背景（background）:鉤端螺旋體（簡(jiǎn)稱鉤體）分為致病性鉤體和非致病性鉤體兩大類,前者引起人或動(dòng)物疾病,后者屬于非致病腐生性原核細(xì)胞型微生物。目前發(fā)現(xiàn),致病性鉤體可分為7個(gè)基因種,其中問號(hào)鉤體基因種（Leptospira interrogans）流行最為廣泛,如黃疸出血群、波摩那群、秋季群等；其次為波帕特森基因種（Leptospira borgpetersenii）,如流感傷寒群。由致病性鉤體感染引起的鉤體病是全球流行的人畜共患傳染病,中國(guó)及東南亞地區(qū)是鉤體病流行的主要疫區(qū),其次是巴西等南美地區(qū)國(guó)家。人類感染致病性鉤體后,首先出現(xiàn)中毒性敗血癥癥狀,如高熱、頭痛、肌痛等；然后因鉤體侵入肺、肝、腎等臟器及中樞神經(jīng)系統(tǒng),出現(xiàn)出血、黃疸及呼吸或腎功能衰竭癥狀,因主要受損臟器不同,臨床上可分為六型,其中肺彌漫性出血（pulmonary diffuse hemorrhage, PHD）型鉤體病死亡率高達(dá)50%～75%。以往生物學(xué)實(shí)驗(yàn)證明,致病性鉤體不產(chǎn)生任何典型的外毒素,業(yè)已公布的2株致病性鉤體全基因組序列中,也未發(fā)現(xiàn)有任何編碼經(jīng)典外毒素基因。致病性鉤體脂多糖（LPS）的內(nèi)毒素毒性僅為大腸桿... 

【文章來源】：浙江大學(xué)浙江省 211工程院校 985工程院校 教育部直屬院校

【文章頁數(shù)】：196 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
Acknowledgement
致謝
Abstract
中文摘要
Introduction
Part Ⅰ:chpK/chpI toxin-antitoxin system
    Chapter 1 #6Construction of pathogenic Leptospira interrogans strain Lai,chpK；chpI and chpIK genes prokaryotic expression systems andrChpK/rChpI rabbit anti-serum preparation #
        1.1 ) Materials
            1.1.1 ) Bacterials cells line used
            1.1.2 ) Plasmid vector used in this work
            1.1.3 ) Kits,solutions and others
            1.1.4 ) Instruments
        1.2 ) Methods
            1.2.1 ) Culture medium preparation
            1.2.2 ) Pathogenic Leptospira interrogans strain Lai culturing
            1.2.3 ) Pathogenic Leptospira interrogans strain Lai,genomic DNA extraction
            1.2.4 ) E.coli DH5a and E.coli BL21(DE3)Competent cells preparation
            1.2.5 ) chpI;chpK and chpIK genes amplification by using standard
            1.2.6 ) Signal peptide prediction
            1.2.7 ) PCR Primers designing
            1.2.8 ) PCR amplication of the chpI;chpK and chpIK genes
            1.2.9 ) PCR cycling conditions
            1.2.10 ) Agarose gel Electropheresis of the pcr product
            1.2.11 ) PCR products gel extraction and purification
            1.2.12 ) Ligation of the purified PCR product to the pMD 18-T plasmid vector(T-Acloning)
            1.2.13 ) Transformation of the ligation product in to the E.coli DH5a competent cellsand plating
            1.2.14 ) pMD18-T-chpI;pMD18-T-chpK and pMD18-T-chpIK plasmid extraction
            1.2.15 ) Double enzyme digestion of the pMD18-T-chpI;pMD18-T-chpK andpMD18-T-chpIK plasmids
            1.2.16 ) Sequencing of the recombinant plasmid transformed into the E.coli DH5a
            1.2.17 ) Construction of chpI;chpK and chpIK genes prokaryotic expression system
            1.2.18 ) pMD18-T-chpI;pMD18-T-chpK;pMD18-T-chpIK and pET42a Plasmids DNAextraction
            1.2.19 ) Double enzyme digestion of the pMD18-T-chpI;pMD18-T-chpK,pMD18-T-chpIK recombinant Plasmids DNA and the pET-42a plasmid vector
            1.2.20 ) pMD18-T-chpI;pMD18-T-chpK;pMD18-T-chpIK and pET42a Plasmids DNApurification
            1.2.21 ) Ligation of the chpI;chpK and chpIK genes to the pET42a plasmid vector
            1.2.22 ) Transformation of the ligation product into the E.coli BL21(DE3)competentcells:sub-cloning
            1.2.23 ) pET42a-chpI;pET42a-chpK and pET42a-chpIK recombinant plasmid DNAextraction
            1.2.24 ) Double enzyme digestion of the recombinant plasmid DNA
            1.2.25 ) Sequencing of the recombinant DNA
            1.2.26 ) Recombinant proteins rChpK and rChpI extraction and purification
            1.2.27 ) Protein expression
            1.2.28 ) SDS PAGE gel preparation
            1.2.29 ) Protein extraction
            1.2.30 ) Protein purification
            1.2.31 ) Rabbit anti-serum preparation
        1.3 ) Results and Discussion
            1.3.1 ) Results
            1.3.2 ) Discussion
        1.4 ) Conclusion
        References
    Chapter 2 #40Study of the chpK/chpI genes expression level after infection of the THP-1cells by the pathogenic Leptospira interrogans strain Lai
        2.1 ) Materials
            2.1.1 ) Bacterial and cell line used in this work
            2.1.2 ) Reagents and kits used in this work
            2.1.3 ) Experimental Instruments
        2.2 ) Methods
