TCR Vα23-Vβ13基因修饰T细胞及其特异性细胞毒活性研究

Specific cytotoxicity of TCR Vα23-Vβ13 gene-modified T-cells

目的:分析弥漫大B细胞淋巴瘤(DLBCL)抗原特异T细胞受体(TCR)Vα23-Vβ13基因修饰T细胞后的体外特异性杀伤活性。方法:扩增并克隆已鉴定的DLBCL患者外周血中单克隆增殖T细胞的TCR Vβ13和TCR Vα23基因全长序列,构建TCR Vβ13-IRES-TCR-Vα23双基因重组质粒,经核转染的方法将其转染正常人T细胞,通过real-time PCR和Western blotting检测TCR Vβ13和TCR Vα23这2个外源基因在mRNA和蛋白水平的体外表达情况,并对TCR转基因T细胞进行体外细胞毒性检测。结果:经酶切分析和核酸序列测定证实重组质粒构建正确,转染正常人T细胞后,TCR Vβ13和TCR Vα23在mRNA及蛋白水平实现了共表达。转染后3 d,TCR转基因T细胞对DLBCL细胞株Toledo细胞的杀伤活性明显高于非特异细胞株Raji细胞和Molt-4细胞,显示出特异性细胞毒活性。结论:成功构建DLBCL相关抗原特异的TCR Vβ13-IRES-TCR Vα23双基因真核表达质粒;TCR基因修饰T细胞可获得特异性细胞毒活性。

AIM ： To analyze the cytotoxicity of TCR Vα23 - Vβ13 gene - modified T - cells specific to diffuse large B- cell lymphoma（DLBCL） associated antigens in vitro. METHODS： The identified full -length TCR Vβ13 and Vα23 genes of monoclonally expanded T - cells in the peripheral blood from a DLBCL patient were amplified and cloned. The bicistronic recombinant plasmid TCR Vβ13 - IRES - TCR Vα23 was constructed and transfected into T - cells isolated from a healthy individual by NucleofectorTM technology. The mRNA expression of antigen - specific TCR Vα23 and Vβ13 genes and the corresponding proteins were determined by real -time PCR and Western blotting, respectively. The specific cytotoxicity of TCR gene - transferred T - cells in vitro was detected. RESULTS ： The recombinant plasmid was constructed successfully and verified by restriction enzyme digestion and nucleotide sequencing. The co - expression of antigen - specific TCR Vα23 and Vβ13 genes at mRNA and protein levels in the transfected healthy T - cells were observed in vitro. Three days after transfection, the cytotoxicity of TCR gene - transduced CD3 ＋ T - cells against Toledo ceils was significantly higher than that against Raji cells or Molt - 4 cells. The DLBCL - specific cytotoxic T - lymphocytes（CTL） were inducted. CONCLUSION ： The bicistronic eukaryotic expression plasmid TCR Vβ13 - IRES - TCR Vα23 specific to DLBCL - asso- ciated antigens is constructed successfully. The TCR gene - transferred T - cells display the ability of DLBCL - specific cytotoxicity.