Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66522
標題: 豬瘟鑑別診斷試劑之研發 II. ELISA診斷試劑之製備
Development of Disciminating Diagnostic Reagents of Classical Swine Fever Virus II. Preparation of ELISA Diagnostic Reagent
作者: 黃千衿
簡茂盛
朱純燕
張天傑
關鍵字: 生物技術, 畜牧獸醫類
應用研究
摘要: 計p畫e目媦標苤:G應野用弇酵疇母懇菌筡Pichia表穛現{系t統庤進i行瞏豬瑍瘟E病f毒r表穛現{蛋J白掑之坐大j量q製s備?與P純瞻化?,A並繹開}發o為蚩ELISA鑑異別O診E斷_試桴劑砥,A以H建堨立艉一@套M優u良}的熄間§接湲ELISA豬瑍瘟E抗凗體橉檢侅測?方隤法k。C而蚑豬瑍瘟E病f毒r醣瑋蛋J白掍亦蟡可i進i一@步B開}發o成足為隻次董單璁位儤標陏識悇疫怑苗],A核硈蛋J白梐產ㄙ物垂則h可i應野用帡做筋為剪鑑異別O診E斷_試桴劑砥,A並繹進i行瑽技瑋術N轉鉦移劓國磥內漪疫怑苗]製s造y廠t商荂。C本誚年~度蚼重垠要n工u作@項等目堣及庣實磟施I方隤法k:G1.豬瑍瘟E病f毒r表穛現{蛋J白掑之坐大j量q生芠產ㄓ及巹純瞻化?選嚝擇靰表穛現{特S性妘最怢佳峇之圻各U重垓組桮菌蒏株镼先?經g隔j夜]培鷎養i於?200ml培鷎養i液G後嵿接絢種堜於騤裝辿有酗二G公膜升仱培鷎養i液G之孝發o酵羹槽恁,A並籀設]定w最抩適A當穜條囓件騥進i行璊大j規W模珛發o酵簸培鷎養i。C分尷泌c性坁表穛現{蛋J白掑之妖純瞻化?可i直蔣接策收炮集偽培鷎養i上W清M液G,A並疇加[入J飽〝和M硫蜓酸躉銨牁溶輔液G或峊以H超W過L濾o(ultrafiltration)進i行瘜蛋J白桵質閮沈H澱?與P濃@縮Y。C2.豬瑍瘟E病f毒r表穛現{蛋J白掑之壯抗颩原鴝性吨分尷析R純瞻化?的漲各U病f毒r蛋J白梐產ㄙ物姣與P適A當磽佐齙劑租混V合X後嵺免K疫怳小p白桯鼠奎進i行瑽免K疫怞血撗清M之宏製s備?,A並癟利Q用庤間§接筆免K疫抰螢疇光?抗凗體擛染V色滫法k檢侅測?這o些?表穛現{蛋J白晥所珨誘今發o產ㄔ生秅之壯抗凗體擗之妖特S異妝性吽。C另t外~將N純瞻化?之余E2表穛現{醣瑋蛋J白掍加[入J適A當磽佐齙劑租混V合X後嵺免K疫?6週g齡硉無L特S定w病f原?(SPF)仔J豬煄,A並矇於韝三T週g後嶆再A補伀強j免K疫怳一@次腹。C於顜免K疫怮前e及峓免K疫怮後嶁每C隔j一@週g採藻血憡並繫測?定w血撗清M中允對鴽抗傰豬瑍瘟E病f毒r之坐中予和M抗凗體擗力O價龤,A以H進i行澅E2表穛現{醣瑋蛋J白掑之壯免K疫怌效臚力O測?定w。C3.ELISA豬瑍瘟E抗凗體橉檢侅測?方隤法k之妨建堨立?經g過L純瞻化?之局豬瑍瘟E病f毒r表穛現{蛋J白掍以H0.1M carbonate buffer (pH 9.2)稀}釋嬰成防適A當篻濃@度蛂,A滴w入J96孔梫微L量q盤L製s成豆抗颩原儠盤L。C經g分嬪別O進i行璊一@連s串窷填騆補?(blocking)、B豬瑍瘟E待搥測?血撗清M及庣過L氧韙化?氫B酵簿素擘標邾幟m之坐山s羊洇抗傰豬牏二G次葷抗凗體曀等尼作@用峆後寣,A最怮後嵽加[入J呈e色熅劑窄進i行瑽呈e色漶。C調桴整膃各U反狨應釣步B驟J之妤條囓件鞳,A以H建堨立蒆最怢佳峇之孜間§接湲ELISA豬瑍瘟E抗凗體橉檢侅測?方隤法k。C4.大j規W模珜野孕外~血撗清M之局調晙查d分尷析R大j規W模狾收炮集偃野孕外~豬瑋場鶡血撗清M,A利Q用峏所珨製s備?之余ELISA抗颩原儠盤L進i行瞏豬瑍瘟E抗凗體擗之尾檢侅測?。C並疇且B與P與P螢疇光?抗凗體擛染V色滫法k所珒測?得o之壯抗凗體擗力O價鬤進i行璊比騆較?,A以H評?估巇ELISA鑑異別O診E斷_試桴劑砟之妖特S異妝性吨及帢敏虓感P性吽。C預w期螳效蠕益q:G所珨製s備?純瞻化?之圻各U豬瑍瘟E病f毒r表穛現{蛋J白梐產ㄙ物咱可i開}發o成足為偯優u良}之余ELISA鑑異別O診E斷_試桴劑砥。C而蚑豬瑍瘟E病f毒r醣瑋蛋J白掍可i進i一@步B開}發o成足為隻次董單璁位儤標陏識悇疫怑苗],A核硈蛋J白梐產ㄙ物垂則h可i應野用帡做筋為剪鑑異別O診E斷_試桴劑砥,A有釦利Q於騣豬瑍瘟E撲雪滅嶺計p畫e之孜進i行獢。C
Classical swine fever virus (CSFV) is the causative agent of the highly contagious infection of swine-classical swine fever (hog cholera). The virion contains four structural proteins : E0, E1 and E2 glycoproteins as well as the nucleocapsid (core) protein C. The purpose of this study is to produce CSFV structural proteins by the yeast Pichia pastoris eukaryotic expression system and for further developing CSFV subunit marker vaccine and companion discriminating ELISA test. The recombinant yeast strains which have been identified to express the CSFV E2 or C protein will be set-up in a large-scale fermentation culture for the preparation of large amounts of expressed CSFV proteins. The immunogenicity of these expressed proteins will be evaluated by immunization of mice and piglets respectively. The ELISA diagnostic reagents developed from this project may be utilized for evaluation and differentiation of the immune reactions induced by CSFV E2 subunit marker vaccine.
URI: http://hdl.handle.net/11455/66522
其他識別: 91農科-3.1.2-檢-B2(4)
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=934663&plan_no=91%E8%BE%B2%E7%A7%91-3.1.2-%E6%AA%A2-B2%284%29&plan_year=91&projkey=PG9306-1582&target=plan&highStr=*&check=0&pnchDesc=%E8%B1%AC%E7%98%9F%E9%91%91%E5%88%A5%E8%A8%BA%E6%96%B7%E8%A9%A6%E5%8A%91%E4%B9%8B%E7%A0%94%E7%99%BC+II.+ELISA%E8%A8%BA%E6%96%B7%E8%A9%A6%E5%8A%91%E4%B9%8B%E8%A3%BD%E5%82%99
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