牛化脓隐秘杆菌性肺炎的病原分离鉴定及诊断方法研究
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摘要
近几年大规模牛场犊牛化脓性肺炎发病率呈上升趋势,经检测其病原以化脓性隐秘杆菌(A. pyogenes)为主。本研究旨在建立快速检测A.pyogenes的PCR诊断方法,并表达部分PLO蛋白,将其作为抗原,初步建立间接ELISA检测方法。
本研究将来源于不同地区的病料,经细菌分离和生化试验初步鉴定为Arcanobacterium,设计4对引物,第一对为16S rRNA通用引物用来扩增A.pyogenes16S rRNA,对分离株进行同源性比较并构建系统进化树;第二对为针对A. pyogenes 16S rRNA设计的特异性引物用来扩增特异性目的基因,建立PCR诊断方法;第三对为扩增PLO全基因引物,分析牛源PLO与GenBank公布的猪源PLO的同源性;第四对针对PLO的生物活性区域设计引物,扩增部分PLO,构建表达载体pQE30-PLO,通过Western Blotting和协同溶血试验(CAMP)检测蛋白生物学活性,并用该蛋白作为抗原,建立间接ELISA检测方法。
从不同地区患有化脓性肺炎的犊牛体内分离到6株菌株,扩增16S rRNA得到1490~1492bp的目的基因,经Blast分析证实分离菌株是A. pyogenes。6株分离株之间同源性在99.7 %~100%。经系统进化分析,6株分离株均为A. pyogenes。特异性引物扩增目的基因大小为927bp,特异性试验结果表明6株分离株均能扩增出927 bp的目的基因,而其他细菌扩增结果均为阴性。敏感性试验结果表明,PCR最低检出量为42个A. pyogenes。扩增牛源PLO大小为1649bp,与GenBank公布的猪源PLO同源性为97.5%。A. pyogenes PLO生物活性区域扩增目的基因大小为585bp,重组蛋白经SDS-PAGE分析,可以在XL1-blue宿主菌中表达,蛋白大小为27.45 kDa。经Western Blotting检测,重组蛋白具有良好的反应原性,用溶血试验检测,重组蛋白具有溶血活性。用建立的间接ELISA检测方法检测以化脓为主的P.multocida、E.coli、S.aureus及S. pneumoniae等四种细菌抗血清,均未发现交叉反应,表明特异性良好,对临床样本进行检测,符合率为86.67%。
本研究成功建立了A. pyogenes PCR诊断方法,为A. pyogenes引起的牛化脓性肺炎的快速诊断及流行病学调查提供了新的手段。同时初步构建了A. pyogenes重组原核表达载体pQE30-PLO585,以该重组蛋白作为包被抗原初步建立了间接ELISA检测方法,为进一步完善A. pyogenes临床监测方法提供了新手段,为疫苗的开发研究奠定了基础。
In recent years, the morbility of bovines suppurative pneumonia has been increasing by Arcanobacterium pyogenes infection. This study was to establish a rapid PCR diagnostic method and to analyze the activity of PLO and indirect ELISA method aginst Arcanobacterium pyogenes.
The clinical samples isolated from different region in Heilongjiang Province, were analyzed by using the bacterial isolated culture and biochemical characteristic, which were identified with Arcanobacterium infection. four pairs of primers were synthesized to conduct further analysis and identification. The first pair of common primers was were used to amplify 16S rRNA, analysised homology and constructing phylogenetic tree;The second pair was were used to amplify a specific gene and established PCR diagnostic methods; The third pairs primers were used to amplify PLO and analyzed homology; The fourth pairs was were used to amplify the activity region of PLO, the target gene was cloned into expression vector PQE-30. Biological activity was identified by Western Blotting and CAMP assay, and use the protein as an antigen, established of indirect ELISA detection methods.
A 1490 ~ 1492bp spedific fragment could be amplified among six isolates, which shared the identity between 99.7%~100%. Moreover, a specific 927 bp DNA fragment could be amplified from all the six isolates however, failed to be done from other bacterial species. The sensitivity of the PCR suggested that 42 A.pyogenes cells still could be detected by using method built in this study. The 1649bp full-lengh PLO was amplified sequenced, which shared 97.5% homology with swine PLO published in GenBank. The 585bp PLO biological activity region was amplified and a recombinant protein with 27.5kD molecular mass was expressed in E.coli XL1-blue host cells.. The result of Western blot showed the recombinant protein had wonderful reaction ogenicity. The result of CAMP showed the recombinant protein had hemolytic activity. The recombinant antigens had no cross reactions with sera of other four bacteria(P.multocida,E.coli,S.aureus,S. pneumoniae) showed that indirect ELISA methods were had highly specificity.Using the indirect ELISA detected clinical sample, positive rate was 86.67% .
