荧光定量PCR检测c⁃Met CAR病毒感染T细胞的效率

作者：张馨予

目的：通过设计特异性引物，应用荧光定量PCR方法检测c?Met CAR病毒感染T细胞的效率。方法：应用基因重组技术构建c?Met CAR（GFP）逆转录病毒质粒。应用病毒包装技术制备c?Met CAR病毒与c?Met CAR（GFP）病毒，感染T细胞制备c?Met CAR?T细胞与c?Met CAR?T（GFP）细胞。Western blot检测c?Met CAR和c?Met CAR（GFP）在293T细胞中表达的外源性CD3ζ蛋白。设计特异性引物应用荧光定量PCR检测CAR病毒对T细胞的感染效率，并与流式细胞术的检测结果进行比较。结果：构建c?Met CAR（GFP）质粒，荧光显微镜可观察到c?Met CAR（GFP）质粒在293T细胞上表达绿色荧光蛋白。c?Met CAR和c?Met CAR（GFP）病毒感染的293T细胞可表达外源性CD3ζ蛋白。荧光定量PCR检测c?Met CAR与c?Met CAR（GFP）的感染效率分别为（52.1 ± 1.7）%、（55.9 ± 2.3）%。流式细胞术检测c?Met CAR（GFP）病毒感染效率为（50.7 ± 3.6）%，两种检测方法比较差异无统计学意义（P > 0.05）。结论：通过设计特异性引物，应用荧光定量PCR方法能够特异性检测 CAR病毒对T细胞的感染效率，该检测方法结果准确、安全，对CAR?T细胞的临床应用具有实用价值。

Abstract:

Objective：To detect the efficiency of c?Met CAR infection in T cells by qRT?PCR using specific primers. Methods：Gene recombination technology was introduced to construct c?Met CAR（GFP）retroviral plasmids. C?Met CAR and c?Met CAR（GFP）viruses were prepared by viral packaging. C?Met CAR?T cells and c?Met CAR?T（GFP）cells were prepared by infection of T cells with virus. The expression of c?Met CAR and c?Met CAR（GFP）virus infected 293T cells was tested by Western blot assay to express exogenous CD3ζ protein. Specific primers were designed to detect the infection efficiency of T cells by qRT?PCR and compared with flow cytometry. Results：c?Met CAR（GFP）plasmid was constructed，and the expression of c?Met CAR（GFP）plasmid on 293T cells was observed by fluorescence microscope. The results showed that c?Met CAR and c?Met CAR（GFP）virus infected 293T cells expressed exogenous CD3ζ protein. The infection efficiency of c?Met CAR and c?Met CAR（GFP）virus were（55.9 ± 2.3）% and（52.1 ± 1.7）% by qRT?PCR. The efficiency of c?Met CAR（GFP）infection was（50.7 ± 3.6）% by flow cytometry. There was no statistical difference between the two methods（P > 0.05）. Conclusion：Through the design of specific primers，the qRT?PCR can detect the infection efficiency of c?Met CAR virus on T cells，which is accurate and security，and have value for the clinical application of CAR?T cell therapy.