靶向GPC3的第4代嵌合体抗原受体慢病毒载体构建及病毒包装滴定  

Construction of the fourth-generation chimeric antigen receptor lentiviral vector targeting GPC3 and virus packaging titration

作　　者：胡想金 王赛仑 郭佳[1] 高耀 谭炳琴 闾军[1] HU Xiangjin;WANG Sailun;GUO Jia;GAO Yao;TAN Bingqin;LYU Jun(Department of Oncology,Beijing YouAn Hospital,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学附属北京佑安医院肿瘤内科,北京100069

出　　处：《胃肠病学和肝病学杂志》2022年第8期873-880,共8页Chinese Journal of Gastroenterology and Hepatology

基　　金：首都卫生发展科研专项项目(2018-1-2181);首都临床特色应用研究(Z181100001718191);北京市属医院科研培育项目(PX2019063)。

摘　　要：目的 构建靶向磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)的第4代慢病毒载体,并进行慢病毒载体鉴定、包装及病毒滴度测定。方法 通过分子克隆技术从本实验室第2代anti-GPC3-CAR的慢病毒载体上得到anti-GPC3-CAR序列,并与人源化IL-15/IL-15Rα序列连接后插入慢病毒载体pCDH,重组质粒经菌落PCR及测序鉴定。将构建的重组质粒分别与辅助质粒共转染293T细胞,48 h后收集上清并进行病毒滴度测定,流式细胞术验证anti-GPC3-CAR的表达。结果 成功构建了第4代anti-GPC3-CAR慢病毒表达载体,并成功包装了慢病毒颗粒。结论 本研究构建第4代anti-GPC3-CAR慢病毒表达载体,可高效包装出含CAR基因的慢病毒颗粒,一定程度上可提高底盘细胞的功能及持久性,为后期CAR修饰的免疫疗法奠定了基础。Objective To construct and identify the fourth-generation lentiviral expression vector targeting glypican-3(GPC3),and to package and prepare the anti-GPC3-CAR lentivirus subsequently.Methods The sequences coding anti-GPC3-CAR were obtained with molecular cloning techniques from the second-generation lentiviral vector carrying anti-GPC3-CAR in our laboratory,and then ligated with humanized IL-15/IL-15Rα sequence.The ligated gene segment was inserted into the lentiviral vector pCDH after enzyme digestion,and the construction was confirmed by colony PCR and sequencing.The fourth-generation CAR lentiviral vector plasmid was co-transfected into 293T cells along with helper plasmid,after 48 hours,the culture supernatant was collected and then the virus titer was measured.The expression of anti-GPC3-CAR was detected by flow cytometry.Results We successfully constructed the fourth-generation antiGPC3-CAR lentivirus expression vector.And corresponding lentiviral particle was also successfully packaged.Conclusion This study constructed the fourth-generation anti-GPC3-CAR lentiviral expression vector,which can efficiently package the lentiviral particles containing the CAR gene,and improve the function as well as persistence of immune cells to a certain extent,laying a foundation for the later study of CAR modified immunotherapy.

关 键 词：GPC3 嵌合抗原受体 慢病毒 肝细胞癌 IL-15 

分 类 号：R735.7[医药卫生—肿瘤]