利用分子遗传标记技术对鸡鲍氏志贺菌的鉴定和菌株起源研究
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摘要
志贺菌为革兰氏阴性兼性细胞内致病菌,临诊上可以引起人畜痢疾。志贺菌属包括4个种群:宋内氏(Shigella sonnei)、福氏(Shigella flexneri)、鲍氏(Shigella boydil)和痢疾志贺氏(Shigella dysenteriae)。按照细菌脂多糖O抗原结构的差异,除宋内氏以外,其它3个种群又可分成若干血清型。在不同的种群间,甚至在同一种群的不同血清型之间,没有明显的交叉保护。作为猕猴肠道中的主要致病菌,志贺菌除可引起猕猴100%发病率、60%以上死亡外,还可感染犊牛、仔猪、小鼠、豚鼠。2004年许兰菊等发现首例鸡志贺菌病例,并对其致病性、微生物学特性、血清流行病学等进行了初步研究。为了进一步了解其分子生物学特性,追溯其起源,为其致病机理研究奠定基础,并初步确定其在公共卫生方面的意义,本文对鸡志贺菌原始分离株开展了下列内容的研究并取得预期结果:
1.利用6条随机引物以随机扩增多态性DNA(RAPD)技术对鸡鲍氏志贺菌、鸡白痢沙门菌、鸡大肠杆菌和痢疾志贺菌进行了鉴别。结果表明,有4条随机引物能较好地在这4种菌中检测到多态性分子标记:4条有效引物共扩增出了64个DNA片段,其中4个菌株共有的谱带有3条,而显示多态性的片段有61条,占95.3%。引物P6的扩增图谱清晰且差异明显,鸡鲍氏志贺菌与人痢疾志贺菌的扩增条带相同率较高达67%,而与鸡致病性大肠杆菌的扩增条带相同率仅为44.4%,与鸡白痢沙门菌的扩增谱带相差甚远,因而可用于三种细菌的鉴别。P4也可用于种的鉴别,但效果稍次于P6。引物P2扩增的志贺菌与大肠杆菌谱带十分相似,但与沙门菌谱带相差很远,因此可将沙门菌鉴别出来,而无法区分志贺菌与大肠杆菌。采用SPSS软件分析了不同细菌间的遗传距离为0.128～0.790,根据遗传距离可将鸡志贺菌等7株菌株分为3个聚类群,鸡志贺菌分离株位于人志贺菌类群中。对克隆的扩增片段测序及同源分析结果显示:个别扩增条带尽管大小一致,实际上所包含基因却还存在差异,也在一定程度上说明RAPD方法存在一定的局限性。另一方面也从基因水平上分析了鸡鲍氏志贺菌的起源的多样性,从而进一步证明志贺菌菌株不存在单一的进化来源,并根据同源关系和进化关系进一步确定鸡鲍氏志贺菌可能是人志贺菌的的一个变异株或新的亚种。
2.建立鸡志贺菌的16S rRNA、gyrB、grpE、recA等四个基因序列(序列登记号分别为DQ229902,DQ995255,DQ995256,DQ995254)的特异分析方法,从分子水平上鉴定鸡鲍氏志贺菌,并分析了该菌株的系统发育起源。结果显示鸡志贺菌与人鲍氏志贺菌同源率达99.6%～100%,几乎所有志贺菌均落入大肠杆菌的分支中。同时发现在进行细菌鉴别时,recA基因和gyrB基因差异比传统方法中使用的16S rRNA基因差异更显著。因而用16S rRNA联合gyrB、grpE、recA三基因进行序列分析来鉴定志贺菌是一种快速、可靠、成本较低的方法。由16S rRNA等四个基因序列构建的系统发育树提示,鸡志贺菌是与人志贺菌密切相关的新种或是它们的亚种。
3.根据GeneBank中登录的志贺菌的八个管家基因thrB/thrC,trpC/trpB,purM/purN,mdh/argR的相应序列,分别设计8对引物。成功地对八个目的基因进行了克隆测序,序列已经登录GeneBank(按照上述顺序登录号分别为DQ995264,DQ995265,DQ995261,DQ995263,DQ995260,DQ995258,DQ995259,DQ995257)。利用BLAST和ClustalX程序分析测序结果,并构建了八个管家基因在染色体上4个区域的相应遗传进化树;由系统进化树可知:志贺菌分为三个主要分支,且均落在大肠杆菌中,鸡鲍氏志贺菌八个管家基因在染色体上4个区域的进化树中分布位置是一致的,均落在志贺菌的第一分支;从整个基因水平上看,鸡鲍氏志贺菌应起源于人志贺菌。通过年龄分析发现,与其它志贺菌相似,该株鸡鲍氏志贺菌的祖先约起源于50,000-270,000年以前。
4.以鸡鲍氏志贺菌为研究对象,分别根据文献报道的O抗原基因簇通用引物以及自行设计的两鉴定基因galF,gnd的相应引物,用长程PCR扩增O抗原基因簇。结果成功地扩增与克隆了O抗原基因簇,并进一步完成了序列测定。同时利用galF基因和GeneBank中的相关序列构建进化树,进一步对鸡志贺菌进行了进化分析,结果显示鸡志贺菌与人鲍氏志贺菌4型(CP000036)的进化关系最近,更近一步显示鸡志贺菌在起源于人鲍氏志贺菌。O抗原基因簇序列的测定为鸡鲍氏志贺菌致病性研究奠定了基础。
