包装细胞转导提高逆转录病毒滴度的研究

Production of High-titer and Helper Virus-free Retrovirus by Transduction ofPackaging Cells with Different Host Ranges

将含标志基因NeoR的逆转录病毒载体pLXSN用脂质体转染法导入包装细胞，然后利用宿生范围不同的包装细胞（PA317／GP＋envAm12与GP＋E86）相互转导来提高病毒滴度，并用敏感方法检测其安全性。结果显示脂质体DOSPER转染法获得生产细胞的病毒滴度为（2．0～8．0）×105CFU／ml，而病毒相互转导方法则为（2．8～16）×106CFU／ml，可提高滴度达10倍以上；以NIH3T3细胞为靶细胞证实NeoR基因可被重组病毒转移至靶细胞基因组，巢式PCR与标志基因补救分析均未能检测到野生型辅助病毒。提示“乒乓效应”为提高病毒滴度的有效方法且无辅助病毒产生，因而该基因转移系统是安全的。

By means of liposome-mediated method,a retroviral vector pLXSN,carrying a selectable marker neomycin resistance gene (NeoR),was transferred into the amphotropic packaging cells PA317 and GP+envAm12 respectively. To produce higher-titer retrovirus for gene transfer,the retrovirus produced by PA317/NeoR was used to cross-infect the ectropic packaging cell GP+E86,and amphotropic packaging cells were transduced with ectrop-ic retrovirus from GP+E86/NeoR ceells to amplify the vector copy number. The titer of DOSPER-transfected PA3l7 and GP+envAm12 pools assayed on NIH3T3 cells was ranged from 2.0×105 to 8. 0×105CFU/ml. When ectropic retrovirus from GP+ E86/NeoR cells was used to cross-infected PA317 and GP+envAm12 packaging cells, the amphotropic retroviruses were obtained with higher-titer ranged from 2.8×106 to 1. 6×107CFU/ml,which are great enough for efficient gene transfer. Successful gene transfer and integration of NeoR into NIH3T3 cells was confirmed by PCR. By both nested PCR and marker rescue assay,supernatant from the producers was found free of helper virus- The efficiency and safety of this system in gene transfer might provide an optimal system for human gene therapy.