猪流行性腹泻病毒变异株与经典疫苗株S蛋白免疫原性差异分析
发布时间：2018-10-23 11:01
【摘要】：2010年10月以来,猪流行性腹泻在中国和世界其他主要养猪国家大面积流行。本论文首先对42株PEDV(2010年以后的PEDV中国流行株24个,美国流行株株7个,韩国毒株6个,参考毒株5个)S蛋白氨基酸序列进行比较,结果显示,与参考毒株相比,2010年以后PEDV流行株的S蛋白的22aa~380aa区域(命名为SA)为突变的集中区域;流行株亲缘关系较近且在一个分支,而疫苗株及早期毒株等参考毒株则位于另一个分支;早期毒株与中国现用疫苗毒株CV777之间的同源性较高,为95.0%~99.1%,而2010年后的流行株与参考毒株间的同源性为92.3%~94.1%,其中与疫苗株CV777的同源性仅为93.3%;对SA区域进行抗原位点与糖基化位点的预测发现,2010年以后流行株与疫苗株及早期毒株之间在22aa~380aa之间抗原位点差异较大,且在60aa~280aa之间差异最显著;2010年以后的毒株在62aa~65aa位多出了一个N糖基化位点。以上结果提示2010年以后的PEDV流行株与经典毒株相比发生了明显变异,在对PEDV S基因进行序列分析的基础上,本论文首先表达了PEDV中国变异株CH-ZMDZY-11与疫苗株CV777的SA区域,表达的SA蛋白具有良好的反应原性,为建立检测抗PEDV抗体的间接ELISA方法、分析PEDV流行株与疫苗株S蛋白免疫反应性差异,以及PED亚单位疫苗研究奠定了基础。以纯化的PEDV中国变异株S蛋白为包被抗原,建立了检测PEDV流行株抗体的间接ELISA方法。建立的间接ELISA方法最佳反应条件为:抗原最适包被量为1ug/孔,包被时间为37℃包被1h后4℃过夜;血清工作浓度为1:100,作用时间为1.5h;二抗选用HRP标记的兔抗猪IgG稀释度为1:4000,作用时间为1.5h;显色液TMB作用时间为15min;判定标准为,样品S/P值大于等于0.058判定为阳性,小于0.045判定为阴性。所建ELISA方法具有良好特异性和重复性。对50份疑似猪流行性腹泻血清样品进行检测,结果表明流行株抗体检测阳性的样品均为PEDV流行株抗原阳性。本研究所建立ELISA方法可用于PEDV流行株抗体检测和疫病监测。为分析PEDV流行株和疫苗株S基因差异对疫苗免疫效果影响,本研究将在表达的PEDV变异株CH/ZMDZY/11与疫苗株CV777 SA蛋白免疫小鼠与成年兔子获得多克隆抗体,并以SA蛋白为抗原,通过Western bloting及间接ELISA的方法检测两种蛋白与多克隆抗体的反应性差异,并通过兔抗PEDV SA蛋白多克隆抗体与CV777中和情况比较抗S蛋白抗体中和活性的差异。结果表明,抗PEDV疫苗株SA蛋白(YSA)多抗与疫苗株SA蛋白的反应性明显高于其与流行株SA蛋白(LSA)的反应性;与此相反,抗PEDV流行株SA蛋白多抗与流行株SA蛋白的反应性明显高于其与疫苗株SA蛋白的反应性;抗体的中和活性比较显示,抗PEDV变异株及疫苗株SA蛋白多克隆抗体在中和PEDV疫苗毒CV777上存在很大的差异,即疫苗株的抗体中和CV777效价明显高于流行株抗体的中和效价。研究结果提示,PEDV S蛋白22aa~380aa区域存在差异性抗原表位,PEDV变异株和疫苗株S基因的差异可能是导致基于CV777疫苗株的PED疫苗免疫效果不好的原因。本研究将为开发针对PEDV变异株的PED亚单位疫苗,有效控制由变异株导致的PED奠定基础。
[Abstract]:Porcine epidemic diarrhea has been prevalent in China and other major pig-raising countries in the world since October 2010. In this paper, 42 strains of PEDV (24 strains of PEDV in China, 7 strains of American epidemic strains, 6 Korean strains and 5 S protein amino acid sequences) were compared. Compared with the reference strain, the 22aa ~ 380aa region (named SA) of the S protein of the PEDV popular strain after 2010 is the concentrated region of the mutation; the genetic relationship of the epidemic strain is close and in one branch, and the reference strain such as the vaccine strain and the early strain is located in the other branch; The homology between the early strain and the vaccine strain CV777 was higher, which was 95.0% ~ 99.1%, and the homology between the epidemic strain and the reference strain after 2010 was 92.3% ~ 94.1%, among which the homology with the vaccine strain CV777 was 93.3%. It was found that there was a significant difference in the antigenic sites between the epidemic strain and the vaccine strain and the early strain at 22aa ~ 380aa after 2010, and the difference between 60aa ~ 280aa was the most significant. The strain after 2010 had an N glycosylation site at 62aa ~ 65aa. The results suggest that the PEDV popular strain after 2010 has a significant variation in comparison with the classical strain. On the basis of sequence analysis