问号钩端螺旋体犬群犬型Gui44株基因组与蛋白质组的研究

发布时间：2018-05-19 14:23

本文选题：问号 + 钩端　；参考：《上海交通大学》2012年博士论文

【摘要】：钩端螺旋体病是一种世界范围内的重要人畜共患疾病，其病原体为致病性钩体。钩端螺旋体病的临床表型轻重不一，轻者可只表现为流感样症状，重者甚至死亡。根据脂多糖组成及结构的差异，致病性钩体可分为230多个血清型。致病性钩体宿主广泛，几乎涉及所有的哺乳动物[1]。某些血清型的问号钩端螺旋体与储存宿主之间具有一定的偏向性，但关键决定因素及分子机制尚未明确。全基因组测序为研究钩体生理、代谢、及致病机制提供了新的方法和视野。目前已完成了两株以鼠类为储存宿主的致病性黄疸出血群问号钩体全基因组序列，犬群问号钩体是中国地区乃至世界范围内引起钩体病的主要血清群之一，Gui44株作为其主要代表菌株，也是我国钩体疫苗株之一。但是目前我们对其血清型特异基因、毒力及致病机制都了解甚少。本研究采用焦磷酸测序与Sanger测序相结合的方法，完成了问号钩体犬群犬型Gui44株全基因组序列。Gui44株和已公布全基因组序列的两株致病性黄疸出血群问号钩端螺旋体56601株和Fiocruz L1-130株一样，都含有两个染色体，但Gui44株还额外含有两个游离于染色体外的环状基因簇。Gui44株染色体（CⅠ、CⅡ）大小分别为4,231,232bp及356,604bp。两个环状基因簇(pGuiⅠ、pGuiⅡ)大小分别为74,981bp和66,851bp。Gui44株的大小染色体G+C%含量均为35.05%，分别含有3385个和292个ORFs。两个环状基因簇G+C%含量分别为34.63%和33.33%，分别含有62个和63个ORFs。我们首次通过碱裂解法结合PFGE、Southern杂交和高通量测序技术从致病性钩体Gui44株中分离并鉴定了这两个环状基因簇，定量PCR的方法证实其拷贝数与大染色体相同。生物信息学分析发现这两个环状基因簇上均含有质粒存在所必需的三要素：复制起始蛋白、Partition系统以及Adiction系统，，但未发现钩体生存所必需的基因。这两个游离于染色体外的环状基因簇的首次发现对全面了解钩端螺旋体基因组结构的多样性及构建适用于致病性钩端螺旋体的穿梭载体和体内定向遗传操作系统的发展具有重要意义。在完成Gui44株全基因组序列的基础上，利用比较基因组学和生物信息学等手段，将问号钩体Gui44株与已公布全基因组序列的高致病性鼠源问号钩体黄疸出血群赖型56601株及哥本哈根型Fiocruz L1-130株全基因组序列进行比较，确定了334个Gui44株特有基因。这些基因绝大多数编码假定蛋白（91%），其余基因涉及O抗原合成、可移动原件、抗毒素等，同时分析了这些基因在不同血清型的19株问号钩体中的保守性，筛选出了26个Gui44特有基因。这26个基因均编码假定蛋白，这些假定蛋白可能是Gui44株血清型、致病性和宿主偏向性差异的关键决定因素。对这些假定蛋白的深入研究将为进一步阐明不同血清型钩体的致病机制、毒力及进化等奠定了基础。我们首次利用无蛋白培养基，获得了问号钩体Gui44株胞外蛋白。全面地分析了Gui44株包括胞外蛋白和外膜蛋白在内的表面暴露蛋白质组。共检测到145个胞外蛋白和502个外膜蛋白，同时通过生物信息学手段验证这些表面暴露蛋白在不同血清群的19株问号钩端螺旋体中的保守性，鉴定到了24个保守的表面暴露蛋白，，为研制跨不同血清型的钩端螺旋体诊断试剂和疫苗候选抗原提供了帮助。
[Abstract]:Leptospirosis is a worldwide important zoonotic disease with pathogenic Leptospira. The clinical phenotype of leptospirosis is different. The leptospirosis may be characterized by influenza like symptoms, heavy or even death. According to the difference in the composition and structure of lipopolysaccharide, the Leptospira can be divided into more than 230 serotypes. Pathogenic hook can be divided into pathogenic hooks. The body and host are extensive and involve almost all mammalian [1]. serotypes. The Leptospira interroponate and the storage host have certain bias, but the key determinants and molecular mechanisms are not clear.
Whole genome sequencing provides a new method and field of vision for the study of Leptospira physiology, metabolism, and pathogenesis. Two strains of the whole genome of the leptospirosis have been completed. The canine interroleo is one of the main serum groups of Leptospira Leptospira (Gui44) in China and even in the world. As the main representative strain, strain is also one of the strains of leptospiral vaccine in China. However, we know little about its specific serotype genes, virulence and pathogenic mechanism.
