華南地區(qū)豬流行性腹瀉病毒分子流行病學(xué)調(diào)查及其ELISA檢測方法的建立
發(fā)布時間：2019-07-05 13:05
【摘要】：自2010年10月PED在中國境內(nèi)爆發(fā)以來,已造成哺乳仔豬的大量死亡,帶來嚴(yán)重的經(jīng)濟(jì)損失。鑒于PED較高的致死率,自爆發(fā)以來華南地區(qū)養(yǎng)殖戶普遍采用疫苗免疫的方法來預(yù)防PED,結(jié)果是華南地區(qū)的PED自爆發(fā)以后一直呈持續(xù)感染狀態(tài),為了解其原因,本實(shí)驗(yàn)對2013~2014年華南地區(qū)各地市送檢的表現(xiàn)腹瀉癥狀的病料共214份進(jìn)行PEDV、TGEV和RotaV抗原檢測,選取30個PED陽性樣品對其M、ORF3和部分S基因進(jìn)行RT-PCR擴(kuò)增、克隆及測序,并對其序列變化、氨基酸位點(diǎn)變化、遺傳進(jìn)化關(guān)系進(jìn)行分析;根據(jù)GenBank中CV777序列設(shè)計一段表達(dá)部分S基因的引物,利用原核表達(dá)系統(tǒng)表達(dá)了S基因蛋白,并將蛋白純化后作為包被抗原建立檢測PEDV-Ab的間接ELISA方法,為檢測豬群中血清抗體提供方法�？乖瓩z測結(jié)果顯示PEDV平均陽性率為74.8%,TGEV的平均陽性率為7.0%,RotaV平均陽性率為12.1%;其中PEDV陽性率在夏季明顯高于冬春季節(jié)。序列比對、氨基酸位點(diǎn)變化、遺傳進(jìn)化關(guān)系分析結(jié)果顯示樣品毒株之間M基因同源性97.5%~100%,氨基酸同源性為97.3%~100%,氨基酸位點(diǎn)僅有個別突變,證明M基因高度保守,系統(tǒng)進(jìn)化分析顯示當(dāng)前毒株形成2個亞型,并與參考毒株的中國株親緣關(guān)系較近,說明當(dāng)前流行毒株基因起源于國內(nèi)的毒株;ORF3基因30個樣品之間核苷酸同源性為95.1%~100%,氨基酸同源性為96.4%~100%,本實(shí)驗(yàn)中30個毒株ORF3基因編碼225個氨基酸,沒有出現(xiàn)氨基酸缺失,在10~30和70~90這兩個區(qū)間氨基酸突變較多,其中有7個毒株在在第85位出現(xiàn)氨基酸突變(L→I),所有氨基酸序列沒有連續(xù)氨基酸突變,說明ORF3基因很保守,系統(tǒng)進(jìn)化分析顯示本實(shí)驗(yàn)毒株形成2個進(jìn)化分支,并且與華南地區(qū)之前分離株高度同源,說明華南地區(qū)毒株呈地方流行性;S基因序列比對發(fā)現(xiàn)30個樣品之間核苷酸同源性為96.5%~100%,與參考疫苗株同源性在88.3%~90.5%之間,說明S基因出現(xiàn)較大變異,系統(tǒng)進(jìn)化分析顯示華南地區(qū)流行毒株與泰國、越南參考毒株親緣關(guān)系較近。結(jié)果表明,PEDV在華南地區(qū)豬場普遍存在,其基因也在不斷的發(fā)生變化。參考GenBank中CV777毒株序列設(shè)計一對用于擴(kuò)增PEDV部分S基因的引物,用RT-PCR方法從豬流行性腹瀉陽性病料中擴(kuò)增目的片段,并將該含有抗原表位的目的片段克隆到原核表達(dá)載體pET32a中,構(gòu)建了包含部分S基因的原核表達(dá)載體pET32a-S,將驗(yàn)證正確的pET32a-S重組質(zhì)粒轉(zhuǎn)化至表達(dá)菌BL21中。包含重組質(zhì)粒的表達(dá)菌BL21在培養(yǎng)基加入IPTG后37℃的條件下進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE電泳結(jié)果顯示表達(dá)的目的蛋白為46 kDa,并且以包涵體蛋白形式存在。Western-blot分析表明,所表達(dá)的蛋白具有良好的反應(yīng)原性。以純化的S蛋白包被酶標(biāo)板建立了PEDV-Ab檢測的間接ELISA檢測方法,方陣滴定法確定抗原最佳包被濃度為2.5μg/mL,血清最佳稀釋度為1:50,二抗最佳稀釋度為1:6000,陽性判定標(biāo)準(zhǔn)為檢測樣品OD450值≥0.24。按照該方法建立的ELISA方法對隨機(jī)抽取的94份血清樣品檢測結(jié)果與商品化試劑盒檢測結(jié)果對比發(fā)現(xiàn),該方法檢測陽性率79.7%,與商品化試劑盒符合率符為87%。結(jié)果果表明本實(shí)驗(yàn)建立的間接ELISA方法可以作為一種有效的PEDV-Ab檢測方法用于臨床免疫監(jiān)測。
[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，
本文編號：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，
本文編號：2510543
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