禽流感病毒（H5N1）血凝素特異性單克隆抗體的制備、鑒定及其ELISA捕獲法的建立
發(fā)布時間：2018-07-24 13:39
【摘要】： 流感病毒屬于正粘病毒科流感病毒屬，是一大類引起人類和禽畜患病的病原體。根據(jù)核蛋白(N)和基質(zhì)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類為主，但流行范圍很窄，而甲型流感病毒可感染人類、禽類及家畜，是引起流感大規(guī)模流行的主要致病原。根據(jù)病毒表面血凝素(Hemagglutinin，HA)和神經(jīng)氨酸酶(Neuraminidase，NA)抗原性的差異，還可將甲型流感病毒進一步分為不同的亞型，目前已發(fā)現(xiàn)的甲型流感病毒包括16個HA亞型(H1-H16)及9個NA亞型(N1-N9)，其中H1、H2、H3及N1、N2亞型是引起人類流感的主要亞型。長期以來，HINl和H3N2亞型流感病毒引發(fā)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，對人類健康造成了巨大的威脅。而自從1997年H5N1亞型禽流感病毒在香港導(dǎo)致18人發(fā)病及6人死亡的事件發(fā)生以后，人們開始高度重視以H5N1和H7N7亞型為代表的禽流感病毒。此后，高致病性禽流感病毒又持續(xù)多次地在東亞、東南亞及歐洲等地感染人類及家禽，為避免疫情擴散，僅亞洲各國政府就宰殺上億只家禽，造成至少上千億美元的損失。更為重要的是，H5N1禽流感病毒正經(jīng)歷迅速的變異，極有可能導(dǎo)致病毒在人際間的傳播。 由于禽流感與其他病毒引發(fā)的急性呼吸道疾病的臨床癥狀類似，僅僅依靠臨床表現(xiàn)難以對禽流感做出準(zhǔn)確的診斷，必須通過實驗室檢測手段進行確診。簡便、快速而且適合基層單位推廣的禽流感早期檢測技術(shù)，可以使臨床一線的醫(yī)生早期、迅速、準(zhǔn)確地作出診斷，及時對患者進行隔離，防止禽流感病毒進一步傳播，在防控禽流感突發(fā)疫情中起著關(guān)鍵作用。目前，已有基于免疫層析法(IC)、酶聯(lián)免疫吸附試驗(ELISA)等免疫檢測技術(shù)的抗原快速檢測試劑盒上市，可用于區(qū)分甲、乙兩型流感病毒，但都無法進一步區(qū)分亞型。研究流感病毒亞型的檢測技術(shù)，將禽流感病毒與普通流感病毒區(qū)分開來，不僅有利于及時開展針對性的抗病毒治療，還有助于研究禽流感病毒亞型的流行和進化規(guī)律，監(jiān)控禽流感病毒新亞型的出現(xiàn)，并且對開展大規(guī)模篩查，即時發(fā)現(xiàn)傳染源和切斷傳染途徑，都具有十分重要的意義。由于甲型流感病毒區(qū)分亞型的標(biāo)準(zhǔn)之一是血凝素的抗原性，而不同亞型病毒血凝素之間同源性不高，因此血凝素是流感病毒亞型檢測的理想靶標(biāo)。到現(xiàn)在為止，實驗室流感病毒亞型檢測方法中，針對血凝素的主要有血清學(xué)檢測、基因檢測及病毒抗原檢測，但血清學(xué)方法檢測的是特異性抗體，不適用于早期診斷；基因檢測對操作者的技術(shù)水平要求較高，無法在基層單位普及使用；而病毒抗原檢測主要是免疫熒光試驗及酶聯(lián)免疫吸附試驗，前者需要配備熒光顯微鏡，只能在較大醫(yī)院或研究所才能開展，，而利用特異性抗體檢測血凝素抗原的ELISA捕獲法，能在病毒感染早期準(zhǔn)確判斷病毒的HA亞型，但是目前國內(nèi)外尚無商品化試劑盒問世。本研究采用多種形式的血凝素抗原免疫小鼠，篩選具有血凝抑制活性和中和活性的特異性抗體，通過競爭抑制試驗分析抗體識別的抗原表位，并利用不同宿主、不同時間分離的病毒株進行考核，最終組建雙抗體夾心ELISA法，檢測H5N1禽流感病毒血凝素抗原。 此外，本研究制備針對血凝素的特異性單克隆抗體，不僅可以發(fā)展區(qū)分流感病毒亞型的檢測方法，還可用于發(fā)展治療性抗體。流感病毒血凝素決定了病毒的血凝活性，與病毒吸附到靶細胞表面唾液酸寡糖受體密切相關(guān)，是病毒重要的毒力決定因子。