【摘要】 目的 比较分别经磷酸钙转染法和脂质体转染法建立的第三代慢病毒包装系统的包装效率。方法制备并纯化完整的重组慢病毒三质粒系统:转移质粒(PXZ171)、包装质粒(△NRF)以及包膜蛋白质粒(VSVG)。分别用磷酸钙法和脂质体法将三质粒共转染包装细胞293T,72 h后收集病毒上清,批量快速测定法测定病毒滴度并进行比较。结果 磷酸钙转染法所产病毒滴度约达到脂质体转染法的1/10。结论 磷酸钙转染法获取的病毒滴度稍低,但价格相对便宜,病毒产量亦可满足实验要求。比较适合在普通实验室中开展。更多还原

【Abstract】 Objective To compare the efficiency of the three-plasmid packaging cell line for recombinant lentivirai vector transfected by calcium phosphate or lipofectamine 2000,respectively.Methods The three-plasmid recombinant lentiviral vector system was prepared and purified:the transfer vector plasmid(pxz171),the packaging plasmid(△NRF) and the envelope plasmid encoding the vesicular stomatitis virus-G glycoprotein(VSV-G).Human embryonic kidney 293T cells were contransfected with the three plasmids by calcium phosphate or lipofectamine 2000,respectively.72 hours after transfection,the viral-supernatant was collected,and large-scale real-time titration(LaSRT)was applied to test the virus titers and compare the titers between these two methods.Results The titer of virus by calcium phosphate was about as 1/10 that by lipofectamine 2000.Conclusion The titer of virus by calcium phosphate is lower than that by Iipofectamine 2000,but it is at a very low cost,and the titer of virus could also meet the requirement of experiments.Thus,it is more appropriate for experiments in ordinary laboratories.更多还原