禽流感病毒（H5N1）血凝素特异性单克隆抗体的制备、鉴定及其ELISA捕获法的建立
发布时间：2018-07-24 13:39
【摘要】： 流感病毒属于正粘病毒科流感病毒属，是一大类引起人类和禽畜患病的病原体。根据核蛋白(N)和基质蛋白(M)的特征，流感病毒可分成甲(A)、乙(B)和丙(C)三型。乙型和丙型流感病毒感染者以人类为主，但流行范围很窄，而甲型流感病毒可感染人类、禽类及家畜，是引起流感大规模流行的主要致病原。根据病毒表面血凝素(Hemagglutinin，HA)和神经氨酸酶(Neuraminidase，NA)抗原性的差异，还可将甲型流感病毒进一步分为不同的亚型，目前已发现的甲型流感病毒包括16个HA亚型(H1-H16)及9个NA亚型(N1-N9)，其中H1、H2、H3及N1、N2亚型是引起人类流感的主要亚型。长期以来，HINl和H3N2亚型流感病毒引发了多次世界性流感流行，如1918年的“西班牙流感”，以及1957年和1968年的流感大流行，对人类健康造成了巨大的威胁。而自从1997年H5N1亚型禽流感病毒在香港导致18人发病及6人死亡的事件发生以后，人们开始高度重视以H5N1和H7N7亚型为代表的禽流感病毒。此后，高致病性禽流感病毒又持续多次地在东亚、东南亚及欧洲等地感染人类及家禽，为避免疫情扩散，仅亚洲各国政府就宰杀上亿只家禽，造成至少上千亿美元的损失。更为重要的是，H5N1禽流感病毒正经历迅速的变异，极有可能导致病毒在人际间的传播。 由于禽流感与其他病毒引发的急性呼吸道疾病的临床症状类似，仅仅依靠临床表现难以对禽流感做出准确的诊断，必须通过实验室检测手段进行确诊。简便、快速而且适合基层单位推广的禽流感早期检测技术，可以使临床一线的医生早期、迅速、准确地作出诊断，及时对患者进行隔离，防止禽流感病毒进一步传播，在防控禽流感突发疫情中起着关键作用。目前，已有基于免疫层析法(IC)、酶联免疫吸附试验(ELISA)等免疫检测技术的抗原快速检测试剂盒上市，可用于区分甲、乙两型流感病毒，但都无法进一步区分亚型。研究流感病毒亚型的检测技术，将禽流感病毒与普通流感病毒区分开来，不仅有利于及时开展针对性的抗病毒治疗，还有助于研究禽流感病毒亚型的流行和进化规律，监控禽流感病毒新亚型的出现，并且对开展大规模筛查，即时发现传染源和切断传染途径，都具有十分重要的意义。由于甲型流感病毒区分亚型的标准之一是血凝素的抗原性，而不同亚型病毒血凝素之间同源性不高，因此血凝素是流感病毒亚型检测的理想靶标。到现在为止，实验室流感病毒亚型检测方法中，针对血凝素的主要有血清学检测、基因检测及病毒抗原检测，但血清学方法检测的是特异性抗体，不适用于早期诊断；基因检测对操作者的技术水平要求较高，无法在基层单位普及使用；而病毒抗原检测主要是免疫荧光试验及酶联免疫吸附试验，前者需要配备荧光显微镜，只能在较大医院或研究所才能开展，，而利用特异性抗体检测血凝素抗原的ELISA捕获法，能在病毒感染早期准确判断病毒的HA亚型，但是目前国内外尚无商品化试剂盒问世。本研究采用多种形式的血凝素抗原免疫小鼠，筛选具有血凝抑制活性和中和活性的特异性抗体，通过竞争抑制试验分析抗体识别的抗原表位，并利用不同宿主、不同时间分离的病毒株进行考核，最终组建双抗体夹心ELISA法，检测H5N1禽流感病毒血凝素抗原。 此外，本研究制备针对血凝素的特异性单克隆抗体，不仅可以发展区分流感病毒亚型的检测方法，还可用于发展治疗性抗体。流感病毒血凝素决定了病毒的血凝活性，与病毒吸附到靶细胞表面唾液酸寡糖受体密切相关，是病毒重要的毒力决定因子。而针对病毒血凝素的具备中和活性的抗体，可以在感染早期特异性结合血凝素，使病毒失去吸附和感染宿主细胞的能力。因此，在获得具有血凝抑制活性的血凝素特异性抗体基础上，本研究进一步评价抗体对禽流感病毒的中和活性，为研究治疗性抗体奠定基础。 本研究主要分为三个部分： 第一部分：H5N1禽流感病毒血凝素抗原的制备和鉴定 本部分研究采用三种方法获得血凝素抗原。(1)利用杆状病毒表达体系，在昆虫细胞High Five中表达重组H5血凝素蛋白(以下简称“重组H5蛋白”)，采用免疫荧光和Western Blot方法检测重组H5蛋白在昆虫细胞中表达情况，血凝试验检测重组H5蛋白的血凝活性。Western Blot鉴定表达重组蛋白分子量约为66kDa，Western Blot和免疫荧光结果证实在昆虫细胞表达的重组H5蛋白能特异性地结合H5N1禽流感病毒免疫的动物血清和H5亚型标准抗血清；血凝试验表明重组H5蛋白能使豚鼠红细胞发生凝集反应，凝集效价为1：128，证明用杆状病毒体系表达的重组H5蛋白具有血凝活性。(2)鉴定携带H5N1禽流感病毒株(A／Hong Kong／482／97)血凝素基因的质粒pVAX1-tpA-97-H5(以下简称“H5基因的质粒”)，利用X-gal染色试验观察pVAXl质粒载体的转染效率，通过免疫荧光试验检测H5基因的质粒在细胞水平的表达情况。