一株呼吸道合胞病毒融合糖蛋白F和EGFP共表达的非复制型腺病毒重组体的构建、鉴定及表达

发布时间：2018-08-05 15:49

【摘要】：目的:呼吸道合胞病毒(Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染的最重要的病毒病原。病毒感染机体后的免疫保护机制尚未明确,也无特异性防治方法。RSV融合糖蛋白(fusion glycoprotein,F)和吸附蛋白(attachment protein,G)是RSV仅有的两种中和抗原。由于G蛋白的差异,RSV有A和B两种抗原亚型。我国流行的RSV主要是以A亚型为主。考虑到RSV病毒结构和感染的特点,研究以共表达RSV A亚型G、F糖蛋白的非复制型重组腺病毒和DNA疫苗进行联合免疫预防RSV感染。采取这种抗原组合的优点在于:结构上类似减毒活疫苗,有助于诱导平衡的免疫反应;提高外源基因转移及表达效率;一次接种产生两种中和抗原,可改善接种效率,减少婴幼儿接种疫苗的创痛,降低疫苗成本。共表达策略目前主要用于以疾病治疗为目的基因治疗领域,或用于表达异源多聚体蛋白的亚单位。用于疫苗研究的报道较少。美国BD Bioscience Clontech公司已经有商品化的真核双表达载体pIRES,但尚缺乏可方便地用于构建双顺反子共表达的腺病毒载体系统。为此,本研究利用内部核糖体进入位点(internal ribosome entry site,IRES)构建可用于双顺反子共表达的腺病毒重组体。方法:本研究依据IRES的碱基序列,设计并合成一对特异性引物,以商品化的真核双表达载体pIRES为模板,扩增IRES及其调控序列IVS,克隆入T载体,获得pGEMT-IRES克隆载体(MCSA、MCVB分别位于IRES序列的上、下游)。根据IRES上、下游多克隆位点,设计并合成两对用于扩增目的基因的PCR引物,分别扩增出F基因和增强型绿色荧光蛋白基因(Enhanced Green Fluorescent Protein,EGFP),分别克隆至pGEMT-IRES,获得重组质粒pGEMT-F/EGFP。为构建共表达RSV F和EGFP的非复制型腺病毒重组体,本研究采用了AdEasy系统。该系统是利用生长周期短,便于操作的E. coli BJ5183细胞完成腺病毒骨架质粒与克隆有外源基因的穿梭载体同源重组。在这一过程中,包括三个依次递进的步骤:将外源性基因(F-IRES-EGFP)亚克隆于穿梭载体pShuttle-CMV;重组穿梭载体与腺病毒骨架载体pAdEasy-1在E. coli BJ5183细胞内同源
[Abstract]:Objective: respiratory syncytial virus (Respiratory Syncytial) is widely distributed all over the world, and it is the most important viral pathogen leading to severe lower respiratory tract infection in infants. The mechanism of immune protection after virus infection is not clear, and there is no specific prevention and treatment method. Fusion glycoprotein F and attachment protein G are the only two neutralizing antigens of RSV. Because of the difference of G protein, RSV has two antigenic subtypes, A and B. The prevalent RSV in China is mainly subtype A. Considering the structure and infection characteristics of RSV virus, the non-replicating recombinant adenovirus and DNA vaccine co-expressing RSV A subtype GnF glycoprotein were studied to prevent RSV infection. The advantages of adopting this antigen combination are that it is structurally similar to a live attenuated vaccine and contributes to the induction of a balanced immune response; increases the efficiency of foreign gene transfer and expression; and produces two neutralizing antigens at one time, which can improve the inoculation efficiency. Reduce the pain of infant vaccination and reduce the cost of vaccine. Coexpression strategy is mainly used in the field of gene therapy for disease treatment or for the expression of heteropolymer protein subunits. There have been few reports of vaccine research. BD Bioscience Clontech company in the United States already has the commercial eukaryotic double expression vector pIRESbut still lacks of the adenovirus vector system which can be used to construct the double cistronic coexpression vector conveniently. In this study, we constructed an adenovirus recombinant which could be used for the co-expression of double cistrons using internal ribosomal entry site (internal ribosome entry sitesires). Methods: according to the base sequence of IRES, a pair of specific primers were designed and synthesized. Using commercial eukaryotic double expression vector pIRES as template, IRES and its regulatory sequence were amplified and cloned into T vector. The pGEMT-IRES clone vector was obtained (MCSA-MCVB was located in the upstream and downstream of the IRES sequence, respectively). According to the polyclonal site downstream of IRES, two pairs of PCR primers were designed and synthesized to amplify the target gene. The F gene and the enhanced green fluorescent protein gene (Enhanced Green Fluorescent protein EGFP) were amplified, respectively, and cloned into pGEMT-IRES. the recombinant plasmid pGEMT-Fr / EGFPwas obtained. In order to construct a non-replicating adenovirus recombinant expressing RSV F and EGFP, AdEasy system was used in this study. In this system, adenovirus skeleton plasmids were homologous recombined with the shuttle vector cloned with foreign genes by using E. coli BJ5183 cells with short growth cycle and easy to operate. In this process, there are three progressive steps: subcloning of exogenous gene (F-IRES-EGFP) into shuttle vector pShuttle-CMV, and homology between recombinant shuttle vector and adenovirus skeleton vector pAdEasy-1 in E. coli BJ5183 cells
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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