結(jié)核�。═B）是分別由結(jié)核分枝桿菌（Mtb）和牛分枝桿菌（牛分枝桿菌）引起的人類和牛的高傳染性疾病。兩種物種都擁有近99%的基因組同一性,并且可以以比感染自己的宿主更少的疾病嚴(yán)重程度感染彼此的宿主。這一觀察結(jié)果導(dǎo)致了針對(duì)人類結(jié)核病的卡介苗芽孢桿菌（BCG）疫苗（牛分枝桿菌的改良形式）的開發(fā)。目前,卡介苗是唯一可用于結(jié)核病的疫苗,對(duì)成人沒有增強(qiáng)作用和功效。有希望的輔助劑或BCG補(bǔ)充劑仍然是克服結(jié)核病威脅的主要挑戰(zhàn)�？紤]到這一事實(shí),使用免疫信息學(xué)方法從Mtb中三個(gè)分泌系統(tǒng)的蛋白質(zhì):雙精氨酸易位（TAT）,Esx和Sec設(shè)計(jì)了亞單位疫苗。分泌蛋白的免疫原性表位被預(yù)測(cè)并用于疫苗方案。然后,通過使用各種在線工具對(duì)疫苗進(jìn)行計(jì)算機(jī)評(píng)估。這些工具宣告了該疫苗是穩(wěn)定的,非過敏性的,具有抗原性（免疫原性）,并且對(duì)通行費(fèi)樣受體2（TLR2）具有結(jié)合親和力。對(duì)于疫苗蛋白的細(xì)胞內(nèi)表達(dá),將氨基酸序列反向翻譯為核苷酸序列,并將其克隆到腺相關(guān)病毒載體（p AAV）中。將沒有和帶有疫苗蛋白的病毒載體包裝到AAV-Dj/8雙順反子表達(dá)系統(tǒng)中。分別通過q PCR和q RT-PCR對(duì)兩種病毒進(jìn)行了體外表征,以估計(jì)其DNA和R... 

【文章頁數(shù)】：141 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
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


【參考文獻(xiàn)】：
期刊論文
[1]卡龍加地區(qū)過去30多年的結(jié)核病研究給我們的啟示是什么?[J]. A.C.Crampin,J.R.Glynn,P.E.M.Fine,胡冬梅,何廣學(xué).  國(guó)際結(jié)核病與肺部疾病雜志(中文版). 2009(03)



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