而針對病毒血凝素的具備中和活性的抗體，可以在感染早期特異性結(jié)合血凝素，使病毒失去吸附和感染宿主細胞的能力。因此，在獲得具有血凝抑制活性的血凝素特異性抗體基礎(chǔ)上，本研究進一步評價抗體對禽流感病毒的中和活性，為研究治療性抗體奠定基礎(chǔ)。 本研究主要分為三個部分： 第一部分：H5N1禽流感病毒血凝素抗原的制備和鑒定 本部分研究采用三種方法獲得血凝素抗原。(1)利用桿狀病毒表達體系，在昆蟲細胞High Five中表達重組H5血凝素蛋白(以下簡稱“重組H5蛋白”)，采用免疫熒光和Western Blot方法檢測重組H5蛋白在昆蟲細胞中表達情況，血凝試驗檢測重組H5蛋白的血凝活性。Western Blot鑒定表達重組蛋白分子量約為66kDa，Western Blot和免疫熒光結(jié)果證實在昆蟲細胞表達的重組H5蛋白能特異性地結(jié)合H5N1禽流感病毒免疫的動物血清和H5亞型標(biāo)準(zhǔn)抗血清；血凝試驗表明重組H5蛋白能使豚鼠紅細胞發(fā)生凝集反應(yīng)，凝集效價為1：128，證明用桿狀病毒體系表達的重組H5蛋白具有血凝活性。(2)鑒定攜帶H5N1禽流感病毒株(A／Hong Kong／482／97)血凝素基因的質(zhì)粒pVAX1-tpA-97-H5(以下簡稱“H5基因的質(zhì)�！�)，利用X-gal染色試驗觀察pVAXl質(zhì)粒載體的轉(zhuǎn)染效率，通過免疫熒光試驗檢測H5基因的質(zhì)粒在細胞水平的表達情況。X-gal染色結(jié)果表明，對照質(zhì)粒pVAX1-1acZ轉(zhuǎn)染293細胞后，能使攜帶的LacZ基因成功表達β-半乳糖苷酶且轉(zhuǎn)染效率較高；免疫熒光試驗證實，質(zhì)粒pVAX1-tpA-97-H5轉(zhuǎn)染293細胞后能與H5N1禽流感病毒免疫的動物血清特異性結(jié)合。(3)對商品化的天然H5血凝素(從H5N1禽流感病毒株A／Goose／Guangdong／1／96濃縮得到，購自哈爾濱獸醫(yī)研究所)進行了血凝滴度測定，證實天然H5血凝素能使豚鼠紅細胞發(fā)生凝集，凝集效價最高達到1∶1024。 本部分研究得到三種不同形式的H5血凝素抗原，并證實轉(zhuǎn)染H5基因的質(zhì)粒的293細胞和重組H5蛋白分別能與禽流感病毒免疫動物血清或H5亞型標(biāo)準(zhǔn)抗血清特異性結(jié)合，而天然H5血凝素具有良好的血凝活性，為制備禽流感病毒(H5N1)血凝素特異性單克隆抗體提供了免疫原。 第二部分：H5N1禽流感病毒血凝素特異性單抗的制備、鑒定及中和活性測定 本部分研究利用多種H5血凝素抗原免疫小鼠，制備抗H5N1禽流感病毒血凝素特異性單抗，并對單抗進行免疫學(xué)性質(zhì)鑒定、抗體識別位點分析、血凝抑制活性鑒定以及中和活性分析。采用三種方案免疫BALB／c小鼠：第一種方案為天然H5血凝素全程免疫；第二種方案為H5基因的質(zhì)粒免疫后再用天然H5血凝素進行加強免疫；第三種方案為重組H5蛋白全程免疫。分別取每一種方案免疫后的小鼠脾細胞與小鼠骨髓瘤細胞進行融合，采用血凝抑制實驗和間接ELISA篩選陽性克隆，并排除與正常雞胚液、A型流感病毒(H1N1和H3N2)、B型流感病毒和A型流感病毒重組核蛋白反應(yīng)陽性的克隆。經(jīng)有限稀釋法連續(xù)克隆化2-3次，獲得32株穩(wěn)定分泌特異性抗H5N1病毒血凝素單抗的雜交瘤細胞株，腹腔接種小鼠后，31株單抗獲得腹水，其中用天然H5血凝素全程免疫后獲得12株，抗體亞類包括IgG1(10株)、IgG2b(1株)和IgM(1株)；用H5基因的質(zhì)粒免疫后獲得9株，抗體亞類除IgG1(1株)、IgG2a(4株)、IgG2b(1株)，另有3株不屬于以上亞類；用重組H5蛋白免疫后獲得10株，抗體亞類包括IgG1(7株)、IgG2a(2株)和IgM(1株)。