羊无浆体感染森林革蜱中肠相关功能分子的鉴定及其相互作用分析
发布时间：2021-01-28 22:32
羊无浆体是经蜱传播的红细胞内寄生的革兰氏阴性菌,是羊无浆体病的病原体。革蜱属的蜱是羊无浆体病的生物学传播媒介。表面蛋白Msp1a、AAAP蛋白、Vir B10和Vir D4蛋白是存在于羊无浆体基因组中的一些重要分子。本研究利用这些分子作诱饵蛋白筛选森林革蜱中肠蛋白,通过酵母双杂交试验研究病原-媒介的相互作用分子机制。将森林革蜱中肠c DNA克隆到p GADT7-Sma I载体（Prey质粒）上构建了酵母双杂交文库,将羊无浆体的蛋白克隆到p GBKT7载体中构建成了诱饵质粒,并对酵母菌株Y2HGold中的自身活化和毒性进行了评估。通过载有诱饵质粒酵母菌株Y2HGold和载有捕获质粒的Y187的杂交开展了酵母双杂交筛选。通过在BLAST,Uni Prot,STRING和SMART数据库对捕获质粒序列分析,多个媒介蜱的蛋白包括一个保守的假定蛋白、核糖体蛋白、被膜蛋白UL36、脂肪酸结合蛋白、蛋白二硫化物异构酶3、涂层亚基δ、细胞包膜完整性内膜蛋白、鞭毛生物合成调节剂F1h F、分子伴侣、剂量补偿调节复合物蛋白和非特征蛋白LOC8036454被鉴定为潜在的相互作用蛋白。由于核糖体（RL12）蛋...
【文章来源】：中国农业科学院北京市
【文章页数】：95 页
【学位级别】：博士
【文章目录】：
摘要
abstract
英文缩略表
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
本文编号：3005810
【文章来源】：中国农业科学院北京市
【文章页数】：95 页
【学位级别】：博士
【文章目录】：
摘要
abstract
英文缩略表
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
本文编号：3005810
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