摘要
目的将T细胞活力响应性启动子(TARP)纳米萤光素酶报告基因系统导入含有嵌合抗原受体(CAR)编码基因的慢病毒质粒中,为CAR⁃T细胞活化水平及功能鉴定提供一种便捷的、定量分析的方案。方法采用全基因合成及分子克隆技术构建重组质粒。慢病毒包装并感染人原代T淋巴细胞,流式细胞术检测慢病毒感染T细胞的阳性率。通过萤光素酶报告基因系统、Western blot法、流式细胞术、小动物活体成像技术鉴定CAR⁃T细胞的功能。结果酶切鉴定和质粒测序结果表明重组质粒的顺利构建,流式细胞术结果显示CAR⁃T细胞的正常制备,萤光素酶活力检测结果表明本系统能够动态响应CAR⁃T细胞的激活,体外功能实验证实本系统能够反映CAR⁃T细胞的耗竭状态,小动物活体成像结果体现了本系统在小鼠体内的示踪功能。结论TARP纳米萤光素酶报告基因系统为评估CAR⁃T细胞活化水平,耗竭状态以及体内示踪等方面提供了更加便捷、灵敏、定量分析的技术手段。
Objective To investigate a convenient and quantitative solution to activation levels and functional characterization of CAR⁃T cells by inserting T cell activity⁃responsive promoter(TARP)nanoluciferase reporter gene system into a lentiviral plasmid containing the gene encoding the chimeric antigen receptor(CAR).Methods The recombinant plasmid was constructed by using whole gene synthesis and molecular cloning techniques.The lentivirus was packaged and was infected with human primary T lymphocytes.Flow cytometry was used to detected the positive rate of lentivirus⁃infected T cells.The functional characterization of CAR⁃T cells was identified by luciferase reporter gene system,Western blot,flow cytometry,and small animal live imaging techniques.Results The results of enzyme digestion identification and the plasmid sequencing showed that the recombinant plasmids were constructed,and flow cytometry displayed the normal preparation of CAR⁃T cells.This system could dynamically respond to the activation of CAR⁃T cells by luciferase reporter gene system.The functional assay in vitro confirmed that the system could reflect the exhaustion of CAR⁃T cells,and the small animal live imaging results demonstrated that the system can be used as a tracer of CAR⁃T cells in mice.Conclusion TARP nanoluciferase reporter gene system provides a more convenient,sensitive and quantitative method for evaluating CAR⁃T cells activation level,exhaustion phenotype and tracing.
作者
梁思辛
郑瑞
赵晓娟
张仪婷
王鹏举
蒙若彤
阎博
杨安钢
LIANG Sixin;ZHENG Rui;ZHAO Xiaojuan;ZHANG Yiting;WANG Pengju;MENG Ruotong;YAN bo;YANG Angang(College of Medical Technology,Xinxiang Medical University,Xinxiang 453003;Department of Biochemistry and Molecular Biology,Air Force Medical University,Xi’an 710032;College of Life Science,Yanan University,Yan’an 716000;Department of Immunology,Air Force Medical University,Xi’an 710032,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2023年第5期397-403,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81972870)
国家肿瘤生物学重点实验室自主课题(CBSKL2022ZZ20)。
