本文選題：登革病毒非結(jié)構(gòu)蛋白1(DENVNS1)　切入點(diǎn)：IgG　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：全球受到登革病毒(Dengue virus,DENV)感染威脅的人群近39億,我國近年感染率增高,僅廣東省2014年就達(dá)45000例。登革病毒有4個(gè)血清型,主要致自限性的登革熱(Dengue Fever,DF),少數(shù)情況下可出現(xiàn)登革出血熱與登革休克綜合征(DHF/DSS),約占DF患者的5%～10%,發(fā)生率雖低但死亡率高。目前對(duì)致命性的DSS的發(fā)病機(jī)制尚未闡明,若干可能的原因,如抗體依賴增強(qiáng)效應(yīng)(ADE),細(xì)胞因子風(fēng)暴,補(bǔ)體依賴的細(xì)胞毒反應(yīng)等。近年人們開始關(guān)注登革病毒非結(jié)構(gòu)蛋白1(DENV-NS1)及其抗體在DHF/DSS的發(fā)病機(jī)制中的作用。因重癥登革患者的血清常含有較高濃度的NS1蛋白及相應(yīng)的IgG抗體水平,我們關(guān)注非結(jié)構(gòu)蛋白1(NS1)與其IgG型抗體結(jié)合形成的免疫復(fù)合物能否引起休克;此外,DHF的發(fā)生可能因NS1蛋白能誘導(dǎo)機(jī)體產(chǎn)生與血小板等血液系統(tǒng)細(xì)胞交叉識(shí)別的抗體,導(dǎo)致血小板減少并引起出血。基于本實(shí)驗(yàn)室長期在登革血清學(xué)診斷方面的關(guān)注積累,尤其是制備并保存了針對(duì)4個(gè)血清型NS1的148株單抗,本文分別對(duì)重癥登革的DSS與DHF,即休克與出血兩個(gè)方面的機(jī)制做了初步的探索。第一章IgG型抗登革病毒NS1抗體介導(dǎo)被動(dòng)系統(tǒng)過敏反應(yīng)的研究通過超濾制備濃縮病毒上清,以親和層析法獲得純化DENV1NS1,并分別與20株IgG型抗DENV1 NS1單抗或單抗組合孵育制備免疫復(fù)合物,然后分別攻擊小鼠,建立被動(dòng)系統(tǒng)性過敏反應(yīng)(passive systemic anaphylaxis,PSA)和被動(dòng)皮膚過敏反應(yīng)(passive cutaneous anaphylaxis,PC A)模型;并觀察體內(nèi)氯化釓(GdC13)和血小板活化因子受體(platelet activating factor receptor,PAFR)拮抗劑CV-3988處理對(duì)PSA的影響。結(jié)果顯示只有5D25+3B1和5D25+3C65兩個(gè)IgG型單克隆抗體組合可以誘發(fā)PSA與PCA;用GdC13抑制單核巨噬細(xì)胞或CV-3988阻斷PAFR處理可抑制或減輕小鼠PSA反應(yīng)。證明DENV1 NS1結(jié)合兩個(gè)IgG型單抗組合的免疫復(fù)合物可誘發(fā)PSA與PCA,這種過敏途徑由免疫復(fù)合物啟動(dòng),以PAF為主要效應(yīng)介質(zhì),以單核/巨噬細(xì)胞為主要的效應(yīng)細(xì)胞。推測(cè)這是發(fā)生重癥登革休克綜合癥(DSS)可能的機(jī)制之一。第二章抗登革病毒NS1抗體的血小板結(jié)合特性研究我們從約113株抗NS1單抗中通過間接ELISA、流式細(xì)胞術(shù)以及免疫印跡技術(shù)篩選鑒定共獲得能結(jié)合人血小板的抗DENV-NS1抗體27株,其中有14株能結(jié)合蛋白質(zhì)二硫鍵異構(gòu)酶(PDI),12株能識(shí)別血小板膜蛋白粗提物的構(gòu)象表位。研究證實(shí)了部分抗DENV-NS1單抗也能結(jié)合人血小板,它們能識(shí)別血小板上多個(gè)抗原表位,包括構(gòu)象表位。證實(shí)抗NS1單抗能夠結(jié)合血小板,但并非所有能夠結(jié)合NS1的單抗都能結(jié)合血小板抗原,該工作可望為后續(xù)研究抗體影響血小板功能,并為疫苗設(shè)計(jì)提供必要的參考。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R512.8
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本文編號(hào)：1608206