牛源结核分枝杆菌复合群的分离鉴定及其分子进化研究

发布时间：2018-11-05 12:17

【摘要】：结核分枝杆菌及牛分枝杆菌是结核分枝杆菌复合群的主要成员,是引起人和动物结核病的主要病原体。结核分枝杆菌主要感染人,而牛分枝杆菌宿主更广,主要感染牛,也可感染其它家畜、鹿、獾等野生动物以及人。结核分枝杆菌感染牛群并可在牛间传播已被证实。但长期以来,人们认为结核分枝杆菌对牛无致病性,但一直存在争论。同时,结核分枝杆菌在牛群中的流行程度,也有待评估。结核分枝杆菌复合群菌株在基因组水平高度相似,达到99.9%。基于差异区的PCR和基于直接重复区的Spoligotyping等分型方法的发展使细分临床分离菌的类型成为可能。基于差异区的PCR分型方法通过扩增分枝杆菌的特异序列及复合群菌株间的差异基因,以鉴定和区分分枝杆菌,基于直接重复区的Spoligotyping等分型方法则能对复合群菌株实施进一步分析。高通量测序技术在基因组学研究方面得到应用后,可以提供更丰富的基因信息,使得在基因组水平上分析菌株间的表型差异成为可能,同时也能为药物开发和疫苗研究等提供数据支持。本课题通过从牛源样品中分离分枝杆菌并对其进行分型鉴定,以调查牛群中结核分枝杆菌的分子流行病学情况。并完成了一株牛源结核分枝杆菌北京型分离株1458株的全基因组测序与比较分析,以期从基因组水平上找得结核分枝杆菌感染牛的分子依据,为深入研究结核分枝杆菌对牛的致病性提供理论数据支持。论文主要得到以下几个方面的结果:1.从497份PPD阳性牛的鼻拭子和O-P液样品中,分离得到5株分枝杆菌,2.完成了24株结核病人分离株的鉴定,PCR分型鉴定结果为23株结核分枝杆菌、Spoligotyping分型鉴定结果为22株结核分枝杆菌,其中北京型19株,占总数86.4%。与Spol DB4数据库比较,17株北京型菌株(000000000003771)为ST1型,2株北京型菌株(000000000003731)为ST190型,另外3株(775767577740771)未找到对应的类型。说明北京型在国内仍是导致结核病的优势种。3.1485株全基因测序及注释本研究利用454 GS-FLX测序方法对1458株进行了全基因组测序工作,后续通过人工填补和序列拼接,完成了其环状基因组图绘制。1458株基因组大小4402033bp,G+C含量为65.6%,测序覆盖度达到29倍,准确度99.99%。1458株注释分析出4349个ORFs,包括3130个有具有生物学功能的基因及1219个假定蛋白质,其中2898个蛋白质能匹配到COG功能聚类。插入元件预测发现57个可信的插入序列,其中IS6110有21个。IS6110在基因组中拷贝数与插入位置的多态性,为研究1458株的致病特性提供依据。4.进化分析从NCBI数据库中获得已完成全基因组测序的结核分枝杆菌、牛分枝杆菌和BCG菌株的基因组信息,通过同源基因聚类分析,再将各菌株的同源基因串联,最终绘制出系统发育树图。结果表明,1458株与国内已公布的北京型分离株CCDC5079与CCDC5180关系最近。5.比较基因组学分析完成了结核分枝杆菌M.tb 1458株、M.tb H37Rv株和牛分枝杆菌M.bovis AF2122_97株的泛基因组分析,其中核心基因簇共3562个,覆盖了结核分枝杆菌生长所必需的所有基因。非同义SNP影响的基因在KEGG通路的富集分析发现,H37Rv株存在一个特异的ace Aa基因。该基因由ace A基因突变而形成,ace A基因编码的异柠檬酸裂合酶参与脂代谢过程并介导广泛的抗生素耐受,推测其可能是影响结核分枝杆菌在牛群传播的一个潜在因素。北京型菌株的SNP比较发现,NC_021054(M.tb Beijing/NITR203)、NC_017523(M.tb CCDC5079)和M.tb 1458株相比于NC_017522(M.tb CCDC5180)和NC_021251(M.tb CCDC5079),存在更多的突变基因,而M.tb Beijing/NITR203则有更多特异的SNP。结果与进化树分析一致。
[Abstract]:Mycobacterium tuberculosis and Mycobacterium bovis are the main members of the Mycobacterium tuberculosis complex, and are the major pathogens causing tuberculosis in humans and animals. Mycobacterium tuberculosis is the main infectious person, while the host of Mycobacterium bovis is more extensive, mainly infected with cattle, but also can be infected with wild animals such as livestock, deer, deer and other wild animals as well as people. Mycobacterium tuberculosis is infected with cattle and can be transmitted between cattle and confirmed. However, it has long been argued that Mycobacterium tuberculosis has no pathogenicity to cattle, but there has been controversy. At the same time, the prevalence of Mycobacterium tuberculosis in cattle remains to be assessed. Mycobacterium tuberculosis complex group strain was similar to the genome level, reaching 99.9%. The development of PCR based on the difference region and the isolation method based on the direct repeat region makes it possible to segment the type of clinically isolated bacteria. According to the PCR typing method based on the difference region, the difference genes between the specific sequence of the mycobacterium and the composite group strain are amplified to identify and differentiate the mycobacteria, and further analysis of the composite group strain can be carried out on the basis of the typing method of the direct repeat region. Since the high-throughput sequencing technology has been applied in genomics research, more abundant gene information can be provided so that phenotypic differences between strains can be analyzed at the genome level, and data support can be provided