X-gal染色结果表明，对照质粒pVAX1-1acZ转染293细胞后，能使携带的LacZ基因成功表达β-半乳糖苷酶且转染效率较高；免疫荧光试验证实，质粒pVAX1-tpA-97-H5转染293细胞后能与H5N1禽流感病毒免疫的动物血清特异性结合。(3)对商品化的天然H5血凝素(从H5N1禽流感病毒株A／Goose／Guangdong／1／96浓缩得到，购自哈尔滨兽医研究所)进行了血凝滴度测定，证实天然H5血凝素能使豚鼠红细胞发生凝集，凝集效价最高达到1∶1024。 本部分研究得到三种不同形式的H5血凝素抗原，并证实转染H5基因的质粒的293细胞和重组H5蛋白分别能与禽流感病毒免疫动物血清或H5亚型标准抗血清特异性结合，而天然H5血凝素具有良好的血凝活性，为制备禽流感病毒(H5N1)血凝素特异性单克隆抗体提供了免疫原。 第二部分：H5N1禽流感病毒血凝素特异性单抗的制备、鉴定及中和活性测定 本部分研究利用多种H5血凝素抗原免疫小鼠，制备抗H5N1禽流感病毒血凝素特异性单抗，并对单抗进行免疫学性质鉴定、抗体识别位点分析、血凝抑制活性鉴定以及中和活性分析。采用三种方案免疫BALB／c小鼠：第一种方案为天然H5血凝素全程免疫；第二种方案为H5基因的质粒免疫后再用天然H5血凝素进行加强免疫；第三种方案为重组H5蛋白全程免疫。分别取每一种方案免疫后的小鼠脾细胞与小鼠骨髓瘤细胞进行融合，采用血凝抑制实验和间接ELISA筛选阳性克隆，并排除与正常鸡胚液、A型流感病毒(H1N1和H3N2)、B型流感病毒和A型流感病毒重组核蛋白反应阳性的克隆。经有限稀释法连续克隆化2-3次，获得32株稳定分泌特异性抗H5N1病毒血凝素单抗的杂交瘤细胞株，腹腔接种小鼠后，31株单抗获得腹水，其中用天然H5血凝素全程免疫后获得12株，抗体亚类包括IgG1(10株)、IgG2b(1株)和IgM(1株)；用H5基因的质粒免疫后获得9株，抗体亚类除IgG1(1株)、IgG2a(4株)、IgG2b(1株)，另有3株不属于以上亚类；用重组H5蛋白免疫后获得10株，抗体亚类包括IgG1(7株)、IgG2a(2株)和IgM(1株)。血凝抑制试验结果表明，用天然H5血凝素或H5基因的质粒免疫后获得单抗对H5血凝素的血凝抑制效价为1∶100～1∶51 200，且与A型流感病毒H1、H3、H7、H9亚型及B型流感病毒的血凝素均无交叉反应；用重组H5蛋白免疫后获得单抗不具有血凝抑制活性。对血凝抑制效价达1∶100以上的18株腹水抗体进行中和活性试验(由香港大学微生物协作完成)，对H5N1禽流感病毒(A／Vietnam／3028／04)中和试验检测的结果表明：18株抗体的中和效价与血凝抑制效价不一致，其中14株抗体的中和效价低于1∶40；另外4株由天然H5血凝素全程免疫得到的抗体中和效价均高于1∶160。 31株单抗腹水经辛酸-硫酸铵沉淀法纯化，共得到28株IgG单抗，SDS-PAGE电泳结果显示单抗纯度均＞90％。采用改良过碘酸钠法对26株已知抗体亚类的单抗(2株单抗未确定抗体亚类除外)标记辣根过氧化物酶，间接EHSA检测酶标抗体的工作浓度在10~(-2)～10~(-5)之间。按是否具有血凝抑制活性将26株酶标记单抗分为两个组，采用竞争抑制试验分别分析单抗识别抗原表位，结果显示具有血凝抑制活性的16株单抗识别2个不同的抗原位点，而且识别位点Ⅰ和Ⅱ的抗体的血凝抑制活性相差较大，分别为1∶3 200～1∶51 200及1∶100～1∶400；无血凝抑制活性的10株单抗识别4个不同的抗原表位，其研究结果为下一步建立双抗体夹心抗原检测方法奠定了基础。 第三部分：针对H5血凝素的双抗体夹心ELISA捕获法的建立 本部分研究通过组合配对，筛选检测H5血凝素灵敏度最高的抗体对，建立针对H5血凝素的双抗体夹心EHSA捕获法。(1)利用10株无血凝抑制活性的单抗进行交叉配对，通过检测梯度稀释的天然H5血凝素、重组H5蛋白及2株H5N1禽流感病毒，最终选定H5M21和H5M15-HRP的抗体对组合，建立的双抗体夹心抗原捕获法检测H5血凝素和H5N1禽流感病毒的灵敏度低于16个血凝单位。(2)利用16株呈血凝抑制阳性的单抗进行交叉配对。为保证检测的敏感性，通过检测梯度稀释的天然H5血凝素及6株不同宿主、不同时间分离的H5N1禽流感病毒，最终选定H5M9和H5M1 1-HRP的抗体对组合用于建立检测方法，检测H5血凝素和H5N1禽流感病毒的灵敏度为1／32血凝单位，而且与A型流感其他亚型及B型流感病毒均无交叉反应，具有良好的特异性。本部分研究表明，采用抗体央心抗原捕获法建立的H5N1病毒检测方法具有较高敏感性和高度特异性，为禽流感病毒感染早期诊断提供了一种快捷、特异的实验室诊断方法。 综合以上三个部分的结果，本研究的结论如下： 一、以重组H5蛋白作为免疫原所获得的10株抗H5单克隆抗体均没有血凝抑制活性，而以H5基因的质粒及天然H5血凝素免疫小鼠后获得了16株具有血凝抑制活性的抗H5单克隆抗体，表明血凝素的天然构象表位对维持抗体的血凝抑制活性起重要的作用。 