马传染性贫血病毒p26蛋白抗原表位鉴定与AC-ELISA方法的建立

发布时间：2018-06-22 06:39

本文选题：马传染性贫血病毒 + p26蛋白　；参考：《中国农业科学院》2015年硕士论文

【摘要】：马传染性贫血(Equine infectious anemia,EIA)是一种能够感染马、驴和斑马等马属动物的动物性传染病。它能够引起马属动物贫血、血小板减少、发热、消瘦和多器官衰竭等症状。EIA由Ligné于1843年在法国首次发现,现已在美洲、欧洲、中东和南非等世界范围都有流行,使当地畜牧业,赛马业和相关产业遭受巨大经济损失。解放前期,我国也曾出现EIA流行,但在20世纪70-80年代由于沈荣显等人研制出EIAV弱毒疫苗,从而使疫病得到有效控制。马传染性贫血病毒(Equine infectious anemia virus,EIAV)是引起EIA的病原体。EIAV属于反转录病毒科慢病毒,同其他慢病毒如人类免疫缺陷病毒(Human immunodeficiency virus,HIV)、猴免疫缺陷病毒(Simian immunodeficiency virus,SIV)和猫免疫缺陷病毒(Feline immunodeficiency virus)类似,EIAV包含有Gag、Pol和Env 3个主要的结构蛋白和Tat、Rev和S2 3个非结构蛋白。Gag蛋白作为多聚蛋白,最终可以剪切为基质蛋白(MA)、衣壳蛋白(CA)、核衣壳蛋白(NC)和p9蛋白,这些蛋白都作为病毒粒子结构的一部分而存在。EIAV衣壳蛋白又称为p26蛋白,它的主要功能是自我组装形成病毒的衣壳结构,其构成的拓扑学结构包裹EIAV的基因组以确保病毒的有效复制。同时作为病毒感染过程中的主要免疫原性蛋白,p26在不同EIAV株的结构与功能高度保守。并且p26蛋白也是病毒粒子中丰度最高的蛋白之一。本研究通过实验室制备的2株EIAV p26单克隆抗体:MAb 1G11和MAb 9H8,利用肽扫描技术,最终通过免疫印迹试验(western blot)鉴定出MAb 1G11和MAb 9H8所针对p26蛋白的线性表位。MAb 1G11针对p26蛋白C端的20个氨基酸:199KNAMRHLRPEDTLEEKMYAC218;MAb 9H8则针对N端的8个氨基酸:73NLDKIAEE80。这对于分析p26蛋白结构与功能以及以表位为基础建立诊断方法提供了依据,也对基于表位的疫苗设计具有指导意义。在国务院颁布的《国家中长期动物疫病防治规划(2012—2020年)》中,明确指出要对EIA开展持续监测,对经济娱乐用马以及高风险区域的马属动物进行重点监测,从而加快推进马传染性贫血消灭行动。根据生产实际和国家发展纲要,建立快速有效的诊断EIA的方法显得尤为迫切与重要。传统检测EIAV的方法是利用p26蛋白检测EIAV抗体的琼脂扩散试验(Agar gel immunodiffusion,AGID),又称为Coggins试验。尽管AGID在检测EIAV高度特异,但是缺乏敏感性。这对于检测处于慢性期和非明显期的临床症状的EIA感染尤为不利。同时只能通过肉眼观察抗原抗体结合的沉淀线粗细来对EIAV含量进行粗略判断,并无法对病毒进行定量分析。再次,由于试验周期较长,无法对疾病进行快速诊断,使得在生产实践无法对疫情做到及时有效防控。本研究所鉴定的线性表位分别针对p26蛋白的N端与C端。其所针对的差异表位特性,为本实验建立抗原捕获ELISA(Antigen Capture-ELISA,AC-ELISA)提供了可能。因此通过利用EIAV(CMV 3-8)感染性克隆或重组p26蛋白作为标准抗原,以MAb 1G11和MAb 9H8分别作为捕获抗体和酶标检测抗体,建立以检测EIAV的AC-ELISA方法。该方法也可以对病毒进行定量,这也为研究病毒复制与其他相关生活周期病毒含量变化,以及EIAV与宿主限制因子(host restriction factors)如TRIM5α,APOBEC3家族和Tetherin相互拮抗作用提供了定量分析平台。综上,本研究利用针对EIAV p26蛋白的两株单克隆抗体MAb 1G11和MAb 9H8,利用肽扫描技术鉴定出p26蛋白在蛋白N端与C端的线性表位;同时利用MAb 1G11和MAb 9H8识别p26蛋白表位的差异性,建立针对不同EIAV病毒株的AC-ELISA方法。已鉴定的线性表位可以为研究p26蛋白的结构与功能以及基于表位建立检测和设计表位疫苗提供依据;通过建立的AC-ELISA方法不仅可以应用于临床样品检测及相关生产实践应用,同时也可以通过病毒定量分析为深入研究病毒本身及病毒-宿主相互作用提供方法学平台。
[Abstract]:Equine infectious anemia (EIA) is an animal infectious disease that can infect horses, donkeys and zebra and other equine animals. It can cause anemia, thrombocytopenia, fever, emaciation and multiple organ failure of the horse..EIA was first discovered in France in France in 1843, and is now in America, Europe, Middle East and South Africa. The world has been popular, making the local animal husbandry, horse racing and related industries suffer huge economic losses. In the early period of liberation, our country also appeared EIA popular, but in the 70-80 years of twentieth Century, the EIAV vaccine was developed by Shen Rongxian et al. So that the epidemic disease was effectively controlled. Equine infectious anemia virus (EIA), EIA V) is the pathogen.EIAV that causes EIA, which belongs to the retrovirus family, similar to other lentiviruses such as the human immunodeficiency virus (Human immunodeficiency virus, HIV), the monkey immunodeficiency virus (Simian immunodeficiency virus, SIV), and the cat immunodeficiency virus (Feline). The structure protein and the 3 non structural protein.Gag proteins, Tat, Rev and S2, as polyproteins, can eventually be cut into matrix protein (MA), capsid protein (CA), nucleocapsid protein (NC) and P9 protein. These proteins all exist as part of the structure of the virus particles and exist in the.EIAV capsid protein, also known as P26 protein. The main function of these proteins is self assembly. The capsid structure of the virus forms the structure of the topology that encapsulate the genome of EIAV to ensure the effective replication of the virus. At the same time, as the main immunogenic protein in the virus infection process, P26 is highly conserved in the structure and function of different EIAV strains. And the P26 protein is also one of the most abundant proteins in the virus particles. 