表达靶向癌胚抗原的嵌合受体LV-EF1α-CEA-2A-第三军医大学学报.DOC

DOI：10.16016/j.1000-5404.201412238 表达靶向癌胚抗原的嵌合受体的慢病毒载体构建及初步应用 余晓玲，杨 智，彭 维，钱 程 （400038 重庆，第三军医大学西南医院病理科） [摘要] 目的 构建表达靶向癌胚抗原（carcinoembryonic antigen，CEA）的嵌合抗原受体（chimeric antigen receptors，CARs）的慢病毒载体LV-EF1α-CEA-2A-GFP-WPRE,研究该载体感染T淋巴细胞后嵌合抗原受体的表达以及对CEA阳性肿瘤细胞系的杀伤作用。方法 利用常规分子克隆技术从含靶向CEA的单链化抗体的载体上得到抗CEA抗体的编码序列，通过测序鉴定，采用酶切将基因片段插入慢病毒载体中。慢病毒载体感染T淋巴细胞后，通过非变性非还原Western blot、流式细胞术检测CEA-CAR在T淋巴细胞中的表达，体外杀伤实验检测对CEA阳性肿瘤细胞系的杀伤效果。 结果 测序结果显示CEA-CAR的序列正确，酶切结果显示慢病毒载体LV-EF1α-CEA-2A-GFP-WPRE构建正确；感染T淋巴细胞后CEA-CAR阳性表达率为62.8%；可有效杀伤CEA阳性肿瘤细胞系（P0.01）。结论 成功构建了表达靶向CEA的嵌合抗原受体LV-EF1α-CEA-2A-GFP-WPRE的慢病毒载体，该嵌合抗原受体可在T细胞中正确表达，并有效杀伤CEA阳性肿瘤细胞。 [关键词] 嵌合抗原受体；肿瘤免疫治疗；CEA；慢病毒 [中图法分类号] [文献标志码] A Construction the lentiviral vector expression chimeric antigen receptor targeted carcinoembryonic antigen Yu Xiaoling,Yang Zhi,Peng Wei，Qian Cheng (Institute of Pathology,Southwest Hospital,Third Military Medical University,Chongqing,400038,China) [Abstract] Objective To construct lentiviral vector LV-EF1α-CEA-2A-GFP-WPRE carrying chimeric antigen receptor（CARs）targeted carcinoembryonic antigen（CEA）,dectection of its expression and cytotoxic effects on the CEA positive tumor cell lines when transfected into T cells. Methods abti-CEA antibody coding sequences were gotten by standard molecular cloning techniques from the plasmid coding antibody of CEA and verified by sequencing.The CEA segment was then inserted into lentiviral vector via enzyme digestion. Native western blot and flow cytometry were used to analyze its expression, detected cytotoxicity against the CEA positive tumor cell lines by cytotoxic assay in vitro when the vector was infected into T cells . Resluts The sequencing result indicated that the CEA-CAR sequences were correct，and the enzyme digestion result exhibited lentiviral vector LV-EF1α-CEA-2A-GFP-WPRE was constructed correctly; when infected into T cells the CEA-CAR+ T cells were 62.8% and effectively killed the CEA positive tumor cells（P0.01）.Conclusions Lentivir