二、本研究获得抗体的中和活性与血凝抑制活性存在不一致，原因可能与检测抗体的中和活性与血凝抑制活性所用的病毒不是同一株病毒有关，血凝抑制试验中使用的血凝素来源于禽流感病毒禽类分离株(A／Goose／Guangdong／1／96)，而中和活性试验中使用的是禽流感病毒人体分离株(a／vietnam／3028／04)，毒株间的差异导致了抗体中和活性与血凝抑制活性的不一致；也可能是由于决定抗体的中和活性与血凝抑制活性的抗原位点是不一致的，其原因还有待于进一步探讨。 三、用具有血凝抑制活性的抗体建立了双抗体夹心捕获EHSA法，检测H5血凝素和H5N1禽流感病毒液的灵敏度明显高于用无血凝抑制活性的抗体建立的检测方法，表明具有血凝抑制活性的抗体才能有效识别血凝素的天然构象表位。
[Abstract]:Influenza viruses belong to the genera of the family of the family of the family of the family. The influenza virus can be divided into a (A), B (B) and C (C) three, based on the characteristics of the nucleoprotein (N) and matrix protein (M). The influenza B and C influenza viruses are mainly human, but the epidemic is very narrow and influenza A virus is susceptible Infected people, poultry and livestock are the main cause of influenza pandemic. According to the difference of the antigenicity of Hemagglutinin (HA) and Neuraminidase (NA), influenza A virus can be further divided into different subtypes. The influenza A virus (influenza A) has been found to include 16 HA subtypes (H1-H16) and the influenza A virus (H1-H16). 9 NA subtypes (N1-N9), of which H1, H2, H3 and N1, the N2 subtype are the main subtypes of human influenza. The HINl and H3N2 subtype influenza viruses have long been causing a number of worldwide influenza pandemic, such as the 1918 "Spanish flu", and the 1957 and 1968 influenza pandemic, causing great threat to human health. And since 1997 H5 After the outbreak of the N1 subtype avian influenza virus (N1) virus in 18 people and 6 deaths, people began to attach great importance to the avian influenza virus represented by the H5N1 and H7N7 subtypes. After that, the highly pathogenic avian influenza virus continued to infect humans and poultry in East Asia, Southeast Asia and Europe, in order to avoid the spread of the epidemic, and only Asia. The government has killed hundreds of millions of poultry, causing at least hundreds of billions of dollars in loss. More importantly, the H5N1 avian influenza virus is experiencing rapid variation, which is likely to lead to the spread of the virus.