2 EIAV P26 monoclonal antibodies, MAb 1G11 and MAb 9H8, were prepared by the peptide scanning technique. Finally, the linear epitopes of MAb 1G11 and MAb 9H8 were identified by the immunoblotting test (Western blot). Acid: 73NLDKIAEE80. provides a basis for the analysis of the structure and function of P26 protein and the establishment of a diagnostic method based on the epitopes. It also has a guiding significance for the design of the epitope based vaccine. In the national long-term animal epidemic prevention and control plan issued by the State Council (2012 to 2020), it is clearly pointed out that the sustainable monitoring of EIA is to be carried out, and the economy is on the economy. It is very urgent and important to establish a rapid and effective method for the diagnosis of EIA based on production practice and national development program. The traditional method for detecting EIAV is to detect the agar diffusion of EIAV antibodies by using P26 protein. The test (Agar gel immunodiffusion, AGID), also known as the Coggins test. Although AGID is highly specific in the detection of EIAV, it lacks sensitivity. This is especially bad for the detection of EIA infection in chronic and non obvious clinical symptoms. At the same time, the content of EIAV is rough by observing the precipitate line of antigen anti body binding by the naked eye. It is impossible to make a quantitative analysis of the virus. Again, because of the long test cycle, it is impossible to quickly diagnose the disease, which makes it impossible to prevent and control the epidemic in time and effectively. The linear epitopes identified in this study are directed against the N and C ends of the P26 protein. It is possible to capture ELISA (Antigen Capture-ELISA, AC-ELISA). Therefore, by using EIAV (CMV 3-8) infectious clones or recombinant P26 proteins as standard antigens, MAb 1G11 and MAb 9H8 are used as capture antibodies and enzyme labeled antibodies to detect EIAV. This method can also be used to quantify the virus, which is also studied. To investigate the changes in virus replication and other related life cycle viruses, and the mutual antagonism of EIAV and host restriction factors, such as TRIM5 alpha, APOBEC3 family and Tetherin, a quantitative analysis platform is provided. In this study, this study uses two monoclonal antibodies against EIAV P26 protein MAb 1G11 and MAb purposes, using peptide scanning. The technique identified the linear epitopes of P26 protein at the N end and the C end, and the identification of P26 protein epitopes by MAb 1G11 and MAb 9H8 to establish AC-ELISA methods for different EIAV virus strains. The identified linear epitopes can provide the basis for the study of the structure and function of P26 protein and the establishment of detection and design of epitope vaccines based on the epitopes. The AC-ELISA method, which is established, can be used not only in clinical sample detection and related production practice, but also by quantitative analysis of virus to provide a methodological platform for the in-depth study of virus and virus host interaction.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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