抗CD20scFv/CD80/CD28/ζ重组基因修饰T细胞的构建与靶向杀伤效果

目的构建表达抗CD20scFv／CD80／CD28／ζ重组基因修饰T细胞，体外观察其特异性清除CD20^＋淋巴瘤细胞的能力，为肿瘤的过继免疫治疗提供新思路。方法采用DNA重组技术将pBULLET上的CD28-ζcDNA插入到已含有抗CD20scFv／CD80的真核表达载体pLNCX质粒上，转染PA317包装细胞，挑取高滴度的包装细胞收获病毒，用收获的病毒感染刺激分裂的人外周血T淋巴细胞，经G418筛选1周后与CD20^＋淋巴瘤细胞株Daudi、Raji在体外共同培养，用非放射性的细胞杀伤试剂盒和ELISA检测试剂盒分别检测T细胞的抗原特异性杀伤和细胞因子分泌的功能。结果酶切及测序鉴定均证实pLNCX／抗CD20seFv／CD80／CD28／ζ构建成功，重组基因修饰的T细胞能在体外杀伤CD20^＋淋巴瘤细胞株Daudi、Raji，而对CD20^＋细胞株K562无杀伤作用，同时CD20^＋靶细胞组上清液中IL-2和IFN-γ水平与CD20^-组相比明显升高。结论重组基因修饰的CD20靶向性T细胞构建成功。该T细胞在体外能特异性杀伤CD20^＋淋巴瘤细胞株。

Construction of anti-CD20scFv/CD80/CD28/ζ recombinant gene modified T cell and research on its targeting cytotoxicity

OBJECTIVE: To construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy. METHODS: CD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 gene. The recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radioactivation cytotoxicity assay and ELISA respectively. RESULTS: The recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro. CONCLUSION: Recombinant gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.