發(fā)布時間：2023-01-09 12:53

　　空腸彎曲桿菌是一種革蘭氏陰性菌,被認為是引起細菌性胃腸炎的主要原因之一�？漳c彎曲桿菌是大約90%彎曲桿菌病的病因。空腸彎曲桿菌病被認為是自我限制的,感染后癥狀在幾天內(nèi)就會解決。然而,抗生素被用來治療免疫受損的病人,特別是患有腸外感染或細菌血癥的患者。抗菌藥物的合理使用對保護食品用動物和人類的健康有著重要的作用,但不適當?shù)氖褂每赡軙䦟е驴咕幬锏哪退幮�。已有報�?彎曲桿菌對氟喹諾酮類、大環(huán)內(nèi)酯類、β-內(nèi)酰胺類和氨基糖苷類產(chǎn)生耐藥。尤其是氟喹諾酮類和大環(huán)內(nèi)酯類藥物的交叉耐藥與空腸彎曲菌的臨床分離株有關(guān)。成簇的規(guī)律間隔短回文重復序列（CRISPR）是一種保護原核微生物免受外來遺傳因素影響的適應性免疫系統(tǒng)�？漳c彎曲桿菌有一個CRISPR-Cas免疫系統(tǒng),它不僅可以識別而且可以干擾入侵者的基因組DNA。該免疫系統(tǒng)可分為三種類型:CRISPR-系統(tǒng)I型、II型和III型。CRISPR-Ⅱ型（Nmeni亞型）存在于空腸彎曲桿菌中,由Cas1、Cas2和Cas9/csn 1蛋白組成。CRISPR-cas系統(tǒng)可以保護細菌免受外來入侵者的影響,但是CRISPR-cas系統(tǒng)在增強抗菌藥耐藥性方面的作用仍存在... 

