H7N9流感病毒神经氨酸酶特异性单克隆抗体的制备及其应用研究

发布时间：2018-03-05 14:44

本文选题：H7N9禽流感病毒　切入点：神经氨酸酶　出处：《中国疾病预防控制中心》2017年硕士论文　论文类型：学位论文

【摘要】：人感染H7N9亚型禽流感病毒于2013年在中国长三角地区首次报道,可引起严重的下呼吸道感染症状。由于病毒每年持续广泛流行于该区域的家禽中,会在一定范围内感染人类,所以成为人们广泛关注的公共卫生问题。截至2017年5月28日,已有1514例人感染H7N9禽流感的实验室确诊病例。2016年底的第五波流行中,在家禽和人中同时检测到了高致病性H7N9病毒的存在,病毒HA抗原发生了变化。简便、高效的病原快速诊断和治疗策略有助于及时采取防控措施以控制疫情扩散,并使患者及时获得抗病毒治疗以改善预后。神经氨酸酶(Neuraminidase,NA)是流感病毒表面重要的抗原之一,以四聚体形式存在,它能够切割唾液酸受体,帮助新生病毒颗粒释放和传播。NA单体分为胞内区、跨膜区、茎部区和头部区四部分,头部含有抗原表位和酶活性位点,在体内可作为抗原诱导体液免疫应答,产生抗体。靶向NA的抗体在体内和体外具有保护作用,部分抗体能够抑制神经氨酸酶活性,通过空间干扰等方式作用于NA,从而减轻流感病毒感染时的临床症状。本研究利用杂交瘤技术制备了多株针对H7N9流感病毒神经氨酸酶(Neuraminidase,NA)的单克隆抗体,并对这些单克隆抗体的功能特性进行了鉴定;基于单抗的特性,又利用其中两株N9特异性单抗3C1和3E9,建立了双抗体夹心ELISA(DAS-ELISA)捕获N9亚型禽流感病毒抗原的特异性快速检测方法,为H7N9禽流感病毒的诊断和防控提供了一个良好的材料。本课题研究结果如下:一、抗H7N9亚型流感病毒神经氨酸酶单克隆抗体的制备及鉴定1.通过小鼠免疫、细胞融合以及间接ELISA筛选方法得到25株H7N9亚型流感病毒神经氨酸酶特异性单克隆抗体。2.25 株单抗分别与 rgH6N2、rgH6N9、rgH9N2 及 A/Environment/Jiangxi/28/2009(H1 1N9)病毒进行神经氨酸酶抑制实验。重组病毒rgH6N2、rgH6N9、rgH9N2的NA分别来自 A/Brisbane/10/2007(H3N2),A/Anhui/1/2013(H7N9)以及 A/Hong Kong/33982/2009(H9N2)。25株抗体对rgH6N9、H11N9、rgH9N2和rgH6N2的神经氨酸酶抑制活性不同,22 株对 rgH6N9 和 H11N9 的 IC50 为1.6～100μg/ml;对 rgH9N2 的 IC50 为15～100μg/ml,对 rgH6N2 则完全没有抑制,IC50 都100μg/ml。3.从25株单抗中挑选3株单抗(1G8、3C4和4E8)进行了详细的功能鉴定,三株单抗为Anhui/1-N9识别特异性的;3C4和4E8具有神经氨酸酶抑制活性,IC50分别为1.45μg/ml 和 8.65μg/ml;竞争 ELISA 显示三株抗体对 A/Anhui/1/2013(H7N9)的 N9蛋白的结合表位各不同。二、禽流感病毒N9抗原检测方法的建立1.通过荧光标记抗体配对平台筛选出2株单抗(3C1和3E9)用于组建基于双抗体夹心ELISA(DAS-ELISA)的N9抗原检测方法。2.DAS-ELISA对N9抗原的检测表现出高敏感性。用重组病毒rgH6N9、野生型病毒A/Environment/Jiangxi/28/2009(H11N9)以及重组 N9 蛋白对 DAS-ELISA 的灵敏度进行了测试,系统对rgH6N9和H11N9的最低检测阈值为0.125血凝单位/50μl;N9标准曲线呈线性反应关系,最低检测限为6.25ng/ml。3.用重组病毒rgH6N1、rgH6N2以及rgH9N2对DAS-ELISA的特异性进行了测试,当固定病毒用量都为8血凝单位/50μl时,检测不出任何阳性信号,表现出高特异性。4.使用咽拭子样本评估DAS-ELISA在临床应用的可行性。DAS-ELISA检测五份Q-PCR确诊的H7N9样本,两份显示阳性信号;同时用商品化H7亚型流感病毒检测试剂盒(胶体金法)检测样本,全部未检出阳性信号。结论1.制备获得25株N9特异性单克隆抗体,其中22株显示出神经氨酸酶抑制活性,IC50 为1.6～15μg/ml。2.用N9特异性抗体组建了双抗体夹心ELISA法,表现出高敏感性和特异性,可用于检测H7N9病毒或N9蛋白和临床样本。
[Abstract]:People infected with H7N9 subtype avian influenza virus was first reported in 2013 in the Yangtze River Delta region Chinese, can cause severe lower respiratory tract infection symptoms. Because the virus last year is widely popular in the area of poultry, infect humans in a certain range, so become a public health problem of concern. As of May 28, 2017, the fifth wave of popular 1514 people have been infected with H7N9 avian influenza cases of laboratory confirmed cases of.2016 at the end of the year, in poultry and people was detected for the highly pathogenic H7N9 virus has changed virus HA antigen. The pathogen is simple, high efficiency and rapid diagnosis and treatment strategies help to take timely prevention and control measures to control the spread of the epidemic, and the patients receive timely antiviral treatment to improve the prognosis. The neuraminidase (Neuraminidase, NA) is one of the major surface antigen of influenza virus, with four dimers form it Can cut sialic acid receptors, help new virus particles release and spread of.NA monomer into the intracellular region, transmembrane region, stem region and head area of four parts, the head containing the epitope and enzyme activity, in vivo can be used as antigen to induce humoral immune response, antibody targeting NA antibody. In vivo and in vitro has a protective effect, part of the antibody can inhibit the activity of neuraminidase, by way of spatial interference in NA, so as to reduce the clinical symptoms of influenza