本文關(guān)鍵詞：利用比較基因組學篩選粗糙型布魯氏菌RM57株致弱相關(guān)基因　出處：《中國獸醫(yī)藥品監(jiān)察所》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 布魯氏菌 粗糙表型 基因組學

【摘要】：布魯氏菌病是一種嚴重的人畜共患傳染病,人、牛、羊、豬、犬等哺乳動物均可以發(fā)生感染而致病,動物會發(fā)生流產(chǎn)、生殖器官炎癥,人感染布魯氏菌病后會引起波浪熱。近年來,隨著我國家畜飼養(yǎng)量不斷增加,動物及其產(chǎn)品流通頻繁,部分地區(qū)布魯氏菌病呈持續(xù)上升勢頭,不僅嚴重影響畜牧業(yè)生產(chǎn),也嚴重危及人民身體健康和公共衛(wèi)生安全。加強對布魯氏菌病的預防和控制具有重要意義。目前在發(fā)達國家消除布魯氏菌病的主要方法是直接撲殺。對于布魯氏菌病疫情比較嚴重的中國而言,各級政府無力負擔過高的撲殺成本,故使用疫苗預防和控制布魯氏菌病疫情是符合我國國情的唯一手段。布魯氏菌根據(jù)表型差異可分為光滑型和粗糙型,兩者在血清學上無交叉反應,但卻有免疫交叉保護性。一般感染牛、羊的布魯氏菌均光滑型,布病疫苗基本上也是光滑型;因此布病疫苗免疫往往會干擾臨床診斷。開發(fā)粗糙型布魯氏菌疫苗株一直是研究的布病研究的熱點。美國上世紀90年代末,在野生動物上開始使用粗糙型牛種布魯氏菌疫苗RB51株。但其安全性和免疫保護效果備受爭議,且對家養(yǎng)動物的真實效果尚無系統(tǒng)的研究報道。尤其是,RB51采用利福平誘導而來,而利福平是治療人布魯氏菌病的特效藥,RB51的廣泛使用對人的安全帶來了潛在危害。本實驗室采用粗糙型抗血清和光滑型抗血清交叉誘導并反復篩選的方式,將一株羊種布魯氏菌分離株(M1981)誘導為粗糙型,命名為RM57。生物學特性研究表明,RM57株是一株毒力穩(wěn)定、安全性高、免疫效果良好的粗糙型疫苗株。本實驗對引起RM57株粗糙表型的脂多糖完整性進行了鑒定,同時根據(jù)國內(nèi)外報道的可能引起布魯氏菌粗糙表型的相關(guān)基因進行了基因完整性鑒定以及轉(zhuǎn)錄水平變化的測定。結(jié)果表明粗糙型RM57株與其親本光滑型M1981株的脂多糖確實存在差異,但是已經(jīng)報道過的脂多糖相關(guān)基因均完整存在且無轉(zhuǎn)錄水平的變化。故必然存在未知的基因參與了布魯氏菌LPS生物學合成過程或存在某種調(diào)節(jié)機制控制著布魯氏菌LPS合成系統(tǒng)。粗糙型羊種布魯氏菌RM57株及其親本光滑型布魯氏菌M1981株遺傳背景清晰,均來自于同一母本菌株,這為我們通過基因組學研究布魯氏菌LPS合成機制提供了寶貴的優(yōu)良素材。本研究以粗糙型羊種布魯氏菌RM57株及其親本光滑型布魯氏菌M1981株為切入點,通過系統(tǒng)的比較基因組學分析,將拓寬人們對于布魯氏菌LPS合成的認知。通過二代高通量測序技術(shù)對粗糙型羊種布魯氏菌RM57株及其親本光滑型布魯氏菌M1981株的全基因組序列進行了測定,然后對獲得的基因組草圖序列補缺口并獲得了兩株菌的基因組完整序列。通過基因預測、重復序列預測等方法獲取兩株菌的基因組組成信息,對包括基因組所含有基因數(shù)量、基因平均長度、串聯(lián)重復序列、小衛(wèi)星序列以及微衛(wèi)星序列等基因組結(jié)構(gòu)信息進行了解析。對布魯氏菌基因組進行ORFs預測并通過基因注釋、基因功能分類、毒力基因預測等,最終繪制了基因組精細圖。