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 CD19 and Fluorescent Protein based on Retroviral Packaging Technology

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CD19 CAR-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。

Objective:To construct SW620 cell line expressing human CD19,enhanced Green fluorescent protein(EGFP)and luciferase(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 CD19 on the cell was detected by flow cytometry,and the expression of luciferase on the cell line surface and cell line function were identified by luciferase assay.Results:The percentage of CD19-positive 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 CD19 in the cell lines was significantly up-regulated compared with the original cell lines.Conclusion:The SW620 cell line that can stably express human CD19 and fluorescent protein was successfully constructed through retroviral vector packaging technology.