本文關(guān)鍵詞： 豬流行性腹瀉病毒 S蛋白 原核表達(dá) 免疫反應(yīng)性 單克隆抗體　出處：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬流行性腹瀉(porcine epidemic diarrhea,PED)是由緒流行性腹瀉病毒(porcine epidemic diarrhea virus,PEDV)引起的豬的一種急性、高度接觸性腸道傳染病,臨床上以腹瀉、嘔吐、脫水為主要特征。PEDV表面的纖突蛋白(S蛋白)是病毒的主要結(jié)構(gòu)蛋白之一,在病毒侵入宿主和誘導(dǎo)機(jī)體產(chǎn)生中和抗體的過(guò)程中起到重要作用。國(guó)內(nèi)外PEDV S基因的遺傳進(jìn)化分析顯示,相比于經(jīng)典毒株(CV777),近年的流行毒株S基因5'端發(fā)生了明顯的插入和缺失。本研究針對(duì)PEDV經(jīng)典株和流行株開(kāi)展S蛋白N段片段免疫反應(yīng)性差異研究,以期為PEDV的遺傳變異分析、鑒別診斷及免疫保護(hù)機(jī)制研究提供參考依據(jù)。選取PEDV經(jīng)典株CV777和流行株NB2011株S基因5'端差異較大的區(qū)段(CV777-S661,NB-S673)進(jìn)行 RT-PCR 擴(kuò)增,克隆至 pET-28a 載體并轉(zhuǎn)化E.coli BL21菌株進(jìn)行表達(dá),分別獲得S661和S673重組蛋白。Western blotting結(jié)果表明S661和S673表達(dá)蛋白均能與豬PEDV陽(yáng)性血清發(fā)生特異性反應(yīng),且不與豬PEDV陰性血清反應(yīng)。說(shuō)明表達(dá)蛋白具有免疫反應(yīng)性。以純化的重組蛋白為免疫原制備鼠多抗血清,對(duì)兩種重組蛋白進(jìn)行交叉Dot-ELISA試驗(yàn)。結(jié)果顯示抗S661多抗血清對(duì)S661和S673蛋白的可見(jiàn)反應(yīng)最低蛋白濃度分別為31.25μg/ml和125μg/ml,抗S673多抗血清對(duì)二種蛋白的可見(jiàn)反應(yīng)最低蛋白濃度均為62.5μg/ml。分別制備了抗S673和抗S661的單克隆抗體,共獲得4株分泌抗S673單克隆抗體的雜交瘤細(xì)胞株,分別命名為1D7、2E11、3G9、4G5;2株分泌抗S661單克隆抗體的雜交瘤細(xì)胞株,分別命名為4A12、5G8。抗體亞型鑒定結(jié)果表明,3G9、5G8和4A12為IgGl型,1D7和2E11為IgG2a型,4G5為IgG2b型�？贵w疊加ELISA試驗(yàn)表明,1D7、2E11、3G9、4G5所識(shí)別的抗原位點(diǎn)相同或相似,4A12、5G8所識(shí)別的抗原位點(diǎn)也沒(méi)有差異。交叉Western-blot結(jié)果表明,抗S673和抗S661的單克隆抗體均能與相應(yīng)重組蛋白發(fā)生特異性反應(yīng),且不與另一毒株蛋白發(fā)生反應(yīng)。IFA結(jié)果顯示,抗S673和S661的單克隆抗體均能與CV777感染的Vero細(xì)胞產(chǎn)生特異性免疫熒光。以上結(jié)果說(shuō)明,PEDV經(jīng)典株和流行株S蛋白N端片段存抗原性差異。
[Abstract]:Porcine epidemic diarrhea (PED) is an acute, highly contact intestinal infection caused by porcine epidemic diarrhea virus (PEDVV) in pigs. Dehydration is one of the main structural proteins of the virus. It plays an important role in invading the host and inducing the body to produce neutralizing antibody. The genetic and evolutionary analysis of PEDV S gene at home and abroad shows that, Compared with the classical virus strain CV777, the S gene 5'terminal insertion and deletion occurred in recent years. In order to analyze the genetic variation of PEDV, the S protein N fragment immunoreactivity of the classical strain and epidemic strain of PEDV was studied in this study. The study of differential diagnosis and immune protection mechanism provided reference for the study of the mechanism of immune protection. The 5'terminal region of S gene of PEDV classic strain CV777 and epidemic strain NB2011 strain S was amplified by RT-PCR and cloned into pET-28a vector and transformed into E. coli BL21 strain for expression. S661 and S673 recombinant proteins were obtained respectively. Western blotting showed that S661 and S673 expressed proteins could react specifically with porcine PEDV positive serum. The expression protein was immunoreactive and purified recombinant protein was used as immunogen to prepare mouse polyclonal antibody serum, which did not react with porcine PEDV negative serum. Two recombinant proteins were tested by cross Dot-ELISA. The results showed that the minimum concentration of visible reaction protein to S661 and S673 was 31.25 渭 g / ml and 125 渭 g / ml, respectively, and the minimum concentration of visible reaction of anti-S673 polyantiserum to the two proteins was found to be 31.25 渭 g / ml and 125 渭 g / ml, respectively. Both were 62.5 渭 g / ml. Monoclonal antibodies against S673 and S661 were prepared, respectively. Four hybridoma cell lines secreting monoclonal antibodies against S673 were obtained, and were named as 1D7O2E11E113G9O4G5H5 2 hybridoma cell lines secreting monoclonal antibodies against S661. The results of antibody subtype identification showed that 3G9G 8 and 4A12 were IgGl 1D7 and 2E11 respectively, and IgG2a 4G5 was IgG2b. The results of antibody superposition ELISA test showed that the antigenic loci recognized by T1D7D7E113G9G9 4G5 were the same or similar, and the antigenic sites recognized by 4A125G8 had no difference. Cross Western-blot results show that, Both anti-S673 and anti-S661 monoclonal antibodies could react specifically with the corresponding recombinant protein, and did not react with another virulent strain of protein. IFA results showed that, Monoclonal antibodies against S673 and S661 could produce specific immunofluorescence with Vero cells infected with CV777, which indicated that the N-terminal antigenicity of S protein fragments in classical and prevalent strains of Vero was different.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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