SARS病毒N蛋白特异性抗体的制备及其B细胞表位研究
发布时间：2018-09-11 15:20
【摘要】：严重急性呼吸道症候群(severe acute respiratory syndrome, SARS)是人类21世纪遭受的一种新型烈性传染病。它严重地危害了人们的身体健康,给社会的经济发展带来了巨大的影响。据报道,截至到2003年7月11日,全球有32个国家和地区,约8437人感染了SARS病毒,其中813人死亡。SARS病毒的致死率大约为10.5%。SARS病毒感染,因起病急、传播快、病死率高,且尚无特效药。故早期诊断、及时隔离病人和合理使用非特异性药物对症治疗是主要的治疗手段。因此,开发针对SARS病毒的灵敏、特异、快速、准确的实验室诊断方法是迫切需要的。这种方法的建立对于病人的早期诊治、药物疗效的评价以及阻断传染源的传播都有极为重要的科学意义。 本研究分别表达了SARS全长N蛋白(nucleocapsid)及其长短不一的四个片段,并用SARS全长N蛋白免疫兔子和BALB/c小鼠制备了特异性的多克隆和单克隆抗体。Western Blotting检测发现,单克隆抗体不仅可以与大肠杆菌表达的N蛋白结合,而且能与SARS病毒感染的细胞裂解液中天然N蛋白发生反应,提示所制备的单抗能够识别天然的SARS N蛋白。为了获得所制备单抗识别表位的信息,用ELISA方法分析了6种N蛋白特异性单抗与N蛋白及其片段的反应性。根据结果可以把这6种单抗分成两组,A组单抗有两个,主要识别N蛋白中249-317氨基酸;B组单抗有四个,主要识别317-422氨基酸。这表明,N蛋白的B细胞表位主要位于其C端的221-422氨基酸区域。在两组抗体中各选择了一株高亲和力单抗作为包被抗体,N蛋白的多克隆抗体偶联辣根过氧化物酶为二抗,组装成双抗体夹心ELISA试剂盒。经验证,该试剂盒能特异性地检测出N蛋白,并且与非SARS病人血清(包括肿瘤患者、系统性红斑狼疮患者、乙肝和丙肝患者)无交叉反应。这些实验结果提示,所构建的N蛋白特异的ELISA试剂盒对SARS感染病人的早期诊断有潜在的应用价值。
[Abstract]:Severe Acute Respiratory Syndrome (severe acute respiratory syndrome, SARS) is a new type of infectious disease in the 21st century. It seriously endangers people's health and brings great influence to social economic development. It is reported that as of July 11, 2003, there are 32 countries and regions in the world, about 8437 people have been infected with SARS virus, of which 813 people died. The mortality rate of 10.5%.SARS virus is about the same as that of 10.5%.SARS virus. And there is no specific drug. Therefore, early diagnosis, timely isolation of patients and rational use of non-specific drugs for symptomatic treatment are the main treatment methods. Therefore, it is urgent to develop a sensitive, specific, rapid and accurate laboratory diagnostic method for SARS virus. The establishment of this method is of great scientific significance for the early diagnosis and treatment of patients, the evaluation of drug efficacy and the interruption of transmission of the source of infection. In this study, SARS full-length N-protein (nucleocapsid) and its four fragments of different length were expressed, and specific polyclones and monoclonal antibodies were prepared by immunizing rabbits and BALB/c mice with SARS full-length N-protein. The monoclonal antibody could not only bind to the N protein expressed in E. coli, but also react with the natural N protein in the cell lysate infected with SARS virus, suggesting that the McAb can recognize the natural SARS N protein. In order to obtain the information of the epitope recognition, the reactivity of six N-protein specific McAbs to N protein and its fragments was analyzed by ELISA method. According to the results, the six kinds of McAbs can be divided into two groups. There are two McAbs in Group A and four McAbs in Group B of 249-317 Amino acids, mainly recognizing 317-422 Amino acids. This indicated that the B cell epitopes of N protein were mainly located in the 221-422 amino acid region of its C-terminal. In the two groups of antibodies, a high affinity monoclonal antibody was selected as a polyclonal antibody conjugated with horseradish peroxidase (horseradish peroxidase) coated with antibody N protein as a second antibody, and a double antibody sandwich ELISA kit was assembled. It was proved that the kit could specifically detect N protein and had no cross reaction with the sera of non SARS patients (including tumor patients, systemic lupus erythematosus patients, hepatitis B and hepatitis C patients). These results suggest that the constructed N-protein specific ELISA kit has potential application value in early diagnosis of SARS infection patients.