Shigella are Gram negative bacilli, and multiply within colonic epithelial cells, which can cause Shigellosis of human and animal. Shigella spp. can be subdivided into four serogroups namely S.sonnei, S.boydii, S.flexneri and S.dysenteriae. These species are subdivided into serotypes on the basis of O-specific polysaccharide of the LPS. There aren't obvious cross- protective among different serogroups or serotypes. Shigella spp. is an main enterobacterium of rhesus monkey causing bacillary dysentery. The significance of the organism in public health has been questioned since the first report of clinical case from chicken infected with shigella spp. by XU Lanju et al. in 2004. In this paper, Shigella boydii isolated from chicken was studied as follows.
1. The genetic diversity among strains of Shigella boydii isolated from chicken, Shigella Dysenteriae, Enteropathogenic E.coli and S. Pullorum was analysed by random amplified polymorphic DNA (RAPD) with 6 random primers. The results shown that four of six 10 bp random primers were efficient to yield PCR product. Sixty four RAPD bands were identified and 3 of them were common in all four strains tested and 61 of them were polymorphic between any two strains. The polymorphic bands accounted for 95.3% of the total amplified bands. The primer P2 could be used to differentiate chicken Shigella boydii and S. Pullorum from other bacterial strains. The result also shown that the common rate of the amplified bands between Shigella boydii and Shigella dysenteriae was 67%, and that between Shigella boydii and Enteroinvasive E.coli was 44.4%. The amplified bands from Shigella and S. Pullorum were much different from each other when primer P6 was used, and this indicated the primer p6 could be used to differentiate different species. The heredity distance of 7 bacterial is 0.128-0.790 which was got by SPSS11.5. According to the heredity distance, 7 strains can be divided into 3 clustering groups. Chicken Shigella boydii and other two Shigella from human were in a same cluster. The sequences of PCR products and evolutionary relationship analysis showed that some PCR products included different genes though they were the same in size, which revealed that RAPD is a local method to a certain extent to differentiate bacteria species. The diversity origin of Chicken Shigella boydii was analysed on gene level, which confirmed Shigella evolved not with a single evolutionary origin. The phylogenetic tree constructed by these four genes revealed that the strain isolated from chicken maybe a new species or subspecies.
