华南地区猪流行性腹泻病毒分子流行病学调查及其ELISA检测方法的建立
发布时间：2019-07-05 13:05
【摘要】：自2010年10月PED在中国境内爆发以来,已造成哺乳仔猪的大量死亡,带来严重的经济损失。鉴于PED较高的致死率,自爆发以来华南地区养殖户普遍采用疫苗免疫的方法来预防PED,结果是华南地区的PED自爆发以后一直呈持续感染状态,为了解其原因,本实验对2013~2014年华南地区各地市送检的表现腹泻症状的病料共214份进行PEDV、TGEV和RotaV抗原检测,选取30个PED阳性样品对其M、ORF3和部分S基因进行RT-PCR扩增、克隆及测序,并对其序列变化、氨基酸位点变化、遗传进化关系进行分析;根据GenBank中CV777序列设计一段表达部分S基因的引物,利用原核表达系统表达了S基因蛋白,并将蛋白纯化后作为包被抗原建立检测PEDV-Ab的间接ELISA方法,为检测猪群中血清抗体提供方法。抗原检测结果显示PEDV平均阳性率为74.8%,TGEV的平均阳性率为7.0%,RotaV平均阳性率为12.1%;其中PEDV阳性率在夏季明显高于冬春季节。序列比对、氨基酸位点变化、遗传进化关系分析结果显示样品毒株之间M基因同源性97.5%~100%,氨基酸同源性为97.3%~100%,氨基酸位点仅有个别突变,证明M基因高度保守,系统进化分析显示当前毒株形成2个亚型,并与参考毒株的中国株亲缘关系较近,说明当前流行毒株基因起源于国内的毒株;ORF3基因30个样品之间核苷酸同源性为95.1%~100%,氨基酸同源性为96.4%~100%,本实验中30个毒株ORF3基因编码225个氨基酸,没有出现氨基酸缺失,在10~30和70~90这两个区间氨基酸突变较多,其中有7个毒株在在第85位出现氨基酸突变(L→I),所有氨基酸序列没有连续氨基酸突变,说明ORF3基因很保守,系统进化分析显示本实验毒株形成2个进化分支,并且与华南地区之前分离株高度同源,说明华南地区毒株呈地方流行性;S基因序列比对发现30个样品之间核苷酸同源性为96.5%~100%,与参考疫苗株同源性在88.3%~90.5%之间,说明S基因出现较大变异,系统进化分析显示华南地区流行毒株与泰国、越南参考毒株亲缘关系较近。结果表明,PEDV在华南地区猪场普遍存在,其基因也在不断的发生变化。参考GenBank中CV777毒株序列设计一对用于扩增PEDV部分S基因的引物,用RT-PCR方法从猪流行性腹泻阳性病料中扩增目的片段,并将该含有抗原表位的目的片段克隆到原核表达载体pET32a中,构建了包含部分S基因的原核表达载体pET32a-S,将验证正确的pET32a-S重组质粒转化至表达菌BL21中。包含重组质粒的表达菌BL21在培养基加入IPTG后37℃的条件下进行诱导表达,SDS-PAGE电泳结果显示表达的目的蛋白为46 kDa,并且以包涵体蛋白形式存在。Western-blot分析表明,所表达的蛋白具有良好的反应原性。以纯化的S蛋白包被酶标板建立了PEDV-Ab检测的间接ELISA检测方法,方阵滴定法确定抗原最佳包被浓度为2.5μg/mL,血清最佳稀释度为1:50,二抗最佳稀释度为1:6000,阳性判定标准为检测样品OD450值≥0.24。按照该方法建立的ELISA方法对随机抽取的94份血清样品检测结果与商品化试剂盒检测结果对比发现,该方法检测阳性率79.7%,与商品化试剂盒符合率符为87%。结果果表明本实验建立的间接ELISA方法可以作为一种有效的PEDV-Ab检测方法用于临床免疫监测。
[Abstract]:Since the outbreak of PED in China in October 2010, a large number of deaths and serious economic losses have been caused. In view of the high level of PED, since the outbreak, the farmers in South China have generally adopted the method of vaccine immunization to prevent the PED, and the result is that the PED in the South China has been continuously infected since the outbreak, so as to understand the reason, In this experiment,214 samples of the symptoms of diarrhea, such as PEDV, TGV and RotaV antigen, were detected in the cities of South China from 2013 to 2014, and 30 PED positive samples were selected to carry out RT-PCR amplification, cloning and sequencing of the M, ORF3 and partial S genes, and the sequence and amino acid sites were changed. The genetic evolution relationship was analyzed. A section of the S gene was designed according to the CV777 sequence in GenBank, the S gene protein was expressed by the prokaryotic expression system, and the protein was purified as a package. The indirect ELISA method for detecting the PEDV-Ab was used to provide a method for detecting the serum antibody in the herd. The results showed that the positive rate of PEDV was 74.8%, the average positive rate of TGEV was 7.0%, and the positive rate of RotaV was 12.1%. The positive rate of PEDV was significantly higher in the summer than in the winter and spring season. The results showed that the M gene of the sample strain was 97.5% ~ 100%, the homology of amino acid was 97.3% ~ 100%, the amino acid site was only mutated, and it was proved that the M gene was highly conserved. The evolution analysis of the system shows that the current strain forms two subtypes and is close to the Chinese strain of the reference strain, indicating that the current epidemic strain gene originated in the domestic strain, and the nucleotide homology between the ORF3 gene and the 30 samples is 95.1% to 100%, and the homology of the amino acid is 96.4% to 100%. in that experiment, the ORF3 gene encode 225 amino acid, no amino acid deletion, more amino acid mutation in the two interval between 10-30 and 70-90,7 of which have amino acid mutation (L-I) in the 85-th position, and all the amino acid sequences do not have continuous amino acid mutation, The results show that the ORF3 gene is very conservative, and the evolution of the system shows that the experimental strain forms two evolutionary branches and is highly homologous to the previous isolates in South China, indicating that the strain of South China is in a local epidemic; the S gene sequence is 96.5% to 100%, The homology of the reference vaccine strain is between 88.3% and 90.5%, indicating that the S gene has a large variation, and the phylogenetic analysis shows that the epidemic strain in South China is close to the reference strain of Thailand and Vietnam. The results