Because avian influenza is similar to the clinical symptoms of acute respiratory diseases caused by other viruses, it is difficult to make accurate diagnosis of avian influenza only depending on clinical manifestations. It is necessary to confirm the diagnosis by laboratory testing. The early detection technique of avian influenza, which is simple, rapid and suitable for the promotion of grass-roots units, can make the doctors in the front line early. A rapid and accurate diagnosis, timely isolation of the patients to prevent the further transmission of avian influenza virus, plays a key role in preventing and controlling the outbreak of avian influenza. At present, the rapid detection kit based on immunochromatography (IC), enzyme linked immunosorbent assay (ELISA) and other immunoassay techniques can be used to distinguish the nail. Type two influenza virus, but can not further distinguish the subtype. Study the detection technology of influenza virus subtype, distinguish the avian influenza virus from the common influenza virus, not only helps to carry out the targeted antiviral treatment in time, but also help to study the epidemic and evolution of the avian influenza virus subtype, and monitor the new avian influenza virus subtype. It is of great significance for the development of large-scale screening, the immediate discovery of the source of infection and the cut off of the transmission route. Because one of the criteria for differentiating the subtype of influenza A virus is the antigenicity of the hemagglutinin, the homology of the different subtypes of hemagglutinin is not high, so the hemagglutinin is the ideal target for the detection of influenza virus subtypes. So far, in the laboratory of detection of influenza virus subtypes in laboratory, there are serological detection, gene detection and virus antigen detection for hemagglutinin, but the serological methods are specific antibodies, which are not suitable for early diagnosis; gene detection has higher technical requirements for operators and can not be widely used in grass-roots units. The detection of virus antigen is mainly by immunofluorescence test and enzyme linked immunosorbent assay. The former needs to be equipped with fluorescence microscopy, which can only be carried out in large hospitals or research institutes, and the ELISA capture method of detecting hemagglutinin antigen by specific antibodies can accurately determine the HA subtype of the virus in the early stage of the virus infection, but it is now at home and abroad. There are no commercialized kits. This study immunized mice with various forms of hemagglutinin antigen to screen specific antibodies with hemagglutination inhibition activity and neutralization activity. The antigenic epitopes identified by antibody were analyzed by competitive inhibition test, and the virus strains isolated from different hosts and different times were used to form a double antibody. Sandwich ELISA method was used to detect the hemagglutinin antigen of H5N1 avian influenza virus.
In addition, the preparation of specific monoclonal antibodies against hemagglutinin can not only develop a detection method to distinguish influenza virus subtypes, but also be used to develop therapeutic antibodies. Influenza virus hemagglutinin determines the blood coagulation activity of the virus and is closely related to the virus adsorbed to the surface of the target cell surface of the salivary acid oligosaccharide receptor, which is an important virus virus. The antibody against viral hemagglutinin, which has neutralizing activity against viral hemagglutinin, can specifically bind hemagglutinin in the early stage of infection and cause the virus to lose its ability to adsorb and infect host cells. Therefore, on the basis of a hemagglutinin specific antibody with hemagglutination inhibition, this study further evaluated the antibody against avian influenza virus. Neutralization activity is the basis for the study of therapeutic antibodies.
This study is divided into three parts:
Part one: preparation and identification of H5N1 avian influenza virus hemagglutinin antigen
In this part, three methods were used to obtain hemagglutinin antigen. (1) the recombinant H5 hemagglutinin protein (hereinafter referred to as "recombinant H5 protein") was expressed in the insect cell High Five by baculovirus expression system. The expression of recombinant H5 protein in insect cells was detected by immunofluorescence and Western Blot, and the recombinant H5 eggs were detected by hemagglutination test. The white blood coagulation activity.Western Blot identified the expression of recombinant protein molecular weight approximately 66kDa, Western Blot and immunofluorescence results confirmed that the recombinant H5 protein expressed in the insect cells could specifically combine the serum and H5 subtype standard antisera of the H5N1 avian influenza virus, and the blood coagulation test showed that the recombinant H5 protein could make the guinea pig red cells hair. The agglutination activity was 1:128, which proved that the recombinant H5 protein expressed in baculovirus system had hemagglutination activity. (2) the identification of plasmid pVAX1-tpA-97-H5 carrying H5N1 avian influenza virus (A / Hong Kong / 482 / 97) hemagglutinin gene (hereinafter referred to as "H5 gene plasmid") was used to observe the pVAXl plasmid carrier by X-gal staining test. Transfection efficiency, the expression of H5 gene plasmid at cell level was detected by immunofluorescence test. The results of.X-gal staining showed that the transfection of the plasmid pVAX1-1acZ to 293 cells could make the LacZ gene successfully express beta galactosidase and transfection efficiency. Immunofluorescence test confirmed that plasmid pVAX1-tpA-97-H5 transfected to 293 cells. Specific binding to the animal serum immunized with H5N1 avian influenza virus. (3) the determination of the hemagglutination of the commercialized natural H5 hemagglutinin (from the H5N1 avian influenza virus strain A / Goose / Guangdong / 1 / 96) was obtained from the Harbin Veterinary Institute. It was confirmed that natural H5 hemagglutinin could agglutinate the red blood cells of the guinea pig and agglutinate the titer most. Up to 1: 1024.