            2.2.1 ) Pathogenic Leptospira interrogans strain Lai culturing
            2.2.2 ) THP-1 cell culturing
            2.2.3 ) Real-Time-PCR Primers designing
            2.2.4 ) Infection of Human THP-1 cells by the pathogenic Leptospira interrogans trainLai
            2.2.5 ) Total RNA extraction of pathogenic Leptospira interrogans strain Lai
            2.2.6 ) Total RNA quantification
            2.2.7 ) Reverse-transcription PCR
            2.2.8 ) Reverse Transcription-PCR-condition
            2.2.9 ) Real-Time PCR Reaction
        2.3 ) Results and Discussion
            2.3.1 ) Results
            2.3.2 ) Discussion
        2.4 ) Conclusion
        References
    Chapter 3 #50Study of pathogenic Leptospira interrogans strain Lai toxic protein rChpKexternal secretion during host cell infection
        3.1 ) Materials
            3.1.1 ) Bacterial and cell line used in this work
            3.1.2 ) Buffers and solutions
        3.2 ) Methods
            3.2.1 ) Sample preparation
            3.2.2 ) SDS-PAGE electrophoresis gel running
            3.2.3 ) Western Bolt
        3.3 ) Results and Discussion
            3.3.1 ) Results
            3.3.2 ) Discussion
        3.4 ) Conclusion
        References
    Chapter 4 #55Study of the chpK gene expression into the eukaryotic cell system
        4.1 ) Materials
            4.1.1 ) Bacterial and Cells line used in this work
            4.1.2 ) Plasmid vector used in this work
            4.1.3 ) Kits and Others
            4.1.4 ) Experimental Instruments
        4.2 ) Methods
            4.2.1 ) Cell culturing
            4.2.2 ) Pathogenic Leptospira interrogans strain Lai,genomic DNA extraction
            4.2.3 ) PCR amplification of the target gene
                4.2.3.1 ) Primers design
                4.2.3.2 ) PCR conditions
                4.2.3.3 ) PCR cycling conditions
                4.2.3.4 ) A garose gel electrophoresis of the pcr product
                4.2.3.5 ) Agarose gel extraction and purification
                4.2.3.6 ) pCMV-Tag2C plasmid extraction
                4.2.3.7 ) Double enzyme digestion of the pCMV-Tag2C plasmid
                4.2.3.8 ) Agarose gel extraction and purification #61Refer to 2.2.5
            4.2.4 ) Liagation of the amplified target gene to the pCMV-Tag2C plasmid
            4.2.5 ) Transformation of the pCMV-Tag2CchpK and pCMV-Tag2CchpI recombinantplasmids into the E.Coli DH5a competent cells and plating
            4.2.6 ) Sequencing of the pCMV-Tag2CchpK and pCMV-Tag2CchpI recombinantplasmids
            4.2.7 ) Recombinant plasmid pCMV-Tag2CchpK and pCMV-Tag2CchpI extraction
            4.2.8 ) HEK 293 cell line transfection
        4.3 ) Results and Discussion
            4.3.1 ) Results
            4.3.2 ) Discussion
        4.4 ) Conclusion
        References
    Chapter 5 Nuclease activity study of recombinant proteins rChpK and rChpI ofpathogenic Leptospira interrogans strain Lai
        5.1 ) Materials
            5.1.1 ) Cells line used in this work
            5.1.2 Kits and others
            5.1.3 ) Instruments
        5.2 ) Method
            5.2.1 ) Pathogenic Leptospira interrogans strain genomic DNA extraction
            5.2.2 ) THP-1 cell genomic DNA extraction
            5.2.3 ) DNA quantification
            5.2.4 ) Enzymatic assay
        5.3 ) Results and Discussion
            5.3.1 ) Results
            5.3.2 ) Discussion
        5.4 ) Conclusion
        References
    Chapter 6 Study of rChpK toxin of pathogenic Leptospira interrogans strain Laiability to internalize the host Cell
        6.1. ) Materials
            6.1.1 ) Bacterial and Cell line used in this work
            6.1.2 ) Kits and others
            6.1.3 ) Experimental Instruments
        6.2 ) Methods
            6.2.1 ) THP-1 preparation
            6.2.2 ) Infection of the THP-1 cell
            6.2.3 ) Laser confocal Microspcope analysis
        6.3 ) Results and Discussion
            6.3.1 ) Results
            6.3.2 ) Discussion
        6.4 ) Conclusion
        References
Part Ⅱ:mazF/mazE toxin-antitoxin system
