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  

Lentiviral vector of chimeric antigen receptor targeting carcinoembryonic antigen and its application

作　　者：余晓玲[1] 杨智[1] 彭维[1] 钱程[1] 

机构地区：[1]第三军医大学西南医院全军临床病理学研究所,重庆400038

出　　处：《第三军医大学学报》2015年第11期1096-1101,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(81301959);第三军医大学临床重大研究项目(2012XLC04)~~

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 receptor,CAR)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CEACAR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(P<0.01)。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。Objective To construct lentiviral vector LV-EF1α-CEA-2A-GFP-WPRE carrying chimeric antigen receptor （CAR） targeting carcinoembryonic antigen （CEA）, and determine its expression and cytotoxic effect on the CEA positive tumor cell lines when the vector are transfected into T cells. Methods The sequences coding anti-CEA antibody were obtained with standard molecular cloning techniques from the plasmid encoding antibody of CEA, and then verified by sequencing. The CEA segment was then inserted into lentiviral vector by enzyme digestion. Native Western blotting and flow cytometry were used to analyze its expression, and cytotoxic assay was employed to detect the cytotoxicity against the CEA positive tumor cell lines when the vector was infected into T cells. Results 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 the vector was infected into T cells, the CEA-CAR^＋ T cells were 62. 8% and effectively killed the CEA positive tumor cells （P 〈 0.01 ）. Conclusion Lentiviral vector LV- EF1α-CEA-2A-GFP-WPRE carrying CAR targeting CEA is constructed successfully. The CAR can be expressed through the vector and efficiently kill the CEA positive tumor cells in T cells.

关 键 词：嵌合抗原受体 肿瘤免疫治疗 CEA 慢病毒 

分 类 号：R392-33[医药卫生—免疫学] R73-36[医药卫生—基础医学]