表达人呼吸道合胞病毒结构蛋白的重组腺病毒及杆状病毒的构建与鉴定

发布时间：2017-12-27 12:01

本文关键词：表达人呼吸道合胞病毒结构蛋白的重组腺病毒及杆状病毒的构建与鉴定　出处：《北京交通大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：人呼吸道合胞病毒(Human respiratory syncytial virus, RSV)广泛分布于世界各地,是导致婴幼儿、老年人和免疫缺陷病人严重下呼吸道感染的最重要的病毒病原。RSV属于副粘病毒科肺病毒属,基因组为15.2 Kb的单股负链RNA,编码11种蛋白,其中黏附蛋白(Glycoprotein, G)和融合蛋白(Fusion protein,F)为RSV主要中和抗原,基质蛋白(Matrix protein, M)为RSV的骨架蛋白。从福尔马林灭活疫苗开始,已研制了多种形式的RSV疫苗,但由于安全性和免疫原性等问题,至今尚没有能够用于预防人RSV感染的疫苗问世。病毒样颗粒疫苗作为一种新型疫苗形式被广泛用于各种病毒疫苗研制,并获得较为理想的免疫效果和保护作用。迄今为止,人们对于RSV病毒样颗粒(Virus-like particles, VLPs)的制备条件尚处探索阶段,仅有的一个关于RSV VLPs见于2008年美国一份专利申请书,但存在产量低、制备困难及未见免疫效果和免疫保护作用研究。本研究拟选用非复制型第一代腺病毒载体(First generation replication deficient recombinant adenoviral vector, FGAd)和杆状病毒表达载体分别构建表达RSV F、G、M的复制缺陷型重组腺病毒及重组杆状病毒,为进一步阐明RSV VLPs形成和出芽机制,以及获得RSV VLPs奠定实验基础。方法：参考GenBank登录的RSV A亚型Long株M、G蛋白基因序列,按照哺乳动物细胞密码子偏性,对M和G基因进行密码子优化,然后分别克隆到穿梭载体pShuttle-CMV,再经同源重组获得重组腺病毒质粒,分别转染293细胞,获得重组腺病毒FGAd-Gsyn和FGAd-Msyn, Western-blot鉴定目的基因表达。参考GenBank登录的RSV A亚型Long株F、G和M蛋白基因序列,按照昆虫细胞密码子偏性,对F、G和M基因进行密码子优化,然后分别克隆到穿梭载体pFast-BacⅠ,转座后获得重组杆状病毒质粒,分别转染sf9细胞,获得重组杆状病毒rBac-Fopt、rBac-Mopt和rBac-Gopt, Western-blot鉴定目的基因表达。结果：重组腺病毒DNA分子转染293细胞后,7d左右细胞出现肿胀,圆缩等典型的致细胞病变效应(Cytopathic effect, CPE), Western blot检测到G和M基因的表达,获得非复制型重组腺病毒FGAd-Gsyn和FGAd-Msyn。重组杆状病毒质粒转染sf9细胞后,3d后出现细胞肿胀、细胞核增大和核内出现空泡等病变,Western blot检测到F、G和M基因的表达,获得重组杆状病毒rBac-Fopt、rBac-Mopt和rBac-Gopt o 结论：成功获得FGAd-Gsyn、FGAd-Msyn、rBac-Fopt、rBac-Mopt和rBac-Gopt,为阐明RSV VLPs形成和出芽机制,以及获得RSV VLPs奠定了实验基础。
[Abstract]:Objective: Human respiratory syncytial virus (RSV) is widely distributed all over the world, and is the most important viral pathogen causing severe lower respiratory tract infection in infants, elderly and immunodeficiency patients. RSV belongs to the paramyxovirus family, belonging to the paramyxovirus family. The genome is a single strand negative chain RNA of 15.2 Kb, encoding 11 proteins. The Glycoprotein G and fusion protein (Fusion protein, F) are the main neutralizing antigens of RSV, and the matrix protein (Matrix protein) is the bone scaffold protein. Since the initiation of formalin inactivated vaccine, various forms of RSV vaccine have been developed, but because of safety and immunogenicity, there is no vaccine available to prevent human RSV infection. As a new type of vaccine, virus like vaccine has been widely used in the development of various virus vaccines, and has obtained more ideal immune effect and protective effect. So far, the RSV virus like particles (Virus-like, particles, VLPs) the preparation conditions is still in the stage of exploration, the only one on the RSV VLPs in 2008 a U.S. patent application, but there are low yield, difficult preparation and no immunological effect and protective effect of. This study intends to use non replicating first generation adenovirus vector (First generation replication deficient recombinant adenoviral vector, FGAd) and baculovirus expression vector were used to construct recombinant adenovirus and recombinant baculovirus expressing RSV F, G, M, VLPs and RSV in order to further clarify the formation mechanism of budding, and get RSV VLPs lay the experimental basis.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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