登革病毒NS1蛋白及其抗体在重症登革发病机制中作用的初步研究

发布时间：2018-03-13 21:30

  本文选题：登革病毒非结构蛋白1(DENVNS1)　切入点：IgG　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：全球受到登革病毒(Dengue virus,DENV)感染威胁的人群近39亿,我国近年感染率增高,仅广东省2014年就达45000例。登革病毒有4个血清型,主要致自限性的登革热(Dengue Fever,DF),少数情况下可出现登革出血热与登革休克综合征(DHF/DSS),约占DF患者的5%～10%,发生率虽低但死亡率高。目前对致命性的DSS的发病机制尚未阐明,若干可能的原因,如抗体依赖增强效应(ADE),细胞因子风暴,补体依赖的细胞毒反应等。近年人们开始关注登革病毒非结构蛋白1(DENV-NS1)及其抗体在DHF/DSS的发病机制中的作用。因重症登革患者的血清常含有较高浓度的NS1蛋白及相应的IgG抗体水平,我们关注非结构蛋白1(NS1)与其IgG型抗体结合形成的免疫复合物能否引起休克;此外,DHF的发生可能因NS1蛋白能诱导机体产生与血小板等血液系统细胞交叉识别的抗体,导致血小板减少并引起出血。基于本实验室长期在登革血清学诊断方面的关注积累,尤其是制备并保存了针对4个血清型NS1的148株单抗,本文分别对重症登革的DSS与DHF,即休克与出血两个方面的机制做了初步的探索。第一章IgG型抗登革病毒NS1抗体介导被动系统过敏反应的研究通过超滤制备浓缩病毒上清,以亲和层析法获得纯化DENV1NS1,并分别与20株IgG型抗DENV1 NS1单抗或单抗组合孵育制备免疫复合物,然后分别攻击小鼠,建立被动系统性过敏反应(passive systemic anaphylaxis,PSA)和被动皮肤过敏反应(passive cutaneous anaphylaxis,PC A)模型;并观察体内氯化钆(GdC13)和血小板活化因子受体(platelet activating factor receptor,PAFR)拮抗剂CV-3988处理对PSA的影响。结果显示只有5D25+3B1和5D25+3C65两个IgG型单克隆抗体组合可以诱发PSA与PCA;用GdC13抑制单核巨噬细胞或CV-3988阻断PAFR处理可抑制或减轻小鼠PSA反应。证明DENV1 NS1结合两个IgG型单抗组合的免疫复合物可诱发PSA与PCA,这种过敏途径由免疫复合物启动,以PAF为主要效应介质,以单核/巨噬细胞为主要的效应细胞。推测这是发生重症登革休克综合症(DSS)可能的机制之一。第二章抗登革病毒NS1抗体的血小板结合特性研究我们从约113株抗NS1单抗中通过间接ELISA、流式细胞术以及免疫印迹技术筛选鉴定共获得能结合人血小板的抗DENV-NS1抗体27株,其中有14株能结合蛋白质二硫键异构酶(PDI),12株能识别血小板膜蛋白粗提物的构象表位。研究证实了部分抗DENV-NS1单抗也能结合人血小板,它们能识别血小板上多个抗原表位,包括构象表位。证实抗NS1单抗能够结合血小板,但并非所有能够结合NS1的单抗都能结合血小板抗原,该工作可望为后续研究抗体影响血小板功能,并为疫苗设计提供必要的参考。
[Abstract]:Nearly 3.9 billion people worldwide are threatened by dengue virus infection. In recent years, the infection rate in China has increased, reaching 45000 cases in Guangdong Province alone in 2014. There are 4 serotypes of dengue virus. DHF / DSS of dengue fever and dengue shock syndrome can be found in a few cases, accounting for 5% of DF patients. The incidence rate is low but the mortality rate is high. At present, the pathogenesis of fatal DSS has not been elucidated. Several possible causes, such as antibody dependent enhancement effects, cytokine storms, In recent years, people began to pay attention to the role of dengue virus nonstructural protein 1DENV-NS1 and its antibodies in the pathogenesis of DHF/DSS. We are concerned about whether the immune complex formed by the binding of nonstructural protein 1 (NS1) with its IgG type antibody can cause shock. In addition, it may be that NS1 protein can induce the body to produce antibodies that are cross-recognized with blood system cells such as platelets. This has led to thrombocytopenia and haemorrhage. Based on our long-term interest in dengue serological diagnosis, 148 monoclonal antibodies against four serotypes of NS1 were prepared and preserved. In this paper, the mechanism of DSS and DHF- shock and haemorrhage in patients with severe dengue was studied. Chapter I. the study of IgG type anti-dengue virus NS1 antibody mediated passive systemic anaphylaxis by ultrafiltration was used to prepare the supernatant of concentrated virus. DENV1 NS1 was purified by affinity chromatography and incubated with 20 strains of IgG monoclonal antibody or monoclonal antibody against DENV1NS1 to prepare immune complex. The models of passive systemic anaphylaxis and passive cutaneous anaphylaxis were established. The effects of GdC13) and platelet activating factor receptor (CV-3988) antagonist CV-3988 on PSA were observed. The results showed that only two IgG monoclonal antibodies, 5D25 3B1 and 5D25 3C65, could induce PSA and PCAand GdC13 inhibited mononuclear cells. Macrophages or CV-3988 block PAFR treatment can inhibit or alleviate the PSA reaction in mice. It is shown that the combination of DENV1 NS1 and two IgG monoclonal antibodies can induce PSA and PCAs, and this allergic pathway is initiated by immune complexes. With PAF as the main effector medium, Mononuclear / macrophages are the main effector cells. We speculate that this is one of the possible mechanisms for the development of severe dengue shock syndrome (DSS). Chapter 2 study on platelet binding characteristics of anti-dengue virus NS1 antibody from about 113 strains of anti-dengue shock syndrome. A total of 27 DENV-NS1 antibody strains were obtained by indirect Elisa, flow cytometry and Western blot analysis. Among them, 14 strains could bind to protein disulfide isomerase and 12 strains could recognize conformational epitopes of the crude extract of platelet membrane protein. It was confirmed that some anti DENV-NS1 monoclonal antibodies could also bind human platelets, and they could recognize multiple antigenic epitopes on platelets. Including conformational epitopes. It has been confirmed that anti NS1 McAbs can bind to platelets, but not all McAbs that can bind to NS1 can bind to platelet antigens. This work is expected to affect platelet function for subsequent studies of antibodies. It also provides the necessary reference for vaccine design.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.8
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本文编号：1608206