for drug development and vaccine research. Objective To investigate the molecular epidemiology of Mycobacterium tuberculosis in cattle by separating and identifying mycobacteria from bovine serum samples. The whole genome sequencing and comparative analysis of 1458 strains of Mycobacterium tuberculosis of Mycobacterium tuberculosis were carried out, with a view to finding the molecular basis of Mycobacterium tuberculosis infected cattle from the genome level, and to provide theoretical data support for further study of the pathogenicity of Mycobacterium tuberculosis to cattle. The paper mainly gets the following results: 1. Five strains of M.tuberculosis were isolated from 497 PPD-positive bovine nasal swab and O-P fluid samples. Twenty-four isolates of tuberculosis were identified. The results of PCR typing were 23 strains of Mycobacterium tuberculosis and 22 strains of Mycobacterium tuberculosis. Among them, 19 strains were Beijing, accounting for 86.4% of total. 17 strains of Beijing type (000000000003771) were ST1 and 2 strains of Beijing type (000000000003731) were ST190 and 3 (775767577740771) did not find the corresponding type. The results showed that Beijing type was still the dominant species leading to tuberculosis in China. The whole genome sequencing of 1458 strains was carried out by 454 GS-FLX sequencing method. The genome size of 1458 strain was 2033bp. The G + C content was 65. 6%, the sequencing coverage reached 29 times, and the accuracy was 99. 99%. 1458 strain notes analyzed 4349 ORFs, including 3130 genes with biological function and 1219 putative proteins, of which 2898 proteins were able to match the COG functional group. The insertion element prediction found 57 credible insertion sequences, of which IS6110 had 21. IS6110 polymorphism of copy number and insertion position in the genome provides a basis for the study of the pathogenic characteristics of 1458 strains. The genome information of Mycobacterium tuberculosis, Mycobacterium bovis and BCG strain with complete genome sequencing was obtained from the NCBI database, the homologous gene cluster analysis was carried out, the homologous gene of each strain was connected in series, and a phylogenetic tree diagram was finally drawn. The results showed that the relationship between CCDC5079 and CCDC5180 in 1458 strains of Beijing-type isolates was closest to that of CCDC5180. Comparative genomics analysis completed the pangenome analysis of M.bovis AF2122 _ 97 strain M.bovis AF2122 _ 97, M.bovis AF2122 _ 97 strain M.bovis AF2122 _ 97, which covered all the genes necessary for the growth of M. bovis AF2122 _ 97. The enrichment and analysis of non-synonymous SNP in KEGG pathway revealed that there was a specific ace Aa gene in H37Rv strain. The gene is formed by the ace A gene mutation, and the ace A gene-encoded isocitrate lyase is involved in the lipid metabolism process and mediates a wide range of antibiotic tolerance, presumably which may be a potential factor that affects the spread of Mycobacterium tuberculosis in the herd. Compared with NC _ 017522 (M., CCDC5079) and NC _ 021251 (M. CCDC5079), there were more mutational genes in NC _ 017522 (M., CCDC5079) and NC _ 021251 (M. CdCCDC5079) compared with NC _ 017522 (M., CCDC5079) and NC _ 021251 (M.CCDC5079). The results were consistent with the tree analysis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.618

【相似文献】