2. Sequence analysis of 16S rRNA, gyrB, grpE, recA genes(accession nos. DQ229902, DQ995255, DQ995256, DQ995254) of Shigella was conducted and phylogenetic origins were analysised for the farther identification of chicken Shigella boydii. Results shown that the nearest homogeneity with chicken Shigella boydii is Shigella boydii isolated from human and the homogeneity is 99.6%～100%, even if almost all Shigella strains are distributed among E.coli strains. All sequences of four genes above from Shigella spp. strains are not identical to that from E.coli strains. On the other hand, recA gene and gyrB gene indicated a greater evolutionary divergence than 16S rRNA genes. It's concluded that using 16S rRNA and gyrB, grpE, recA gene sequences analysis may be a reliable, rapid, and low costs way for identification of Shigella strains. The phylogenetic tree constructed by these four genes revealed that the strain isolated from chicken maybe a new species or subspecies.
3. According to the related sequences of Shigella deposited in the GenBank database, eight pairs of specific primers for eight housekeeping genes were designed and used to amplify thrB/thrC, trpC/trpB, purM/purN and mdh/argR genes of Shigella boydii successfully. The PCR products were cloned into PMD18-T vector and sequenced. The sequences reported in this paper have been deposited in the GenBank database (accession nos. DQ995264, DQ995265, DQ995261, DQ995263, DQ995260, DQ995258, DQ995259, DQ995257). By using the BLAST and Clustalx software, the sequences were analyzed and the phyligenetic tree for eight housekeeping genes in four regions of chromosome was established. Three clusters of Shigella strains were identified and nonetheless, are clearly within Escherichia coli. The locations of eight housekeeping genes in chicken Shigella boydii are identically distributed in four regions in chromosome, which are all contained in cluster 1. Chicken Shigella boydii probably originated from human Shigella boydii. It is estimated that the isolated strain maybe have evolved about 50,000-270,000 years.
4. According to the related references, some specific primers for O antigen gene cluster and galF, gnd genes were designed and used to amplify O antigen genes cluster by using long PCR system. The results show O antigen gene cluster was amplified and cloned successfully. The recombinant plasmid was sequenced after identified and the sequences have been deposited in the GenBank database. On the other hand, the sequene of galF gene obtained in this paper was compared with the related sequences reported in GeneBank and phylogenetic tree were constructed. Results showed that Shigetta boydii isolated from chicken has the nearest homogeneity with Shigella boydii 4 isolated from human (CP000036), so Shigella boydii isolated from chicken is probably originated from Shigella boydii isolated from human. Some pathogenic of Shigella boydii isolated from chicken can be studied based Sequence of O antigen genes cluster.
