豬流行性腹瀉病毒主要結(jié)構(gòu)基因遺傳變異分析及ELISA檢測方法的建立
發(fā)布時間：2019-02-23 18:29
【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是由豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)引起的一種豬的急性、高度接觸性傳染病,以腹瀉、嘔吐、脫水等為主要臨床特征特征,給我國養(yǎng)豬業(yè)造成了巨大損失。臨床上該病與豬傳染性胃腸炎、輪狀病毒病癥狀相似,較難進(jìn)行鑒別診斷。為了探究PEDV的分子流行病學(xué)特點(diǎn)并建立有效的診斷方法,本研究主要開展了以下幾個方面的工作:1.PEDV主要結(jié)構(gòu)基因的遺傳變異分析2013-2014年于北京、河南、陜西、廣東、山東5個省市采集的豬流行性腹瀉(PED)陽性病料,設(shè)計(jì)特異性引物對結(jié)構(gòu)基因S、M、N進(jìn)行克隆及測序,與國內(nèi)外主要毒株進(jìn)行比較,分析序列變化和遺傳變異情況。序列比對結(jié)果顯示,1株為弱毒株,其余4個樣品株的S基因與國內(nèi)疫苗株CV777差異較大,突變主要存在于S1區(qū)。S基因氨基酸序列存在5個氨基酸的插入(59QGVN62 and 140N)和2個氨基酸的缺失(163NI164),S1區(qū)的2個中和表位(499~638aa和764~771aa)有7處氨基酸突變。M、N基因的核苷酸序列相對保守,只存在部分氨基酸突變。遺傳進(jìn)化分析結(jié)果顯示,4個樣品株與亞洲主要疫苗株(中國疫苗株CV777、韓國弱毒疫苗株DR13以及日本弱毒疫苗株83P-5)同源性較低(93.8%~94.7%),親緣關(guān)系較遠(yuǎn);與2007~2009年韓國毒株,2011年日本毒株以及中國近年流行毒株同源性較高(96.0%~99.6%),親緣關(guān)系密切。2.PEDV BJ-1株M、N、S1融合蛋白的表達(dá)和抗原性分析將樣品株BJ-1的M、N和S1基因克隆至原核表達(dá)載體p GEX-6p-1,轉(zhuǎn)化感受態(tài)細(xì)胞BL21,經(jīng)IPTG誘導(dǎo)表達(dá),SDS-PAGE電泳結(jié)果顯示M、N、S1重組蛋白獲得表達(dá),大小分別為50KDa、80KDa、110KDa,以包涵體表達(dá)形式為主。Western blot分析結(jié)果表明3種重組蛋白能被PEDV豬陽性血清特異性識別,具有良好的抗原性,可以作為診斷抗原用于特異性檢測PEDV感染。3.PEDV N蛋白間接ELISA抗體檢測方法的建立以純化重組PEDV N蛋白作為包被抗原,優(yōu)化各項(xiàng)反應(yīng)條件,初步建立了可以檢測PEDV血清中N蛋白抗體的間接ELISA檢測方法。本研究中最終優(yōu)化的反應(yīng)條件為:N蛋白的最佳包被濃度為0.5?g/m L,最佳包被時間為37℃2h后4℃過夜(10~16h),5%脫脂牛奶37℃封閉3h,血清樣品最佳稀釋度為1:400,37℃作用1h,兔抗豬酶標(biāo)二抗最佳稀釋度為1:40,000,37℃作用1h,抗體臨界值為OD450nm?0.40判為陽性,OD450nm0.40判為陰性。試驗(yàn)證明該方法具有較好的敏感性、特異性、重復(fù)性和符合率,表明PEDV N蛋白間接ELISA抗體檢測方法初步建立,可以用于豬PEDV血清抗體的檢測和流行病學(xué)調(diào)查。綜上所述,本研究分析了PEDV樣品株結(jié)構(gòu)基因中M、N、S的序列變化和遺傳變異情況,為研究PEDV毒株的變異情況提供理論參考;成功表達(dá)了M、N、S1重組蛋白,具有良好的反應(yīng)原性,為建立針對結(jié)構(gòu)蛋白的檢測方法奠定基礎(chǔ);用純化的重組N蛋白,成功建立了PEDV N蛋白間接ELISA抗體檢測方法,為PEDV的血清學(xué)診斷和流行病學(xué)調(diào)查提供了一種簡便的檢測手段。
[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，
本文編號：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，
本文編號：2429089
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2429089.html
最近更新
教材專著
