利用基因组生物信息学预测和蛋白组学对牛支原体分泌蛋白组的研究及分泌性抗原MbovP0581的验证
发布时间：2024-05-20 01:52
牛支原体是牛重要的病原体之一,可引起牛的多种感染,包括肺炎、关节炎、中耳炎、乳腺炎、角膜结膜炎、生殖器疾病和各种临床相关疾病,造成了重大的经济损失。其发病机制认识不足对其早期诊断和有效疫苗的研制提出了挑战。由于分泌蛋白在细菌发病机制中具有重要的作用,因此本研究旨在系统地揭示牛支原体分泌组及其在致病力中的潜在作用。我们结合基因组预测和蛋白质组学验证开展研究。利用生物信息学工具,共预测出246个牛支原体分泌蛋白。其中14个蛋白属于经典通路,154个蛋白属于非经典通路,78个蛋白属于经典和非经典通路。接着采用双向凝胶电泳(2-DE)和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术,共检测到169个蛋白。其中60个蛋白属于先前被预测的分泌蛋白,包括3个经典通路蛋白,43个非经典通路蛋白,14个经典和非经典通路蛋白。亚细胞定位预测显示,8.3%(5/60)位于细胞膜,5.0%(3/60)位于胞外区,31.6%(19/60)位于胞浆,55.0%(33/60)位置未知。对这60种分泌蛋白进行KEGG富集了GO和蛋白质相互作用分析。利用VFDB数据库进行毒力相关因子分析,其中8个蛋白...
【文章页数】：129 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Abbreviations
1.Introduction
1.1 Background of this research
1.1.1 Bovine Mycoplasmosis in cattle population of China
1.1.2 Prevention and control strategies of M.bovis
1.1.3 Role of genomics and proteomics in the study of bacterial cell secretome
1.1.4 Statement of problems
1.2 Review of Literature
1.2.1 Biological characteristics of Mycoplasmas
1.2.2 Pathogenesis of Mycoplasma bovis
1.2.3 Diagnosis
1.2.4 Vaccination
1.2.5 Bacterial cell secretome
1.2.6 Secretome of Mycoplasmas
1.2.7 Methodology to study secretome of Mycoplasmas
2.Aims and objectives
3.Identification of antigenic secreted proteins of Mycoplasma bovis with genome and proteome analyses
3.1 Introduction
3.2 Materials and methods
3.2.1 Ethical statement
3.2.2 Strain and culture conditions
3.2.3 Extraction of secretome
3.2.4 Protocol for using2-D clean-up kit
3.2.5 2-Dimensional Gel Electrophoresis
3.2.6 Matrix-assisted laser desorption/ionization time of flight(MALDI-TOF)mass spectrometry(MS)
3.2.7 Bioinformatics analysis
3.2.8 Immunoinformatics analysis of secreted proteins
3.2.9 Cloning and expression of selected secreted proteins
3.2.10 Transformation of ligated sample into BL21
3.2.11 Checking protein expression
3.2.12 Checking solubility of proteins
3.2.13 Purification of r Mbov581 from supernatant
3.2.14 Purification of r Mbov674 from pellet
3.2.15 Extraction of whole cell proteins
3.2.16 Production of mouse polyclonal antibodies against the selected secreted proteins
3.2.17 Preparation of cattle antisera to M.bovis
3.2.18 Western blot assays for verification of secreted nature and antigenicity of selected proteins
3.2.19 Structure prediction and identification of conserved regions
3.2.20 Adhesion assay
3.2.21 Quantitative assay for adhesion
3.2.22 Statistical analysis
3.3 Results
3.3.1 Secretome analysis using M.bovis whole genome
3.3.2 Identification of secretome using proteomics approach
3.3.3 Proteins involved in various virulence-associated pathways
3.3.4 Gene Ontology(GO)Analysis
3.3.5 Protein-Protein interaction
3.3.6 Virulence-related factors of proteins identified by VFDB
3.3.7 Conserved domain Analysis
3.3.8 Identification of conformational B cell epitopes
3.3.9 Prediction of T cell epitopes
3.3.10 Cloning,expression and purification of selected proteins
3.3.11 Validation of secreted nature and antigenicity
3.3.12 Structural and functional prediction showed conserved regions in Mbov P0581
3.3.13 Adhesion of Mbov P581 to EBL cells
3.4 Discussion
3.4.1 The number of secreted proteins of M.bovis