    Chapter 7 #82Construction of pathogenic Leptospira interrogans strain Lai,mazF；mazEand mazEF genes prokaryotic expression systems and rChpK/rChpI rabbitanti-serum preparation
        7.1 ) Materials
            7.1.1 ) Bacterials cells line used
            7.1.2 ) Plasmid vector used in this work
            7.1.3 ) Kits,solutions and others
            7.1.4 ) Instruments
        7.2 ) Methods
            7.2.1 ) Culture medium
            7.2.2 ) Pathogenic Leptospira interrogans strain Lai culturing
            7.2.3 ) Pathogenic Leptospira interrogans strain Lai, genomic DNA extraction
            7.2.4 ) E.Coli DH5a and E.Coli BL2 (DE3) Competent cells preparation
            7.2.5 ) mazF/mazE genes amplification by using standard PCR protocol
                7.2.5.1 ) Signal peptide prediction
                7.2.5.2 ) PCR Primers designing
                7.2.5.3 ) PCR amplication of the mazF/mazEgenes
                7.2.5.4 ) PCR cycling conditions
                7.2.5.5 ) Agarose gel Electropheresis of the pcr product
                7.2.5.6 ) Pcr product gel extraction and purification
            7.2.6 ) Ligation of the purified PCR product to the PMD18-T plasmid vector (T-Acloning)
                7.2.6.1 ) Transformation of the ligation product in to the E. coli DH5a competent cellsand plating
                7.2.6.2 ) PMD18-T-mazF; PMD18-T-mazE plasmids extraction
                7.2.6.3 ) Double enzyme digestion of the PMD18-T-mazF; PMD18-T-mazE plasmid
                7.2.6.4 ) Sequencing of the recombinant plasmid transform into the E. coli DH5a
                7.2.6.5 ) mazF/mazE genes prokaryotic expression system construction
                7.2.6.6 ) PMD18-T-mazF; PMD18-T-mazE and pET42a Plasmid DNA extraction
                7.2.6.7 ) Double enzyme digestion of the PMD18-T-mazE;PMD18-T-mazFrecombinant Plasmid DNA and the pET-42a plasmid vector
                7.2.6.8 ) PMD18-T-mazE；PMD18-T-mazF and pET42a Plasmid DNA extraction andpurification
                7.2.6.9 ) Ligation of the mazF and mazE gene to the pET42a plasmid vector:sub-cloning
            7.2.7 ) Transformation of the ligation product into the E.coli BL21(DE3)competentcells:sub-cloning
            7.2.8 ) pET42a-mazF and pET42a-mazE recombinant plasmid DNA extraction
            7.2.9 ) Double enzyme digestion of the recombinant plasmid DNA
            7.2.10 ) Sequencing of the recombinant DNA
            7.2.11 ) Recombinant proteins rMazF and rMazE expression and purification
            7.2.12 ) Protein expression
            7.2.13 ) SDS-PAGE gel preparation
            7.2.14 ) Protein extraction
            7.2.15 ) Protein purification
            7.2.16 ) rMazF/rMazE toxin-antitoxin rabbit anti-serum preparation
        7.3 ) Results and Discussion
            7.3.1 ) Results
            7.3.2 ) Discussion
        7.4 ) Conclusion
        References
    CHAPTER 8 Study of the mazF/mazE genes expression level after infection ofthe THP-1 cells by the pathogenic Leptospira interrogans strainLai
        8.1 ) Materials
            8.1.1 ) Bacterial and cell line used in this work
            8.1.2 ) Reagents and kits used in this work
            8.1.3 ) Experimental Instruments
        8.2 ) Methods
            8.2.1 ) Pathogenic Leptospira interrogans strain Lai culturing
            8.2.2 ) THP-1 cell culturing
            8.2.3 ) Real-Time-PCR Primers designing
            8.2.4 ) Infection of THP-1 cells by the pathogenic Leptospira interrogans strain Lai
            8.2.5 ) Leptospires Total RNA extraction
            8.2.6 ) Total RNA quantification
            8.2.7 ) Reverse-transcription PCR
            8.2.8 ) Reverse Transcription-PCR-condition
            8.2.9 ) Real time PCR Reaction
        8.3 ) Results and Discussion
            8.3.1 ) Results
            8.3.2 ) Discussion
        8.4 ) Conclusion
        References
    Chapter 9 #119Study of pathogenic Leptospira interrogans strain Lai toxic protein rMazFexternal secretion during host cell infection
        9.1 ) Materials
            9.1.1 ) Bacterial and cell line used in this work
