高滴度慢病毒制备及其感染人原代T细胞的条件优化

Preparation of High-titer Lentivirus and Optimization of the Infection Conditions for Human Primary T cells

目的为提高T细胞的感染率,探究高滴度慢病毒的制备、保存及感染T细胞的较佳条件。方法本研究使用二代和三代慢病毒包装系统搭配二代和三代慢病毒载体、相同载体不同启动子制备慢病毒浓缩后,用不同保存条件的慢病毒感染Jurkat细胞和不同活化程度的T细胞,然后用流式细胞仪检测,进一步计算出病毒滴度和感染率。结果三代慢病毒包装系统搭配三代慢病毒载体pLVX-EGFP包装慢病毒滴度较高;EF-1a启动子的载体优于CMV启动子的载体;病毒在4℃存储1d和在-80℃冻融1次滴度下降低于10%,在4℃存储2d以上和在-80℃冻融两次以上滴度下降超过35%;T细胞活化后才能被慢病毒感染;T细胞活化后较佳感染时相点为活化后24h。结论 EF-1a启动子的载体优于CMV启动子的载体;病毒不要反复冻融;T细胞活化后24h转导慢病毒转到效率较高。

Objective To explore the optimal conditions for the preparation,preservation and infection of T cells with high-titer lentivirus for the improvement of the infection rate of T cells.Methods The second-and third-generation lentiviral packaging systems with the second-and third-generation lentiviral vectors were used to prepare the lentivirus concentrate by different promoters with the same vector.Then Jurkat cells and T cells with different activation degrees were infected with the lentivirus of different storage conditions.The flow cytometry was used to detect the infection and the virus titer and infection rate were calculated.Results The lentiviral titer was higher by the third-generation lentiviral packaging system with the third-generation lentiviral vector pLVX-EGFP.The vector of EF-1a promoter was superior to the vector of CMV promoter.The titer decreased by less than 10% when the virus was stored at 4℃ for one day or freeze-thawed once at-80 ℃,and it decreased by more than 35% when the virus was stored at 4℃ for no less than two days or freeze-thawed for more than once.T cells were infected by lentivirus only after being activated and the optimal infection phase point is 24 hours after activation.Conclusion The vector of EF-1a promoter is superior to the vector of CMV promoter.Lentivirus should be freeze-thawed repeatedly.The transduction efficiency of lentivirus is the highest 24 hours after activation of T cells.