靶向磷脂酰肌醇蛋白聚糖3的嵌合体抗原受体慢病毒载体构建及病毒包装滴定的实验研究  

Construction of 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科,100069

出　　处：《北京医学》2021年第9期905-909,共5页Beijing Medical Journal

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

摘　　要：目的构建靶向磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)的慢病毒载体,并进行慢病毒载体的鉴定、包装及病毒滴度测定。方法设计靶向GPC3抗原的嵌合性抗原受体(chimeric antigen receptor,CAR)分子重组基因及特异性引物,用聚合酶链反应(polymerase chain reaction,PCR)对CAR分子重组基因进行体外扩增及PCR反应片段搭桥,双酶切后定向插入到pCDH质粒,构建pCDH-anti-GPC3-CAR慢病毒表达载体,经菌落PCR、DNA凝胶电泳及测序鉴定。将目的质粒与包装质粒共转染293T细胞,收集上清液按不同比例稀释后感染293T细胞,流式细胞术检测anti-GPC3-CAR表达并计算病毒滴度。结果经菌落PCR及测序证实,正确构建重组慢病毒载体pCDH-anti-GPC3-CAR,包装后的病毒滴度为(1.50~3.24)×10^(6)TU/ml。结论本研究成功构建携带不同共刺激分子的pCDH-anti-GPC3-CAR慢病毒表达载体,可在293T细胞中表达并产毒,为后续构建CAR免疫细胞提供技术储备。Objective To construct the lentiviral vector targeting glypican-3(GPC3),and perform identification,packaging and titer determination of the lentiviral vector.Methods The chimeric antigen receptor(CAR)motif against GPC3 and specific primers were designed,and the polymerase chain reaction(PCR)was used for in vitro amplification of CAR fragments and ligation of PCR reaction fragments.Furthermore,the target gene fragment was inserted into pCDH plasmid after restriction double enzyme digestion reaction to construct pCDH-anti-GPC3-CAR lentiviral expression vector,which was confirmed by colony PCR,DNA gel electrophoresis and sequencing.The target plasmid and the packaging plasmid were transfected into 293 T cells.293 T cells were infected with the above supernatant collected and diluted,the expression of anti-GPC3-CAR was detected by flow cytometry,and the viral titer was calculated.Results The colony PCR and sequencing confirmed that the recombinant plasmid pCDH-anti-GPC3-CAR was successfully constructed,and the virus titer after packaging was(1.50-3.24)×10^(6) TU/ml without concentration.Conclusions Lentiviral vector pCDH-anti-GPC3-CAR carrying different costimulatory molecules was constructed successfully,which can be expressed in 293 T cells and produce lentiviral particles,providing technical reserves for the subsequent construction of CAR-engineered immune cells.

关 键 词：磷脂酰肌醇蛋白聚糖3 嵌合抗原受体 免疫治疗 原发性肝癌 

分 类 号：Q78[生物学—分子生物学] R730.51[医药卫生—肿瘤]