【摘要】：結核分枝桿菌及牛分枝桿菌是結核分枝桿菌復合群的主要成員,是引起人和動物結核病的主要病原體。結核分枝桿菌主要感染人,而牛分枝桿菌宿主更廣,主要感染牛,也可感染其它家畜、鹿、獾等野生動物以及人。結核分枝桿菌感染牛群并可在牛間傳播已被證實。但長期以來,人們認為結核分枝桿菌對牛無致病性,但一直存在爭論。同時,結核分枝桿菌在牛群中的流行程度,也有待評估。結核分枝桿菌復合群菌株在基因組水平高度相似,達到99.9%�；诓町悈^(qū)的PCR和基于直接重復區(qū)的Spoligotyping等分型方法的發(fā)展使細分臨床分離菌的類型成為可能�；诓町悈^(qū)的PCR分型方法通過擴增分枝桿菌的特異序列及復合群菌株間的差異基因,以鑒定和區(qū)分分枝桿菌,基于直接重復區(qū)的Spoligotyping等分型方法則能對復合群菌株實施進一步分析。高通量測序技術在基因組學研究方面得到應用后,可以提供更豐富的基因信息,使得在基因組水平上分析菌株間的表型差異成為可能,同時也能為藥物開發(fā)和疫苗研究等提供數(shù)據(jù)支持。本課題通過從牛源樣品中分離分枝桿菌并對其進行分型鑒定,以調查牛群中結核分枝桿菌的分子流行病學情況。并完成了一株牛源結核分枝桿菌北京型分離株1458株的全基因組測序與比較分析,以期從基因組水平上找得結核分枝桿菌感染牛的分子依據(jù),為深入研究結核分枝桿菌對牛的致病性提供理論數(shù)據(jù)支持。論文主要得到以下幾個方面的結果:1.從497份PPD陽性牛的鼻拭子和O-P液樣品中,分離得到5株分枝桿菌,2.完成了24株結核病人分離株的鑒定,PCR分型鑒定結果為23株結核分枝桿菌、Spoligotyping分型鑒定結果為22株結核分枝桿菌,其中北京型19株,占總數(shù)86.4%。與Spol DB4數(shù)據(jù)庫比較,17株北京型菌株(000000000003771)為ST1型,2株北京型菌株(000000000003731)為ST190型,另外3株(775767577740771)未找到對應的類型。說明北京型在國內(nèi)仍是導致結核病的優(yōu)勢種。3.1485株全基因測序及注釋本研究利用454 GS-FLX測序方法對1458株進行了全基因組測序工作,后續(xù)通過人工填補和序列拼接,完成了其環(huán)狀基因組圖繪制。1458株基因組大小4402033bp,G+C含量為65.6%,測序覆蓋度達到29倍,準確度99.99%。1458株注釋分析出4349個ORFs,包括3130個有具有生物學功能的基因及1219個假定蛋白質,其中2898個蛋白質能匹配到COG功能聚類。插入元件預測發(fā)現(xiàn)57個可信的插入序列,其中IS6110有21個。IS6110在基因組中拷貝數(shù)與插入位置的多態(tài)性,為研究1458株的致病特性提供依據(jù)。4.進化分析從NCBI數(shù)據(jù)庫中獲得已完成全基因組測序的結核分枝桿菌、牛分枝桿菌和BCG菌株的基因組信息,通過同源基因聚類分析,再將各菌株的同源基因串聯(lián),最終繪制出系統(tǒng)發(fā)育樹圖。結果表明,1458株與國內(nèi)已公布的北京型分離株CCDC5079與CCDC5180關系最近。5.比較基因組學分析完成了結核分枝桿菌M.tb 1458株、M.tb H37Rv株和牛分枝桿菌M.bovis AF2122_97株的泛基因組分析,其中核心基因簇共3562個,覆蓋了結核分枝桿菌生長所必需的所有基因。非同義SNP影響的基因在KEGG通路的富集分析發(fā)現(xiàn),H37Rv株存在一個特異的ace Aa基因。該基因由ace A基因突變而形成,ace A基因編碼的異檸檬酸裂合酶參與脂代謝過程并介導廣泛的抗生素耐受,推測其可能是影響結核分枝桿菌在牛群傳播的一個潛在因素。北京型菌株的SNP比較發(fā)現(xiàn),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),存在更多的突變基因,而M.tb Beijing/NITR203則有更多特異的SNP。結果與進化樹分析一致。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.618

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