羊無漿體是經(jīng)蜱傳播的紅細(xì)胞內(nèi)寄生的革蘭氏陰性菌,是羊無漿體病的病原體。革蜱屬的蜱是羊無漿體病的生物學(xué)傳播媒介。表面蛋白Msp1a、AAAP蛋白、Vir B10和Vir D4蛋白是存在于羊無漿體基因組中的一些重要分子。本研究利用這些分子作誘餌蛋白篩選森林革蜱中腸蛋白,通過酵母雙雜交試驗(yàn)研究病原-媒介的相互作用分子機(jī)制。將森林革蜱中腸c DNA克隆到p GADT7-Sma I載體（Prey質(zhì)粒）上構(gòu)建了酵母雙雜交文庫,將羊無漿體的蛋白克隆到p GBKT7載體中構(gòu)建成了誘餌質(zhì)粒,并對(duì)酵母菌株Y2HGold中的自身活化和毒性進(jìn)行了評(píng)估。通過載有誘餌質(zhì)粒酵母菌株Y2HGold和載有捕獲質(zhì)粒的Y187的雜交開展了酵母雙雜交篩選。通過在BLAST,Uni Prot,STRING和SMART數(shù)據(jù)庫對(duì)捕獲質(zhì)粒序列分析,多個(gè)媒介蜱的蛋白包括一個(gè)保守的假定蛋白、核糖體蛋白、被膜蛋白UL36、脂肪酸結(jié)合蛋白、蛋白二硫化物異構(gòu)酶3、涂層亞基δ、細(xì)胞包膜完整性內(nèi)膜蛋白、鞭毛生物合成調(diào)節(jié)劑F1h F、分子伴侶、劑量補(bǔ)償調(diào)節(jié)復(fù)合物蛋白和非特征蛋白LOC8036454被鑒定為潛在的相互作用蛋白。由于核糖體（RL12）蛋... 

【文章來源】：中國農(nóng)業(yè)科學(xué)院北京市

【文章頁數(shù)】：95 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
abstract
英文縮略表
CHAPTER Ⅰ Introduction
    1.1 Anaplasma ovis
        1.1.1 Genus Anaplasma taxonomy
        1.1.2 Anaplasma infectivity
        1.1.3 Anaplasma life cycle
        1.1.4 Genomic highlights
        1.1.5 Veterinary and public health importance
    1.2 Ovine anaplasmosis
        1.2.1 Etiology and distribution
        1.2.2 Signs and symptoms
        1.2.3 Transmission and diagnosis
        1.2.4 Treatment, control and prevention
    1.3 Taxonomy and life cycle of Dermacentor silvarum
    1.4 Development of Anaplasma in the tick vector
    1.5 Y2H introduction and principle
    1.6 Introduction to Type IV Secretion System（T4SS）
    1.7 Rationale
CHAPTER Ⅱ Screening of D.silvarum midgut proteins interacting with the proteinsrelated to A.ovis infection
    2.1 Materials and methods
        2.1.1 Reagents
    2.2 Bait plasmid construction
        2.2.1 AAAP bait construction
        2.2.2 Msp1a bait construction
        2.2.3 VirB10 bait construction
        2.2.4 VirD4 bait construction
    2.3 Expression vector pGBKT7
    2.4 Purification of amplified products（AAAP,Msp1a,VirB10 and VirD4）after gelelectrophoresis
    2.5 Ligation of the purified PCR products in pGEM T-easy vector
    2.6 Transformation into competent cells
    2.7 Spreading on the LB-agar plates（Amp+）
    2.8 Plasmid extraction
    2.9 Digestion reaction
    2.10 Ligation with T4 DNA ligase
    2.11 Transformation into competent cells
    2.12 Transfer to LB-agar plates（Kan+）
    2.13 Colony PCR
    2.14 Sequence analysis
    2.15 Extraction of bait plasmids from bacterial culture
    2.16 Auto-activation and toxicity tests of bait plasmid
CHAPTER Ⅲ
    3.1 Construction of yeast two-hybrid c DNA library of tick midgut
        3.1.1 Isolation of the midguts from the ticks
    3.2 cDNA library
    3.3 Yeast-two-hybrid screening by mating bait with prey
        3.3.1 Y2H screening
        3.3.2 Preparation of negative and positive control vectors
        3.3.3 Selection of the positive prey plasmids
    3.4 Positive prey analysis
        3.4.1 Glutathione S-transferase（GST）pull-down
        3.4.2 Western Blot
    3.5 Results
        3.5.1 Construction of yeast two-hybrid c DNA library of midgut
    3.6 Bait preparation
        3.6.1 Construction of p GBKT7-AAAP bait plasmid
        3.6.2 Construction of p GBKT7-Msp1a bait plasmid
        3.6.3 Construction of p GBKT7-Vir D4 bait plasmid
        3.6.4 Transformation of control vectors
        3.6.5 Auto-activation and toxicity test of the bait plasmids
    3.7 Identification of positive prey from the c DNA library via Y2H mating assay
        3.7.1 Mating of AAAP bait plasmid and prey
        3.7.2 Mating of Msp1a bait plasmid and prey
        3.7.3 Mating of VirB10 bait plasmid and prey
        3.7.4 Mating of Vir D4 bait plasmid and prey
    3.8 Sequencing and analysis of positive prey
        3.8.1 In vitro evaluation of the interaction between RL12 and VirD4
Chapter Ⅳ Discussion
Chapter Ⅴ Conclusion
References
Appendices
Acknowledgements
Author’s Resume



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