3.4.2 Identification of critical secreted proteins for M.bovis
3.4.3 Detection of Mbov P0581 as an adhesin to EBL cells
4.Conclusion
5.Statement of novelty of this study
References
Supplementary data
List of publications
Acknowledgement
本文编号：3978778
【文章页数】：129 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Abbreviations
1.Introduction
1.1 Background of this research
1.1.1 Bovine Mycoplasmosis in cattle population of China
1.1.2 Prevention and control strategies of M.bovis
1.1.3 Role of genomics and proteomics in the study of bacterial cell secretome
1.1.4 Statement of problems
1.2 Review of Literature
1.2.1 Biological characteristics of Mycoplasmas
1.2.2 Pathogenesis of Mycoplasma bovis
1.2.3 Diagnosis
1.2.4 Vaccination
1.2.5 Bacterial cell secretome
1.2.6 Secretome of Mycoplasmas
1.2.7 Methodology to study secretome of Mycoplasmas
2.Aims and objectives
3.Identification of antigenic secreted proteins of Mycoplasma bovis with genome and proteome analyses
3.1 Introduction
3.2 Materials and methods
3.2.1 Ethical statement
3.2.2 Strain and culture conditions
3.2.3 Extraction of secretome
3.2.4 Protocol for using2-D clean-up kit
3.2.5 2-Dimensional Gel Electrophoresis
3.2.6 Matrix-assisted laser desorption/ionization time of flight(MALDI-TOF)mass spectrometry(MS)
3.2.7 Bioinformatics analysis
3.2.8 Immunoinformatics analysis of secreted proteins
3.2.9 Cloning and expression of selected secreted proteins
3.2.10 Transformation of ligated sample into BL21
3.2.11 Checking protein expression
3.2.12 Checking solubility of proteins
3.2.13 Purification of r Mbov581 from supernatant
3.2.14 Purification of r Mbov674 from pellet
3.2.15 Extraction of whole cell proteins
3.2.16 Production of mouse polyclonal antibodies against the selected secreted proteins
3.2.17 Preparation of cattle antisera to M.bovis
3.2.18 Western blot assays for verification of secreted nature and antigenicity of selected proteins
3.2.19 Structure prediction and identification of conserved regions
3.2.20 Adhesion assay
3.2.21 Quantitative assay for adhesion
3.2.22 Statistical analysis
3.3 Results
3.3.1 Secretome analysis using M.bovis whole genome
3.3.2 Identification of secretome using proteomics approach
3.3.3 Proteins involved in various virulence-associated pathways
3.3.4 Gene Ontology(GO)Analysis
3.3.5 Protein-Protein interaction
3.3.6 Virulence-related factors of proteins identified by VFDB
3.3.7 Conserved domain Analysis
3.3.8 Identification of conformational B cell epitopes
3.3.9 Prediction of T cell epitopes
3.3.10 Cloning,expression and purification of selected proteins
3.3.11 Validation of secreted nature and antigenicity
3.3.12 Structural and functional prediction showed conserved regions in Mbov P0581
3.3.13 Adhesion of Mbov P581 to EBL cells
3.4 Discussion
3.4.1 The number of secreted proteins of M.bovis
3.4.2 Identification of critical secreted proteins for M.bovis
3.4.3 Detection of Mbov P0581 as an adhesin to EBL cells
4.Conclusion
5.Statement of novelty of this study
References
Supplementary data
List of publications
Acknowledgement
本文编号：3978778
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