抗SARS-CoV單克隆抗體的鑒定及應(yīng)用研究
發(fā)布時間：2019-03-31 10:08
【摘要】：嚴重急性呼吸系統(tǒng)綜合征(Severe acute respiratory syndrome,SARS)是一種烈性新發(fā)傳染病,其病原體為一種新型冠狀病毒(SARS-CoV)。盡管目前SARS的流行已經(jīng)得到控制,但迄今為止,SARS的起源、動物宿主、傳播途徑及致病機理等仍不完全清楚,SARS對人類的威脅依然存在。由于從臨床癥狀及其發(fā)病體征很難與其它病原體引起的發(fā)熱性呼吸系統(tǒng)疾病區(qū)分,所以更顯得早期特異實驗診斷的重要。對于該病的治療目前主要依賴對癥和支持治療,沒有特效藥物。利用恢復(fù)期病人血清,對感染者進行被動免疫治療,已被臨床證明是一種有效的治療方法,所以開發(fā)被動免疫制劑受到廣泛的重視。 為開發(fā)SARS早期、特異診斷實驗室檢測方法及應(yīng)急被動免疫制劑,我室用滅活純化SARS-CoV(BJ01株)免疫BALB/C小鼠,通過細胞融合技術(shù),建立了12株能穩(wěn)定分泌SARS-CoV單克隆抗體的雜交瘤細胞株。對這些單克隆抗體的免疫學(xué)鑒定證明:1.IFA對單克隆抗體效價測定,12份單克隆抗體,除1D4效價稍低一些外,其余IFA效價均在1:2560—20480之間,顯示出這些單抗都有較高的抗SARS—CoV活性,而且與不同地區(qū)分離毒株均有良好反應(yīng);2.采用雙向瓊脂擴散和酶聯(lián)免疫吸附試驗對12株單克隆抗體免疫球蛋白類型及其亞類特異性鑒定,結(jié)果5株(2D4、1A2、2C5、2A3、384)單克隆抗體重鏈為IgG1,5株(184、2B1、1C5、1A5、2A2)為IgG2a,1株(3A3)為IgG2b,另一株(1D4)為IgM,輕鏈全部為k型;3.采用微量細胞培養(yǎng)中和試驗,對12株抗SARS冠狀病毒單克隆抗體進行抗SARS—CoV中和活性測定,6株(1A2、1A5、2D4、2A3、2C5、3B4)具有中和活性,其中1A5和2C5的中和效價分別達1:20480和1:5957。 在對這些單克隆抗體免疫學(xué)特性鑒定的基礎(chǔ)上,對它們的抗原結(jié)合表位進行了分析。首先用重組表達的SARS-CoV S蛋白和N對這些單抗進行測定,其中6株(1A5、2C5、2A3、1B4、2B1、3A3)與S蛋白反應(yīng),2株(2D4,1A2)與N蛋白反應(yīng),說明它們結(jié)合的抗原表位分別位于S蛋白和N蛋白上。另外4株單抗與S和N蛋白均不結(jié)合,說明它們可能針對S、N以外的結(jié)構(gòu)蛋白。對6株結(jié)合S蛋白的單抗用不同長度的S肽段進一步分析它們結(jié)合的抗原位點,其中3株(2A3、
[Abstract]:Severe acute respiratory syndrome (Severe acute respiratory syndrome,SARS) is a new severe infectious disease, and its pathogen is a new type of coronavirus (SARS-CoV). Although the prevalence of SARS has been controlled up to now, the origin, animal host, transmission pathway and pathogenic mechanism of SARS are still unclear, and the threat of SARS to human remains. Because it is difficult to distinguish febrile respiratory diseases caused by other pathogens from clinical symptoms and signs, it is more important to diagnose febrile respiratory diseases by early specific experiments. Treatment for the disease is currently mainly dependent on symptomatic and supportive treatment, with no specific drugs. Passive immunotherapy for infected patients with convalescent serum has been proved to be an effective therapy. Therefore, the development of passive immune agents has been paid more and more attention. In order to develop an early, specific diagnostic laboratory assay for SARS and an emergency passive immune preparation, BALB/C mice were immunized with inactivated and purified SARS-CoV (BJ01 strain) by cell fusion technique. Twelve hybridoma cell lines stably secreting monoclonal antibodies against SARS-CoV were established. The immunologic identification of these monoclonal antibodies showed that the titers of 1.IFA to monoclonal antibodies were lower than those of 1D4, and the titers of IFA were between 1 脳 2560 and 20480, but the titer of McAbs was lower than that of McAbs. The results showed that these McAbs had high anti-SARS-CoV activity and had good reaction with the strains isolated from different regions. 2. The immunoglobulin type and subclass specificity of 12 monoclonal antibodies were identified by two-way Agar diffusion and enzyme-linked immunosorbent assay. The results showed that the heavy chain of monoclonal antibodies (2D4, 1A2, 2C5, 2A3384) was IgG1,5 strain (184, 2B1, 1C5, 1A5, 2A2) and IgG2a,1 strain (3A3) was IgG2b,. The other strain (1D4) was all K-type of IgM, light chain. 3. The neutralization activity of 12 anti-SARS coronavirus monoclonal antibodies (1A2, 1A5, 2D4, 2A3, 2C5, 3B4) was determined by micro cell culture neutralization assay. The neutralization titers of 1A5 and 2C5 were 1? 20480 and 1? 5957, respectively, and the neutralization activity of 1A5 and 2C5 were 1A2, 1A5, 2D4, 2A3, 2C5, 3B4, respectively. Based on the immunological characteristics of these monoclonal antibodies, their antigen binding epitopes were analyzed. These monoclonal antibodies were first detected by recombinant SARS-CoV S protein and N, among which 6 strains (1A5, 2C5, 2A3, 1B4, 2B1, 3A3) reacted with S protein and 2 strains (2D4, 1A2) reacted with N protein, indicating that the antigen epitopes of these monoclonal antibodies were located on S protein and N protein, respectively. The other four McAbs did not bind to S and N proteins, suggesting that they may target structural proteins other than S and N. Six monoclonal antibodies binding to S protein were further analyzed by using different length S peptide fragments, of which 3 strains (2A3, 2A3, 2A3, 2A3, 2A3, 2A3, 2A3, 2A3,
