表达呼吸道合胞病毒（RSV）融合蛋白（F）的重组杆状病毒构建与特性

发布时间：2018-01-02 09:39

本文关键词：表达呼吸道合胞病毒（RSV）融合蛋白（F）的重组杆状病毒构建与特性　出处：《武汉大学》2015年博士论文　论文类型：学位论文

【摘要】：呼吸道合胞病毒(Respiratory syncytial virus, RSV)是引起婴幼儿下呼吸道感染的重要病原,导致支气管炎和肺炎,严重时可致患者死亡,给婴幼儿的健康和生命安全带来巨大威胁。接种疫苗是预防病毒性疾病的有效手段。为了有效预防RSV感染性疾病,人们一直在研制RSV疫苗,但是目前仍没有疫苗获得许可上市。因此,研发安全有效的RSV疫苗是预防RSV感染的首要任务。位于RSV病毒粒子表面的糖蛋白——融合蛋白(fusion protein, F)是其主要的保护性抗原,也是研制RSV亚单位疫苗的首选组分之一。本研究构建了表达RSV F蛋白的重组杆状病毒疫苗,探讨了其免疫原性和抗RSV感染免疫应答情况,以及先天免疫信号通路接头蛋白分子VISA在抗感染免疫中的作用。1.以RSV A2株基因组RNA为模板,RT-PCR扩增F蛋白的F1亚基(包含第155到524位氨基酸的F蛋白片段)编码序列,克隆至原核表达载体pET-28a中,在大肠杆菌E.coli中诱导表达重组蛋白,经分离纯化后,免疫新西兰大白兔,制备抗F蛋白的多克隆抗体。ELISA检测表明,制备的多克隆抗体效价大于106；免疫荧光染色证实,抗F蛋白多克隆抗体能与重组杆状病毒表达的F蛋白产生特异性反应。2.以RSV A2株基因组RNA为模板,RT-PCR分别扩增全长F蛋白(第1到574位氨基酸)编码基因和包含F蛋白胞外区的截短F蛋白(tF)(第22到524位氨基酸)编码序列,采用MultiBac系统构建获得以不同形式表达F蛋白的三种重组杆状病毒-Bac-tF64、Bac-CF、Bac-CF/tF64。其中,Bac-tF64能将F蛋白胞外区展示在杆状病毒包膜上；Bac-CF能在转导哺乳动物细胞后,内源性地表达全长F蛋白。Bac-CF/tF64既能将F蛋白胞外区展示在杆状病毒包膜上,又能在哺乳动物细胞中表达F蛋白。3.以BALB/c小鼠为模型,对三种重组杆状病毒在机体中诱导的免疫应答和抗感染免疫进行了研究。特异性抗体及细胞因子ELISA检测结果显示,Bac-tF/64免疫组小鼠血清IgG2a/IgG11, Th2类细胞因子(IL-4, IL-5, IL-10)水平较高：Bac-CF免疫组小鼠血清IgG2a/IgG11, Thl类细胞因子(IFN-γ和TNF-α)水平较高；Bac-CF/tF64免疫组小鼠血清IgG2a/IgGl1,但是Thl与Th2类细胞因子水平均较高,表明该组诱导Thl和Th2混合型免疫应答,但是更偏向于Thl类。RSV攻毒感染试验结果表明,Bac-tF64和Bac-CF免疫组小鼠肺组织内均能检测到滴度约为102pfu per lung的RSV,但在Bac-CF/tF64免疫组小鼠肺组织内未能检测到RSV。肺研磨液细胞因子分析显示,RSV感染后,Bac-tF64免疫组小鼠肺组织具有较高的Th2类细胞因子含量,Bac-CF免疫组小鼠肺组织具有较高的Thl类细胞因子含量,而Bac-CF/tF64组Thl与Th2类细胞因子含量均较高。病理分析显示,RSV感染后,Bac-tF64免疫组小鼠肺内产生了严重的炎症反应,是疫苗增强性疾病(Vaccine Enhanced Disease,VED)的重要标志,而Bac-CF和Bac-CF/tF64组只有轻微的炎症。4.将先天免疫信号通路接头蛋白VISA编码序列插入到Bac-CF/tF64基因组中,构建了共表达VISA的重组杆状病毒Bac-CF/tF64/VISA。用Bac-CF/tF64/VISA免疫小鼠后,共表达VISA对F蛋白诱导的抗体水平没有显著影响(p0.05)。但是,能够显著降低Th2类细胞因子水平(p0.05),降低Th2类反应。病理分析显示,共表达VISA能明显减弱免疫小鼠在RSV攻毒感染时出现的VED现象。我们的研究成果将为研发基于F蛋白的RSV疫苗和发展新型疫苗佐剂奠定了基础。
[Abstract]:Respiratory syncytial virus (Respiratory syncytial, virus, RSV) is an important pathogen of lower respiratory tract infection in children, lead to bronchitis and pneumonia, severe cases can cause death in patients, brings a huge threat to children's health and life safety. Vaccination is the effective means of preventing viral diseases. In order to effectively prevent RSV infection has been in the development of RSV vaccine, but there is still no vaccine licensed market. Therefore, the development of safe and effective RSV vaccine is the primary task of prevention of RSV infection. At the surface of RSV virion glycoprotein - fusion protein (fusion protein F) is the main protective antigen, but also the development of the preferred group RSV subunit vaccine one of the points. This study constructed recombinant baculovirus vaccine expressing RSV F protein, investigate the immunogenicity and immune response against RSV infection, and congenital Immune signaling adaptor protein molecule VISA in anti infection effect in RSV.1. immune A2 strain genomic RNA as template, F1 amplification of the F subunit of RT-PCR protein (containing 155th to 524 amino acid fragment of F protein) encoding sequence was cloned into the prokaryotic expression vector pET-28a and expression of recombinant protein in Escherichia coli E.coli in the purified and immunized New