1.利用6条随机引物以随机扩增多态性DNA(RAPD)技术对鸡鲍氏志贺菌、鸡白痢沙门菌、鸡大肠杆菌和痢疾志贺菌进行了鉴别。结果表明,有4条随机引物能较好地在这4种菌中检测到多态性分子标记:4条有效引物共扩增出了64个DNA片段,其中4个菌株共有的谱带有3条,而显示多态性的片段有61条,占95.3%。引物P6的扩增图谱清晰且差异明显,鸡鲍氏志贺菌与人痢疾志贺菌的扩增条带相同率较高达67%,而与鸡致病性大肠杆菌的扩增条带相同率仅为44.4%,与鸡白痢沙门菌的扩增谱带相差甚远,因而可用于三种细菌的鉴别。P4也可用于种的鉴别,但效果稍次于P6。引物P2扩增的志贺菌与大肠杆菌谱带十分相似,但与沙门菌谱带相差很远,因此可将沙门菌鉴别出来,而无法区分志贺菌与大肠杆菌。采用SPSS软件分析了不同细菌间的遗传距离为0.128～0.790,根据遗传距离可将鸡志贺菌等7株菌株分为3个聚类群,鸡志贺菌分离株位于人志贺菌类群中。对克隆的扩增片段测序及同源分析结果显示:个别扩增条带尽管大小一致,实际上所包含基因却还存在差异,也在一定程度上说明RAPD方法存在一定的局限性。另一方面也从基因水平上分析了鸡鲍氏志贺菌的起源的多样性,从而进一步证明志贺菌菌株不存在单一的进化来源,并根据同源关系和进化关系进一步确定鸡鲍氏志贺菌可能是人志贺菌的的一个变异株或新的亚种。
2.建立鸡志贺菌的16S rRNA、gyrB、grpE、recA等四个基因序列(序列登记号分别为DQ229902,DQ995255,DQ995256,DQ995254)的特异分析方法,从分子水平上鉴定鸡鲍氏志贺菌,并分析了该菌株的系统发育起源。结果显示鸡志贺菌与人鲍氏志贺菌同源率达99.6%～100%,几乎所有志贺菌均落入大肠杆菌的分支中。同时发现在进行细菌鉴别时,recA基因和gyrB基因差异比传统方法中使用的16S rRNA基因差异更显著。因而用16S rRNA联合gyrB、grpE、recA三基因进行序列分析来鉴定志贺菌是一种快速、可靠、成本较低的方法。由16S rRNA等四个基因序列构建的系统发育树提示,鸡志贺菌是与人志贺菌密切相关的新种或是它们的亚种。
3.根据GeneBank中登录的志贺菌的八个管家基因thrB/thrC,trpC/trpB,purM/purN,mdh/argR的相应序列,分别设计8对引物。成功地对八个目的基因进行了克隆测序,序列已经登录GeneBank(按照上述顺序登录号分别为DQ995264,DQ995265,DQ995261,DQ995263,DQ995260,DQ995258,DQ995259,DQ995257)。利用BLAST和ClustalX程序分析测序结果,并构建了八个管家基因在染色体上4个区域的相应遗传进化树;由系统进化树可知:志贺菌分为三个主要分支,且均落在大肠杆菌中,鸡鲍氏志贺菌八个管家基因在染色体上4个区域的进化树中分布位置是一致的,均落在志贺菌的第一分支;从整个基因水平上看,鸡鲍氏志贺菌应起源于人志贺菌。通过年龄分析发现,与其它志贺菌相似,该株鸡鲍氏志贺菌的祖先约起源于50,000-270,000年以前。
4.以鸡鲍氏志贺菌为研究对象,分别根据文献报道的O抗原基因簇通用引物以及自行设计的两鉴定基因galF,gnd的相应引物,用长程PCR扩增O抗原基因簇。结果成功地扩增与克隆了O抗原基因簇,并进一步完成了序列测定。同时利用galF基因和GeneBank中的相关序列构建进化树,进一步对鸡志贺菌进行了进化分析,结果显示鸡志贺菌与人鲍氏志贺菌4型(CP000036)的进化关系最近,更近一步显示鸡志贺菌在起源于人鲍氏志贺菌。O抗原基因簇序列的测定为鸡鲍氏志贺菌致病性研究奠定了基础。
Shigella are Gram negative bacilli, and multiply within colonic epithelial cells, which can cause Shigellosis of human and animal. Shigella spp. can be subdivided into four serogroups namely S.sonnei, S.boydii, S.flexneri and S.dysenteriae. These species are subdivided into serotypes on the basis of O-specific polysaccharide of the LPS. There aren't obvious cross- protective among different serogroups or serotypes. Shigella spp. is an main enterobacterium of rhesus monkey causing bacillary dysentery. The significance of the organism in public health has been questioned since the first report of clinical case from chicken infected with shigella spp. by XU Lanju et al. in 2004. In this paper, Shigella boydii isolated from chicken was studied as follows.
