結(jié)核分枝桿菌（M.tuberculosis）是一種胞內(nèi)寄生菌,全世界約有三分之一的人口感染M.tuberculosis,但是,大多數(shù)人在肺中無害地攜帶細(xì)菌,而沒有任何臨床癥狀,被稱為潛伏性結(jié)核�。↙TB）。目前,LTB在全球范圍內(nèi)廣泛活化和傳播,且尚不清楚其清除的具體機(jī)制。此外,結(jié)核分枝桿菌對目前用于治療結(jié)核病的抗生素有極大的耐藥性,這為結(jié)核分枝桿菌感染和在人群中的傳播提供了基礎(chǔ)。此外,沒有良好的診斷工具可以及早發(fā)現(xiàn)該疾病。目前可用的結(jié)核病疫苗僅對兒童有效。要了解M.tuberculosis的抗藥性、毒力及再活化的機(jī)制并開發(fā)有效的抗結(jié)核藥物和疫苗,人們應(yīng)該了解M.tuberculosis的功能基因組學(xué)。但是,M.tuberculosis的功能基因組學(xué)研究受到復(fù)雜且耗時的遺傳操作技術(shù)的阻礙�；谝陨蠁栴},本課題主要集中在如下兩個方面進(jìn)行研究:（1）我們對M.tuberculosis內(nèi)源性III-A型CRISPR-Cas10系統(tǒng)進(jìn)行了重新編輯,僅僅通過轉(zhuǎn)入帶有mini-CRISPR序列的質(zhì)粒來簡單有效地進(jìn)行基因編輯、RNA干擾和全基因組CRISPR干擾（CRISPRi）的篩選,同時避免了引... 

【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁數(shù)】：109 頁

【學(xué)位級別】：博士

【文章目錄】：
摘要
Abstract
1. Literature review and introduction
    1.1 Tuberculosis
    1.2 Latent TB
    1.3 Epidemiology of TB
    1.4 Diagnosis of TB
    1.5 Treatment of TB
    1.6 Control and Prevention of TB
    1.7 Mycobacterium tuberculosis (M. tuberculosis) the causative agent of TB
    1.8 Functional Genomics of M. tuberculosis
    1.9 Genome complexity and diversity
    1.10 Genome editing In M. tuberculosis
    1.11 Genome editing In M. tuberculosis
    1.12 The CRISPR system
    1.13 Effects of stress hormones on M. tuberculosis
2. Aims and Objectives
3. Material and Methods
    3.1 Chemicals and reagents
    3.2 Bacterial strains, Cell lines and Culture conditions
    3.3 Competence cell preparation and electro transformation
    3.4 Bacteria treatment and in-vitro growth analysis
    3.5 Plasmid construction and Cloning
    3.6 DNA Isolation, PCR amplification, purification and plasmid isolation
    3.7 RNA extraction and quantitative real-time PCR
    3.8 g RNA designing for single gene knockdown
    3.9 g RNA library designing and construction
    3.10 Library screening and high throughput sequencing
    3.11 High throughput sequencing data analysis
    3.12 THP-1Cell infection and Treatment with hormones
    3.13 Isolation and quantification of Intracellular bacteria
    3.14 Other Insilco studies
    3.15 Molecular docking
    3.16 Statistical analysis
    3.17 Reagents Preparation
        3.17.1 PBS preparation
        3.17.2 LB medium Preparation
        3.17.3 7H9broth 7H10 preparation
    3.18. Other Protocols
        3.18.1 Mt.b DNA Extraction
        3.18.2 Protocol for g RNA strands Annealing
        3.18.3 Anneal in a thermo cycler using the following parameters
        3.18.4 Bbs I Digestion of p MV-261
        3.18.5 BbsI Digestion of p MV-261
        3.18.6 Ligation reaction
        3.18.7 Different hormones preparation
4. Results
    Part I
        4.1 Genome-wide CRISPR interference screening in M. tuberculosis
        4.2 Discussion
    Part II
        4.3. Elucidation the response of M. tuberculosis toward host stress hormones
        4.4. Discussion
5. Conclusion
References
ACKNOWLEDGEMENTS
Supplementary Materials


【參考文獻(xiàn)】：
期刊論文
[1]細(xì)菌中CRISPR/Cas系統(tǒng)的應(yīng)用和優(yōu)化[J]. 傅俊豪,楊發(fā)譽(yù),謝海華,谷峰.  生物工程學(xué)報. 2019(03)
[2]Advances in the development of molecular genetic tools for Mycobacterium tuberculosis[J]. Chiranjibi Chhotaray,Yaoju Tan,Julius Mugweru,Md Mahmudul Islam,H.M.Adnan Hameed,Shuai Wang,Zhili Lu,Changwei Wang,Xinjie Li,Shouyong Tan,Jianxiong Liu,Tianyu Zhang.  Journal of Genetics and Genomics. 2018(06)



本文編號：3586825