利用比较基因组学筛选粗糙型布鲁氏菌RM57株致弱相关基因

发布时间：2017-12-30 17:38

本文关键词：利用比较基因组学筛选粗糙型布鲁氏菌RM57株致弱相关基因　出处：《中国兽医药品监察所》2017年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌病是一种严重的人畜共患传染病,人、牛、羊、猪、犬等哺乳动物均可以发生感染而致病,动物会发生流产、生殖器官炎症,人感染布鲁氏菌病后会引起波浪热。近年来,随着我国家畜饲养量不断增加,动物及其产品流通频繁,部分地区布鲁氏菌病呈持续上升势头,不仅严重影响畜牧业生产,也严重危及人民身体健康和公共卫生安全。加强对布鲁氏菌病的预防和控制具有重要意义。目前在发达国家消除布鲁氏菌病的主要方法是直接扑杀。对于布鲁氏菌病疫情比较严重的中国而言,各级政府无力负担过高的扑杀成本,故使用疫苗预防和控制布鲁氏菌病疫情是符合我国国情的唯一手段。布鲁氏菌根据表型差异可分为光滑型和粗糙型,两者在血清学上无交叉反应,但却有免疫交叉保护性。一般感染牛、羊的布鲁氏菌均光滑型,布病疫苗基本上也是光滑型;因此布病疫苗免疫往往会干扰临床诊断。开发粗糙型布鲁氏菌疫苗株一直是研究的布病研究的热点。美国上世纪90年代末,在野生动物上开始使用粗糙型牛种布鲁氏菌疫苗RB51株。但其安全性和免疫保护效果备受争议,且对家养动物的真实效果尚无系统的研究报道。尤其是,RB51采用利福平诱导而来,而利福平是治疗人布鲁氏菌病的特效药,RB51的广泛使用对人的安全带来了潜在危害。本实验室采用粗糙型抗血清和光滑型抗血清交叉诱导并反复筛选的方式,将一株羊种布鲁氏菌分离株(M1981)诱导为粗糙型,命名为RM57。生物学特性研究表明,RM57株是一株毒力稳定、安全性高、免疫效果良好的粗糙型疫苗株。本实验对引起RM57株粗糙表型的脂多糖完整性进行了鉴定,同时根据国内外报道的可能引起布鲁氏菌粗糙表型的相关基因进行了基因完整性鉴定以及转录水平变化的测定。结果表明粗糙型RM57株与其亲本光滑型M1981株的脂多糖确实存在差异,但是已经报道过的脂多糖相关基因均完整存在且无转录水平的变化。故必然存在未知的基因参与了布鲁氏菌LPS生物学合成过程或存在某种调节机制控制着布鲁氏菌LPS合成系统。粗糙型羊种布鲁氏菌RM57株及其亲本光滑型布鲁氏菌M1981株遗传背景清晰,均来自于同一母本菌株,这为我们通过基因组学研究布鲁氏菌LPS合成机制提供了宝贵的优良素材。本研究以粗糙型羊种布鲁氏菌RM57株及其亲本光滑型布鲁氏菌M1981株为切入点,通过系统的比较基因组学分析,将拓宽人们对于布鲁氏菌LPS合成的认知。通过二代高通量测序技术对粗糙型羊种布鲁氏菌RM57株及其亲本光滑型布鲁氏菌M1981株的全基因组序列进行了测定,然后对获得的基因组草图序列补缺口并获得了两株菌的基因组完整序列。通过基因预测、重复序列预测等方法获取两株菌的基因组组成信息,对包括基因组所含有基因数量、基因平均长度、串联重复序列、小卫星序列以及微卫星序列等基因组结构信息进行了解析。对布鲁氏菌基因组进行ORFs预测并通过基因注释、基因功能分类、毒力基因预测等,最终绘制了基因组精细图。根据布鲁氏菌基因组信息,挖掘了与病原菌生长、代谢相关的基因,构建了布鲁氏菌的代谢通路,一共包括22大门类:染色体结构和动力学、能量产生及传递、细胞周期调控、细胞分裂、染色体分离、氨基酸转运和代谢、核酸转运和代谢、碳水化合物转运和代谢、辅酶转运和代谢、脂类转运和代谢、翻译、核糖体结构和生物发生、转录、复制、重组和修饰、细胞壁/细胞膜生物发生、细胞活性、翻译后修饰、蛋白翻转、伴侣、无机离子转运和代谢、次生代谢物生物合成、转运和代谢、只有一般功能预测、未知功能、信号传递机制、细胞间运输、分泌物和囊泡运动、防御机制、胞外结构。根据布鲁氏菌基因组信息,挖掘了与毒力因子相关的基因,包括:尿素酶辅助蛋白、尿素酰胺水解酶、尿素酰胺水解酶、铁III、ABC转运蛋白,ATP结合蛋白、环状β1-2葡聚糖合成酶、血红素出口蛋白、磷酸葡萄糖变位酶、假定蛋白Rv0981、内肽酶Clp ATP结合链C、ABC转运蛋白的可能的ATP结合组分、ABC转运蛋白的可能的ATP结合组分、icl/ace A、脂多糖生物合成蛋白、溶血素B、ATP依赖性蛋白酶、双组分响应调节器、ATP依赖性Clp蛋白酶蛋白水解亚基、UDP-葡萄糖4-差向异构酶、具有Pho Q的双组分调节系统中的应答调节因子,转录低Mg2+浓度表达的基因、sod B超氧化物歧化酶、甘露糖-6-磷酸异构酶、甘露糖-1-磷酸鸟苷酰转移酶、man B-磷酸甘露糖变性酶、甘露糖基转移酶、perosamine合成酶、GDP-甘露糖4,6-脱水酶/GDP-4-氨基-4,6-二脱氧-D-甘露糖甲酰转移酶、脂多糖生物合成蛋白、pan C、4'-磷酸泛酰乙酰转移酶、2,3-二氢-2,3-二羟基苯甲酸脱氢酶、Isochorismatase、肠杆菌素合成酶组分E、等离子体合成酶、Mg2+转运蛋白、mgt C、ATPase Vir B11同系物、通道蛋白Vir B10同系物、通道蛋白Vir B9同源物、Vir B8、通道蛋白Vir B6同系物、Vir B5、ATPase Vir B4同系物、Vir B3、Vir B2、附着介导蛋白Vir B1同源物、UDP-葡萄糖焦磷酸化酶、Kps F蛋白、鞭毛马达开关蛋白、III型分泌系统ATP酶、鞭毛基底杆蛋白、鞭毛基底杆蛋白、鞭毛P蛋白前体、鞭毛生物合成蛋白、葡萄糖/半乳糖转运蛋白、Hsp60,60K热休克蛋白、nar H、nar G、磷酸甘露糖异构酶/鸟苷5'-二磷酸-D-甘露糖焦磷酸化酶、GDP-岩藻糖合成酶、GDP-甘露糖4,6-脱水酶、ferric enterobactin转运ATP结合蛋白、Gifsy-2原噬菌体:超氧化物歧化酶前体Cu-Zn、、ahp C、内膜ABC转运体、推定型III型分泌蛋白。以粗糙型羊种布鲁氏菌RM57株和其亲本光滑型羊种布鲁氏菌M1981株为研究对象,从基因组全长差异、氨基酸水平上的差异对功能基因的影响等方面,系统分析了M1981株在粗糙型抗血清和光滑型抗血清反复交叉诱导过程中LPS表型变化的分子机制,最终挖掘了26个遗传变异位点。
[Abstract]:Brucellosis is a serious zoonotic infectious disease, human, cattle, sheep, pigs, dogs and other mammals can be infected and pathogenic, animal happens abortion, genital inflammation, infection of brucellosis will cause heat wave. In recent years, with China's livestock and animal is increasing. The product circulate frequently, some areas of brucellosis sustained upward momentum, not only affects the production of animal husbandry, people's health and public health safety and seriously endangered. It is of great significance to strengthen the prevention and control of brucellosis. Is the main method to eliminate direct killing of brucellosis in developed countries at present. The more serious outbreak of brucellosis the China, all levels of government cannot afford the high cost of the vaccine culling, prevention and control