小鼠DND1慢病毒载体的构建及病毒生产

发布时间：2018-02-22 08:52

  本文关键词： 慢病毒 载体 DND1 基因　出处：《湖南师范大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：在本研究中,将用PCR法从小鼠10.5d胚胎cDNA文库中扩增出的Dnd1基因引入慢病毒载体系统,并生产Dnd1慢病毒颗粒。为进一步明确Dnd1体外生物学功能奠定基础。 方法： 1.Dnd1基因的获得 根据GenBank中小鼠Dnd1基因序列(BC034897),使用DNAstar软件设计基因的特异引物Dnd1-Age IF和Dnd1-AgeI-R,并且引入Age I酶切位点。用PCR法从小鼠10.5d胚胎cDNA文库中扩增出Dndl基因。PCR法筛选阳性克隆。 2.重组慢病毒载体转移质粒pGC-FU-Dndl的构建采用In-Fusion技术,将Age I酶切回收后的PCR产物交换连接入Age I酶切的pGC-FU真核表达载体,产生pGC-FU-Dnd1质粒。连接产物质粒转化大肠杆菌E.coli DH5a感受态细胞,扩增质粒。 3连接产物pGC-FU-Dnd1的酶切鉴定 收集部分扩增后连接产物质粒,酶切后经电泳证实所得片段长度均与预期相符,说明Dnd1基因已克隆成功。基因测序结果亦证实成功克隆Dnd1基因。 4连接产物pGC-FU-Dnd1质粒转染293T细胞 用脂质体Lipofectamine 2000包裹构建的pGC-FU-dnd1和辅助包装载体,共转染293T细胞,产生含有表达Dnd1蛋白的Lentivirus病毒颗粒。 结果： 1成功获得Dnd1目的基因片段。通过PCR筛选得到Dnd1目的基因片段。 2连接产物pGC-FU-Dnd1的测序结果与GenBank中dnd1基因序列(BC034897)比较,表明Dnd1基因序列及读码框均正确。 3三质粒转染293 T细胞后荧光显微镜观察结果及Western-blot检测鉴定表明成功建立Dnd1慢病毒表达载体系统。 结论： 1成功克隆小鼠Dndl基因和成功构建pGC-FU-Dnd1质粒 2 pGC-FU-Dnd1表达载体经过转染293T细胞,48小时后观察到绿色荧光蛋白的表达；经Western bloting检测到Dnd1蛋白在293T细胞中的表达,从而证明我们成功建立了Dnd1重组慢病毒,对其进行纯化后采用逐孔稀释法测定滴度,滴度测定结果为2E+9TU/ml。这为更深入的探讨Dnd1基因抑制细胞增殖中等体外细胞生物学功能提供实验基础。
[Abstract]:Aim: in this study, the Dnd1 gene amplified from mouse embryo cDNA library by PCR method was introduced into the lentivirus vector system to produce Dnd1 lentivirus particles, which laid a foundation for further clarifying the biological function of Dnd1 in vitro. Methods:. 1. Acquisition of Dnd1 gene. According to the mouse Dnd1 gene sequence BC034897 in GenBank, the specific primers Dnd1-Age IF and Dnd1-AgeI-Rx were designed by DNAstar software, and the AgeI restriction site was introduced. The Dndl gene was amplified by PCR from the cDNA library of mouse embryo at day 10.5. 2.Recombinant lentivirus vector transfer plasmid pGC-FU-Dndl was constructed by using In-Fusion technique. The PCR products recovered by Age I digestion were exchanged and ligated into Age I digested pGC-FU eukaryotic expression vector. PGC-FU-Dnd1 plasmids were produced. The plasmids were transformed into E. coli DH5a cells and the plasmids were amplified. Identification of pGC-FU-Dnd1 by enzyme digestion. After partial amplification, the plasmids of ligated products were collected, and the length of the fragments was confirmed by electrophoretic electrophoresis, which indicated that the Dnd1 gene had been cloned successfully, and the gene sequencing confirmed that the Dnd1 gene was cloned successfully. Transfection of pGC-FU-Dnd1 plasmid into 293T cells. The pGC-FU-dnd1 and auxiliary packaging vector were encapsulated with liposome Lipofectamine 2000 and co-transfected into 293T cells to produce Lentivirus virus particles expressing Dnd1 protein. Results:. 1 the Dnd1 target gene fragment was successfully obtained, and the Dnd1 gene fragment was obtained by PCR screening. 2Compared with the dnd1 gene sequence BC034897 in GenBank, the sequencing results of the ligated product pGC-FU-Dnd1 showed that the Dnd1 gene sequence and the reading frame were correct. 3After the transfection of the three plasmids into 293T cells, the results of fluorescence microscopy and Western-blot detection showed that the Dnd1 lentivirus expression vector system was successfully established. Conclusion:. 1 successful cloning of mouse Dndl gene and construction of pGC-FU-Dnd1 plasmid. (2) the expression of green fluorescent protein was observed in 293T cells 48 hours after transfection with pGC-FU-Dnd1 expression vector, and the expression of Dnd1 protein in 293T cells was detected by Western bloting, which proved that we successfully established Dnd1 recombinant lentivirus. After purification, the titer was determined by one-hole dilution method, and the titer was 2E9TU / ml, which provided the experimental basis for further study on the biological function of Dnd1 gene inhibiting cell proliferation in vitro.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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