SARS病毒N蛋白特異性抗體的制備及其B細(xì)胞表位研究
發(fā)布時間：2018-09-11 15:20
【摘要】：嚴(yán)重急性呼吸道癥候群(severe acute respiratory syndrome, SARS)是人類21世紀(jì)遭受的一種新型烈性傳染病。它嚴(yán)重地危害了人們的身體健康,給社會的經(jīng)濟發(fā)展帶來了巨大的影響。據(jù)報道,截至到2003年7月11日,全球有32個國家和地區(qū),約8437人感染了SARS病毒,其中813人死亡。SARS病毒的致死率大約為10.5%。SARS病毒感染,因起病急、傳播快、病死率高,且尚無特效藥。故早期診斷、及時隔離病人和合理使用非特異性藥物對癥治療是主要的治療手段。因此,開發(fā)針對SARS病毒的靈敏、特異、快速、準(zhǔn)確的實驗室診斷方法是迫切需要的。這種方法的建立對于病人的早期診治、藥物療效的評價以及阻斷傳染源的傳播都有極為重要的科學(xué)意義。 本研究分別表達了SARS全長N蛋白(nucleocapsid)及其長短不一的四個片段,并用SARS全長N蛋白免疫兔子和BALB/c小鼠制備了特異性的多克隆和單克隆抗體。Western Blotting檢測發(fā)現(xiàn),單克隆抗體不僅可以與大腸桿菌表達的N蛋白結(jié)合,而且能與SARS病毒感染的細(xì)胞裂解液中天然N蛋白發(fā)生反應(yīng),提示所制備的單抗能夠識別天然的SARS N蛋白。為了獲得所制備單抗識別表位的信息,用ELISA方法分析了6種N蛋白特異性單抗與N蛋白及其片段的反應(yīng)性。根據(jù)結(jié)果可以把這6種單抗分成兩組,A組單抗有兩個,主要識別N蛋白中249-317氨基酸;B組單抗有四個,主要識別317-422氨基酸。這表明,N蛋白的B細(xì)胞表位主要位于其C端的221-422氨基酸區(qū)域。在兩組抗體中各選擇了一株高親和力單抗作為包被抗體,N蛋白的多克隆抗體偶聯(lián)辣根過氧化物酶為二抗,組裝成雙抗體夾心ELISA試劑盒。經(jīng)驗證,該試劑盒能特異性地檢測出N蛋白,并且與非SARS病人血清(包括腫瘤患者、系統(tǒng)性紅斑狼瘡患者、乙肝和丙肝患者)無交叉反應(yīng)。這些實驗結(jié)果提示,所構(gòu)建的N蛋白特異的ELISA試劑盒對SARS感染病人的早期診斷有潛在的應(yīng)用價值。
[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.
【學(xué)位授予單位】：中國科學(xué)院研究生院（上海生命科學(xué)研究院）
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：中國科學(xué)院研究生院（上海生命科學(xué)研究院）
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392
【共引文獻】
相關(guān)博士學(xué)位論文 前4條
1 陳蘇紅;四種重要傳染病病原體檢測新技術(shù)研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2005年
2 李振勇;新型冠狀病毒實驗室診斷方法學(xué)研究[D];鄭州大學(xué);2005年
3 劉懷然;SARS冠狀病毒S糖蛋白基因片段的表達及初步應(yīng)用[D];東北農(nóng)業(yè)大學(xué);2005年
4 梁云飛;病毒抗原蛋白的B細(xì)胞表位生物學(xué)研究[D];第三軍醫(yī)大學(xué);2006年
相關(guān)碩士學(xué)位論文 前4條
1 常昭瑞;SARS-CoV結(jié)構(gòu)蛋白血清IgG抗體變化規(guī)律及恢復(fù)期SARS患者排毒監(jiān)測[D];山西醫(yī)科大學(xué);2004年
2 周浩;SARS冠狀病毒S2基因的克隆表達和S2蛋白抗原活性的初步研究[D];第一軍醫(yī)大學(xué);2006年
3 楊海燕;利用噬菌體肽庫技術(shù)篩選SARS冠狀病毒N蛋白B細(xì)胞表位的研究[D];第一軍醫(yī)大學(xué);2006年
4 陳亮;SARS-CoV N蛋白與細(xì)胞色素P450（CYP4F3）相互作用的研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2007年
，本文編號：2237073
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2237073.html
最近更新
教材專著