In this study, using pyrosequencing and Sanger sequencing, the whole genome sequence.Gui44 of the canine Gui44 strain of the Leptospira canine group and two pathogenic jaundice haemorrhage groups, 56601 strains of leptospirosis and Fiocruz L1-130, all contain two chromosomes, but the Gui44 strain also contains extra Two chromosomes (C I, C II) of the ring gene cluster.Gui44 (C I, C II) are respectively 4231232bp and 356604bp. two ring gene clusters (pGui I, pGui II), the size of 74981bp and 66851bp.Gui44, respectively, is 35.05%, containing 3385 and 292 ORFs. two ring gene clusters, respectively. The quantities were 34.63% and 33.33%, respectively, containing 62 and 63 ORFs. respectively.
We first isolated and identified two cyclic gene clusters from pathogenic Leptospira Gui44 strains by combining PFGE, Southern hybridization and high throughput sequencing technology. The quantitative PCR method confirmed that the number of copies was the same as the large chromosome. Bioinformatics analysis found that all of the two ring genes contained the necessary three of the existence of plasmid. Factors: replicating starting protein, Partition system, and Adiction system, but no genes necessary for Leptospira survival were found. The first discovery of these two unstained ring gene clusters for the first time to understand the diversity of the genome structure of Leptospira and the construction of shuttle vectors and in vivo orientation for pathogenic Leptospira The development of genetic operating system is of great significance.
On the basis of complete genome sequence of Gui44 strain, using comparative genomics and bioinformatics, the Gui44 strain of the Leptospira interrolea was compared with the complete genome sequence of the 56601 highly pathogenic Leptospira jaundice haemorrhage group and the Copenhagen type Fiocruz L1-130 strain, and 334 Gui were determined. 44 endemic genes, most of which encode the hypothetical protein (91%), and the other genes involved in the synthesis of O antigen, mobile original, antitoxin, etc., and analyzed the conservatism of these genes in 19 interroleas of different serotypes, and screened out 26 Gui44 endemic groups. These 26 genes encode the presumed protein, these hypothetical proteins It may be the key determinant of the serotypes, pathogenicity and host bias of Gui44 strains. The in-depth study of these hypothesized proteins will provide a basis for further clarifying the pathogenesis, virulence and evolution of different serotype Leptospira.
We obtained the extracellular protein of the Gui44 strain of the Leptospira of the Leptospira for the first time using a protein free medium. The surface exposed protein groups, including extracellular protein and outer membrane protein, were analyzed comprehensively. 145 extracellular proteins and 502 outer membrane proteins were detected, and the surface exposed proteins were tested by bioinformatics in different blood. The conservatism in the 19 interroleo leptospirosis of the Qing group identified 24 conservative surface exposed proteins, which helped to develop the diagnostic reagents for Leptospira and vaccine candidate antigens across different serotypes.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R377

【参考文献】