慢病毒载体包装细胞系的建立

发布时间：2018-06-26 12:42

本文选题：第三代慢病毒载体 + 包装细胞系　；参考：《沈阳药科大学》2006年硕士论文

【摘要】：慢病毒载体因为可以介导外源基因在非分裂细胞中稳定高效地表达而成为转基因动物和基因治疗研究的重要工具。为了能够制备安全的慢病毒，慢病毒的元件被构建在多个质粒上以减少因重组产生可复制病毒的可能性，其中最具代表性的是基于HIV-1的第三代慢病毒载体系统：包括(1)编码病毒核心蛋白(Gag)和病毒复制所需酶类(Pol)的质粒；(2)编码Rev蛋白的质粒；(3)编码水泡性口炎病毒G蛋白(VSVG)的包膜蛋白的质粒；(4)携带目的基因的慢病毒载体，通过瞬时转染上述元件到293T细胞可以产生复制缺陷型的慢病毒，但费时费力是这种方法的主要缺点。本研究的目的在于为第三代慢病毒载体建立一个包装细胞系以优化慢病毒的产出。由于包膜蛋白VSVG的过量表达可能会引起细胞形态的改变甚至细胞的死亡，我们将包膜蛋白VSVG构建在了一个单质粒的四环素诱导表达系统上，使VSVG的表达可以人为地调节，保证细胞的稳定传代。首先将编码慢病毒包装蛋白的质粒pLP1-zeo、pLP2、pTRE2rtTAG共转染到293FT细胞，经过筛选，得到了79个Zeocin抗性细胞克隆，然后进行PCR鉴定以验证DNA已经整合到细胞基因组上，得到了6个gag／pol、rev、VSVG和rtTA全部呈阳性的细胞克隆，，进一步RT-PCR分析得到了3个具有所有慢病毒元件转录产物的细胞克隆。最后用Northern确定VSVG的mRNA是否可以诱导产生，结果显示，3个细胞克隆在没有强力霉素诱导的条件下都没有信号，而经过强力霉素诱导后有两个细胞克隆检测到了mRNA，因此这两个细胞克隆就是我们所需要的稳定细胞系。用携带增强型绿色荧光蛋白(EGFP)的慢病毒载体pLenti4-EGFP来转染一个生长状况良好的细胞克隆，收集细胞上清液感染新鲜的293FT细胞，在荧光显微镜下检测到了EGFP的表达，说明有慢病毒产生。通过计数表达EGFP的细胞个数来计算慢病毒的滴度，大约为10~4IU／mL，同时检测不到可复制病毒的产生，因而是安全的。总之，我们建立了一个四环素诱导的包装细胞系，这个细胞系可以很方便地产生复制缺陷型的慢病毒，为转基因动物的生产打下了基础。
[Abstract]:Lentivirus vector has become an important tool for transgenic animals and gene therapy because of its ability to mediate the stable and efficient expression of foreign genes in non-mitotic cells. In order to be able to prepare a safe lentivirus, the lentivirus elements are constructed on multiple plasmids to reduce the possibility of replicating viruses due to recombination. One of the most representative is the third generation lentivirus vector system based on HIV-1: (1) plasmids encoding viral core protein (Gag) and required enzymes for viral replication (Pol); (2) plasmids encoding Rev protein; (3) the plasmid encoding the envelope protein of vesicular stomatitis virus G protein (VSVG); (4) the lentivirus vector carrying the target gene, which can produce replication-deficient lentivirus by transient transfection of the above elements into 293T cells. But time-consuming and laborious is the main disadvantage of this method. The aim of this study was to establish a packaging cell line for the third generation lentivirus vector to optimize the production of lentivirus. Because the overexpression of VSVG may cause cell morphology change and even cell death, we constructed the envelope protein VSVG on a tetracycline inducible expression system of a single plasmid, so that the expression of VSVG can be artificially regulated. Ensure stable passage of cells. Firstly, the plasmid pLP1-zeotTAG encoding lentivirus packaging protein was cotransfected into 293FT cells. After screening, 79 Zeocin-resistant cell clones were obtained, and then PCR was performed to verify that the DNA had been integrated into the cell genome. Six cell clones with positive expression of VSVG and RTTA were obtained, and three cell clones with all the transcription products of lentivirus elements were obtained by RT-PCR. Finally, Northern was used to determine whether VSVG mRNA could be induced. The results showed that all three cell clones had no signal without doxycycline induction. MRNAs were detected in two cell clones induced by doxycycline, so these two cell clones are the stable cell lines we need. A lentivirus vector pLenti4-EGFP carrying enhanced green fluorescent protein (EGFP) was used to transfect a well growing cell clone. The supernatant was collected and infected with fresh 293FT cells. The expression of EGFP was detected under fluorescence microscope. This indicates that a lentivirus is produced. The titer of lentivirus was calculated by counting the number of cells expressing EGFP. The titer of lentivirus was about 10 ~ (4) U / m ~ (L), and the production of replicable virus could not be detected at the same time, so it was safe. In conclusion, we have established a tetracycline induced packaging cell line, which can easily produce replication-deficient lentivirus and lay the foundation for the production of transgenic animals.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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