1. The genetic diversity among strains of Shigella boydii isolated from chicken, Shigella Dysenteriae, Enteropathogenic E.coli and S. Pullorum was analysed by random amplified polymorphic DNA (RAPD) with 6 random primers. The results shown that four of six 10 bp random primers were efficient to yield PCR product. Sixty four RAPD bands were identified and 3 of them were common in all four strains tested and 61 of them were polymorphic between any two strains. The polymorphic bands accounted for 95.3% of the total amplified bands. The primer P2 could be used to differentiate chicken Shigella boydii and S. Pullorum from other bacterial strains. The result also shown that the common rate of the amplified bands between Shigella boydii and Shigella dysenteriae was 67%, and that between Shigella boydii and Enteroinvasive E.coli was 44.4%. The amplified bands from Shigella and S. Pullorum were much different from each other when primer P6 was used, and this indicated the primer p6 could be used to differentiate different species. The heredity distance of 7 bacterial is 0.128-0.790 which was got by SPSS11.5. According to the heredity distance, 7 strains can be divided into 3 clustering groups. Chicken Shigella boydii and other two Shigella from human were in a same cluster. The sequences of PCR products and evolutionary relationship analysis showed that some PCR products included different genes though they were the same in size, which revealed that RAPD is a local method to a certain extent to differentiate bacteria species. The diversity origin of Chicken Shigella boydii was analysed on gene level, which confirmed Shigella evolved not with a single evolutionary origin. The phylogenetic tree constructed by these four genes revealed that the strain isolated from chicken maybe a new species or subspecies.
2. Sequence analysis of 16S rRNA, gyrB, grpE, recA genes(accession nos. DQ229902, DQ995255, DQ995256, DQ995254) of Shigella was conducted and phylogenetic origins were analysised for the farther identification of chicken Shigella boydii. Results shown that the nearest homogeneity with chicken Shigella boydii is Shigella boydii isolated from human and the homogeneity is 99.6%～100%, even if almost all Shigella strains are distributed among E.coli strains. All sequences of four genes above from Shigella spp. strains are not identical to that from E.coli strains. On the other hand, recA gene and gyrB gene indicated a greater evolutionary divergence than 16S rRNA genes. It's concluded that using 16S rRNA and gyrB, grpE, recA gene sequences analysis may be a reliable, rapid, and low costs way for identification of Shigella strains. The phylogenetic tree constructed by these four genes revealed that the strain isolated from chicken maybe a new species or subspecies.
3. According to the related sequences of Shigella deposited in the GenBank database, eight pairs of specific primers for eight housekeeping genes were designed and used to amplify thrB/thrC, trpC/trpB, purM/purN and mdh/argR genes of Shigella boydii successfully. The PCR products were cloned into PMD18-T vector and sequenced. The sequences reported in this paper have been deposited in the GenBank database (accession nos. DQ995264, DQ995265, DQ995261, DQ995263, DQ995260, DQ995258, DQ995259, DQ995257). By using the BLAST and Clustalx software, the sequences were analyzed and the phyligenetic tree for eight housekeeping genes in four regions of chromosome was established. Three clusters of Shigella strains were identified and nonetheless, are clearly within Escherichia coli. The locations of eight housekeeping genes in chicken Shigella boydii are identically distributed in four regions in chromosome, which are all contained in cluster 1. Chicken Shigella boydii probably originated from human Shigella boydii. It is estimated that the isolated strain maybe have evolved about 50,000-270,000 years.
4. According to the related references, some specific primers for O antigen gene cluster and galF, gnd genes were designed and used to amplify O antigen genes cluster by using long PCR system. The results show O antigen gene cluster was amplified and cloned successfully. The recombinant plasmid was sequenced after identified and the sequences have been deposited in the GenBank database. On the other hand, the sequene of galF gene obtained in this paper was compared with the related sequences reported in GeneBank and phylogenetic tree were constructed. Results showed that Shigetta boydii isolated from chicken has the nearest homogeneity with Shigella boydii 4 isolated from human (CP000036), so Shigella boydii isolated from chicken is probably originated from Shigella boydii isolated from human. Some pathogenic of Shigella boydii isolated from chicken can be studied based Sequence of O antigen genes cluster.
引文
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