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392.1
本文編號：2450811
[Abstract]:Severe acute respiratory syndrome (Severe acute respiratory syndrome,SARS) is a new severe infectious disease, and its pathogen is a new type of coronavirus (SARS-CoV). Although the prevalence of SARS has been controlled up to now, the origin, animal host, transmission pathway and pathogenic mechanism of SARS are still unclear, and the threat of SARS to human remains. Because it is difficult to distinguish febrile respiratory diseases caused by other pathogens from clinical symptoms and signs, it is more important to diagnose febrile respiratory diseases by early specific experiments. Treatment for the disease is currently mainly dependent on symptomatic and supportive treatment, with no specific drugs. Passive immunotherapy for infected patients with convalescent serum has been proved to be an effective therapy. Therefore, the development of passive immune agents has been paid more and more attention. In order to develop an early, specific diagnostic laboratory assay for SARS and an emergency passive immune preparation, BALB/C mice were immunized with inactivated and purified SARS-CoV (BJ01 strain) by cell fusion technique. Twelve hybridoma cell lines stably secreting monoclonal antibodies against SARS-CoV were established. The immunologic identification of these monoclonal antibodies showed that the titers of 1.IFA to monoclonal antibodies were lower than those of 1D4, and the titers of IFA were between 1 脳 2560 and 20480, but the titer of McAbs was lower than that of McAbs. The results showed that these McAbs had high anti-SARS-CoV activity and had good reaction with the strains isolated from different regions. 2. The immunoglobulin type and subclass specificity of 12 monoclonal antibodies were identified by two-way Agar diffusion and enzyme-linked immunosorbent assay. The results showed that the heavy chain of monoclonal antibodies (2D4, 1A2, 2C5, 2A3384) was IgG1,5 strain (184, 2B1, 1C5, 1A5, 2A2) and IgG2a,1 strain (3A3) was IgG2b,. The other strain (1D4) was all K-type of IgM, light chain. 3. The neutralization activity of 12 anti-SARS coronavirus monoclonal antibodies (1A2, 1A5, 2D4, 2A3, 2C5, 3B4) was determined by micro cell culture neutralization assay. The neutralization titers of 1A5 and 2C5 were 1? 20480 and 1? 5957, respectively, and the neutralization activity of 1A5 and 2C5 were 1A2, 1A5, 2D4, 2A3, 2C5, 3B4, respectively. Based on the immunological characteristics of these monoclonal antibodies, their antigen binding epitopes were analyzed. These monoclonal antibodies were first detected by recombinant SARS-CoV S protein and N, among which 6 strains (1A5, 2C5, 2A3, 1B4, 2B1, 3A3) reacted with S protein and 2 strains (2D4, 1A2) reacted with N protein, indicating that the antigen epitopes of these monoclonal antibodies were located on S protein and N protein, respectively. The other four McAbs did not bind to S and N proteins, suggesting that they may target structural proteins other than S and N. Six monoclonal antibodies binding to S protein were further analyzed by using different length S peptide fragments, of which 3 strains (2A3, 2A3, 2A3, 2A3, 2A3, 2A3, 2A3, 2A3,
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392.1
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1 李佳明;抗SARS-CoV單克隆抗體的鑒定及應(yīng)用研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2005年
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