本文選題：問號 + 鉤端　； 參考：《上海交通大學》2012年博士論文

【摘要】：鉤端螺旋體病是一種世界范圍內的重要人畜共患疾病，其病原體為致病性鉤體。鉤端螺旋體病的臨床表型輕重不一，輕者可只表現(xiàn)為流感樣癥狀，重者甚至死亡。根據(jù)脂多糖組成及結構的差異，致病性鉤體可分為230多個血清型。致病性鉤體宿主廣泛，幾乎涉及所有的哺乳動物[1]。某些血清型的問號鉤端螺旋體與儲存宿主之間具有一定的偏向性，但關鍵決定因素及分子機制尚未明確。 全基因組測序為研究鉤體生理、代謝、及致病機制提供了新的方法和視野。目前已完成了兩株以鼠類為儲存宿主的致病性黃疸出血群問號鉤體全基因組序列，犬群問號鉤體是中國地區(qū)乃至世界范圍內引起鉤體病的主要血清群之一，Gui44株作為其主要代表菌株，也是我國鉤體疫苗株之一。但是目前我們對其血清型特異基因、毒力及致病機制都了解甚少。 本研究采用焦磷酸測序與Sanger測序相結合的方法，完成了問號鉤體犬群犬型Gui44株全基因組序列。Gui44株和已公布全基因組序列的兩株致病性黃疸出血群問號鉤端螺旋體56601株和Fiocruz L1-130株一樣，都含有兩個染色體，但Gui44株還額外含有兩個游離于染色體外的環(huán)狀基因簇。Gui44株染色體（CⅠ、CⅡ）大小分別為4,231,232bp及356,604bp。兩個環(huán)狀基因簇(pGuiⅠ、pGuiⅡ)大小分別為74,981bp和66,851bp。Gui44株的大小染色體G+C%含量均為35.05%，分別含有3385個和292個ORFs。兩個環(huán)狀基因簇G+C%含量分別為34.63%和33.33%，分別含有62個和63個ORFs。 我們首次通過堿裂解法結合PFGE、Southern雜交和高通量測序技術從致病性鉤體Gui44株中分離并鑒定了這兩個環(huán)狀基因簇，定量PCR的方法證實其拷貝數(shù)與大染色體相同。生物信息學分析發(fā)現(xiàn)這兩個環(huán)狀基因簇上均含有質粒存在所必需的三要素：復制起始蛋白、Partition系統(tǒng)以及Adiction系統(tǒng)，，但未發(fā)現(xiàn)鉤體生存所必需的基因。這兩個游離于染色體外的環(huán)狀基因簇的首次發(fā)現(xiàn)對全面了解鉤端螺旋體基因組結構的多樣性及構建適用于致病性鉤端螺旋體的穿梭載體和體內定向遺傳操作系統(tǒng)的發(fā)展具有重要意義。 在完成Gui44株全基因組序列的基礎上，利用比較基因組學和生物信息學等手段，將問號鉤體Gui44株與已公布全基因組序列的高致病性鼠源問號鉤體黃疸出血群賴型56601株及哥本哈根型Fiocruz L1-130株全基因組序列進行比較，確定了334個Gui44株特有基因。這些基因絕大多數(shù)編碼假定蛋白（91%），其余基因涉及O抗原合成、可移動原件、抗毒素等，同時分析了這些基因在不同血清型的19株問號鉤體中的保守性，篩選出了26個Gui44特有基因。這26個基因均編碼假定蛋白，這些假定蛋白可能是Gui44株血清型、致病性和宿主偏向性差異的關鍵決定因素。對這些假定蛋白的深入研究將為進一步闡明不同血清型鉤體的致病機制、毒力及進化等奠定了基礎。 我們首次利用無蛋白培養(yǎng)基，獲得了問號鉤體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內的表面暴露蛋白質組。共檢測到145個胞外蛋白和502個外膜蛋白，同時通過生物信息學手段驗證這些表面暴露蛋白在不同血清群的19株問號鉤端螺旋體中的保守性，鑒定到了24個保守的表面暴露蛋白，，為研制跨不同血清型的鉤端螺旋體診斷試劑和疫苗候選抗原提供了幫助。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R377

【參考文獻】

相關期刊論文 前1條

1 張英;鮑朗;朱海龍;黃畢;章樂;張會東;;賴型鉤端螺旋體Loa22重組質粒的蛋白表達和對巨噬細胞的毒性作用[J];四川大學學報(醫(yī)學版);2008年03期

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