PCR diagnostic method has established in this study, and provided a new means about bovine suppurative pneumonia for A.pyogenes. while A.pyogenes recombinant prokaryotic expression vector pQE-PLO585 was constructed in this study, the recombinant protein was used to establish an indirect ELISA method, a new means was provided in order to further perfect the clinical monitor methods and laied basis for vaccines study and development.
本研究将来源于不同地区的病料,经细菌分离和生化试验初步鉴定为Arcanobacterium,设计4对引物,第一对为16S rRNA通用引物用来扩增A.pyogenes16S rRNA,对分离株进行同源性比较并构建系统进化树;第二对为针对A. pyogenes 16S rRNA设计的特异性引物用来扩增特异性目的基因,建立PCR诊断方法;第三对为扩增PLO全基因引物,分析牛源PLO与GenBank公布的猪源PLO的同源性;第四对针对PLO的生物活性区域设计引物,扩增部分PLO,构建表达载体pQE30-PLO,通过Western Blotting和协同溶血试验(CAMP)检测蛋白生物学活性,并用该蛋白作为抗原,建立间接ELISA检测方法。
从不同地区患有化脓性肺炎的犊牛体内分离到6株菌株,扩增16S rRNA得到1490~1492bp的目的基因,经Blast分析证实分离菌株是A. pyogenes。6株分离株之间同源性在99.7 %~100%。经系统进化分析,6株分离株均为A. pyogenes。特异性引物扩增目的基因大小为927bp,特异性试验结果表明6株分离株均能扩增出927 bp的目的基因,而其他细菌扩增结果均为阴性。敏感性试验结果表明,PCR最低检出量为42个A. pyogenes。扩增牛源PLO大小为1649bp,与GenBank公布的猪源PLO同源性为97.5%。A. pyogenes PLO生物活性区域扩增目的基因大小为585bp,重组蛋白经SDS-PAGE分析,可以在XL1-blue宿主菌中表达,蛋白大小为27.45 kDa。经Western Blotting检测,重组蛋白具有良好的反应原性,用溶血试验检测,重组蛋白具有溶血活性。用建立的间接ELISA检测方法检测以化脓为主的P.multocida、E.coli、S.aureus及S. pneumoniae等四种细菌抗血清,均未发现交叉反应,表明特异性良好,对临床样本进行检测,符合率为86.67%。
本研究成功建立了A. pyogenes PCR诊断方法,为A. pyogenes引起的牛化脓性肺炎的快速诊断及流行病学调查提供了新的手段。同时初步构建了A. pyogenes重组原核表达载体pQE30-PLO585,以该重组蛋白作为包被抗原初步建立了间接ELISA检测方法,为进一步完善A. pyogenes临床监测方法提供了新手段,为疫苗的开发研究奠定了基础。
In recent years, the morbility of bovines suppurative pneumonia has been increasing by Arcanobacterium pyogenes infection. This study was to establish a rapid PCR diagnostic method and to analyze the activity of PLO and indirect ELISA method aginst Arcanobacterium pyogenes.
The clinical samples isolated from different region in Heilongjiang Province, were analyzed by using the bacterial isolated culture and biochemical characteristic, which were identified with Arcanobacterium infection. four pairs of primers were synthesized to conduct further analysis and identification. The first pair of common primers was were used to amplify 16S rRNA, analysised homology and constructing phylogenetic tree;The second pair was were used to amplify a specific gene and established PCR diagnostic methods; The third pairs primers were used to amplify PLO and analyzed homology; The fourth pairs was were used to amplify the activity region of PLO, the target gene was cloned into expression vector PQE-30. Biological activity was identified by Western Blotting and CAMP assay, and use the protein as an antigen, established of indirect ELISA detection methods.
A 1490 ~ 1492bp spedific fragment could be amplified among six isolates, which shared the identity between 99.7%~100%. Moreover, a specific 927 bp DNA fragment could be amplified from all the six isolates however, failed to be done from other bacterial species. The sensitivity of the PCR suggested that 42 A.pyogenes cells still could be detected by using method built in this study. The 1649bp full-lengh PLO was amplified sequenced, which shared 97.5% homology with swine PLO published in GenBank. The 585bp PLO biological activity region was amplified and a recombinant protein with 27.5kD molecular mass was expressed in E.coli XL1-blue host cells.. The result of Western blot showed the recombinant protein had wonderful reaction ogenicity. The result of CAMP showed the recombinant protein had hemolytic activity. The recombinant antigens had no cross reactions with sera of other four bacteria(P.multocida,E.coli,S.aureus,S. pneumoniae) showed that indirect ELISA methods were had highly specificity.Using the indirect ELISA detected clinical sample, positive rate was 86.67% .
PCR diagnostic method has established in this study, and provided a new means about bovine suppurative pneumonia for A.pyogenes. while A.pyogenes recombinant prokaryotic expression vector pQE-PLO585 was constructed in this study, the recombinant protein was used to establish an indirect ELISA method, a new means was provided in order to further perfect the clinical monitor methods and laied basis for vaccines study and development.
引文
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