This section studies three different forms of H5 hemagglutinin antigen, and confirmed that 293 cells and recombinant H5 proteins transfected with H5 gene can be specifically combined with the standard antisera of avian influenza virus immune animal or H5 subtype, and natural H5 hemagglutinin has good hemagglutination activity for preparation of avian influenza virus (H5N1) hemagglutinin. Heterosexual monoclonal antibodies provide an immunogen.
The second part: preparation, identification and neutralization activity assay of H5N1 avian influenza virus hemagglutinin specific monoclonal antibody.
In this part, a variety of H5 hemagglutinin antigens were used to immunize mice to prepare the specific monoclonal antibodies against H5N1 avian influenza virus, and to identify the immunological properties of the monoclonal antibodies, the analysis of antibody recognition sites, the identification of blood coagulation inhibition activity and the neutralization activity analysis. Three kinds of immunization BALB / c mice were adopted: the first scheme was natural H5 hemagglutination The second schemes were immunized with the natural H5 hemagglutinin for the plasmid immunization of the H5 gene, and the third schemes were immunized with the recombinant H5 protein. The spleen cells of each immunized mouse were fused with the murine myeloma cells respectively, and the hemagglutination inhibition test and indirect ELISA were used to screen the positive clones. The clone with positive chicken embryo fluid, influenza A virus (H1N1 and H3N2), B influenza virus and A virus recombinant nucleoprotein were cloned continuously by finite dilution method for 2-3 times, and 32 hybridoma cell lines that secreted the specific anti H5N1 virus hemagglutinin monoclonal antibody were obtained. After inoculation in mice, 31 monoclonal antibodies obtained ascites. 12 strains were obtained with natural H5 hemagglutinin, including IgG1 (10 strains), IgG2b (1 strains) and IgM (1 strains); 9 strains were immunized with H5 gene, antibody subclass IgG1 (1 strains), IgG2a (4), IgG2b (1), and 3 were not in the upper subclass; 10 strains were obtained with recombinant H5 protein, and the antibody subclass included IgG1 (7 strain), Ig. G2a (2 strains) and IgM (1 strains). The result of hemagglutination inhibition test showed that the inhibition titer of monoclonal antibody against H5 hemagglutinin was 1: 100~1: 51200 after immunization with the plasmid of natural H5 hemagglutinin or H5 gene, and there was no cross reaction with the hemagglutinin of influenza virus H1, H3, H7, H9 subtype and B type influenza virus. The neutralization activity test of 18 ascitic antibodies against hemagglutination inhibition titer above 1: 100 (completed by University of Hong Kong microorganism), the results of the neutralization test of H5N1 avian influenza virus (A / Vietnam / 3028 / 04) showed that the neutralization titer of the 18 strains was not consistent with the hemagglutination inhibition titer, of which 14 The neutralizing titer of the antibody was lower than 1: 40; the neutralizing titers of the other 4 strains were all higher than that of 1: 160., which were obtained by whole H5 immunization.
31 mAb ascites were purified by octanic acid ammonium sulfate precipitation, and 28 IgG monoclonal antibodies were obtained. The results of SDS-PAGE electrophoresis showed that the purity of McAbs was > 90%., using the improved sodium periodate method to mark the horseradish peroxidase enzyme of 26 monoclonal antibodies (except for 2 undetermined antibody subclasses) of the known antibody subclasses, and to detect the working concentration of the enzyme labeled antibody by indirect EHSA Between 10~ (-2) to 10~ (-5). According to the activity of hemagglutination inhibition, 26 strains of enzyme labeled monoclonal antibody were divided into two groups, and the antigenic epitopes of McAbs were analyzed by competitive inhibition test respectively. The results showed that 16 monoclonal antibodies with hemagglutination inhibition activity identified 2 different antigen sites and the hemagglutination activity of antibody of identification site I and II. The difference was 1: 3200~1: 51200 and 1: 100~1: 400, and 10 McAbs without hemagglutination inhibitory activity identified 4 different epitopes. The results laid the foundation for the next step to establish the double antibody sandwich antigen detection method.