of the PEDV S gene, this paper first expresses the SA region of the strain CH-ZMDZY-11 of PEDV and the vaccine strain CV777, and the expressed SA protein has good reactivity. In order to establish an indirect ELISA method for detecting anti-PEDV antibody, the difference of immune reactivity between PEDV and vaccine strain S protein was analyzed, and the basis was laid for the research of PED subunit vaccine. An indirect ELISA method for detecting the antibody of PEDV was established by using purified PEDV Chinese mutant S protein as envelope antigen. The optimal reaction conditions of indirect ELISA were as follows: the optimal coating amount of antigen was 1ug/ hole, the coating time was 37 鈩，
本文编号：2289041
[Abstract]:Porcine epidemic diarrhea has been prevalent in China and other major pig-raising countries in the world since October 2010. In this paper, 42 strains of PEDV (24 strains of PEDV in China, 7 strains of American epidemic strains, 6 Korean strains and 5 S protein amino acid sequences) were compared. Compared with the reference strain, the 22aa ~ 380aa region (named SA) of the S protein of the PEDV popular strain after 2010 is the concentrated region of the mutation; the genetic relationship of the epidemic strain is close and in one branch, and the reference strain such as the vaccine strain and the early strain is located in the other branch; The homology between the early strain and the vaccine strain CV777 was higher, which was 95.0% ~ 99.1%, and the homology between the epidemic strain and the reference strain after 2010 was 92.3% ~ 94.1%, among which the homology with the vaccine strain CV777 was 93.3%. It was found that there was a significant difference in the antigenic sites between the epidemic strain and the vaccine strain and the early strain at 22aa ~ 380aa after 2010, and the difference between 60aa ~ 280aa was the most significant. The strain after 2010 had an N glycosylation site at 62aa ~ 65aa. The results suggest that the PEDV popular strain after 2010 has a significant variation in comparison with the classical strain. On the basis of sequence analysis of the PEDV S gene, this paper first expresses the SA region of the strain CH-ZMDZY-11 of PEDV and the vaccine strain CV777, and the expressed SA protein has good reactivity. In order to establish an indirect ELISA method for detecting anti-PEDV antibody, the difference of immune reactivity between PEDV and vaccine strain S protein was analyzed, and the basis was laid for the research of PED subunit vaccine. An indirect ELISA method for detecting the antibody of PEDV was established by using purified PEDV Chinese mutant S protein as envelope antigen. The optimal reaction conditions of indirect ELISA were as follows: the optimal coating amount of antigen was 1ug/ hole, the coating time was 37 鈩，
本文编号：2289041
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2289041.html
