本文選題：豬流行性腹瀉病毒　切入點：N蛋白　出處：《河北農業(yè)大學》2015年碩士論文

【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是由豬流行性腹瀉病毒(PEDV)引起的以腹瀉、嘔吐、脫水和哺乳仔豬高致死率為主要特征的一種高度接觸性腸道傳染病。其臨床癥狀與豬傳染性胃腸炎、輪狀病毒感染等其他腸道傳染病極易混淆,制備PEDV特異性診斷試劑,并建立快速檢測方法具有重要的實踐意義。核蛋白(nucleoprotein,N蛋白)是PEDV的主要結構蛋白,保守性強,是病毒感染早期診斷和疫病監(jiān)測的主要靶蛋白。本論文應用雜交瘤技術,制備針對N蛋白的特異性單克隆抗體(monoclonal antibody,Mc Ab),旨在以Mc Ab為材料建立檢測PEDV的膠體金免疫層析檢測方法,為進一步研發(fā)特異、便捷的診斷工具搭建必要的技術平臺。本研究以純化的重組N蛋白作為抗原,免疫4只雌性BALB/c小鼠,利用雜交瘤技術,經過3~5次亞克隆和篩選,獲得了4株分泌抗PEDV Mc Ab的雜交瘤細胞,分別命名為A7、E10、F7、C1株雜交瘤細胞,其染色體數目在97~103之間。經亞類鑒定,4株雜交瘤細胞分泌的Mc Ab均為Ig M類,其輕鏈均為κ鏈,分別識別4個不同的抗原表位。Western blot和免疫組化鑒定顯示,4株Mc Ab均能夠與PEDV N蛋白,或與PEDV感染豬小腸絨毛上皮細胞內的PEDV特異結合。A7、E10、F7和C1株雜交瘤細胞的培養(yǎng)上清抗體效價分別為1:1600、1:100、1:100和1:100,腹水抗體效價分別為1:10~6、1:10~5、1:10~3和1:10~2。將4株雜交瘤細胞連續(xù)傳代30代,具有良好的抗體分泌穩(wěn)定性。應用檸檬酸三鈉還原法制備粒徑為20 nm的酒紅色膠體金懸液,用其標記純化的Mc Ab A7。將40μg/m L p H 8.0的膠體金標記抗體Mc Ab A7吸附于玻璃纖維素膜上,制備金標墊;以1 mg/m L的Mc Ab F7和羊抗鼠Ig M,按照5μL/cm的包被量,印跡于硝酸纖維膜,分別作為檢測線與質控線,組裝試紙條。該試紙條能夠特異地與PEDV感染豬小腸組織中的病毒反應,在檢測線與質控線處分別出現一條紅色線。同時用組裝的試紙條和RT-PCR檢測13份來自不同豬場的腹瀉仔豬小腸內容物,試紙條與RT-PCR的陽性檢出率分別為38.46%(5/13)和46.15%(6/13),兩者的總符合率為83.3%,表明利用抗PEDV N蛋白單克隆抗體建立的PEDV膠體金免疫層析試紙條能夠快速、特異地檢測PEDV,為進一步開發(fā)應用PEDV膠體金免疫層析試紙條檢測試劑盒搭建了必要的技術平臺。
[Abstract]:Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection caused by porcine epidemic diarrhea virus (PEDVV), which is characterized by high mortality of diarrhea, vomiting, dehydration and breast-feeding piglets.Its clinical symptoms are easily confused with other enteric diseases such as porcine infectious gastroenteritis rotavirus infection and so on. It is of great practical significance to prepare PEDV specific diagnostic reagent and establish a rapid detection method.Nucleoprotein N (nucleoprotein N) is the main structural protein of PEDV, and is the main target protein for the early diagnosis of virus infection and the surveillance of epidemic disease.In this paper, a monoclonal antibody (McAb) against N protein was prepared by using hybridoma technique. The aim of this study was to establish a colloidal gold immunochromatographic assay for the detection of PEDV using McAb as a material, so as to develop a specific method for further development.Convenient diagnostic tools to build the necessary technical platform.In this study, 4 female BALB/c mice were immunized with purified recombinant N protein as antigen. Four hybridoma cells secreting anti PEDV McAb were obtained by using hybridoma technique.Its chromosome number is between 97 and 103.The McAb secreted by four hybridoma strains were identified as Ig M, and the light chain was 魏 chain, which recognized four different epitopes. Western blot and immunohistochemical analysis showed that Mc-Ab of the four strains could be associated with PEDV N protein.鎴栦笌PEDV鎰熸煋鐚皬鑲犵粧姣涗笂鐨粏鑳?yōu)鍐呯殑PEDV鐗瑰紓緇撳悎.A7,E10,F7鍜孋1鏍潅浜ょ槫緇嗚優(yōu)鐨勫煿鍏諱笂娓呮姉浣撴晥浠峰垎鍒負1:1600,1:100,1:100鍜，

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