            9.1.2 Buffers and solutions
        9.2 ) Methods
            9.2.1 ) Sample preparation
            9.2.2 ) SDS-PAGE electrophoresis gel running
            9.2.3 ) Western blot analysis
        9.3 ) Results and Discussion
            9.3.1 ) Results
            9.3.2 ) Discussion
        9.4 ) Conclusion
        References
    Chapter 10 #126Study of the mazF/mazE gene expression in the eukaryotic cells system
        10.1 ) Materials
            10.1.1 ) Bacterial and Cells line used in this work
            10.1.2 ) Plasmid vector used in this work
            10.1.3 ) Kits and Others
            10.1.4 ) Instruments
        10.2 ) Methods
            10.2.1 ) Cell culturing
            10.2.2 ) Pathogenic leptospira genomic DNA extraction
            10.2.3 ) PCR amplification of the target gene
                10.2.3.1 ) Primers design
                10.2.3.2 ) PCR condition
                10.2.3.3 ) PCR cycling conditions
                10.2.3.4 ) Agarose gel electrophoresis of the pcr product
                10.2.3.5 ) Agarose gel extraction and purification
                10.2.3.6 ) pCMV-Tag2C plasmid extraction
                10.2.3.7 ) Double enzyme digestion of the pCMV-Tag2C plasmid
                10.2.3.8 ) Agarose gel extraction and purification
                10.2.3.9 ) Liagation of the amplified target gene to the pCMV-Tag2C plasmid
                10.2.3.10 ) Transformation of the pCMV-Tag2C~(mazF) and pCMV-Tag2C~(mazE) recombinantplasmids in to the E.Coli DH5a competent cells and plating
                10.2.3.11 ) Sequencing of the the pCMV-Tag2C~(mazF) and pCMV-Tag2C~(mazE) recombinantplasmids
                10.2.3.12 ) Recombinant plasmid pCMV-Tag2C~(mazF) and pCMV-Tag2C~(mazE) extraction
                10.2.3.13 ) HEK 293 cell line transfection
        10.3 ) Results and Discussion
            10.3.1 ) Results
            10.3.2 ) Discussion
        10.4 ) Conclusion
        References
    Chapter11 #140Nuclease activity study of recombinant proteins rMazF and rMazE ofpathogenic Leptospira interrogans strain Lai
        11.1 ) Materials
            11.1.1 ) Bacterials and Cells line used in this work
            11.1.2 ) Kits and others
            11.1.3 ) Experimental Instruments
        11.2 ) Methods
            11.2.1 ) Pathogenic Leptospira interrogans strain,genomic DNA extraction
            11.2.2 ) THP-1 cell genomic DNA extraction
            11.2.3 ) DNA quantification
            11.2.4 ) Cleavage of Total RNA and genomic DNA by MazF
        11.3 ) Results and Discussion
            11.3.1 ) Results
            11.3.2 ) Discussion
        11.4 ) Conclusion
        References
    Chapter 12 Study of rMazF, toxin of pathogenic Leptospira interrogans strainLai, ability to internalize the host Cell
        12.1 ) Materials
            12.1.1 ) Bacterial and Cell line used in this work
            12.1.2 ) Kits and others
            12.1.3 ) Experimental Instruments
        12.2 ) Methods
            12.2.1 ) THP-1 preparation
            12.2.2 ) Pathogenic Leptospira interrogans strain Lai preparation
            12.2.3 ) Infection of the THP-1 cell
            12.2.4 ) Laser confocal Microspcope analysis
        12.3 ) Results and Discussions
            12.3.1 ) Results
            12.3.2 ) Discussion
        12.4 ) Conclusion
        12.5 ) General conclusion and perspectives
        References
Glossary
英文綜述
    References


【參考文獻(xiàn)】：
期刊論文
[1]Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome[J]. Yi Xuan ZHANG, Xiao Kui GUO, Chuan WU, Bo BI, Shuang Xi REN, Chun Fu WU, Guo Ping ZHAO Pharmaceutical Department, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China. Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, Shanghai 200233, China. Department of Microbiology and Parasitology, Shanghai Second Medical University, 280 Chongqingnan Road, Shanghai 200025, China. Chinese National Human Genome Center at Shanghai (CHGCS), 250 Bibo Road, ZhangJiang High Tech Park, Shanghai, 201203, China..  Cell Research. 2004(03)



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