乙脑病毒和西尼罗病毒单克隆抗体的制备及鉴定

发布时间：2019-05-07 05:34

【摘要】：乙型脑炎病毒(Japancse encephlitic virus,JEV)和西尼罗病毒(West Nile Virus, WNV)同属于虫媒病毒黄病毒科,黄病毒属、乙型脑炎病毒抗原复合组(Japanese Encephalitis Virus Serocomplex Group)的成员。该复合组成员病毒属于蚊媒病毒,主要由鸟类携带,经蚊虫传播给人,也可以感染其它哺乳动物,引起多种临床症状,包括发热,甚至脑炎及脑膜炎,重者可致死。近年来,该组成员中的西尼罗病毒流行已经给人类的健康造成严重的损害和威胁。国外将西尼罗热归于新发传染病(emerging infectious disease)范畴,我国国家卫生部将西尼罗热归于可能传入的新发传染病。早期发现和预防WNV的传播是控制西尼罗热爆发流行的主要措施,检测蚊虫体内的WNV感染率是西尼罗病毒传播预警的一个直观指标。从人员和设备需求来看,快速免疫检测方法具有明显的优势。黄病毒是正链RNA病毒。以西尼罗病毒为例,它由10962个核苷酸组成,其开放读码框编码病毒3种结构蛋白和7种非结构蛋白。其中包膜蛋白(E)和膜蛋白(M)是病毒主要的结构蛋白,可能与病毒的毒力及侵袭性有关,也是病毒的主要抗原。但是,由于黄病毒属成员间在病毒表面尤其是E蛋白上存在许多共同的抗原表位,所以在血清学检测时容易出现交叉反应,使反应结果特异性不高。文献报道,针对登革病毒M蛋白的前体prM蛋白的单克隆抗体与乙脑病毒抗血清无交叉反应,提示在prM蛋白上可能存在病毒特异性抗原表位。为了探索高效、灵敏的检测WNV方法,本研究分别克隆了JEV和WNVprM基因,并原核表达,制备分别针对JEV和WNVprM蛋白的特异性单克隆抗体,用于JEV复合组成员感染的鉴定、鉴别诊断和流行病学研究。首先,本研究采用JEV疫苗株免疫BALB/c小鼠,制备单克隆抗体,经过五次亚克隆筛选,获得分泌针对JEV疫苗株的单抗的杂交瘤细胞株V6B9,抗体亚型为IgG1,抗体滴度为10~5。ELISA、Western Blot和免疫组化检测结果证实该株单抗能够与JEV感染鼠脑上清中的病毒,以及与WNV反应。该抗体可用于JEV复合组病
[Abstract]:Japanese Encephalitis virus (Japancse encephlitic virus,JEV) and West Nile virus (West Nile Virus, WNV) are both members of (Japanese Encephalitis Virus Serocomplex Group), which belong to the Genus Flavor virus, Genus Flavor virus and Japanese Encephalitis virus Antigen complex group. This composite group of members of the virus is a mosquito-borne virus, mainly carried by birds, transmitted by mosquitoes to humans, but also can infect other mammals, causing a variety of clinical symptoms, including fever, and even encephalitis and meningitis, serious people can die. In recent years, the West Nile virus epidemic among the members of the group has caused serious damage and threat to human health. West Nile fever has been classified as a new infectious disease in foreign countries, and the Ministry of Health of our country has classified West Nile fever as a new infectious disease which may be introduced into China. Early detection and prevention of WNV transmission are the main measures to control the outbreak of West Nile fever. Detection of WNV infection rate in mosquitoes is an intuitive indicator for early warning of West Nile virus transmission. From the personnel and equipment requirements, the rapid immune detection method has obvious advantages. Yellow virus is a positive-stranded RNA virus. Taking West Nile virus as an example, it consists of 10962 nucleotides, and its open reading frame encodes three structural proteins and seven non-structural proteins of the virus. The envelope protein (E) and membrane protein (M) are the main structural proteins of the virus, which may be related to the virulence and invasiveness of the virus, and are also the main antigens of the virus. However, because there are many common antigenic epitopes on the surface of the virus, especially on the E protein, there are many common antigenic epitopes among the members of the genus flavivirus, so it is easy to appear cross-reaction in serological detection, which makes the reaction result less specific. It has been reported that monoclonal antibodies against the precursor prM protein of dengue virus M protein did not cross-react with Japanese encephalitis virus antiserum, suggesting that there may be virus specific epitopes on the prM protein. In order to explore an efficient and sensitive method for the detection of WNV, JEV and WNVprM genes were cloned and expressed in E. coli to prepare specific monoclonal antibodies against JEV and WNVprM proteins, respectively, for identification of the infection of JEV complex group members. Differential diagnosis and epidemiological study. Firstly, monoclonal antibodies were prepared by immunizing BALB/c mice with JEV vaccine strain. After five subclones screening, hybridoma cell line V6B9 secreting monoclonal antibody against JEV vaccine strain V6B9 was obtained. The antibody subtype was IgG1, antibody titer of 10? The results of Western Blot and immunohistochemistry showed that the monoclonal antibody could react with the virus in the supernatant of the brain infected with JEV and with WNV. The antibody can be used in JEV complex group.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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