呼吸道合胞病毒疫苗增强性疾病（VED）模型建立及G蛋白基因重组质粒免疫特征
发布时间：2018-07-25 17:19
【摘要】： 目的呼吸道合胞病毒(respiratory syncytial virus,RSV)是婴幼儿下呼吸道感染的主要病原体,其感染引起婴幼儿毛细支气管炎和肺炎,并与儿童哮喘的发生发展有着密切关系。接种疫苗是预防和控制传染病最为有效的措施,但RSV至今尚无安全有效的疫苗产生。以福尔马林灭活RSV疫苗(FI-RSV)预防接种的婴儿或学龄前儿童,自然感染RSV后反而导致更为严重的疫苗增强性疾病(Vaccineenhanced disease,VED)的产生。有研究表明,生化方法提纯的RSV G蛋白免疫和重组G蛋白免疫均有明显的诱导VED效应。另有研究者报道,尽管G蛋白和FI-RSV在免疫病理学方面引起的免疫应答相似,但T细胞介导的应答却不同,即两者引起疾病加重的机理不同。提示G蛋白与VED相关性有待于进一步研究。 本研究中我们通过建立福尔马林灭活RSV(FI-RSV)疫苗相关VED模型并定义其主要特征,为相关免疫调控机理研究提供平台;构建编码RSV G蛋白基因的重组质粒,观察其对RSV感染小鼠的保护性及特异性免疫效应,探讨表达的G蛋白抗原与VED的相关性,为探索RSV疫苗接种后疾病加重的相关机制以及为研制安全、有效的RSV疫苗提供线索和实验依据。 方法制备RSVA1型Long株FI-RSV疫苗;以FI-RSV免疫BALB/c小鼠后以RSV攻击,收集攻毒前后肺组织、血清标本;采用荧光定量RT-PCR检测肺组织标本中病毒滴度,HE染色法检查肺组织病理改变;采用ELISA检测血清标本中RSV特异性IgG水平。利用基因重组方法构建含RSV G蛋白编码基因的重组质粒,测序和酶切鉴定,Western Blot检测目的基因经体外表达后的免疫原性。重组质粒免疫小鼠后以RSV感染,采用上述相同方法分别检测肺组织标本中RSV滴度、肺组织病理改变和血清抗体水平,同时采用双抗体夹心ELISA测定肺泡灌洗液(BAL)内Th1/Th2细胞因子表达量,流式细胞术检测BAL中T淋巴细胞亚群数量及活化状态。 结果与对照组相比,FI-RSV免疫小鼠感染RSV后肺组织病毒滴度降低,但出现更为严重的肺组织病变,主要是以肺炎、间质性肺炎以及支气管周围炎为主要特征的广泛炎症反应,伴有局部肺气肿。免疫小鼠血清RSV特异性IgG抗体滴度增高,但感染RSV后其滴度明显降低。同时成功构建了编码RSV G蛋白基因的重组质粒pcDNA3.1~G。Western Blot证实目的基因表达蛋白在体外具有免疫原性。被免疫小鼠感染RSV后肺组织病毒滴度降低,肺组织中未见明显的炎性细胞浸润。小鼠血清中产生较高滴度的抗RSV-G IgG。小鼠BAL细胞中CD4+CD25+T淋巴细胞比例显著增加,并且IFN-γ(Th1)、IL-4(Th2)两类细胞因子表达显示平衡。 结论本研究成功地建立了FI-RSV免疫BALB/c小鼠感染RSV后VED模型,其主要特征为肺组织中出现与病毒滴度、血清抗体水平不一致的严重病理改变;编码呼吸道合胞病毒(RSV)G蛋白基因的重组质粒pcDNA3.1~G能诱导产生CD4+CD25+T细胞亚群,对小鼠具有明显的保护作用,而未见明显的VED现象产生。
[Abstract]:Objective Respiratory syncytial virus (RSV) is the main pathogen of infantile lower respiratory tract infection, which causes bronchiolitis and pneumonia in infants, and is closely related to the occurrence and development of asthma in children. Vaccination is the most effective measure to prevent and control infectious diseases, but there is no safe and effective vaccine for RSV. Infants or preschool children vaccinated with formalin inactivated RSV vaccine (FI-RSV) naturally infected with RSV resulted in more serious Vaccineenhanced disease (VED). Some studies showed that RSV G protein and recombinant G protein immunized by biochemical method could induce VED obviously. Other researchers reported that although G protein and FI-RSV have similar immune responses in immunopathology, T cell mediated responses are different, that is, the mechanisms of disease aggravation are different between them. It suggests that the correlation between G protein and VED needs further study. In this study, we established a formalin inactivated RSV (FI-RSV) vaccine related VED model and defined its main characteristics to provide a platform for the study of the related immune regulation mechanism, and to construct the recombinant plasmid encoding the RSV G protein gene. To observe the protective and specific immune effects of RSV on mice infected with RSV, to explore the correlation between G protein antigen expressed and VED, and to explore the mechanism of disease aggravation after RSV vaccination and the safety of its development. Effective RSV vaccine provides clues and experimental evidence. Methods the FI-RSV vaccine of RSVA1 Long strain was prepared, and the lung tissues and serum samples were collected after BALB/c mice were immunized with FI-RSV before and after attack with RSV, and the pathological changes of lung tissues were detected by fluorescence quantitative RT-PCR and HE staining. ELISA was used to detect the level of RSV specific IgG in serum samples. The recombinant plasmid containing RSV G protein encoding gene was constructed by gene recombination method. The immunogenicity of the target gene expressed in vitro was determined by sequencing and restriction endonuclease digestion. Mice immunized with recombinant plasmids were infected with RSV. The titer of RSV, the pathological changes of lung tissue and the level of serum antibody were detected by the same method mentioned above. The expression of Th1/Th2 cytokines in alveolar lavage fluid (BAL) was determined by double antibody sandwich ELISA, and the number and activation of T lymphocyte subsets in BAL were detected by flow cytometry. Results compared with the control group, the viral titers of lung tissue in mice immunized with FI-RSV decreased after infection with RSV, but more serious lung tissue lesions occurred, mainly characterized by extensive inflammatory reactions characterized by pneumonia, interstitial pneumonia and bronchitis. Accompanied by local emphysema. The titer of serum RSV specific IgG antibody increased in immunized mice, but decreased significantly after RSV infection. At the same time, the recombinant plasmid pcDNA3.1~G.Western Blot encoding RSV G protein gene was successfully constructed to confirm the immunogenicity of the expressed protein in vitro. The viral titer of lung tissue decreased after RSV infection in immunized mice, and no inflammatory cell infiltration was found in the lung tissue. A high titer of anti RSV-G IgG was produced in mouse serum. The proportion of CD4 CD25 T lymphocytes in mouse BAL cells increased significantly, and the expression of IFN- 纬 (Th1) IL-4 (Th2) cytokines showed a balance. Conclusion the VED model of FI-RSV immunized BALB/c mice after infection with RSV was successfully established. The main characteristics of the model were severe pathological changes in lung tissues which were inconsistent with virus titer and serum antibody level. The recombinant plasmid pcDNA3.1~G encoding the (RSV) G protein gene of respiratory syncytial virus can induce the production of CD4 CD25 T cell subsets, which has obvious protective effect on mice, but no obvious VED phenomenon.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R186
