森林革蜱唾液腺与羊无浆体VirD4和VirB10相互作用分子鉴定及其功能分析

发布时间：2022-07-15 16:25

　　羊无浆体（Anapolasma ovis）是一种红细胞内专性寄生、经蜱传播的革兰氏阴性病原体,是引起羊无浆体病的病原。该病原在世界范围分别广泛,分布范围包括亚洲、欧洲、非洲和北美等地区。羊无浆体主要经硬蜱传播,革蜱属的蜱是其最常见的传播媒介。VirD4和VirB10是羊无浆体IV型分泌系统（T4SS）的重要组成成分。T4SS系统对细胞的转运、存活和致病起着至关重要的作用。致病菌常利用T4SS系统直接将毒力因子、DNA和蛋白质转运到宿主细胞。为了揭示羊无浆体和森林革蜱（Dermacentor silvarum）的相互作用的分子基础,本研究旨在以羊无浆体VirD4和VirB10构建诱饵质粒,通过酵母双杂交系统（Y2H）从森林革蜱的唾液腺cDNA文库中筛选与病原体蛋白相互作用的宿主蛋白。本研究将森林革蜱唾液腺的cDNA克隆到pGADT7-SmaI载体（捕获载体）中,构建Y2H cDNA文库。通过对酵母菌Y2H Gold的自激活和毒性试验后,将VirD4和VirB10克隆到pGBKT7载体中构建诱饵质粒。利用酵母双杂交筛选,筛选到的阳性质粒经测序后采用BLAST、Gene Ontology、U... 

【文章页数】：82 页

【学位级别】：博士

【文章目录】：
博士学位论文评阅人、答辩委员会签名表
摘要
abstract
英文缩略表
CHAPTER Ⅰ Introduction
    1.Anaplasma ovis biology
        1.1 Anaplasma taxonomy and bacteriology
        1.2 Genomic structure
        1.3 Life cycle of Anaplasma
        1.4 Anaplasma infection
    2.Veterinary and public health importance
    3.Ovine anaplasmosis
        3.1 Etiology and distribution
        3.2 Signs and symptoms
        3.3 Transmission and diagnosis
        3.4 Treatment,control and prevention
    4.Dermacentor silvarum taxonomy and life cycle
    5.Development of Anaplasma in ticks
    6. Rationale
CHAPTER Ⅱ Yeast-two-hybrid screening(Y2H)of c DN A library of Dermacentor silvarum salivary gland to identify protein interacting with Vir D4 and Vir B10 of Anaplasma ovis
    1.Yeast two-hybrid assay introduction and principle
    2.Introduction to Type IV Secretion System(T4SS)
    3.Materials and methods
        3.1 Reagents
        3.2 Research design/Methodology
        3.3 Methodology flowchart
    4.Bait plasmid construction
        4.1 Vir B10 bait construction
        4.2 Vir D4 bait construction
        4.3 Expression vector p GBKT7
        4.4 Gel extraction of Vir D4 and Vir B10 amplified product
        4.5 Ligation of Vir B10 and Vir D4 in p GEM T-easy vector
        4.6 Transformation into competent cells
        4.7 Transfer to LB-agar plates(Amp+)
        4.8 Plasmid extraction
        4.9 Digestion reaction
        4.10 Terminating digestion reaction
        4.11 Ligation with T4 DNA ligase
        4.12 Transformation into competent cells
        4.13 Transfer to LB-agar plates(Kan+)
        4.14 PCR confirmation
        4.15 Sequence analysis
        4.16 Vir D4-p GBKT7 and Vir B10-p GBKT7 plasmid extraction
    5.Auto-activation and toxicity tests of bait plasmids
    6.Yeast-two-hybrid screening by mating bait with prey
        6.1 Y2H screening by Vir D4
        6.2 Y2H screening by Vir B10
    7.Isolation of the salivary glands from the ticks
    8.Construction of yeast two-hybrid c DNA library of salivary glands
    9.Preparation of negative and positive control vectors
    10.Selection of the positive prey plasmids
        10.1 Positive prey analysis
    11.Results
        11.1 Bait Construction
        11.2 Auto-activation and toxicity test of the Vir B10 bait plasmid
        11.3 Auto-activation and toxicity test of the Vir D4 bait plasmid
        11.4 Transformation of negative and positive controls
        11.5 Cons truction of Y2H cDNA library of D .sil varum salivary gland
        11.6 Y2H screening and confirmation of the VirB10 interactions
        11.7 Sequencing and analysis of Vir B10 positive prey
        11.8 Y2H screening and confirmation of the Vir D4 interactions
        11.9 Sequencing and analysis of Vir D4 positive preys
CHAPTER Ⅲ Discussion
CHAPTER Ⅳ Graphical summarization
CHAPTER Ⅴ Conclusion
References
Appendices
    Appendices1.1 Buffers,media?s and solutions
        Appendix1.1.1:PCR master mix
        Appendix1.1.2:1XTEA buffer
        Appendix1.1.3:1X agarose gel
        Appendix1.1.4:LB liquid medium
        Appendix1.1.5:LB-agar solid medium
        Appendix1.1.6:100 mg/ml ampicillin
        Appendix1.1.7:50 mg/ml kanamycin
        Appendix1.1.8:1×PBS buffer composition
        Appendix1.1.9:1 M IPTG composition
        Appendix1.1.10:20 mg/ml X-Gal composition
        Appendix1.1.11:20 mg/ml X- α -Gal composition
        Appendix1.1.12:Aureobasidin A stock solution
    Appendices1.2 Yeast growth media and supplements
        Appendix1.2.1:SDO agar plates
        Appendix1.2.2:SDO/X agar plates
        Appendix1.2.3:SDO/X/A agar plates
        Appendix1.2.4:DDO agar plates
        Appendix1.2.5:DDO/X agar plates
        Appendix1.2.6:DDO/X/A agar plates
        Appendix1.2.7:QDO/X/A agar plates
        Appendix1.2.8:Freezing medium
        Appendix1.2.9:0.5X YPDA broth
        Appendix1.2.10:0.5X YPDA broth
    Appendices1.3 Yeast handling and plasmid isolation
        Appendix1.3.1:1.1X TE/Li Ac solution
        Appendix1.3.2:PEG/Li Ac solution
        Appendix1.3.3:Preparation of competent yeast cells
        Appendix1.3.4:Yeast plasmid isolation protocol
    Appendices1.4 Yeast two-hybrid library and screening
        Appendix1.4.1:Construction of normalized Y2H AD library
        Appendix1.4.2:Y2H library screening using yeast mating
        Appendix1.4.3:Y2H library titration
    Appendices1.5 Control plasmid information
        Appendix1.5.1:Map of p GBKT7-53 DNA-BD control plasmid
        Appendix1.5.2:Map of p GADT7-T AD control plasmid
致谢(Acknowledgements)
作者简历(Resume)



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