The third part: the establishment of double antibody sandwich ELISA capture method for H5 hemagglutinin.
In this part, a pair of antibody pairs with the highest sensitivity to H5 hemagglutinin was screened by combination matching, and a double antibody sandwich EHSA capture method for H5 hemagglutinin was established. (1) cross matching by 10 strains of hemagglutinin activity, and by detecting gradient diluted natural H5 hemagglutinin, recombinant H5 protein and 2 strains of H5N1 avian influenza virus, finally selected. The sensitivity of H5M21 and H5M15-HRP in combination, the sensitivity of the established double antibody sandwich antigen capture method to detect H5 hemagglutinin and H5N1 avian influenza virus was lower than 16 hemagglutinating units. (2) cross matching of 16 monoclonal antibodies with hemagglutination inhibition positive, to ensure the sensitivity of detection, through the detection of natural H5 hemagglutinin and 6 strains of gradient dilution. Homoclinic, H5N1 avian influenza virus isolated at different times, the final selection of antibodies against H5M9 and H5M1 1-HRP was used in combination to establish a detection method, and the sensitivity of H5 hemagglutinin and H5N1 avian influenza virus was 1 / 32 hemagglutination unit, and no cross reaction with other subtypes of influenza and B influenza virus, which had good specificity. The study shows that the detection method of H5N1 virus based on the antibody central heart antigen capture method has high sensitivity and high specificity. It provides a quick and specific laboratory diagnosis for the early diagnosis of avian influenza virus infection.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2141558
[Abstract]:Influenza viruses belong to the genera of the family of the family of the family of the family. The influenza virus can be divided into a (A), B (B) and C (C) three, based on the characteristics of the nucleoprotein (N) and matrix protein (M). The influenza B and C influenza viruses are mainly human, but the epidemic is very narrow and influenza A virus is susceptible Infected people, poultry and livestock are the main cause of influenza pandemic. According to the difference of the antigenicity of Hemagglutinin (HA) and Neuraminidase (NA), influenza A virus can be further divided into different subtypes. The influenza A virus (influenza A) has been found to include 16 HA subtypes (H1-H16) and the influenza A virus (H1-H16). 9 NA subtypes (N1-N9), of which H1, H2, H3 and N1, the N2 subtype are the main subtypes of human influenza. The HINl and H3N2 subtype influenza viruses have long been causing a number of worldwide influenza pandemic, such as the 1918 "Spanish flu", and the 1957 and 1968 influenza pandemic, causing great threat to human health. And since 1997 H5 After the outbreak of the N1 subtype avian influenza virus (N1) virus in 18 people and 6 deaths, people began to attach great importance to the avian influenza virus represented by the H5N1 and H7N7 subtypes. After that, the highly pathogenic avian influenza virus continued to infect humans and poultry in East Asia, Southeast Asia and Europe, in order to avoid the spread of the epidemic, and only Asia. The government has killed hundreds of millions of poultry, causing at least hundreds of billions of dollars in loss. More importantly, the H5N1 avian influenza virus is experiencing rapid variation, which is likely to lead to the spread of the virus.
Because avian influenza is similar to the clinical symptoms of acute respiratory diseases caused by other viruses, it is difficult to make accurate diagnosis of avian influenza only depending on clinical manifestations. It is necessary to confirm the diagnosis by laboratory testing. The early detection technique of avian influenza, which is simple, rapid and suitable for the promotion of grass-roots units, can make the doctors in the front line early. A rapid and accurate diagnosis, timely isolation of the patients to prevent the further transmission of avian influenza virus, plays a key role in preventing and controlling the outbreak of avian influenza. At present, the rapid detection kit based on immunochromatography (IC), enzyme linked immunosorbent assay (ELISA) and other immunoassay techniques can be used to distinguish the nail. Type two influenza virus, but can not further distinguish the subtype. Study the detection technology of influenza virus subtype, distinguish the avian influenza virus from the common influenza virus, not only helps to carry out the targeted antiviral treatment in time, but also help to study the epidemic and evolution of the avian influenza virus subtype, and monitor the new avian influenza virus subtype. It is of great significance for the development of large-scale screening, the immediate discovery of the source of infection and the cut off of the transmission route. Because one of the criteria for differentiating the subtype of influenza A virus is the antigenicity of the hemagglutinin, the homology of the different subtypes of hemagglutinin is not high, so the hemagglutinin is the ideal target for the detection of influenza virus subtypes. So far, in the laboratory of detection of influenza virus subtypes in laboratory, there are serological detection, gene detection and virus antigen detection for hemagglutinin, but the serological methods are specific antibodies, which are not suitable for early diagnosis; gene detection has higher technical requirements for operators and can not be widely used in grass-roots units. The detection of virus antigen is mainly by immunofluorescence test and enzyme linked immunosorbent assay. The former needs to be equipped with fluorescence microscopy, which can only be carried out in large hospitals or research institutes, and the ELISA capture method of detecting hemagglutinin antigen by specific antibodies can accurately determine the HA subtype of the virus in the early stage of the virus infection, but it is now at home and abroad. There are no commercialized kits. This study immunized mice with various forms of hemagglutinin antigen to screen specific antibodies with hemagglutination inhibition activity and neutralization activity. The antigenic epitopes identified by antibody were analyzed by competitive inhibition test, and the virus strains isolated from different hosts and different times were used to form a double antibody. Sandwich ELISA method was used to detect the hemagglutinin antigen of H5N1 avian influenza virus.
