猪流行性腹泻病毒的流行病学调查及快速检测方法的初步建立

发布时间：2017-12-31 03:37

  本文关键词：猪流行性腹泻病毒的流行病学调查及快速检测方法的初步建立　出处：《扬州大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 猪流行性腹泻病毒 流行病学调查 单克隆抗体 快速检测方法

【摘要】：由猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)引起的猪流行性腹泻(Porcine epidemic diarrhea, PED)是一种急性胃肠炎疾病,其主要症状为水样腹泻和呕吐,现在已经成为一种世界性传染病。2010年我国南部大面积爆发猪流行性腹泻疫情,各个年龄段的猪均发病,新生仔猪的发病率和死亡率都很高。为了了解近两年该病在我国的流行病学特点和建立快速检测方法,我们主要开展了以下研究：1.猪流行性腹泻病毒的遗传变异分析为了调查我国猪流行性腹泻病毒的遗传变异情况,本研究通过RT-PCR方法对从江苏、吉林、上海、广东、河南五省市规模猪场采集来的314份样本进行病原学检测,检测出PEDV阳性病料76份,阳性率达24.2%,其中广东省几个猪场的PEDV阳性率最高,高达94.7%,吉林省几个猪场没检出PEDV阳性,另外江苏省、河南省和上海市的PEDV阳性率分别为17.8%、31.6%和27.6%,表明PED在我国部分地区仍流行严重,有必要对PED采取更加严格的防范措施。对部分阳性样品中PEDV的S1、M、ORF3基因进行克隆、测序和比对分析,结果表明我国大部分流行株与韩国、美国流行株同源性较近,与欧洲流行株以及疫苗株CV777同源性较远,只有HN13和YM毒株与欧洲流行株和疫苗株CV777同源性较近。比对发现S1蛋白氨基酸发生变异明显,M蛋白和ORF3蛋白的氨基酸较为保守,另外HN13和YM毒株的ORF3基因与疫苗株DR13的ORF3基因相同,有49个核苷酸缺失。2.猪流行性腹泻病毒单克隆抗体的研制及特性鉴定为了获得可用于建立PEDV快速检测方法的单克隆抗体,本研究将超速离心法纯化的PEDV作为免疫原,免疫6-8周龄Balb/c小鼠,利用免疫荧光法(IFA)筛选出9株能够稳定分泌抗PEDV抗体的杂交瘤细胞株,分别命名为PEDV-1C12、PEDV-2A9、PEDV-2C11、 PEDV-2E5、PEDV-2F1、PEDV-3E9、PEDV-5B12、PEDV-6D1、PEDV-6E10;随后对单克隆抗体的腹水荧光效价、亚类以及特异性等生物学特性进行鉴定。单克隆抗体亚类鉴定结果显示,7株单克隆抗体亚类为ⅠgG,2株为ⅠgM；其腹水的IFA效价分别为：5.12×104、1.08×104、5.12×104、2.16×104、1.08×104、2.048×105、3.2×103、1.024×105、1.024×105：Western-blot结果表明,8株单克隆抗体是PEDVN蛋白特异性抗体,PEDV-2A9有可能为针对构象表位的抗体。3.猪流行性腹泻病毒快速检测方法的初步建立为了建立猪流行性腹泻病毒快速检测方法,本研究利用柠檬酸三钠还原法制备了粒径30nm的胶体金溶液,采用双抗体夹心的原理制备PEDV胶体金试纸条。即将金标抗体PEDV-2C11喷于金标垫上；将兔抗鼠ⅠgG和单抗PEDV-1C12分别喷于NC膜的C线和T线。本试纸条能够特异性的针对PEDV,而不与正常组织或细胞反应。最低能够检测到310个TCID50的病毒量,能够用于临床检测。
[Abstract]:Porcine epidemic diarrhea virus. Porcine epidemic diarrhea (PEDV) is an acute gastroenteritis disease. Its main symptoms are watery diarrhea and vomiting, now it has become a worldwide infectious disease. In 2010, a large area of swine epidemic diarrhea outbreak in southern China, all ages of pig disease. The morbidity and mortality of newborn piglets are very high. In order to understand the epidemiological characteristics of the disease in China in recent two years and establish a rapid detection method. In order to investigate the genetic variation of porcine epidemic diarrhea virus (ECDV) in China, we conducted the following research: 1. In order to investigate the genetic variation of porcine epidemic diarrhea virus (ECDV) in China, this study was carried out from Jiangsu Province by RT-PCR method. In Jilin, Shanghai, Guangdong and Henan provinces and cities, there were 314 samples collected from pig farms, 76 samples of PEDV positive venereal diseases were detected, the positive rate was 24.2%. The positive rate of PEDV in several pig farms in Guangdong Province was the highest, as high as 94.70.The PEDV positive rate was not detected in several pig farms in Jilin Province, and in Jiangsu Province. The positive rates of PEDV in Henan and Shanghai were 17.8% and 27.6% respectively, which indicated that PED was still prevalent in some parts of China. It is necessary to take more strict precautions against PED. Cloning, sequencing and comparative analysis of the S1MN ORF3 gene of PEDV in some positive samples were carried out. The results showed that most of the epidemic strains in China had close homology with Korean and American epidemic strains, and far homology with European epidemic strains and vaccine strains. Only HN13 and YM strains had close homology with CV777 of European epidemic strains and vaccine strains. The amino acids of S1 protein and ORF3 protein were conserved by comparing the amino acid variation of S1 protein. In addition, the ORF3 gene of HN13 and YM strains was the same as the ORF3 gene of vaccine strain DR13. Preparation and characterization of monoclonal antibodies against porcine epidemic diarrhea virus in order to obtain monoclonal antibodies for rapid detection of PEDV. In this study, PEDV purified by ultracentrifugation was used as immunogen to immunize 6-8 week old Balb/c mice. Nine hybridoma cell lines which could stably secrete anti PEDV antibody were selected by immunofluorescence assay and named PEDV-1C12 PEDV-2A9. PEDV-2C11, PEDV-2E5, PEDV-2F1, PEDV-3E9, PEDV-5B12, PEDV-6D1, PEDV-6E10; Then the fluorescence titers, subclasses and specificity of monoclonal antibodies were identified. The results showed that the monoclonal antibody subclasses of 7 strains were 鈪，

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