CD19-CAR慢病毒生产方法的比较分析与优化  

Comparative Analysis and Optimization of Cd19 Car Lentiviral Production Methods

作　　者：詹思建 张帆 段海潇 汪洋 胡翰 刘滨磊 ZHAN Sijian;ZHANG Fan;DUAN Haixiao;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

出　　处：《湖北工业大学学报》2025年第1期65-69,共5页Journal of Hubei University of Technology

摘　　要：为优化CD19-CAR慢病毒的生产方法,以获得更优的生产条件,采用慢病毒三质粒包装系统(pLVX,psPAX2,pMD2.G),并利用转染试剂(lipo8000^(TM),Lipofectamine■3000,PEIpro■)进行慢病毒的包装,通过超速离心和超滤技术对慢病毒进行浓缩。并利用流式细胞仪测定慢病毒滴度。研究发现,使用Lipofectamine■3000转染试剂包装的慢病毒原液滴度最高;在pLVX、psPAX2与pMD2.G质量比为4∶3∶1,质粒总量为5μg,质粒总量(m/μg)与转染试剂体积比(V/μL)为1∶5时,慢病毒的滴度达到最高。此外,使用超滤方法浓缩的慢病毒滴度高于超速离心法。尽管使用Lipofectamine■3000包装慢病毒原液滴度最高,但考虑到扩大生产的成本,选择性价比更高的PEIpro■更为适宜。因此,本研究得出的最佳慢病毒生产方法是:使用转染试剂PEIpro■包装慢病毒,在pLVX、psPAX2与pMD2质量比为4∶3∶1,质粒总量为5μg,质粒总量(m/μg)与转染试剂体积比(V/μL)为1∶5,收集48 h和72 h的病毒上清液,并通过超滤方法对慢病毒进行浓缩。The production method of CD19 CAR lentivirus was optimized to obtain better production conditions.Lentiviruses were packaged using a lentiviral three-plasmid packaging system(pLVX,psPAX2,pMD2.G)with transfection reagents(lipo8000 TM,Lipofectamine 3000,PEIpro),and concentrated by ultracentrifugation and ultrafiltration.The lentivirus titer was determined by flow cytometry.The highest titer of lentivirus was obtained by packaging lentivirus with Lipofectamine 3000.When pLVX:psPAX2:pMD2.G=4∶3∶1,the total amount of plasmid was 5μg.The ratio of transfection reagent volume and total plasmid was 5∶1;the titer of lentivirus was the highest.The titer of lentivirus concentrated by ultrafiltration was higher than that by ultracentrifugation.The original titer of lentivirus packaged with Lipofectamine 3000 was the highest,but considering the cost of expanding production,PEIpro with higher cost performance was more suitable.Therefore,the best method for lentivirus production in this study is to use the transfection reagent PEIpro to package the lentivirus.The virus supernatant was collected at 48 h and 72 h under the conditions of pLVX∶psPAX2∶pMD2.G=4∶3∶1,the total amount of plasmid was 5μg,and the ratio of transfection reagent volume to the total amount of plasmid was 5∶1.

关 键 词：慢病毒 病毒包装 病毒浓缩 滴度 

分 类 号：R153[医药卫生—营养与食品卫生学]