结核分枝杆菌主要分泌蛋白的功能和免疫原性分析

发布时间：2022-11-03 23:19

　　结核病（TB）是分别由结核分枝杆菌（Mtb）和牛分枝杆菌（牛分枝杆菌）引起的人类和牛的高传染性疾病。两种物种都拥有近99%的基因组同一性,并且可以以比感染自己的宿主更少的疾病严重程度感染彼此的宿主。这一观察结果导致了针对人类结核病的卡介苗芽孢杆菌（BCG）疫苗（牛分枝杆菌的改良形式）的开发。目前,卡介苗是唯一可用于结核病的疫苗,对成人没有增强作用和功效。有希望的辅助剂或BCG补充剂仍然是克服结核病威胁的主要挑战。考虑到这一事实,使用免疫信息学方法从Mtb中三个分泌系统的蛋白质:双精氨酸易位（TAT）,Esx和Sec设计了亚单位疫苗。分泌蛋白的免疫原性表位被预测并用于疫苗方案。然后,通过使用各种在线工具对疫苗进行计算机评估。这些工具宣告了该疫苗是稳定的,非过敏性的,具有抗原性（免疫原性）,并且对通行费样受体2（TLR2）具有结合亲和力。对于疫苗蛋白的细胞内表达,将氨基酸序列反向翻译为核苷酸序列,并将其克隆到腺相关病毒载体（p AAV）中。将没有和带有疫苗蛋白的病毒载体包装到AAV-Dj/8双顺反子表达系统中。分别通过q PCR和q RT-PCR对两种病毒进行了体外表征,以估计其DNA和R... 

【文章页数】：141 页

【学位级别】：博士

【文章目录】：
摘要
ABSTRACT
ABBREVIATION
CHAPTER 1:INTRODUCTION
    1.1 INTRODUCTION
    1.2 Literature review
        1.2.1 History
        1.2.2 Transmission and Pathogenesis
        1.2.3 Epidemiology and topographical spread
        1.2.4 Reverse zoonosis: Mtb infection in bovines
        1.2.5 Vaccines for TB
        1.2.6 Peptide vaccine(s)for TB
        1.2.7 Genomic organization features
        1.2.8 Functional genetics of Mtb
        Objectives
CHAPTER 2:SUBUNIT VACCINE DESIGN AND IN SILICO ANALYSIS
    2.1 MATERIALS AND METHODS
        2.1.1 Collection of protein sequences for vaccine design
        2.1.2 MHC-I/-II binding and B cell epitopes prediction
        2.1.3 Multi-epitope subunit vaccine construct
        2.1.4 Prediction of physiochemical parameter,allergenicity,antigenicity analysis
        2.1.5 Docking of SV with mouse TLR2 receptor
        2.1.6 Reverse translation,codon optimization and in silico cloning
    2.2 RESULTS
        2.2.1 Construction of multi-epitope vaccine sequence
        2.2.2 Physiochemical properties,Allergenicity and antigenicity analysis
        2.2.3 Molecular docking
        2.2.4 Reverse translation,codon optimization and in silico cloning
CHAPTER 3:IN VITRO WORKING OF SUBUNIT VACCINE
    3.1 MATERIALS AND METHODS
        3.1.1 In vitro cloning
        3.1.2 Intracellular SV expression
        3.1.3 AAV virus generation
        3.1.4 DNA and RNA copy number determination
    3.2 RESULTS
        3.2.1 Removal of adjuvant and docking of vaccine protein with mouse TLR9
        3.2.2 Cloning confirmation
        3.2.3 Subunit vaccine protein expression
        3.2.4 Calculated DNA and RNA copy number
CHAPTER 4:RV3444C-RV3445C CHARACTERIZATION
    4.1 MATERIALS AND METHODS
        4.1.1 Hypothesis of work
        4.1.2 Vaccine candidate characterization(DNA binding prediction)
        4.1.3 Subcellular localization prediction and in silico structure modeling
        4.1.4 Plasmid design,cell culture and IFA
        4.1.5 Co-localization of Rv3444c/5c in nucleus
        4.1.6 Co-immunoprecipitation(Co-IP)
        4.1.7 Bacterial culture
        4.1.8 In vitro and intracellular expression of Rv3444c and Rv3445c
    4.2 RESULTS
        4.2.1 Shortlisting for DNA-binding protein
        4.2.2 Sub-cellular localization prediction,in silico docking
        4.2.3 IFA analysis
        4.2.4 Nuclear co-localization
        4.2.5 Co-IP interaction analysis
        4.2.6 Relative transcriptional expression(in vitro and intracellular)
CHAPTER 5:RV3444C-RV3445C FUNCTIONAL ANALYSIS IN THP1 STABLE CELL LINE
    5.1 MATERIALS AND METHODS
        5.1.1 Lentivirus generation
        5.1.2 Stable cell line generation
        5.1.3 qPCR and IFA for expression analysis of both genes
        5.1.4 Chromatin immunoprecipitation and sequencing(Ch IP-seq)
        5.1.5 Transcriptional profiling(RNA-seq)of stable cell lines
        5.1.6 Cytokines analysis of stable cell lines
        5.1.7 Viable bacilli assay in stable cell lines
    5.2 RESULTS
        5.2.1 Genes expression detection in stable cell line
        5.2.2 Global map of histone tri-methylation and acetylation using stable cell lines
        5.2.3 Transcriptional profile(RNA-seq)of stable cell lines
            5.2.3.1 Nucleic acid binding protein class and q RT-PCR validation
        5.2.4 Stable cell line cytokines analysis
        5.2.6 Viable bacilli assay in stable cell lines
CHAPTER 6:KNOCK DOWN OF RV3444C-RV3445C AND FUNCTIONAL ANALYSIS
    6.1 MATERIALS AND METHODS
        6.1.1 Knock down strain generation
        6.1.2 In vitro/Planktonic growth analysis
        6.1.3 WT/KD infected THP1 cells
    6.2 RESULTS
        6.2.1 Knock down(KD)strain evaluation by q RT-PCR
        6.2.2 In vitro growth curve analysis of both strains
        6.2.3 Viable bacilli assay in WT/KD infected THP1 cells
        6.2.4 Transcriptional profile of WT/KD infected cells
        6.2.5 Cytokines analysis of WT/KD infected THP1 cells
CHAPTER 7:WT/KD INFECTION:IN VIVO STUDY
    7.1 MATERIALS AND METHODS
    7.2 Results
        7.2.1 Bacilli viability in lung and spleen samples
        7.2.2 Histopathology
        7.2.3 Cytokines analysis of WT/KD infected mouse lungs
CHAPTER 8:DISCUSSION
CONCLUSION
REFERENCES
APPENDIX-Ⅰ
APPENDIX-Ⅱ
ACKNOWLEDGEMENTS


【参考文献】：
期刊论文
[1]卡龙加地区过去30多年的结核病研究给我们的启示是什么?[J]. A.C.Crampin,J.R.Glynn,P.E.M.Fine,胡冬梅,何广学.  国际结核病与肺部疾病杂志(中文版). 2009(03)



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