γ-逆转录病毒载体包装及浓缩方法对比研究  

Comparative Study on Packaging and Concentration Methods ofγ-Retrovirus Vector

作　　者：张帆 程奕宁 沈齐 詹思建 汪洋 胡翰 刘滨磊 ZHANG Fan;CHENG Yining;SHEN Qi;ZHAN Sijian;WANG Yang;HU Han;LIU Binlei(Biological Engineering and Food,Hubei Univ.of Tech.,Wuhan 430068,China;Wuhan Binhui Biotechnology Co.,Ltd,Wuhan 436030,China)

机构地区：[1]湖北工业大学生物工程与食品学院,湖北武汉430068 [2]武汉滨会生物科技股份有限公司,湖北武汉436030

出　　处：《湖北工业大学学报》2024年第5期74-79,共6页Journal of Hubei University of Technology

摘　　要：为选择合适的包装条件与安全高效的浓缩方法生产用于CAR-T制备的γ-逆转录病毒载体,使用两种包装条件γ-逆转录病毒表达载体(pMIG与pMIGⅡ)和包装质粒总量(20μg与40μg)分别生产逆转录病毒载体,利用流式细胞术检测不同条件下生产的病毒滴度;使用超速离心与超滤离心两种浓缩方法得到逆转录病毒浓缩液,运用流式细胞术,检测两种病毒浓缩液滴度及其对T细胞的转导效率,并监测CAR-T细胞增殖与存活率。数据显示,pMIGⅡ载体包装病毒滴度较高(p<0.05),20μg与40μg质粒总量对病毒滴度无显著影响(p>0.05),超滤离心法与超速离心法浓缩后逆转录病毒滴度无显著差异(p>0.05)。研究表明,pMIGⅡ载体与20μg质粒总量更适合包装γ-逆转录病毒,超滤离心法可用于实验室小规模浓缩逆转录病毒,满足CAR-T研究的需要。For the optimization of packaging conditions and concentration methods forγ-retroviral vector production for CAR-T production,two packaging vectors(pMIG and pMIG II)and different amounts of plasmids(20μg and 40μg)were used to produce retrovirus,respectively,and the virus titers were detected by flow cytometry;Ultracentrifugation and ultrafiltration were used to concentrate the retrovirus,the titer of the two viruses and their transduction efficiency on T cells were detected by flow cytometry,the proliferation and survival rate of CAR-T cells were monitored.The data showed that the virus titer was higher using pMIG II vector(p<0.05),the different amounts of plasmids(20μg and 40μg)did not affect the virus titer(p>0.05),ultrafiltration and ultracentrifugation concentration methods did not affect the virus titer(p>0.05).The results showed that pMIG II vector and the total amount of 20μg plasmid were suitable for packagingγ-retrovirus,and ultrafiltration can be used for small-scale concentration of retrovirus in the laboratory to content the needs of CAR-T research.

关 键 词：γ-逆转录病毒载体 病毒包装 超速离心 超滤离心 CAR-T细胞 

分 类 号：R153[医药卫生—营养与食品卫生学]