Zealand rabbits, showed that the polyclonal antibody against F protein by.ELISA detection system, the titer of polyclonal antibody is greater than 106; immunofluorescence staining confirmed that anti F polyclonal antibody with recombinant baculovirus expressed F protein had specific reaction with.2. RSV A2 genomic RNA as template, RT-PCR amplification of full-length F protein respectively (first to 574 amino acids) F protein encoding gene and F protein contains the extracellular domain of truncated (tF) (twenty-second to 524 amino acid sequence encoding), constructed by MultiBac system Three kinds of recombinant baculovirus -Bac-tF64 and F protein expression in different forms of Bac-CF, Bac-CF/tF64., Bac-tF64 to F protein extracellular display in the baculovirus envelope; Bac-CF in the transduction of mammalian cells, endogenous expression of full-length F protein.Bac-CF/tF64 protein extracellular F can be displayed in the baculovirus envelope, and in mammalian cells the expression of F.3. protein in BALB/c mouse model of immune response of three recombinant baculovirus induced in the body and anti infection immunity were studied. Specific antibody and cytokine ELISA assay showed that Bac-tF/64 immunized mice serum IgG2a/IgG11, Th2 cytokines (IL-4, IL-5, IL-10 high level: Bac-CF) immunized mice serum IgG2a/IgG11, Thl cytokines (IFN- and TNF-) with high levels of Bac-CF/tF64 in serum of mice; immune IgG2a/IgGl1, But Thl and Th2 cytokines levels were higher, showed that the group induced by Thl and Th2 mixed type immune response, but in favor of Thl.RSV infection test results showed that Bac-tF64 and Bac-CF immunized mice were detected in lung tissue was approximately 102pfu per lung RSV, but failed to display in the analysis Bac-CF/tF64 immunized mice lung tissue RSV. was detected in lung grinding fluid cytokine, RSV infection, Bac-tF64 immunized mice lung tissue with higher levels of Th2 cytokines, Bac-CF immunized mice lung tissue with Thl cytokine content was higher, while the Bac-CF/tF64 group of Thl and Th2 cytokines pathological content were higher. The analysis showed that RSV infection, immune Bac-tF64 mice lung had a severe inflammatory response, vaccine enhanced disease (Vaccine Enhanced, Disease, VED) the important symbol, while the Bac-CF and Bac-CF/tF64 group Only mild inflammation.4. innate immune signaling pathway VISA adaptor protein encoding sequence inserted into the genome of Bac-CF/tF64, were constructed recombinant baculovirus expressing Bac-CF/tF64/VISA. VISA mice immunized with Bac-CF/tF64/VISA, co expression of F protein induced by VISA antibody levels had no significant effect (P0.05). However, can significantly reduce the level of Th2 type cytokines (P0.05), lower class Th2 reaction. Pathological analysis showed that co expression of VED VISA can significantly weaken the immune mice appeared in RSV infection. The results of our research will be to research and development of new vaccine adjuvant RSV vaccine based on F protein of the foundation.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392

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