猪流行性腹泻病毒主要结构基因遗传变异分析及ELISA检测方法的建立
发布时间：2019-02-23 18:29
【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的一种猪的急性、高度接触性传染病,以腹泻、呕吐、脱水等为主要临床特征特征,给我国养猪业造成了巨大损失。临床上该病与猪传染性胃肠炎、轮状病毒病症状相似,较难进行鉴别诊断。为了探究PEDV的分子流行病学特点并建立有效的诊断方法,本研究主要开展了以下几个方面的工作:1.PEDV主要结构基因的遗传变异分析2013-2014年于北京、河南、陕西、广东、山东5个省市采集的猪流行性腹泻(PED)阳性病料,设计特异性引物对结构基因S、M、N进行克隆及测序,与国内外主要毒株进行比较,分析序列变化和遗传变异情况。序列比对结果显示,1株为弱毒株,其余4个样品株的S基因与国内疫苗株CV777差异较大,突变主要存在于S1区。S基因氨基酸序列存在5个氨基酸的插入(59QGVN62 and 140N)和2个氨基酸的缺失(163NI164),S1区的2个中和表位(499~638aa和764~771aa)有7处氨基酸突变。M、N基因的核苷酸序列相对保守,只存在部分氨基酸突变。遗传进化分析结果显示,4个样品株与亚洲主要疫苗株(中国疫苗株CV777、韩国弱毒疫苗株DR13以及日本弱毒疫苗株83P-5)同源性较低(93.8%~94.7%),亲缘关系较远;与2007~2009年韩国毒株,2011年日本毒株以及中国近年流行毒株同源性较高(96.0%~99.6%),亲缘关系密切。2.PEDV BJ-1株M、N、S1融合蛋白的表达和抗原性分析将样品株BJ-1的M、N和S1基因克隆至原核表达载体p GEX-6p-1,转化感受态细胞BL21,经IPTG诱导表达,SDS-PAGE电泳结果显示M、N、S1重组蛋白获得表达,大小分别为50KDa、80KDa、110KDa,以包涵体表达形式为主。Western blot分析结果表明3种重组蛋白能被PEDV猪阳性血清特异性识别,具有良好的抗原性,可以作为诊断抗原用于特异性检测PEDV感染。3.PEDV N蛋白间接ELISA抗体检测方法的建立以纯化重组PEDV N蛋白作为包被抗原,优化各项反应条件,初步建立了可以检测PEDV血清中N蛋白抗体的间接ELISA检测方法。本研究中最终优化的反应条件为:N蛋白的最佳包被浓度为0.5?g/m L,最佳包被时间为37℃2h后4℃过夜(10~16h),5%脱脂牛奶37℃封闭3h,血清样品最佳稀释度为1:400,37℃作用1h,兔抗猪酶标二抗最佳稀释度为1:40,000,37℃作用1h,抗体临界值为OD450nm?0.40判为阳性,OD450nm0.40判为阴性。试验证明该方法具有较好的敏感性、特异性、重复性和符合率,表明PEDV N蛋白间接ELISA抗体检测方法初步建立,可以用于猪PEDV血清抗体的检测和流行病学调查。综上所述,本研究分析了PEDV样品株结构基因中M、N、S的序列变化和遗传变异情况,为研究PEDV毒株的变异情况提供理论参考;成功表达了M、N、S1重组蛋白,具有良好的反应原性,为建立针对结构蛋白的检测方法奠定基础;用纯化的重组N蛋白,成功建立了PEDV N蛋白间接ELISA抗体检测方法,为PEDV的血清学诊断和流行病学调查提供了一种简便的检测手段。
[Abstract]:Porcine epidemic diarrhea (Porcine epidemic diarrhea,PED) is an acute and highly contagious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus,PEDV). It is characterized by diarrhea, vomiting, dehydration and so on. Great losses have been caused to the pig industry in our country. The clinical symptoms of the disease are similar to those of porcine infectious gastroenteritis and rotavirus disease, so it is difficult to differentiate diagnosis. In order to explore the molecular epidemiological characteristics of PEDV and to establish an effective diagnostic method, the following aspects were carried out in this study: genetic variation analysis of major structural genes of 1.PEDV was conducted in Beijing, Henan, Shaanxi, Guangdong from 2013-2014. (PED) positive venereal materials collected from five provinces and cities of Shandong Province were cloned and sequenced by designing specific primers. The sequence changes and genetic variations of the structural gene were compared with the main strains at home and abroad. The results of sequence alignment showed that one strain was virulent, and the other four strains had different S genes from domestic vaccine strain CV777. 5 amino acid insertions (59QGVN62 and 140N) and 2 amino acid deletions (163NI164) were found in the amino acid sequence of S gene, and 7 amino acid mutations were found in the two neutralization epitopes (499~638aa and 764~771aa) of S1 region. The nucleotide sequence of N gene is relatively conserved and only some amino acid mutations exist. The results of genetic evolution analysis showed that the homology of the four strains with the main Asian vaccine strains (Chinese CV777, Korean attenuated vaccine DR13 and Japanese attenuated vaccine 83P-5) was lower (93.880% and 94.7%) and the phylogenetic relationship between the four strains was far away from that of the Asian main vaccine strains (Chinese vaccine strain CV777, Korean attenuated vaccine strain and Japanese attenuated vaccine strain 83P-5). There was a high homology (99.6%) with the Korean strain from 2007 to 2009, the Japanese strain from 2011 and the epidemic strain from China in recent years (96.00.99. 