本文關(guān)鍵詞：豬流行性腹瀉病毒的流行病學(xué)調(diào)查及快速檢測(cè)方法的初步建立　出處：《揚(yáng)州大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 豬流行性腹瀉病毒 流行病學(xué)調(diào)查 單克隆抗體 快速檢測(cè)方法

【摘要】：由豬流行性腹瀉病毒(Porcine epidemic diarrhea virus, PEDV)引起的豬流行性腹瀉(Porcine epidemic diarrhea, PED)是一種急性胃腸炎疾病,其主要癥狀為水樣腹瀉和嘔吐,現(xiàn)在已經(jīng)成為一種世界性傳染病。2010年我國(guó)南部大面積爆發(fā)豬流行性腹瀉疫情,各個(gè)年齡段的豬均發(fā)病,新生仔豬的發(fā)病率和死亡率都很高。為了了解近兩年該病在我國(guó)的流行病學(xué)特點(diǎn)和建立快速檢測(cè)方法,我們主要開(kāi)展了以下研究：1.豬流行性腹瀉病毒的遺傳變異分析為了調(diào)查我國(guó)豬流行性腹瀉病毒的遺傳變異情況,本研究通過(guò)RT-PCR方法對(duì)從江蘇、吉林、上海、廣東、河南五省市規(guī)模豬場(chǎng)采集來(lái)的314份樣本進(jìn)行病原學(xué)檢測(cè),檢測(cè)出PEDV陽(yáng)性病料76份,陽(yáng)性率達(dá)24.2%,其中廣東省幾個(gè)豬場(chǎng)的PEDV陽(yáng)性率最高,高達(dá)94.7%,吉林省幾個(gè)豬場(chǎng)沒(méi)檢出PEDV陽(yáng)性,另外江蘇省、河南省和上海市的PEDV陽(yáng)性率分別為17.8%、31.6%和27.6%,表明PED在我國(guó)部分地區(qū)仍流行嚴(yán)重,有必要對(duì)PED采取更加嚴(yán)格的防范措施。對(duì)部分陽(yáng)性樣品中PEDV的S1、M、ORF3基因進(jìn)行克隆、測(cè)序和比對(duì)分析,結(jié)果表明我國(guó)大部分流行株與韓國(guó)、美國(guó)流行株同源性較近,與歐洲流行株以及疫苗株CV777同源性較遠(yuǎn),只有HN13和YM毒株與歐洲流行株和疫苗株CV777同源性較近。比對(duì)發(fā)現(xiàn)S1蛋白氨基酸發(fā)生變異明顯,M蛋白和ORF3蛋白的氨基酸較為保守,另外HN13和YM毒株的ORF3基因與疫苗株DR13的ORF3基因相同,有49個(gè)核苷酸缺失。2.豬流行性腹瀉病毒單克隆抗體的研制及特性鑒定為了獲得可用于建立PEDV快速檢測(cè)方法的單克隆抗體,本研究將超速離心法純化的PEDV作為免疫原,免疫6-8周齡Balb/c小鼠,利用免疫熒光法(IFA)篩選出9株能夠穩(wěn)定分泌抗PEDV抗體的雜交瘤細(xì)胞株,分別命名為PEDV-1C12、PEDV-2A9、PEDV-2C11、 PEDV-2E5、PEDV-2F1、PEDV-3E9、PEDV-5B12、PEDV-6D1、PEDV-6E10;隨后對(duì)單克隆抗體的腹水熒光效價(jià)、亞類(lèi)以及特異性等生物學(xué)特性進(jìn)行鑒定。單克隆抗體亞類(lèi)鑒定結(jié)果顯示,7株單克隆抗體亞類(lèi)為ⅠgG,2株為ⅠgM；其腹水的IFA效價(jià)分別為：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結(jié)果表明,8株單克隆抗體是PEDVN蛋白特異性抗體,PEDV-2A9有可能為針對(duì)構(gòu)象表位的抗體。3.豬流行性腹瀉病毒快速檢測(cè)方法的初步建立為了建立豬流行性腹瀉病毒快速檢測(cè)方法,本研究利用檸檬酸三鈉還原法制備了粒徑30nm的膠體金溶液,采用雙抗體夾心的原理制備PEDV膠體金試紙條。即將金標(biāo)抗體PEDV-2C11噴于金標(biāo)墊上；將兔抗鼠ⅠgG和單抗PEDV-1C12分別噴于NC膜的C線和T線。本試紙條能夠特異性的針對(duì)PEDV,而不與正常組織或細(xì)胞反應(yīng)。最低能夠檢測(cè)到310個(gè)TCID50的病毒量,能夠用于臨床檢測(cè)。
[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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