第三代慢病毒高效率包装系统的建立.PDFVIP

基因时代()为生命科学提供源动力！ 基因组学与应用生物学，2009 年，第 28 卷，第 2 期，第 326-330 页 Genomics and Applied Biology, 2009, Vol.28, No.2, 326-330 实验技术与方法 Techniques and Methodology 第三代慢病毒高效率包装系统的建立 张磊 * 刘庆友 * 胡天 张晓溪 崔奎青 陆凤花 石德顺 ** 广西大学动物繁殖研究所, 南宁, 530005 * 同等贡献作者 ** 通讯作者, ardsshi@ 摘 要 慢病毒介导法是最有前途的转基因动物生产方法之一，高滴度慢病毒颗粒的包装是慢病毒转基因 动物技术的关键。本研究应用含有增强型绿色荧光蛋白基因(eGFP)的第 3 代慢病毒载体系统，用脂质体转染 法将慢病毒系统 4 质粒共转染 293T 包装细胞，培养 48~72 h 收集病毒上清液，通过超速离心进行浓缩，采用 批量快速测定法(LaSRT)测定病毒滴度。结果显示，用脂质体转染法包装的慢病毒能成功地感染 293T 细胞， 经检测病毒滴度达到 5×108 IU/mL 以上，初步建成了高滴度慢病毒包装平台，为慢病毒介导制作转基因动物 奠定了良好的研究基础。 关键词 慢病毒, 病毒包装, 转基因动物 The Construction of High-efficiency Packaging System of the 3rd Lentivirus Zhang Lei * Liu Qingyou * Hu Tian Zhang Xiaoxi Cui Kuiqing Lu Fenghua Shi Deshun ** Animal Reproduction Institute, Guangxi University, Nanning, 530005 * The authors who contribute equally ** Corresponding author, ardsshi@ DOI：10.3969/gab.028.000326 Abstract Lentivirus-mediated method is one of the most promising methods of producing transgenic animals, high-titer lentiviral particles packaging is the key of lentivirus-mediated transgenic animal technology. In this study, 293T cells were co-transfected with four plasmids, which are the 3rd generation of lentiviral vector systems contain- ing enhanced green fluorescent protein gene (eGFP), using the Lipofectamine 2 000 mediated transfection method. Virus supernate was collected after 48~72 hours, and then concentrated by ultracentrifuge. Large-scale real-time titration (LaSRT) was applied to test the titer of virus. The results showed that 293T cells could be successfully in- fected by the virus packaged using Lipofectamine-mediated transfection, as evidenced of that the virus titer reached higher than 5×108 IU/mL, indicating that a high-titer lentiviral packaging platform was preliminary estabilished. This result will be beneficial for