【作者】 李小月；

【导师】 沈继龙； 何金生；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2005， 硕士

【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细胞内同源更多还原

【Abstract】 Objective: Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract illness in infants and young children. This virus is distributed worldwidely. The mechanism of host defense against RSV infection remains unknown. Currentl, no licensed vaccine is available. The fusion glycoprotein (F) and attachment protein (G) are the only two neutralizing antigens of RSV. Due to the diversity of G protein, there are two antigenic subgroups, A and B, and subgroup A is more prevalent in China. Because of its structural and infective characteristic, we intend to combine the replication-defective adenoviral recombinant and DNA vaccine expressing both G and F glycoprotein of RSV to prevent RSV infection. The advantages of the F and G expressed in combination are as follows: they can induce a balanced immune response similar to live attenuated RSV vaccine; the transfection and the expression efficiency of foreign genes will be improved; a more efficient vaccination with less pain and reduced expense can be achived because two kinds of neutralizing antibodies will be produced by one shot of inoculation.At present, the coexpressive strategy are mainly used in gene therapy, or used to express the subunit of heterogenous polyprotein, but few reports have come from vaccine research. American BD Bioscience Clontech Company has commercialized eukaryotic vector pIRES, yet there is still no adenoviral vector system for bicistronic coexpression. We tried to utilize internal ribosome entry site (IRES) to construct adenoviral recombinant that can be used for bicistronic expression.Methods: According to the sequence of IRES, a pair of specific primers was disigned and synthesized. The commercial eukaryotic co-expressive vector pIRES was used to amplify IRES and its regulative sequences of IVS. Then, both IRES and IVS were cloned into T vector and the recombinant T vector pGEMT-IRES (IRES was inserted between MCSA and MCSB) was constructed. Based on the sequence of MCSA and MCSB, F gene and enhanced green fluorescence protein (EGFP) were amplified and cloned into pGEMT-IRES to construct更多还原