原核表达PEDV经典株和流行株S基因N端片段的抗原性比较及单克隆抗体制备

发布时间：2018-02-12 00:07

本文关键词： 猪流行性腹泻病毒 S蛋白原核表达免疫反应性单克隆抗体　出处：《南京农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪流行性腹泻(porcine epidemic diarrhea,PED)是由绪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)引起的猪的一种急性、高度接触性肠道传染病,临床上以腹泻、呕吐、脱水为主要特征。PEDV表面的纤突蛋白(S蛋白)是病毒的主要结构蛋白之一,在病毒侵入宿主和诱导机体产生中和抗体的过程中起到重要作用。国内外PEDV S基因的遗传进化分析显示,相比于经典毒株(CV777),近年的流行毒株S基因5'端发生了明显的插入和缺失。本研究针对PEDV经典株和流行株开展S蛋白N段片段免疫反应性差异研究,以期为PEDV的遗传变异分析、鉴别诊断及免疫保护机制研究提供参考依据。选取PEDV经典株CV777和流行株NB2011株S基因5'端差异较大的区段(CV777-S661,NB-S673)进行 RT-PCR 扩增,克隆至 pET-28a 载体并转化E.coli BL21菌株进行表达,分别获得S661和S673重组蛋白。Western blotting结果表明S661和S673表达蛋白均能与猪PEDV阳性血清发生特异性反应,且不与猪PEDV阴性血清反应。说明表达蛋白具有免疫反应性。以纯化的重组蛋白为免疫原制备鼠多抗血清,对两种重组蛋白进行交叉Dot-ELISA试验。结果显示抗S661多抗血清对S661和S673蛋白的可见反应最低蛋白浓度分别为31.25μg/ml和125μg/ml,抗S673多抗血清对二种蛋白的可见反应最低蛋白浓度均为62.5μg/ml。分别制备了抗S673和抗S661的单克隆抗体,共获得4株分泌抗S673单克隆抗体的杂交瘤细胞株,分别命名为1D7、2E11、3G9、4G5;2株分泌抗S661单克隆抗体的杂交瘤细胞株,分别命名为4A12、5G8。抗体亚型鉴定结果表明,3G9、5G8和4A12为IgGl型,1D7和2E11为IgG2a型,4G5为IgG2b型。抗体叠加ELISA试验表明,1D7、2E11、3G9、4G5所识别的抗原位点相同或相似,4A12、5G8所识别的抗原位点也没有差异。交叉Western-blot结果表明,抗S673和抗S661的单克隆抗体均能与相应重组蛋白发生特异性反应,且不与另一毒株蛋白发生反应。IFA结果显示,抗S673和S661的单克隆抗体均能与CV777感染的Vero细胞产生特异性免疫荧光。以上结果说明,PEDV经典株和流行株S蛋白N端片段存抗原性差异。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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