文档介绍：产生重组逆转录病毒包装细胞系建立及病毒滴度影响因素
作者:张惠中范清宇杨安钢
【关键词】逆转录病毒载体
关键词: 逆转录病毒载体;包装细胞;集落形成单位
摘要:目的探讨产生重组逆转录病毒包装细胞系建立及其病毒滴度(以cfu表示)的影响因素. 方法用逆转录病毒载体pSLXCMV,以磷酸钙共沉淀法转染PA317包装细胞,挑选抗性集落(PA317/pSLXCMV)扩大培养后,进行PA317/pSLXCMV细胞接种密度、培养温度(37℃,32℃)、纯化方法等对其培养上清中重组病毒cfu影响的比较性研究. 结果包装细胞的密度是影响cfu滴度的关键因素;降低培养温度需延长培养时间方可提高cfu滴度;对比离心纯化与过滤纯化,并未发现两者之间的差异. 结论该实验结果为应用逆转录病毒载体进行的ex vivo基因治疗实验研究提供重要参考指标.
Keywords:retroviral vector;packaging cell;colony-forming units
Abstract:AIM To optimize the methods of manufacturing retrovirus containing supernatant of PA317packaging cell line for the use in the retroviral-mediated gene Studies were conducted using pSLXCMV retro-viral colony forming unit(cfu)influence
factors including packaging cell density,incubation temperature(32℃and37℃),incubation time and different purification method pared, Results demonstrated ×106 PA317 packaging cells seeded in100mm plate with the incubation condition at37℃for24hours could get the highest cfu titer of retroviral containing for purification,there was no significant dif-ference between filter and centrifugation ┐SION These results will be useful for those who use retrovi-ral vector for ex vivo gene therapy experiment.
0 引言
在目前众多的基因治疗临床试验方案中,逆转录病毒载体仍是被广泛应用的载体之一[1-4] .作为目的基因转运过程中的逆转录病毒载体和包装细胞,其本身的分子生物学特性已被广泛研究,但对于包装后这些含目的基因的重组逆转