六邻体嵌合型rAd5载体的构建与功能研究

发布时间：2018-05-24 16:10

本文选题：重组腺病毒 + 嵌合　；参考：《吉林大学》2012年硕士论文

【摘要】：重组5型腺病毒载体已被广泛应用于人类免疫缺陷病毒（HIV）、乙肝病毒、结核杆菌等疫苗和肿瘤基因治疗的研究。然而，人体内普遍存在的针对载体本身的预存免疫会显著地降低病毒载体的转导效率和转基因表达水平。本论文首先根据目前对57种亚型的人腺病毒的血清阳性率调查结果，然后通过分析所有稀有型腺病毒六邻体超变区（HVR）的基因和氨基酸序列，选择出理想的替换血清型Ad26，Ad37和Ad43。再次，结合相关文献报道的将稀有亚型人腺病毒六邻体与Ad5进行替换的研究结果，，设计以Ad26，Ad37和Ad43的全长HVR5和HVR7基因以及Loop4基因替换rAd5相应区域来构建六邻体嵌合型rAd5载体。我们最终成功构建了6种六邻体嵌合型rAd5载体rAd5HVR26(5+7)、rAd5HVR26(5+7)Loop4、 rAd5HVR37(5+7)、 rAd5HVR(5+7)37Loop4、rAd5HVR43(5+7)和rAd5HVR43(5+7)Loop4。为方便对比评价嵌合载体的包装、复制能力以及转基因传代稳定性和表达水平，我们将GFP和Luciferase报告基因分别克隆到穿梭载体pDC316上。与改造的嵌合腺病毒载体共转293cell和Hela细胞，通过荧光显微镜下绿色荧光的观察或Luciferase活性检测来确认嵌合载体的相关性质。本论文最终得到了一种有利于包装，传代性质稳定，并且复制能力和转基因表达水平都显著提高的嵌合载体Ad5HVR43(5+7)，为后续在体内实验中评价其免疫水平，探索腺病毒免疫机制打下基础，期望得到免疫水平较高且能很好的避免预存免疫的重组腺病毒载体，以应用于肿瘤基因治疗和人类免疫缺陷病毒、结核等疫苗的研究。本论文的研究对于腺病毒载体修饰后的包装有一定意义，同时也为腺病毒载体的进一步修饰改造提供了重要依据。
[Abstract]:Recombinant adenovirus vector 5 has been widely used in the study of human immunodeficiency virus (HIV), hepatitis B virus (HBV), tuberculosis vaccine and tumor gene therapy. However, the preexisting immunization against the vector itself can significantly reduce the transduction efficiency and transgenic expression level of the virus vector. In this paper, the seropositive rate of 57 subtypes of human adenovirus was investigated. Then, by analyzing the gene and amino acid sequence of all rare adenovirus, the ideal replacement serotypes Ad26AAd37 and Ad43were selected. Thirdly, based on the reported results of replacing the rare subtype of human adenovirus with Ad5, we designed to replace the rAd5 region with the full-length HVR5 and HVR7 genes of Ad26Ad37 and Ad43 and Loop4 gene to construct the hexagonal chimeric rAd5 vector. We successfully constructed six kinds of hexagonal chimeric rAd5 vectors, rAd5HVR26(5 7rAd5HVR2657 Loop4, rAd5HVR37(5 7, rAd5HVR(5 7 7Loop4, rAd37Loop4 rAd5HVR4357) and rAd5HVR43(5 7 Loop4. In order to compare and evaluate the packaging, replication ability, stability and expression level of chimeric vectors, GFP and Luciferase reporter genes were cloned into shuttle vector pDC316 respectively. The chimeric vector was co-transfected with the modified chimeric adenovirus vector to 293cell and Hela cells. The properties of the chimeric vector were confirmed by the observation of green fluorescence under fluorescence microscope or the detection of Luciferase activity. In this paper, a chimeric vector, Ad5HVR43(5 7, which is favorable to packaging, stable in passage, and significantly improved in replication and transgenic expression, was obtained to evaluate its immune level in vivo. To explore the immune mechanism of adenovirus, we hope to obtain recombinant adenovirus vector with high immune level and avoid preexisting immunity, which can be used in tumor gene therapy, human immunodeficiency virus, tuberculosis and other vaccines. The research in this paper has some significance for the packaging of adenovirus vector, and also provides an important basis for the further modification of adenovirus vector.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.11

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