根據(jù)布魯氏菌基因組信息,挖掘了與病原菌生長、代謝相關(guān)的基因,構(gòu)建了布魯氏菌的代謝通路,一共包括22大門類:染色體結(jié)構(gòu)和動力學、能量產(chǎn)生及傳遞、細胞周期調(diào)控、細胞分裂、染色體分離、氨基酸轉(zhuǎn)運和代謝、核酸轉(zhuǎn)運和代謝、碳水化合物轉(zhuǎn)運和代謝、輔酶轉(zhuǎn)運和代謝、脂類轉(zhuǎn)運和代謝、翻譯、核糖體結(jié)構(gòu)和生物發(fā)生、轉(zhuǎn)錄、復制、重組和修飾、細胞壁/細胞膜生物發(fā)生、細胞活性、翻譯后修飾、蛋白翻轉(zhuǎn)、伴侶、無機離子轉(zhuǎn)運和代謝、次生代謝物生物合成、轉(zhuǎn)運和代謝、只有一般功能預測、未知功能、信號傳遞機制、細胞間運輸、分泌物和囊泡運動、防御機制、胞外結(jié)構(gòu)。根據(jù)布魯氏菌基因組信息,挖掘了與毒力因子相關(guān)的基因,包括:尿素酶輔助蛋白、尿素酰胺水解酶、尿素酰胺水解酶、鐵III、ABC轉(zhuǎn)運蛋白,ATP結(jié)合蛋白、環(huán)狀β1-2葡聚糖合成酶、血紅素出口蛋白、磷酸葡萄糖變位酶、假定蛋白Rv0981、內(nèi)肽酶Clp ATP結(jié)合鏈C、ABC轉(zhuǎn)運蛋白的可能的ATP結(jié)合組分、ABC轉(zhuǎn)運蛋白的可能的ATP結(jié)合組分、icl/ace A、脂多糖生物合成蛋白、溶血素B、ATP依賴性蛋白酶、雙組分響應調(diào)節(jié)器、ATP依賴性Clp蛋白酶蛋白水解亞基、UDP-葡萄糖4-差向異構(gòu)酶、具有Pho Q的雙組分調(diào)節(jié)系統(tǒng)中的應答調(diào)節(jié)因子,轉(zhuǎn)錄低Mg2+濃度表達的基因、sod B超氧化物歧化酶、甘露糖-6-磷酸異構(gòu)酶、甘露糖-1-磷酸鳥苷酰轉(zhuǎn)移酶、man B-磷酸甘露糖變性酶、甘露糖基轉(zhuǎn)移酶、perosamine合成酶、GDP-甘露糖4,6-脫水酶/GDP-4-氨基-4,6-二脫氧-D-甘露糖甲酰轉(zhuǎn)移酶、脂多糖生物合成蛋白、pan C、4'-磷酸泛酰乙酰轉(zhuǎn)移酶、2,3-二氫-2,3-二羥基苯甲酸脫氫酶、Isochorismatase、腸桿菌素合成酶組分E、等離子體合成酶、Mg2+轉(zhuǎn)運蛋白、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、附著介導蛋白Vir B1同源物、UDP-葡萄糖焦磷酸化酶、Kps F蛋白、鞭毛馬達開關(guān)蛋白、III型分泌系統(tǒng)ATP酶、鞭毛基底桿蛋白、鞭毛基底桿蛋白、鞭毛P蛋白前體、鞭毛生物合成蛋白、葡萄糖/半乳糖轉(zhuǎn)運蛋白、Hsp60,60K熱休克蛋白、nar H、nar G、磷酸甘露糖異構(gòu)酶/鳥苷5'-二磷酸-D-甘露糖焦磷酸化酶、GDP-巖藻糖合成酶、GDP-甘露糖4,6-脫水酶、ferric enterobactin轉(zhuǎn)運ATP結(jié)合蛋白、Gifsy-2原噬菌體:超氧化物歧化酶前體Cu-Zn、、ahp C、內(nèi)膜ABC轉(zhuǎn)運體、推定型III型分泌蛋白。以粗糙型羊種布魯氏菌RM57株和其親本光滑型羊種布魯氏菌M1981株為研究對象,從基因組全長差異、氨基酸水平上的差異對功能基因的影響等方面,系統(tǒng)分析了M1981株在粗糙型抗血清和光滑型抗血清反復交叉誘導過程中LPS表型變化的分子機制,最終挖掘了26個遺傳變異位點。
[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.

【學位授予單位】：中國獸醫(yī)藥品監(jiān)察所
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61

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