6%). The expression and antigenicity analysis of S1 fusion protein were used to clone Mon N and S1 genes of BJ-1 into the transformant BL21, of prokaryotic expression vector p GEX-6p-1,. The results of SDS-PAGE electrophoresis showed that Mon was expressed by IPTG. S1 recombinant protein was expressed in 50 KDa-80KDa-110KDa. the results of. Western blot analysis showed that the three recombinant proteins could be specifically recognized by PEDV positive serum and had good antigenicity. It can be used as diagnostic antigen for the specific detection of PEDV infection. The establishment of indirect ELISA antibody detection method of 3.PEDV N protein using purified recombinant PEDV N protein as coating antigen to optimize the reaction conditions. An indirect ELISA method was established for the detection of antibody to N protein in serum of PEDV. The optimal reaction conditions were as follows: the optimal coating concentration of N protein was 0.5?g/m L, the optimal coating time was 4 鈩，
本文编号：2429089
[Abstract]:Porcine epidemic diarrhea (Porcine epidemic diarrhea,PED) is an acute and highly contagious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus,PEDV). It is characterized by diarrhea, vomiting, dehydration and so on. Great losses have been caused to the pig industry in our country. The clinical symptoms of the disease are similar to those of porcine infectious gastroenteritis and rotavirus disease, so it is difficult to differentiate diagnosis. In order to explore the molecular epidemiological characteristics of PEDV and to establish an effective diagnostic method, the following aspects were carried out in this study: genetic variation analysis of major structural genes of 1.PEDV was conducted in Beijing, Henan, Shaanxi, Guangdong from 2013-2014. (PED) positive venereal materials collected from five provinces and cities of Shandong Province were cloned and sequenced by designing specific primers. The sequence changes and genetic variations of the structural gene were compared with the main strains at home and abroad. The results of sequence alignment showed that one strain was virulent, and the other four strains had different S genes from domestic vaccine strain CV777. 5 amino acid insertions (59QGVN62 and 140N) and 2 amino acid deletions (163NI164) were found in the amino acid sequence of S gene, and 7 amino acid mutations were found in the two neutralization epitopes (499~638aa and 764~771aa) of S1 region. The nucleotide sequence of N gene is relatively conserved and only some amino acid mutations exist. The results of genetic evolution analysis showed that the homology of the four strains with the main Asian vaccine strains (Chinese CV777, Korean attenuated vaccine DR13 and Japanese attenuated vaccine 83P-5) was lower (93.880% and 94.7%) and the phylogenetic relationship between the four strains was far away from that of the Asian main vaccine strains (Chinese vaccine strain CV777, Korean attenuated vaccine strain and Japanese attenuated vaccine strain 83P-5). There was a high homology (99.6%) with the Korean strain from 2007 to 2009, the Japanese strain from 2011 and the epidemic strain from China in recent years (96.00.99. 6%). The expression and antigenicity analysis of S1 fusion protein were used to clone Mon N and S1 genes of BJ-1 into the transformant BL21, of prokaryotic expression vector p GEX-6p-1,. The results of SDS-PAGE electrophoresis showed that Mon was expressed by IPTG. S1 recombinant protein was expressed in 50 KDa-80KDa-110KDa. the results of. Western blot analysis showed that the three recombinant proteins could be specifically recognized by PEDV positive serum and had good antigenicity. It can be used as diagnostic antigen for the specific detection of PEDV infection. The establishment of indirect ELISA antibody detection method of 3.PEDV N protein using purified recombinant PEDV N protein as coating antigen to optimize the reaction conditions. An indirect ELISA method was established for the detection of antibody to N protein in serum of PEDV. The optimal reaction conditions were as follows: the optimal coating concentration of N protein was 0.5?g/m L, the optimal coating time was 4 鈩，
本文编号：2429089
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