In addition, the preparation of specific monoclonal antibodies against hemagglutinin can not only develop a detection method to distinguish influenza virus subtypes, but also be used to develop therapeutic antibodies. Influenza virus hemagglutinin determines the blood coagulation activity of the virus and is closely related to the virus adsorbed to the surface of the target cell surface of the salivary acid oligosaccharide receptor, which is an important virus virus. The antibody against viral hemagglutinin, which has neutralizing activity against viral hemagglutinin, can specifically bind hemagglutinin in the early stage of infection and cause the virus to lose its ability to adsorb and infect host cells. Therefore, on the basis of a hemagglutinin specific antibody with hemagglutination inhibition, this study further evaluated the antibody against avian influenza virus. Neutralization activity is the basis for the study of therapeutic antibodies.
This study is divided into three parts:
Part one: preparation and identification of H5N1 avian influenza virus hemagglutinin antigen
In this part, three methods were used to obtain hemagglutinin antigen. (1) the recombinant H5 hemagglutinin protein (hereinafter referred to as "recombinant H5 protein") was expressed in the insect cell High Five by baculovirus expression system. The expression of recombinant H5 protein in insect cells was detected by immunofluorescence and Western Blot, and the recombinant H5 eggs were detected by hemagglutination test. The white blood coagulation activity.Western Blot identified the expression of recombinant protein molecular weight approximately 66kDa, Western Blot and immunofluorescence results confirmed that the recombinant H5 protein expressed in the insect cells could specifically combine the serum and H5 subtype standard antisera of the H5N1 avian influenza virus, and the blood coagulation test showed that the recombinant H5 protein could make the guinea pig red cells hair. The agglutination activity was 1:128, which proved that the recombinant H5 protein expressed in baculovirus system had hemagglutination activity. (2) the identification of plasmid pVAX1-tpA-97-H5 carrying H5N1 avian influenza virus (A / Hong Kong / 482 / 97) hemagglutinin gene (hereinafter referred to as "H5 gene plasmid") was used to observe the pVAXl plasmid carrier by X-gal staining test. Transfection efficiency, the expression of H5 gene plasmid at cell level was detected by immunofluorescence test. The results of.X-gal staining showed that the transfection of the plasmid pVAX1-1acZ to 293 cells could make the LacZ gene successfully express beta galactosidase and transfection efficiency. Immunofluorescence test confirmed that plasmid pVAX1-tpA-97-H5 transfected to 293 cells. Specific binding to the animal serum immunized with H5N1 avian influenza virus. (3) the determination of the hemagglutination of the commercialized natural H5 hemagglutinin (from the H5N1 avian influenza virus strain A / Goose / Guangdong / 1 / 96) was obtained from the Harbin Veterinary Institute. It was confirmed that natural H5 hemagglutinin could agglutinate the red blood cells of the guinea pig and agglutinate the titer most. Up to 1: 1024.
This section studies three different forms of H5 hemagglutinin antigen, and confirmed that 293 cells and recombinant H5 proteins transfected with H5 gene can be specifically combined with the standard antisera of avian influenza virus immune animal or H5 subtype, and natural H5 hemagglutinin has good hemagglutination activity for preparation of avian influenza virus (H5N1) hemagglutinin. Heterosexual monoclonal antibodies provide an immunogen.
The second part: preparation, identification and neutralization activity assay of H5N1 avian influenza virus hemagglutinin specific monoclonal antibody.
