抗SARS-CoV单克隆抗体的鉴定及应用研究
发布时间：2019-03-31 10:08
【摘要】：严重急性呼吸系统综合征(Severe acute respiratory syndrome,SARS)是一种烈性新发传染病,其病原体为一种新型冠状病毒(SARS-CoV)。尽管目前SARS的流行已经得到控制,但迄今为止,SARS的起源、动物宿主、传播途径及致病机理等仍不完全清楚,SARS对人类的威胁依然存在。由于从临床症状及其发病体征很难与其它病原体引起的发热性呼吸系统疾病区分,所以更显得早期特异实验诊断的重要。对于该病的治疗目前主要依赖对症和支持治疗,没有特效药物。利用恢复期病人血清,对感染者进行被动免疫治疗,已被临床证明是一种有效的治疗方法,所以开发被动免疫制剂受到广泛的重视。 为开发SARS早期、特异诊断实验室检测方法及应急被动免疫制剂,我室用灭活纯化SARS-CoV(BJ01株)免疫BALB/C小鼠,通过细胞融合技术,建立了12株能稳定分泌SARS-CoV单克隆抗体的杂交瘤细胞株。对这些单克隆抗体的免疫学鉴定证明:1.IFA对单克隆抗体效价测定,12份单克隆抗体,除1D4效价稍低一些外,其余IFA效价均在1:2560—20480之间,显示出这些单抗都有较高的抗SARS—CoV活性,而且与不同地区分离毒株均有良好反应;2.采用双向琼脂扩散和酶联免疫吸附试验对12株单克隆抗体免疫球蛋白类型及其亚类特异性鉴定,结果5株(2D4、1A2、2C5、2A3、384)单克隆抗体重链为IgG1,5株(184、2B1、1C5、1A5、2A2)为IgG2a,1株(3A3)为IgG2b,另一株(1D4)为IgM,轻链全部为k型;3.采用微量细胞培养中和试验,对12株抗SARS冠状病毒单克隆抗体进行抗SARS—CoV中和活性测定,6株(1A2、1A5、2D4、2A3、2C5、3B4)具有中和活性,其中1A5和2C5的中和效价分别达1:20480和1:5957。 在对这些单克隆抗体免疫学特性鉴定的基础上,对它们的抗原结合表位进行了分析。首先用重组表达的SARS-CoV S蛋白和N对这些单抗进行测定,其中6株(1A5、2C5、2A3、1B4、2B1、3A3)与S蛋白反应,2株(2D4,1A2)与N蛋白反应,说明它们结合的抗原表位分别位于S蛋白和N蛋白上。另外4株单抗与S和N蛋白均不结合,说明它们可能针对S、N以外的结构蛋白。对6株结合S蛋白的单抗用不同长度的S肽段进一步分析它们结合的抗原位点,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,
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1
本文编号：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,
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1
【相似文献】
相关硕士学位论文 前1条
1 李佳明;抗SARS-CoV单克隆抗体的鉴定及应用研究[D];中国人民解放军军事医学科学院;2005年
，本文编号：2450811
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2450811.html
最近更新
教材专著
