论文摘要
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、UniProt、SMART、STRING等方法进行序列分析。本研究发现森林革蜱的4种唾液腺蛋白:核糖体蛋白L12,保守的预测蛋白cadherin repeats和FlhF,以及肿瘤坏死因子（TNF）受体因子样蛋白与VirD4相互作用;发现衣壳蛋白UL36与VirB10相互作用。与VirD4、VirB10相互作用的宿主蛋白经功能分析发现,这些蛋白参与细胞黏附、凋亡调控、细胞增殖、信号转导、存活、后期复制、泛素化调控等细胞过程。本研究首次筛选了与羊无浆体VirD4和VirB10的相互作用的森林革蜱唾液腺蛋白,研究发现5种蛋白,核糖体蛋白L12,保守的预测蛋白cadherin repeats和FlhF,肿瘤坏死因子（TNF）受体因子样蛋白和衣壳蛋白UL36为羊无浆体和森林革蜱相互作用关键分子。本研究结果为进一步深入了解羊无浆体和森林革蜱的相互作用关系奠定了基础。
论文目录
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. RationaleCHAPTER 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 preysCHAPTER Ⅲ DiscussionCHAPTER Ⅳ Graphical summarizationCHAPTER Ⅴ ConclusionReferencesAppendices 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）
文章来源
类型: 博士论文
作者: Muhammad Uzair Mukhtar
导师: 殷宏
关键词: 森林革蜱,唾液腺,羊无浆体,酵母双杂交
来源: 中国农业科学院
年度: 2019
分类: 基础科学,农业科技
专业: 生物学,畜牧与动物医学
单位: 中国农业科学院
基金: the National Key R&D Program of China (2017YFD0501200,2018YFD0502305),NSFC(№31502054,№31502091),ASTIP (CAAS-ASTIP-2016-LVRI),NBCIS (CARS-37),973 Program (2015CB150300),Jiangsu Co-innovation Center program for Prevention and Control of Important Animal Infectious Disease and Zoonosis
分类号: S852.64
DOI: 10.27630/d.cnki.gznky.2019.000037
总页数: 82
文件大小: 4952k
下载量: 2
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标签：森林革蜱论文; 唾液腺论文; 羊无浆体论文; 酵母双杂交论文;
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
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