基于逆转录病毒载体构建稳定表达人CD19和荧光蛋白的SW620细胞株
Construction of a SW620 Cell line Stably Expressing Human CD 19 and Fluorescent Protein based on Retroviral Packaging Technology
冯义超 1刘秀盈 1刘静静 1朱晶晶 1胡周 1史渊源 2王建勋2
作者信息
- 1. 北京中医药大学生命科学学院 北京 100029
- 2. 北京中医药大学生命科学学院 北京 100029;深圳细胞谷生物医药有限公司 广东深圳 518118
- 折叠
摘要
目的:构建表达人CD19和增强型绿色荧光蛋白(EGFP)和荧光素酶(LUC)的SW620细胞株.方法:构建MFG-CD19质粒,进行逆转录病毒载体包装,与MFG-EGFP-P2A-LUC逆转录病毒载体共同转导SW620细胞,筛选出稳定且强表达的单克隆细胞株SW620-CD19-EGFP-LUC并扩大培养.通过流式细胞术与qPCR技术检测细胞CD19的表达、通过荧光素酶法对细胞系表面荧光素酶的表达和细胞系功能进行鉴定.结果:流式检测构建完成的细胞株中CD19阳性的细胞占比为99.9％,EGFP阳性的细胞占比为99.2％;荧光显微镜下能够明显观察到细胞的绿色荧光;qPCR检测结果表明细胞株中CD19的表达水平相比原始细胞株明显上调,并且能够激活CD19CAR-T细胞对其进行杀伤.结论:成功构建了能够稳定表达人CD19和荧光蛋白的SW620细胞株.
Abstract
Objective:To construct SW620 cell line expressing human CD 19,enhanced Green fluorescent protein(EGFP)and lu-ciferase(LUC).Methods:Construct the MFG-CD19 plasmid,package it with a retroviral vector,and co transfect it with the MFG-EGFP-P2A-LUC retroviral vector to SW620 cells.Select a stable and strongly expressed monoclonal cell line SW620-CD19-EGFP-LUC and expand the culture.The expression of CD 19 on the cell was detected by flow cytometry,and the expres-sion of luciferase on the cell line surface and cell line function were identified by luciferase assay.Results:The percentage of CD19-posi-tive cells and EGFP-positive cells was 99.9％and 99.2％,respectively,and the green fluorescence could be observed in the Fluorescence microscope The results of qPCR showed that the expression level of CD 19 in the cell lines was significantly up-regulated compared with the original cell lines.Conclusions:The SW620 cell line that can stably express human CD 19 and fluorescent protein was successfully constructed through retroviral vector packaging technology.
关键词
人CD19/SW620细胞/逆转录病毒载体/结直肠癌Key words
HumanCD19/SW620 cell/Retroviral vector/Colorectal cancer引用本文复制引用
基金项目
高层次人才科研启动经费项目(9011451310032)
出版年
2024
NETL
NSTL
万方数据