应用CRISPR/Cas9技术构建Raji-Luc CD19 KO淋巴瘤细胞系  

Construction of Raji-Luc CD19 KO Lymphoma Cell Line Using CRISPR/Cas9 Technology

作　　者：刘静静 刘秀盈 冯娅茹 冯义超 于梦圆 王建勋 LIU Jingjing;LIU Xiuying;FENG Yaru;FENG Yichao;YU Mengyuan;WANG Jianxun(School of Life Sciences,Beijing University of Chinese Medicine,Beijing 102488,China;Shenzhen Cell Valley Biopharmaceutical Co.Ltd,Guangdong Shenzhen 518118,China;Shenzhen Research Institute,Beijing University of Chinese Medicine,Guangdong Shenzhen 518118,China)

机构地区：[1]北京中医药大学生命科学学院,北京102488 [2]深圳细胞谷生物医药有限公司,广东深圳518118 [3]深圳北京中医药大学研究院,广东深圳518118

出　　处：《现代检验医学杂志》2024年第1期10-15,99,共7页Journal of Modern Laboratory Medicine

基　　金：北京中医药大学高层次人才科研启动经费项目(9011451310032):中医药调节肿瘤免疫的生物学机制研究。

摘　　要：目的应用CRISPR/cas9技术构建敲除CD19的Raji-Luc淋巴瘤细胞,并对其免疫逃逸能力进行初步验证。方法构建PB-CRISPR-CD19小向导RNA(small guide RNA,sgRNA)质粒,筛选最优的sgRNA序列,用pCAG-PBase转座酶、PB-CD19 sgRNA及PB-CRISPR-Cas9共同电转染Raji-Luc细胞,采用流式分选和极限稀释法筛选得到稳定敲除的单克隆细胞株。经过基因序列检测对敲除效果进行验证;酶标仪检测细胞系表面荧光素酶的表达情况;以实验室前期构建的CD19 CAR-T和CD38 CAR-T作为效应细胞,用荧光素酶化学发光法验证Raji-Luc CD19 KO细胞株的免疫逃逸能力。结果电转染制备的Raji-Luc CD19 KO细胞转染效率较高,筛选所得两组单克隆细胞敲除效率均达到99%以上,与原始Raji-Luc细胞的荧光素酶表达无显著差异,且不能激活CD19 CAR-T细胞对其进行杀伤。结论成功构建了Raji-Luc CD19 KO淋巴瘤细胞系。Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

关 键 词：嵌合抗原受体-T细胞 白细胞分化抗原19 KO 电转染 淋巴瘤 Raji-Luc细胞 

分 类 号：R392-33[医药卫生—免疫学]