慢病毒的超滤浓缩及细胞感染效应

Ultrafiltration concentration and cell infection of Lentivirus

目的构建pLVX-CD63-mNeptune-Puro重组质粒,使用HEK293T细胞包装慢病毒,探讨慢病毒经超滤浓缩后的纯化效果极其对MGC-803细胞的转染效应。方法使用重叠PCR方法构建CD63-mNeptune融合基因,并插入pLVX-Puro载体构建转移质粒。转移质粒、包装质粒和外膜蛋白质粒共转染HEK293T细胞包装慢病毒颗粒。收集转染后48 h及72 h细胞培养液,4℃下2500×g离心10 min去除细胞沉淀和碎片,上清液通过0.45μm PVDF Millex-HV滤器过滤,收集滤液于15 mL离心管。取1 mL滤液于1.5 mL EP管,余下滤液用截留分子量100000超滤管进行浓缩。使用P24检测试剂盒检测浓缩前后细胞培养液中病毒颗粒数。分别使用未浓缩慢病毒液与超滤浓缩慢病毒液感染MGC-803细胞。通过观察MGC-803细胞荧光信号来比较慢病毒的感染率。结果成功构建慢病毒转移质粒并使用HEK293T细胞包装出慢病毒颗粒,细胞上清液中的慢病毒颗粒数经超滤浓缩后浓度提高了264倍,使用超滤浓缩的慢病毒液感染细胞后,荧光显微镜观察到携带荧光信号的活细胞多于未浓缩慢病毒液。结论超滤浓缩可以提高慢病毒颗粒浓度,并提高细胞感染率。采用超滤浓缩慢病毒的方法适合大多数普通实验室操作。

Objective To construct the recombinant plasmid pLVX-CD63-mNeptune-Puro,producing Lentivirus in HEK293T cells,and compare the infection efficiency of un-concentrated Lentivirus and ultrafiltration concentrated Lentivirus.Methods The CD63-mNeptune fusion gene was constructed by overlapping PCR.The fusion gene was inserted into the lentiviral expression vector pLVX-Puro to construct the transferred plasmid.Transferred plasmid,packaging plasmid and envelope plasmid were transfected into HEK293T cells.The cell culture medium was collected 48 h and 72 h after transfection.Cell debris and cells in the culture medium are removed by centrifugation at 2500×g for 10 min in 4℃.The supernatant was filtered through a 0.45μm pore PVDF Millex-HV filter(Millipore).The filtrate was collected in a 15 mL centrifuge tube.We took 1 mL of Lentivirus filtrate in a 1.5 mL EP tube.The remaining filtrate was concentrated by using ultrafiltration(with a 100000 cutoff membrane).Then MGC-803 cells were infected with un-concentrated Lentivirus and ultrafiltration concentrated Lentivirus.Efficiency of Lentivirus infection was compared by observing fluorescence signal of MGC-803 cells.ResultsThe Lentivirus transferred plasmid was successfully constructed,and Lentivirus particles were produced by transfection of HEK293T cells.The concentration of lentiviral particles was increased 264 times after ultrafiltration concentration.When cells were infected with the ultrafiltration concentration of Lentivirus,the fluorescence microscopy showed that there were more living cells carrying the fluorescence signal than the un-concentrated Lentivirus.ConclusionThe ultrafiltration concentration method can increase the concentration of virus particles and improve the cell infection efficiency.The method of ultrafiltration to concentrate Lentivirus is suitable for most laboratories.