In this part, a variety of H5 hemagglutinin antigens were used to immunize mice to prepare the specific monoclonal antibodies against H5N1 avian influenza virus, and to identify the immunological properties of the monoclonal antibodies, the analysis of antibody recognition sites, the identification of blood coagulation inhibition activity and the neutralization activity analysis. Three kinds of immunization BALB / c mice were adopted: the first scheme was natural H5 hemagglutination The second schemes were immunized with the natural H5 hemagglutinin for the plasmid immunization of the H5 gene, and the third schemes were immunized with the recombinant H5 protein. The spleen cells of each immunized mouse were fused with the murine myeloma cells respectively, and the hemagglutination inhibition test and indirect ELISA were used to screen the positive clones. The clone with positive chicken embryo fluid, influenza A virus (H1N1 and H3N2), B influenza virus and A virus recombinant nucleoprotein were cloned continuously by finite dilution method for 2-3 times, and 32 hybridoma cell lines that secreted the specific anti H5N1 virus hemagglutinin monoclonal antibody were obtained. After inoculation in mice, 31 monoclonal antibodies obtained ascites. 12 strains were obtained with natural H5 hemagglutinin, including IgG1 (10 strains), IgG2b (1 strains) and IgM (1 strains); 9 strains were immunized with H5 gene, antibody subclass IgG1 (1 strains), IgG2a (4), IgG2b (1), and 3 were not in the upper subclass; 10 strains were obtained with recombinant H5 protein, and the antibody subclass included IgG1 (7 strain), Ig. G2a (2 strains) and IgM (1 strains). The result of hemagglutination inhibition test showed that the inhibition titer of monoclonal antibody against H5 hemagglutinin was 1: 100~1: 51200 after immunization with the plasmid of natural H5 hemagglutinin or H5 gene, and there was no cross reaction with the hemagglutinin of influenza virus H1, H3, H7, H9 subtype and B type influenza virus. The neutralization activity test of 18 ascitic antibodies against hemagglutination inhibition titer above 1: 100 (completed by University of Hong Kong microorganism), the results of the neutralization test of H5N1 avian influenza virus (A / Vietnam / 3028 / 04) showed that the neutralization titer of the 18 strains was not consistent with the hemagglutination inhibition titer, of which 14 The neutralizing titer of the antibody was lower than 1: 40; the neutralizing titers of the other 4 strains were all higher than that of 1: 160., which were obtained by whole H5 immunization.
31 mAb ascites were purified by octanic acid ammonium sulfate precipitation, and 28 IgG monoclonal antibodies were obtained. The results of SDS-PAGE electrophoresis showed that the purity of McAbs was > 90%., using the improved sodium periodate method to mark the horseradish peroxidase enzyme of 26 monoclonal antibodies (except for 2 undetermined antibody subclasses) of the known antibody subclasses, and to detect the working concentration of the enzyme labeled antibody by indirect EHSA Between 10~ (-2) to 10~ (-5). According to the activity of hemagglutination inhibition, 26 strains of enzyme labeled monoclonal antibody were divided into two groups, and the antigenic epitopes of McAbs were analyzed by competitive inhibition test respectively. The results showed that 16 monoclonal antibodies with hemagglutination inhibition activity identified 2 different antigen sites and the hemagglutination activity of antibody of identification site I and II. The difference was 1: 3200~1: 51200 and 1: 100~1: 400, and 10 McAbs without hemagglutination inhibitory activity identified 4 different epitopes. The results laid the foundation for the next step to establish the double antibody sandwich antigen detection method.
The third part: the establishment of double antibody sandwich ELISA capture method for H5 hemagglutinin.
In this part, a pair of antibody pairs with the highest sensitivity to H5 hemagglutinin was screened by combination matching, and a double antibody sandwich EHSA capture method for H5 hemagglutinin was established. (1) cross matching by 10 strains of hemagglutinin activity, and by detecting gradient diluted natural H5 hemagglutinin, recombinant H5 protein and 2 strains of H5N1 avian influenza virus, finally selected. The sensitivity of H5M21 and H5M15-HRP in combination, the sensitivity of the established double antibody sandwich antigen capture method to detect H5 hemagglutinin and H5N1 avian influenza virus was lower than 16 hemagglutinating units. (2) cross matching of 16 monoclonal antibodies with hemagglutination inhibition positive, to ensure the sensitivity of detection, through the detection of natural H5 hemagglutinin and 6 strains of gradient dilution. Homoclinic, H5N1 avian influenza virus isolated at different times, the final selection of antibodies against H5M9 and H5M1 1-HRP was used in combination to establish a detection method, and the sensitivity of H5 hemagglutinin and H5N1 avian influenza virus was 1 / 32 hemagglutination unit, and no cross reaction with other subtypes of influenza and B influenza virus, which had good specificity. The study shows that the detection method of H5N1 virus based on the antibody central heart antigen capture method has high sensitivity and high specificity. It provides a quick and specific laboratory diagnosis for the early diagnosis of avian influenza virus infection.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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10 孙学辉;高效表达H5亚型禽流感病毒HA基因的重组鸡痘病毒的构建及其免疫效力[D];扬州大学;2005年
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