of brucellosis epidemic is the only way to meet the situation of our country. Brucella can be divided into smooth and rough type according to the phenotypic differences both in serological cross reaction, but the immune cross protection. General infection in cattle, sheep brucellosis were smooth, brucellosis vaccine is basically smooth; therefore brucellosis vaccine often interfere with clinical diagnosis. The development of rough Brucella vaccine strain has been a hot research of brucellosis research. The last century the United States at the end of 90s, in the wild animal began to use rough type Brucella vaccine RB51 strain. But the safety and protective efficacy of the controversial, research reports and real effect on the domestic animal no system. Especially, the RB51 of rifampicin induced by. But rifampicin is a cure for brucellosis, bring potential harm to the widespread use of RB51 for the safety of people. Rough type antiserum and smooth type used in this laboratory The antiserum cross induction and repeated screening method, a strain of Brucella melitensis isolates (M1981) induced by rough type, named RM57. biological characteristics research showed that RM57 strain was a stable virulence, high safety, rough type vaccine strain good immune effect. This experiment for the identification of integrity caused by lipopolysaccharide RM57 strains rough phenotype, and according to the determination of reported at home and abroad may be caused by the rough phenotype of Brucella gene gene integrity identification and transcriptional level changes. The results show that the rough type RM57 strain and its parent smooth LPS M1981 strain differences do exist, but the changes of lipopolysaccharide related genes had been reported to have complete existence and there is no transcriptional level. So there must be unknown genes involved in the biological synthesis process of Brucella LPS or the existence of a regulatory mechanism to control brucellosis Strain LPS synthesis system. The rough type of Brucella melitensis strain RM57 and its parental smooth Brucella strain M1981 clear genetic background, are from the same parent strains, which provides excellent material for us to learn valuable research on the mechanism of synthesis of LPS by Brucella genome. In this study, the rough type of Brucella melitensis strain RM57 and its parental smooth Brucella strain M1981 as the starting point, through comparative genomic analysis of the system, will broaden the people for Brucella LPS synthesis of cognition. Through the two generation high-throughput sequencing of the whole genome sequence of Brucella melitensis strain RM57 and its parental smooth Brucella strain M1981 rough type were determined, and then the genome sequence obtained and fill the gap the complete genome sequences of two strains. The gene prediction and genome repeat sequence prediction method to obtain two strains As information, including the number of genes contained in the genome, the average length of genes, tandem repeats, small satellite sequences and microsatellite sequences of genome structure information were analyzed. The genome of ORFs and prediction of Brucella by gene annotation, gene function classification, virulence gene prediction, finally draw the genome according to Brucella genome. Mining information, growth and metabolism related genes of pathogenic bacteria, and construct the metabolic pathways of Brucella, including a total of 22 categories: chromosome structure and dynamics, energy production and transmission, cell cycle regulation, cell division, chromosome segregation, amino acid transport and metabolism, nucleic acid transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, translation, ribosome structure and biogenesis, transcription, replication, recombination and modification, cell wall / fine Cell membrane biogenesis, cell activity, posttranslational modification, protein turnover, mate, inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport and metabolism, only general function prediction, unknown function, signal transduction, cellular transport, secretion and vesicle movement, defense mechanism, extracellular structure according to Brucella genome information. Mining, associated with virulence factor genes, including urease accessory protein, urea amidohydrolase, urea amidohydrolase, iron III, ABC transporter, ATP binding protein, 1-2 synthase beta cyclic glucose, heme protein export, Phosphoglucomutase, hypothetical protein Rv0981, endopetidase Clp ATP with C chain may, ABC transporter ATP binding component of ABC transporters may be ATP with the component of icl/ace, A, lipopolysaccharide biosynthesis protein, hemolysin B, ATP dependent protease, two-component response regulation For ATP, Clp dependent protease hydrolysis of protein subunits, UDP- glucose 4- epimerase, Pho Q is a two-component regulatory system in the regulation of transcription factor response, low concentration of Mg2+ gene expression, SOD B superoxide dismutase, mannose phosphate isomerase -6-, mannose phosphate -1- guanyltransferase. Man B- phosphomannose denatured enzyme, mannosyltransferase, perosamine synthase, GDP- 4,6- mannose dehydratase /GDP-4- two amino -4,6- deoxy -D- mannose formyltransferase, lipopolysaccharide biosynthesis protein, pan C, 4'- acyl phosphate acetyltransferase, 2,3- two hydrogen -2,3- two hydroxy benzoic acid dehydrogenase, Isochorismatase, enterobactin synthetase component E, plasma synthase, Mg2+ transporter, Mgt C, ATPase Vir B11 homolog, Vir channel protein B10 homolog, Vir channel protein B9 homolog, Vir B8, Vir channel protein B6 homolog, Vir B5, ATPase Vir B4 Vir B3, Vir homologue, B2, adhesion mediated protein Vir B1 homolog, UDP- glucose pyrophosphorylase, Kps F protein, flagellar motor switch protein, type III secretion system of ATP enzyme, the flagellar basal rod protein, flagellar basal pole flagellum protein, P protein precursor, biological synthesis of flagellar protein, glucose / galactose sugar transporter Hsp60,60K, heat shock protein, NAR H, NAR G, phosphomannose isomerase / guanosine 5'- phosphate two -D- mannose pyrophosphorylase, GDP- fucose synthetase, GDP- 4,6- mannose dehydratase, ferric enterobactin transporter ATP binding protein, Gifsy-2 prophage: superoxide dismutase precursor Cu-Zn, AHP, C. Endometrial type III type ABC transporter, presumed secretory protein. The rough type of Brucella melitensis strain RM57 and its parental smooth Brucella melitensis strain M1981 as the research object, from the genome differences in amino acid level differences of functional groups The molecular mechanism of LPS phenotypic variation in M1981 strain during repeated cross induction of rough antiserum and smooth antiserum was systematically analyzed, and 26 genetic variation sites were finally excavated.

【学位授予单位】：中国兽医药品监察所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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