Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66412
標題: 以原核系統表現水禽小病毒之外殼蛋白及非結構蛋白並應用於抗體之快速檢測及次單位疫苗之研發
Expression of Capsid Proteins and Nonstructural Proteins of Waterfowl Parvoviruses in Escherichia coli and Their Use in Serological Assay and Development of Subunit Vaccine
作者: 汪佳穎
Wang, Chia-Ying
關鍵字: goose parvovirus
鴨鵝小病毒
Muscovy duck parvovirus
出版社: 獸醫微生物學研究所
摘要: 水禽小病毒感染症在鴨鵝間具有高致死率及高罹病率,台灣地區曾經發生兩次大流行,造成養禽業者經濟上重大損失。雖然目前已有許多血清學診斷技術應用於水禽小病毒抗體的檢測,然而大部份診斷技術皆需要培養病毒作為抗原,且無法區分外殼蛋白及非結構蛋白之抗體。另一方面,目前在防疫上主要以耐過病毒感染或免疫過之鴨鵝高免血清進行被動免疫,但被動免疫所使用之高免血清抗體力價不易判定,使免疫成效難以評估,此外,田間感染或活毒疫苗免疫後可引發抗各病毒蛋白之抗體,在檢疫上無法區別自然感染或以疫苗免疫之動物。故本研究主題分為兩部分,第一部分利用原核系統E. coli表現正番鴨小病毒 (Muscovy duck parvovirus;MDPV) 及鵝小病毒 (goose parvovirus;GPV) 重組之外殼蛋白VP3、裁切過的外殼蛋白VP1N,和非結構蛋白NS,並以這些重組蛋白為抗原進行Western blotting,檢測GPV人工感染正番鴨之血清,及2002-2003年間於台灣地區收集之田間血清。第二部分則利用這些重組蛋白作為次單位疫苗進行免疫與攻毒試驗。在田間血清的檢測結果中,我們發現台灣地區鵝及鴨飼養場中,水禽小病毒之抗體場陽性率分別達94.7 %及90 %,此外,我們將各場血清對VP3、VP1N及NS呈現之不同反應型加以歸類,推估這些血清之不同反應可能與病毒感染的時程相關。而在GPV感染鴨隻的血清檢測結果中,我們則發現NS抗體出現在感染的最早期,其後依序出現的是VP3及VP1N抗體。總結而言,本研究首度以E. coli表現水禽小病毒之重組蛋白,並發現重組NS、VP3及VP1N蛋白皆具有良好之抗原性。利用Western blotting分析方法則可區別水禽小病毒外殼蛋白及非結構蛋白之抗體,藉此我們希望發展快速之血清診斷技術,將來可運用在區分自然感染與免疫次單位疫苗之鴨鵝,以利於水禽小病毒之監控。另一方面,在重組次單位疫苗之免疫試驗結果部分,我們初步認為E. coli表現之重組蛋白所引發的抗體,可能具有部分保護力,但仍需進一步實驗加以評估。
The infection of waterfowl parvovirus could lead to high mortality and morbidity of ducklings and goslings and cause severe economic losses. While there are a number of methods available for detection of antibodies against waterfowl parvoviruses, none is able to differentiate responses against the capsid and nonstructural proteins. Moreover, current vaccination program is using the traditional passive immunization, but it is difficult to evalute the titers of antibody of the sera used for vaccination. Both field infection and vaccination using live virus elicit antibodies against the capsid and nonstructural protein and it is impossible to differentiate field-infected from vaccinated birds. To enable this, the capsid and nonstructural proteins of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) were expressed in E. coli. These proteins were purified and used as antigens in Western blotting assays and as subunit vaccine. The goal of this study can be divided into two parts; the first part is using Western blotting assays to evalute the prevalence of antibody against GPV and MDPV in the field. The second part is to develop a subunit vaccine for the control of GPV and MDPV. Western blotting assays showed that 94.7% of the goose and 90.0% of the duck sera collected from the field contained antibodies against GPV or MDPV. Moreover, these sera could be classified into distinct groups based on differences in patterns of Western blot reactivity. These different patterns might indicate different stages in infection. Western blotting assays of sera collected from experimentally infected ducks showed that antibodies against the nonstructural protein appeared first after infection, followed by antibodies against the capsid protein. It was concluded that the recombinant capsid and nonstructural proteins might serve as useful antigens for assays for antibodies against GPV and MDPV. Moreover, because these assays could discriminate between antibodies against the nonstructural protein and those against the capsid protein, they may be useful in differentiating vaccinated from infected birds when recombinant capsid protein is used as the vaccine. Vaccination of ducks by recombinant VP3, VP1N and NS showed that three recombinant proteins could provide partial protection against the challenge of GPV and MDPV, but this result needs to be confirmed by further experiments.
URI: http://hdl.handle.net/11455/66412
Appears in Collections:微生物暨公共衛生學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.