基孔肯雅病毒免疫学检测方法的建立与初步评价

发布时间：2018-06-01 14:37

本文选题：基孔肯雅病毒(CHIKV) + 双抗体夹心ELISA　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：基孔肯雅病毒(Chikungunyavirus,CHIKV)是单股正链RNA病毒,为披膜病毒科,甲病毒属;病毒颗粒呈T=4正二十面体结构,直径约60-70nm,在电镜下呈圆形。病毒主要有五个结构蛋白(C、E2、E3、6K、E1),E1与E2形成异二聚体,每三个E1/E2异二聚体形成一个微小的凸起,每个病毒颗粒的表面共有80个相同的凸起。在包膜之下E1/E2异二聚体同C蛋白构成的衣壳相连。除结构蛋白外,还有四个非结构蛋白(NS1、NS2、NS3、NS4)主要在病毒复制过程中起辅助调节作用。人感染CHIKV后可引起以关节痛、发热、皮疹为主要症状的基孔肯雅热。大多数患者症状轻,其他症状数周之内可以自行痊愈,但关节痛往往持续存在,影响正常的生活、产生经济负担。少数患者会并发脑炎等重症危及生命。CHIKV通过伊蚊传播,伊蚊分布范围广泛;在2014年CHIKV传入美洲大陆后,在全球大部分地区均有流行报道。同我国临近的东南亚地区一直是基孔肯雅热的主要流行地区之一。我国于1986年首次成功分离CHIKV,2008年首次报道输入性基孔肯雅热,2010年首次由于输入性病例导致的本土基孔肯雅热的暴发流行。随着CHIKV在全球范围内扩大流行,我国仍有再次暴发基孔肯雅热的可能性。在疾病流行的预防控制工作当中,早期诊断十分重要,本研究基于以上目的拟建立血清CHIKV抗原检测的方法。通过杆状病毒表达系统生产CHIKV病毒样颗粒(VLP),该VLP包含CHIKV的全部结构蛋白,在电镜下可以见到圆形颗粒。VLP与灭活的CHIKV有一致的抗原性,可以与抗CHIKV抗体良好结合。VLP由于不含有核酸不具有感染性,相比灭活病毒更加安全,可以在生物安全一级实验室中进行操作,降低了研究的生物安全风险。使用VLP免疫小鼠和家兔制备抗CHIKV的鼠免疫腹水和兔免疫血清。通过免疫荧光方法检测,显示抗体与病毒结合良好;通过ELISA方法检测抗体效价均在1:10万以上。1、以纯化鼠免疫腹水作为包被抗体,兔免疫血清作为捕获抗体,建立了检测CHIKV抗原的双抗体夹心ELISA方法。,该方法法具有较好的特异性和敏感性,在0.1ml中含50TCID50以上CHIKV的样本均可以用本方法检测出;模拟病人血清可被成功检出;登革热、肾综合征出血热病人及健康人血清进行CHIKV抗原检测,均为阴性;重复性良好板间变异小于10%,板内变异小于5%。2、用VLP为抗原建立了检测抗CHIKV IgG抗体的间接法ELISA,VLP拥有的良好抗原性可以替代灭活病毒作为抗原对IgG进行捕获检测。完善抗CHIKV IgM抗体的捕获法ELISA,检测病人急性期血清检测均呈阳性。两种方法重复性良好板间变异小于10%,板内变异小于5%。本研究建立了 CHIKV抗原和抗体检测方法,并进行初步评价,对于临床诊断以及流行病学调查有积极意义。
[Abstract]:Chikungunya virus of Chikungunya virus (Chikungunya virus) is a single-stranded RNA virus belonging to the genus Arbovirus belonging to the family Erythroviridae. The virus particles have a Tap-4 regular icosahedron structure with a diameter of about 60-70 nm and are circular under electron microscope. The virus mainly has five structural proteins, Con E2E _ 3, E _ 3N _ 6K, E _ 1, E _ 1, and E _ 2. Each three E1/E2 heterodimers form a tiny protuberance, and each virus particle has 80 identical protuberances on the surface. Under the capsule, E1/E2 heterodimer is connected to the capsid of C protein. In addition to structural proteins, there are four nonstructural proteins, NS1, NS2, NS3, NS4, which play a coregulatory role in viral replication. Human infection with CHIKV can cause Kikungunya fever with arthralgia, fever and rash as the main symptoms. Most patients have mild symptoms and other symptoms can heal themselves within a few weeks, but joint pain often persists, affecting normal life and creating financial burdens. A few patients with encephalitis and other serious life-threatening. CHIKV spread through the Aedes mosquito, Aedes is widely distributed. After CHIKV was introduced to the American continent in 2014, there are popular reports in most parts of the world. Southeast Asia, which is close to our country, has been one of the main endemic areas of Kikungunya fever. CHIKV was isolated successfully in China in 1986, the imported Kikungunya fever was first reported in 2008, and the outbreak of local Kikungunya fever caused by imported cases was first reported in 2010. With the spread of CHIKV in the world, there is still the possibility of a further outbreak of Kikungunya fever in China. Early diagnosis is very important in the prevention and control of epidemic diseases. This study aims to establish a method for detection of serum CHIKV antigen. CHIKV virus-like particles were produced by baculovirus expression system. The VLP contains all the structural proteins of CHIKV. Under electron microscope, circular particles. VLP have the same antigenicity as inactivated CHIKV. It is more safe than inactivated virus because it does not contain nucleic acid. It can be operated in biosafety primary laboratory and reduce the risk of biosafety. VLP was used to immunize mice and rabbits to prepare anti-CHIKV ascites and rabbit immune serum. Immunofluorescence assay showed that the antibody was well bound to the virus, and the titers of the antibody were all above 1: 100,000 by ELISA method. The purified mouse ascites were used as the coated antibody, and the rabbit immune serum was used as the capture antibody. A sandwich ELISA method for the detection of CHIKV antigen was established. The method has good specificity and sensitivity. All samples containing CHIKV above 50TCID50 in 0.1ml can be detected by this method; the simulated patient serum can be successfully detected; dengue fever can be detected successfully. Serum CHIKV antigen was negative in patients with hemorrhagic fever with renal syndrome (HFRS) and healthy subjects. The good inter-plate variation was less than 10% and the intraplate variation was less than 5.2%. Using VLP as antigen, an indirect method for detection of anti CHIKV IgG antibodies, ELISAN VLP with good antigenicity, could be used as antigen instead of inactivated virus to capture and detect IgG. The capture method of anti-CHIKV IgM antibody was improved to detect positive serum of patients in acute phase. The reproducibility of the two methods was better than 10, and the intra-plate variation was less than 5. In this study, a method for the detection of CHIKV antigen and antibody was established and evaluated, which has positive significance for clinical diagnosis and epidemiological investigation.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.6;R511

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