virus infection. This study was prepared for H7N9 strains of influenza virus neuraminidase by hybridoma technology (Neuraminidase, NA) monoclonal antibody, and functional properties of the monoclonal antibodies were identified; monoclonal antibody based on the characteristics of the two strains of N9 specific monoclonal antibody 3C1 and 3E9, a double antibody sandwich ELISA (DAS-ELISA) acquisition of N9 subtype avian influenza virus antigen Method for rapid detection of specificity, provides a good material for the diagnosis and prevention of H7N9 avian influenza virus. The results of this study are as follows: first, the monoclonal antibody of H7N9 subtype influenza virus neuraminidase preparation and identification of 1. mice by immunization, cell fusion and indirect ELISA screening method of 25 strains of H7N9 subtype influenza virus neuraminidase specific monoclonal antibody McAb.2.25 respectively with rgH6N2, rgH6N9, rgH9N2 and A/Environment/Jiangxi/28/2009 (H1 1N9) virus neuraminidase inhibition assay. The recombinant virus rgH6N2, rgH6N9, rgH9N2 NA from A/Brisbane/10/2007 (H3N2), A/Anhui/1/2013 (H7N9) and A/Hong Kong/33982/2009 (H9N2) H11N9.25 antibodies to rgH6N9, neuraminidase. RgH9N2 and rgH6N2 inhibitory activity, 22 strains of rgH6N9 and H11N9 IC50 was 1.6 ~ 100 g/ml; rgH9N2 I C50 is 15~100 g/ml, the rgH6N2 has no inhibition of IC50 100 g/ml.3. from the 25 mAbs selected 3 monoclonal antibodies (1G8,3C4 and 4E8) were identified with the function, the three strains of monoclonal antibodies to Anhui/1-N9 specific identification; 3C4 and 4E8 with neuraminidase inhibitory activity, g/ml and IC50 were 1.45 8.65 g/ml; competitive ELISA showed that three strains of antibodies to A/Anhui/1/2013 (H7N9) N9 protein binding to different epitopes. Two, detection of avian influenza virus N9 antigen by 1. fluorescent antibody pairing platform screened 2 strains of monoclonal antibody (3C1 and 3E9) for the formation of double antibody sandwich ELISA (DAS-ELISA) based on.2.DAS-ELISA detection of N9 antigen of N9 antigen detection showed Gao Min sensibility. With the recombinant virus rgH6N9, wild type virus A/Environment/Jiangxi/28/2009 (H11N9) and the sensitivity of the recombinant N9 protein of DAS-ELISA were measured Try, the lowest detection threshold of rgH6N9 and H11N9 0.125 blood coagulation unit /50 L; linear response relationship N9 standard curve, the lowest detection limit for the recombinant virus with 6.25ng/ml.3. rgH6N1 specific rgH6N2 and rgH9N2 on DAS-ELISA were measured, when the dosage is 8 fixed virus hemagglutination unit /50 L NO, not a positive signal detection, showed high specificity to.4. using swab samples to assess feasibility of DAS-ELISA in.DAS-ELISA clinical application of detection of five Q-PCR H7N9 were two samples showed positive signals; simultaneously with the commercialization of H7 subtype influenza virus detection kit (colloidal gold) of all test samples. No positive signal was detected. Conclusion 1. obtained 25 strains of N9 specific monoclonal antibodies, of which 22 strains showed neuraminidase inhibitory activity, IC50 was 1.6 ~ 15 g/ml.2. with N9 specific antibody and a double antibody sandwich ELISA method, It shows Gao Min's sensibility and specificity, which can be used to detect H7N9 virus or N9 protein and clinical samples.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R511.7;R446.6

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