prM抗独特型抗体对登革病毒感染的免疫保护性研究

发布时间：2018-01-26 10:12

本文关键词： 登革病毒中和作用抗体依赖增强作用 prM抗体 prM抗独特型抗体　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：登革病毒(dengue virus,DENV)是一种正链RNA病毒,在分类学上属于黄病毒属,分为4种血清型(DENV1-4)。四种不同血清型的登革病毒可以共循环在同一地区。初次感染登革病毒的患者出现轻微的登革热症状,其体内产生的抗体能够对同一类型的病毒产生长时间的保护作用,但是二次异型感增加患者出现登革重症疾病的风险,出现威胁生命的登革出血热(DHF)和登革休克(DSS)症状。异型交叉反应的抗体介导了病毒与Fcγ受体细胞的结合,促进了登革病毒进入Fcγ受体细胞,这个过程被成为抗体依赖增强作用(antibody dependent enhancement,ADE)。许多研究表明人对登革病毒产生免疫应答的抗原主要是登革病毒的前膜prM蛋白,体内外研究证实prM抗体增加了登革病毒的感染能力。在这篇研究中,通过空斑中和实验,我们发现了一种能够中和DENV-1的兔源性多抗。荧光定量PCR结果证明了DENV-2人免疫血清和鼠源性prM单抗显著增加培养上清中的DENV-1的核酸拷贝数,流式细胞仪显示DENV-2人免疫血清和鼠源性prM单抗显著增加DENV-1对K562细胞的感染率。随后,我们通过向Balb/c小鼠免疫鼠源性prM单抗获得了较高滴度的prM抗独特型抗体(prM-AIDs)。荧光定量PCR结果表明prM-AIDs能够显著地抑制鼠源性prM单抗介导的DENV-1对K562细胞的感染,其抑制率最高达到90%。为了进一步探究是否prM-AIDs也能在体内抑制ADE作用,我们建立了以一种干扰素-α和γ受体缺陷的AG6小鼠为背景的登革病毒ADE模型。荧光相对定量PCR检测AG6小鼠外周血细胞中的病毒变化,空斑实验检测血浆中的活病毒粒子。显微镜计数血小板数量,ELISA试剂盒测定感染感染第三天时AG6小鼠的IL-10和ALT水平。实验结果显示鼠源性prM单抗增加了小鼠体内的病毒滴度,升高了血浆中的白介素IL-10和转氨酶ALT,减少了血小板的数量。然而,注射prM-AIDs降低了小鼠体内的病毒滴度、IL-10和ALT水平。重要的是在感染第三天时小鼠的血小板数量也显著回升。实验结果证明prM-AIDs能够有效地在体内外抑制prM单抗介导的登革病毒ADE效应。为治疗登革重症提供了一种有效的新策略。
[Abstract]:Dengue virus (DENVV) is a positive stranded RNA virus belonging to the genus yellow virus in taxonomy. There are four serotypes of DENV1-4. Four different serotypes of dengue virus can be circulated in the same area. Patients with initial dengue virus infection develop mild symptoms of dengue fever. Antibodies produced in the body can protect the same type of virus for a long time, but secondary allelopathy increases the risk of developing severe disease. The presence of life-threatening DHFs and DSSs. Heterogeneous cross-reaction antibodies mediate the binding of the virus to FC 纬 receptor cells. It promotes the entry of dengue virus into FC 纬 receptor cells, and this process is called antibody dependent enhancement antibody dependent enhancement. Many studies have shown that the antigen of human immune response to dengue virus is mainly the promembrane prM protein of dengue virus. In vivo and in vitro studies have confirmed that prM antibodies increase the ability to infect dengue viruses. In this study, plaque neutralization was used. We found a rabbit polyclonal antibody capable of neutralizing DENV-1. The results of fluorescence quantitative PCR showed that DENV-2 human immune serum and murine derived prM monoclonal antibody significantly increased DENV in culture supernatant. Copy number of nucleic acid. Flow cytometry showed that DENV-2 human immune serum and murine prM monoclonal antibody significantly increased the infection rate of DENV-1 on K562 cells. We obtained a higher titer of prM anti-idiotypic antibody (PRM-AIDs) by immunizing murine prM monoclonal antibody to Balb/c mice. The results of fluorescence quantitative PCR showed that prM-AIDs could significantly inhibit the infection of K562 cells by DENV-1 mediated by murine prM monoclonal antibody. In order to further explore whether prM-AIDs can also inhibit the effect of ADE in vivo. We established a ADE model of dengue virus (DV) in AG6 mice with interferon- 伪 and 纬 receptor deficiency. Fluorescence relative quantitative PCR was used to detect the viral changes in peripheral blood cells of AG6 mice. Plaque test was used to detect live virus particles in plasma. Microscopically counted platelet count. ELISA kit was used to measure the IL-10 and ALT levels of AG6 mice on the third day of infection. The results showed that the murine prM monoclonal antibody increased the virus titer in mice. Increased plasma levels of interleukin IL-10 and transaminase alt reduced platelet count. However, injection of prM-AIDs reduced the titer of the virus in mice. The levels of IL-10 and ALT. Importantly, the platelet count of mice also increased significantly on the third day of infection. The results show that prM-AIDs can effectively inhibit prM monoclonal antibody mediated in vivo and in vitro. The ADE effect of dengue virus provides an effective new strategy for the treatment of severe dengue disease.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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