【学位授予单位】：中国科学院研究生院（上海生命科学研究院）
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2237073
[Abstract]:Severe Acute Respiratory Syndrome (severe acute respiratory syndrome, SARS) is a new type of infectious disease in the 21st century. It seriously endangers people's health and brings great influence to social economic development. It is reported that as of July 11, 2003, there are 32 countries and regions in the world, about 8437 people have been infected with SARS virus, of which 813 people died. The mortality rate of 10.5%.SARS virus is about the same as that of 10.5%.SARS virus. And there is no specific drug. Therefore, early diagnosis, timely isolation of patients and rational use of non-specific drugs for symptomatic treatment are the main treatment methods. Therefore, it is urgent to develop a sensitive, specific, rapid and accurate laboratory diagnostic method for SARS virus. The establishment of this method is of great scientific significance for the early diagnosis and treatment of patients, the evaluation of drug efficacy and the interruption of transmission of the source of infection. In this study, SARS full-length N-protein (nucleocapsid) and its four fragments of different length were expressed, and specific polyclones and monoclonal antibodies were prepared by immunizing rabbits and BALB/c mice with SARS full-length N-protein. The monoclonal antibody could not only bind to the N protein expressed in E. coli, but also react with the natural N protein in the cell lysate infected with SARS virus, suggesting that the McAb can recognize the natural SARS N protein. In order to obtain the information of the epitope recognition, the reactivity of six N-protein specific McAbs to N protein and its fragments was analyzed by ELISA method. According to the results, the six kinds of McAbs can be divided into two groups. There are two McAbs in Group A and four McAbs in Group B of 249-317 Amino acids, mainly recognizing 317-422 Amino acids. This indicated that the B cell epitopes of N protein were mainly located in the 221-422 amino acid region of its C-terminal. In the two groups of antibodies, a high affinity monoclonal antibody was selected as a polyclonal antibody conjugated with horseradish peroxidase (horseradish peroxidase) coated with antibody N protein as a second antibody, and a double antibody sandwich ELISA kit was assembled. It was proved that the kit could specifically detect N protein and had no cross reaction with the sera of non SARS patients (including tumor patients, systemic lupus erythematosus patients, hepatitis B and hepatitis C patients). These results suggest that the constructed N-protein specific ELISA kit has potential application value in early diagnosis of SARS infection patients.
【学位授予单位】：中国科学院研究生院（上海生命科学研究院）
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
【共引文献】
相关博士学位论文 前4条
1 陈苏红;四种重要传染病病原体检测新技术研究[D];中国人民解放军军事医学科学院;2005年
2 李振勇;新型冠状病毒实验室诊断方法学研究[D];郑州大学;2005年
3 刘怀然;SARS冠状病毒S糖蛋白基因片段的表达及初步应用[D];东北农业大学;2005年
4 梁云飞;病毒抗原蛋白的B细胞表位生物学研究[D];第三军医大学;2006年
相关硕士学位论文 前4条
1 常昭瑞;SARS-CoV结构蛋白血清IgG抗体变化规律及恢复期SARS患者排毒监测[D];山西医科大学;2004年
2 周浩;SARS冠状病毒S2基因的克隆表达和S2蛋白抗原活性的初步研究[D];第一军医大学;2006年
3 杨海燕;利用噬菌体肽库技术筛选SARS冠状病毒N蛋白B细胞表位的研究[D];第一军医大学;2006年
4 陈亮;SARS-CoV N蛋白与细胞色素P450（CYP4F3）相互作用的研究[D];中国人民解放军军事医学科学院;2007年
，本文编号：2237073
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2237073.html
最近更新
教材专著