show that the gene of PEDV is common in the South China, and its gene is changing continuously. a pair of primers for amplifying the S gene of the PEDV part are designed according to the sequence of the CV777 strain in the GenBank, the target fragment is amplified from the pig epidemic diarrhea positive disease material by the RT-PCR method, and the target fragment containing the antigen epitope is cloned into the prokaryotic expression vector pET32a, The prokaryotic expression vector pET32a-S containing the partial S gene was constructed, and the correct pET32a-S recombinant plasmid was transformed into the expression strain BL21. The expression of the recombinant plasmid was induced by IPTG at 37 鈩，
本文编号：2510543
[Abstract]:Since the outbreak of PED in China in October 2010, a large number of deaths and serious economic losses have been caused. In view of the high level of PED, since the outbreak, the farmers in South China have generally adopted the method of vaccine immunization to prevent the PED, and the result is that the PED in the South China has been continuously infected since the outbreak, so as to understand the reason, In this experiment,214 samples of the symptoms of diarrhea, such as PEDV, TGV and RotaV antigen, were detected in the cities of South China from 2013 to 2014, and 30 PED positive samples were selected to carry out RT-PCR amplification, cloning and sequencing of the M, ORF3 and partial S genes, and the sequence and amino acid sites were changed. The genetic evolution relationship was analyzed. A section of the S gene was designed according to the CV777 sequence in GenBank, the S gene protein was expressed by the prokaryotic expression system, and the protein was purified as a package. The indirect ELISA method for detecting the PEDV-Ab was used to provide a method for detecting the serum antibody in the herd. The results showed that the positive rate of PEDV was 74.8%, the average positive rate of TGEV was 7.0%, and the positive rate of RotaV was 12.1%. The positive rate of PEDV was significantly higher in the summer than in the winter and spring season. The results showed that the M gene of the sample strain was 97.5% ~ 100%, the homology of amino acid was 97.3% ~ 100%, the amino acid site was only mutated, and it was proved that the M gene was highly conserved. The evolution analysis of the system shows that the current strain forms two subtypes and is close to the Chinese strain of the reference strain, indicating that the current epidemic strain gene originated in the domestic strain, and the nucleotide homology between the ORF3 gene and the 30 samples is 95.1% to 100%, and the homology of the amino acid is 96.4% to 100%. in that experiment, the ORF3 gene encode 225 amino acid, no amino acid deletion, more amino acid mutation in the two interval between 10-30 and 70-90,7 of which have amino acid mutation (L-I) in the 85-th position, and all the amino acid sequences do not have continuous amino acid mutation, The results show that the ORF3 gene is very conservative, and the evolution of the system shows that the experimental strain forms two evolutionary branches and is highly homologous to the previous isolates in South China, indicating that the strain of South China is in a local epidemic; the S gene sequence is 96.5% to 100%, The homology of the reference vaccine strain is between 88.3% and 90.5%, indicating that the S gene has a large variation, and the phylogenetic analysis shows that the epidemic strain in South China is close to the reference strain of Thailand and Vietnam. The results show that the gene of PEDV is common in the South China, and its gene is changing continuously. a pair of primers for amplifying the S gene of the PEDV part are designed according to the sequence of the CV777 strain in the GenBank, the target fragment is amplified from the pig epidemic diarrhea positive disease material by the RT-PCR method, and the target fragment containing the antigen epitope is cloned into the prokaryotic expression vector pET32a, The prokaryotic expression vector pET32a-S containing the partial S gene was constructed, and the correct pET32a-S recombinant plasmid was transformed into the expression strain BL21. The expression of the recombinant plasmid was induced by IPTG at 37 鈩，
本文编号：2510543
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