钩端螺旋体ChpK/ChpI和MazF/MazE毒素—抗毒素系统致病性及作用机制研究

发布时间：2021-09-28 15:13

　　背景（background）:钩端螺旋体（简称钩体）分为致病性钩体和非致病性钩体两大类,前者引起人或动物疾病,后者属于非致病腐生性原核细胞型微生物。目前发现,致病性钩体可分为7个基因种,其中问号钩体基因种（Leptospira interrogans）流行最为广泛,如黄疸出血群、波摩那群、秋季群等；其次为波帕特森基因种（Leptospira borgpetersenii）,如流感伤寒群。由致病性钩体感染引起的钩体病是全球流行的人畜共患传染病,中国及东南亚地区是钩体病流行的主要疫区,其次是巴西等南美地区国家。人类感染致病性钩体后,首先出现中毒性败血症症状,如高热、头痛、肌痛等；然后因钩体侵入肺、肝、肾等脏器及中枢神经系统,出现出血、黄疸及呼吸或肾功能衰竭症状,因主要受损脏器不同,临床上可分为六型,其中肺弥漫性出血（pulmonary diffuse hemorrhage, PHD）型钩体病死亡率高达50%～75%。以往生物学实验证明,致病性钩体不产生任何典型的外毒素,业已公布的2株致病性钩体全基因组序列中,也未发现有任何编码经典外毒素基因。致病性钩体脂多糖（LPS）的内毒素毒性仅为大肠杆... 

【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校

【文章页数】：196 页

【学位级别】：博士

【文章目录】：
Acknowledgement
致谢
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
英文综述
    References


【参考文献】：
期刊论文
[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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