2] 

机构地区：[1]桂林医学院,广西桂林541004 [2]解放军302医院全军传染病研究所肝衰竭研究中心病毒性肝炎研究室,北京100039 [3]桂林市第三人民医院肝病科,广西桂林541004

出　　处：《细胞与分子免疫学杂志》2013年第5期453-457,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30771898);军队"十一五"医药卫生科研杰出人才项目(06J024)

摘　　要：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 express T lymphocyte receptors （TCRs） on hepatitis B virus （HBV）-specific cytotoxic T lym- phocytes （CTLs） mediated by retrovirus and investigate their binding affinity. Methods Peripheral blood mononuclear cells were isolated from acute hepatitis B patients with HI_A-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 cc and 13 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-A2 ~ healthy subjects. Results Two paired TCR Va and VI3, respectively named a2]1313 and cd513]3, were obtained from one patient with acute hepatitis B and HLA-A2 ＊. The titers of packaged recombinant retroviruses were ]. 5 x t05 - 5.0 x ]05 IU/mL. Immunofluorescence staining by anti-VIS13 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 VISt3 on Jurkat cells, 1.03%-2.06% for Vβ13 chain and 1. 0.5%-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 V13 or the pentamer. Conclusion 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.

关 键 词：乙型肝炎病毒 细胞毒性T淋巴细胞 T细胞受体 表位特异性 表达 

分 类 号：R392.11[医药卫生—免疫学] R373.21[医药卫生—基础医学]