本文编号：2144505
[Abstract]:Objective Respiratory syncytial virus (RSV) is the main pathogen of infantile lower respiratory tract infection, which causes bronchiolitis and pneumonia in infants, and is closely related to the occurrence and development of asthma in children. Vaccination is the most effective measure to prevent and control infectious diseases, but there is no safe and effective vaccine for RSV. Infants or preschool children vaccinated with formalin inactivated RSV vaccine (FI-RSV) naturally infected with RSV resulted in more serious Vaccineenhanced disease (VED). Some studies showed that RSV G protein and recombinant G protein immunized by biochemical method could induce VED obviously. Other researchers reported that although G protein and FI-RSV have similar immune responses in immunopathology, T cell mediated responses are different, that is, the mechanisms of disease aggravation are different between them. It suggests that the correlation between G protein and VED needs further study. In this study, we established a formalin inactivated RSV (FI-RSV) vaccine related VED model and defined its main characteristics to provide a platform for the study of the related immune regulation mechanism, and to construct the recombinant plasmid encoding the RSV G protein gene. To observe the protective and specific immune effects of RSV on mice infected with RSV, to explore the correlation between G protein antigen expressed and VED, and to explore the mechanism of disease aggravation after RSV vaccination and the safety of its development. Effective RSV vaccine provides clues and experimental evidence. Methods the FI-RSV vaccine of RSVA1 Long strain was prepared, and the lung tissues and serum samples were collected after BALB/c mice were immunized with FI-RSV before and after attack with RSV, and the pathological changes of lung tissues were detected by fluorescence quantitative RT-PCR and HE staining. ELISA was used to detect the level of RSV specific IgG in serum samples. The recombinant plasmid containing RSV G protein encoding gene was constructed by gene recombination method. The immunogenicity of the target gene expressed in vitro was determined by sequencing and restriction endonuclease digestion. Mice immunized with recombinant plasmids were infected with RSV. The titer of RSV, the pathological changes of lung tissue and the level of serum antibody were detected by the same method mentioned above. The expression of Th1/Th2 cytokines in alveolar lavage fluid (BAL) was determined by double antibody sandwich ELISA, and the number and activation of T lymphocyte subsets in BAL were detected by flow cytometry. Results compared with the control group, the viral titers of lung tissue in mice immunized with FI-RSV decreased after infection with RSV, but more serious lung tissue lesions occurred, mainly characterized by extensive inflammatory reactions characterized by pneumonia, interstitial pneumonia and bronchitis. Accompanied by local emphysema. The titer of serum RSV specific IgG antibody increased in immunized mice, but decreased significantly after RSV infection. At the same time, the recombinant plasmid pcDNA3.1~G.Western Blot encoding RSV G protein gene was successfully constructed to confirm the immunogenicity of the expressed protein in vitro. The viral titer of lung tissue decreased after RSV infection in immunized mice, and no inflammatory cell infiltration was found in the lung tissue. A high titer of anti RSV-G IgG was produced in mouse serum. The proportion of CD4 CD25 T lymphocytes in mouse BAL cells increased significantly, and the expression of IFN- 纬 (Th1) IL-4 (Th2) cytokines showed a balance. Conclusion the VED model of FI-RSV immunized BALB/c mice after infection with RSV was successfully established. The main characteristics of the model were severe pathological changes in lung tissues which were inconsistent with virus titer and serum antibody level. The recombinant plasmid pcDNA3.1~G encoding the (RSV) G protein gene of respiratory syncytial virus can induce the production of CD4 CD25 T cell subsets, which has obvious protective effect on mice, but no obvious VED phenomenon.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R186
【参考文献】
相关期刊论文 前1条
1 Harvey Cantor;;Generation and Regulation of CD8~+ Regulatory T Cells[J];Cellular & Molecular Immunology;2008年06期
，本文编号：2144505
本文链接：https://www.wllwen.com/yixuelunwen/liuxingb/2144505.html