血凝抑制試驗結(jié)果表明，用天然H5血凝素或H5基因的質(zhì)粒免疫后獲得單抗對H5血凝素的血凝抑制效價為1∶100～1∶51 200，且與A型流感病毒H1、H3、H7、H9亞型及B型流感病毒的血凝素均無交叉反應(yīng)；用重組H5蛋白免疫后獲得單抗不具有血凝抑制活性。對血凝抑制效價達1∶100以上的18株腹水抗體進行中和活性試驗(由香港大學(xué)微生物協(xié)作完成)，對H5N1禽流感病毒(A／Vietnam／3028／04)中和試驗檢測的結(jié)果表明：18株抗體的中和效價與血凝抑制效價不一致，其中14株抗體的中和效價低于1∶40；另外4株由天然H5血凝素全程免疫得到的抗體中和效價均高于1∶160。 31株單抗腹水經(jīng)辛酸-硫酸銨沉淀法純化，共得到28株IgG單抗，SDS-PAGE電泳結(jié)果顯示單抗純度均＞90％。采用改良過碘酸鈉法對26株已知抗體亞類的單抗(2株單抗未確定抗體亞類除外)標(biāo)記辣根過氧化物酶，間接EHSA檢測酶標(biāo)抗體的工作濃度在10~(-2)～10~(-5)之間。按是否具有血凝抑制活性將26株酶標(biāo)記單抗分為兩個組，采用競爭抑制試驗分別分析單抗識別抗原表位，結(jié)果顯示具有血凝抑制活性的16株單抗識別2個不同的抗原位點，而且識別位點Ⅰ和Ⅱ的抗體的血凝抑制活性相差較大，分別為1∶3 200～1∶51 200及1∶100～1∶400；無血凝抑制活性的10株單抗識別4個不同的抗原表位，其研究結(jié)果為下一步建立雙抗體夾心抗原檢測方法奠定了基礎(chǔ)。 第三部分：針對H5血凝素的雙抗體夾心ELISA捕獲法的建立 本部分研究通過組合配對，篩選檢測H5血凝素靈敏度最高的抗體對，建立針對H5血凝素的雙抗體夾心EHSA捕獲法。(1)利用10株無血凝抑制活性的單抗進行交叉配對，通過檢測梯度稀釋的天然H5血凝素、重組H5蛋白及2株H5N1禽流感病毒，最終選定H5M21和H5M15-HRP的抗體對組合，建立的雙抗體夾心抗原捕獲法檢測H5血凝素和H5N1禽流感病毒的靈敏度低于16個血凝單位。(2)利用16株呈血凝抑制陽性的單抗進行交叉配對。為保證檢測的敏感性，通過檢測梯度稀釋的天然H5血凝素及6株不同宿主、不同時間分離的H5N1禽流感病毒，最終選定H5M9和H5M1 1-HRP的抗體對組合用于建立檢測方法，檢測H5血凝素和H5N1禽流感病毒的靈敏度為1／32血凝單位，而且與A型流感其他亞型及B型流感病毒均無交叉反應(yīng)，具有良好的特異性。本部分研究表明，采用抗體央心抗原捕獲法建立的H5N1病毒檢測方法具有較高敏感性和高度特異性，為禽流感病毒感染早期診斷提供了一種快捷、特異的實驗室診斷方法。 綜合以上三個部分的結(jié)果，本研究的結(jié)論如下： 一、以重組H5蛋白作為免疫原所獲得的10株抗H5單克隆抗體均沒有血凝抑制活性，而以H5基因的質(zhì)粒及天然H5血凝素免疫小鼠后獲得了16株具有血凝抑制活性的抗H5單克隆抗體，表明血凝素的天然構(gòu)象表位對維持抗體的血凝抑制活性起重要的作用。 二、本研究獲得抗體的中和活性與血凝抑制活性存在不一致，原因可能與檢測抗體的中和活性與血凝抑制活性所用的病毒不是同一株病毒有關(guān)，血凝抑制試驗中使用的血凝素來源于禽流感病毒禽類分離株(A／Goose／Guangdong／1／96)，而中和活性試驗中使用的是禽流感病毒人體分離株(a／vietnam／3028／04)，毒株間的差異導(dǎo)致了抗體中和活性與血凝抑制活性的不一致；也可能是由于決定抗體的中和活性與血凝抑制活性的抗原位點是不一致的，其原因還有待于進一步探討。 三、用具有血凝抑制活性的抗體建立了雙抗體夾心捕獲EHSA法，檢測H5血凝素和H5N1禽流感病毒液的靈敏度明顯高于用無血凝抑制活性的抗體建立的檢測方法，表明具有血凝抑制活性的抗體才能有效識別血凝素的天然構(gòu)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.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392
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