【文章頁數(shù)】：184 頁

【學位級別】：博士

【文章目錄】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
1.INTRODUCTION
    1.1 GENERAL CHARACTERISTICS OF CAMPYLOBACTER SPECIES
    1.2 EPIDEMIOLOGICAL SIGNIFICANCE
    1.3 ANTIBIOTIC RESISTANCE IN CAMPYLOBACTER
        1.3.1 Quinolone resistance
        1.3.2 Resistance to tetracycline
        1.3.3 Resistance to macrolides
        1.3.4 Resistance to aminoglycosides
    1.4 CRISPR-CAS SYSTEM
    1.5 CRISPR-CAS SYSTEM INVOLVEMENT IN ANTIMICROBIAL RESISTANCE
    1.6 THE CRISPR-CAS SYSTEM IN C.JEJUNI
    1.7 RNA-SEQ BASED TRANSCRIPTOME SEQUENCING
    1.8 AIMS AND OBJECTIVES OF STUDY
2.MATERIALS AND METHODS
    2.1 CHEMICALS,MEDIA AND REAGENTS
    2.2 INSTRUMENTS
    2.3 ANTIMICROBIAL AGENTS
    2.4 TEST SOLUTIONS PREPARATION
    2.5 BACTERIAL STRAINS
    2.6 ISOLATION AND GROWTH CONDITIONS OF CLINICAL ISOLATES OF C.JEJUNI
        2.6.1 Sample collection and transport
        2.6.2 Isolation and confirmation of clinical isolates of C.jejuni by PCR
        2.6.3 Antimicrobial susceptibility testing of clinical isolates of C.jejuni
    2.7 DETECTION OF CRISPR-SPACER FROM THE POULTRY ISOLATES OF C.JEJUNI
    2.8 Recovery of C.jejuni NCTC11168 and C.coli ATCC33559
    2.9 DETECTION OF CRISPR GENES EXPRESSION BY RT-QPCR AFTER EXPOSURE TO DIFFERENT ANTIMICROBIALS
        2.9.1 Reverse transcription
        2.9.2 Quantitative PCR
    2.10 CONSTRUCTION OF CJ1523C(CAS9)DELETION MUTANT
        2.10.1 Extraction of C.jejuni NCTC11168 genomic DNA
        2.10.2 Vector construction
        2.10.3 Construction of mutant vector
        2.10.4 Fusion PCR
        2.10.5 Ligation with vector or plasmid
        2.10.6 Plasmid extraction
        2.10.7 Construction ofΔcas9 mutant
        2.10.8 Confirmation of mutant
    2.11 Complementation of Δcas9 mutant strain
    2.12 ANTIMICROBIAL SUSCEPTIBILITY TESTING OFΔCAS9 MUTANT STRAIN
    2.13 IN VITRO RESISTANCE DEVELOPMENT AGAINST DIFFERENT ANTIMICROBIALS
    2.14 DETERMINATION OF STANDARD GROWTH CURVE AND GROWTH CURVE RESPONSE AGAINST DIFFERENT ANTIMICROBIAL AGENTS
    2.15 NATURAL TRANSFORMATION
    2.16 RNA-SEQ BASED TRANSCRIPTOME ANALYSIS
        2.16.1 RNA extraction and purification
        2.16.2 RNA-Seq protocol
        2.16.3 Validation of library
        2.16.4 Differential gene expression analysis
        2.16.5 Bioinformatics analysis
        2.16.6 Validation by RT-qPCR
        2.16.7 Quantitative PCR
    2.17 STATISTICAL ANALYSIS
3.RESULTS
    3.1 CONFIRMATION OF C.JEJUNI FROM POULTRY ISOLATES
    3.2 ANTIMICROBIAL SUSCEPTIBILITY OF POULTRY ISOLATES
    3.3 SPACER IDENTIFICATION AND ANALYSIS FROM THE POULTRY ISOLATES OF C.JEJUNI
    3.4 CRISPR-CAS GENES EXPRESSION IN STANDARD STRAINS AFTER DRUG TREATMENT
    3.5 EXTRACTED BACTERIAL DNA RESULTS
    3.6 CONSTRUCTION OFΔCAS9 MUTANT VECTORS
        3.6.1 Homologous arm and amplification of screening genes
        3.6.2 Fragments fusion by fusion PCR
        3.6.3 Verification of recombinant plasmid
        3.6.4 Recombinant plasmid extraction results
        3.6.5 Δcas9 Mutant validation
    3.7 ROLE OF CAS9 IN ANTIMICROBIAL SUSCEPTIBILITY
    3.8 ROLE OF CAS9 IN RESISTANCE DEVELOPMENT
    3.9 ROLE OF CAS9 ON GROWTH UNDER DRUG EXPOSURE
    3.10 ROLE OF CAS9 ON TRANSFORMATION OF RESISTANCE
    3.11 RNA-SEQ BASED TRANSCRIPTOME ANALYSIS
        3.11.1 RNA extraction and quality
        3.11.2 Quality of raw reads
        3.11.3 Differential gene analysis
        3.11.4 Bioinformatics analysis
    3.12 VALIDATION OF RNA-SEQ RESULTS BY RT-QPCR
4.DISCUSSION
5.SUMMARY
6.PUBLISHED REVIEW ARTICLE 1
    6.1 AVERTING PHAGE ADSORPTION/RECEPTOR MUTATION
    6.2 BLOCKAGE OF INVADER DNA
    6.3 RESTRICTION–MODIFICATION SYSTEMS
    6.4 ABORTIVE INFECTION
    6.5 INTERFERENCE DURING ASSEMBLY
    6.6 CRISPR:BACTERIA ADAPTIVE IMMUNE SYSTEM
    6.7 COUNTER ATTACK OF INVADERS AGAINST THE CRISPR-CAS SYSTEM
    6.8 FUNCTION OF ANTI-CRISPR PROTEINS EXHIBITED BY VARIOUS MECHANISMS
    6.9 ANTI-CRISPR GENES IN MGES
    6.10 SUMMARY
    6.11 FUTURE PERSPECTIVES
7.PUBLISHED REVIEW ARTICLE 2
    7.1 HISTORY OF CRISPR-CAS SYSTEM
    7.2 FATE OF CRISPR SYSTEM AGAINST INVADING DNA
    7.3 CLASSIFICATION OF CRISPR-CAS SYSTEM
        7.3.1 Type I CRISPR system
        7.3.2 Type II CRISPR system
        7.3.3 Type III CRISPR system
    7.4 ROLE OF CRISPR BEYOND ADAPTIVE IMMUNITY OF PROKARYOTES
    7.5 THE EVOLUTION OF CRISPR SYSTEM PLAYING AS A DEFENSE
    7.6 CRISPR SYSTEM AND ITS REGULATION
    7.7 GENE REGULATION BY CRISPR AND ITS ROLE IN PATHOGENESIS
    7.8 CRISPR-CAS SYSTEM FACILITATES EVOLUTION OF THE GENOME
    7.9 CRISPR BASED APPLICATIONS
        7.9.1 Genome engineering
        7.9.2 Use of CRISPR-cas9 as a tool of genome editing in disease
        7.9.3 Use of CRISPR-cas9 in curing human genetic diseases
        7.9.4 CRISPR-cas9 potential to target diseases developed by epigenetic alterations
    7.10 FUTURE DIMENSIONS
    7.11 CONCLUSION
REFERENCE
APPENDIX
    SEQUENCES DETAIL OF3H,KAN AND5H
    SEQUENCING MAP OF RECOMBINANT PLASMID P3HKAN5H-3
        M13-F primer sequencing results
        M13-R primer sequencing results
        W1-F primer sequencing results
        W1-R primer sequencing results:
        W2-R primer sequencing results:
        W3-R primer sequencing results:
    RAW DATA
    RNA-SEQ RAW DATA
ACKNOWLEDGEMENT
CURRICULUM VITAE


【參考文獻】：
期刊論文
[1]DNA甲基轉(zhuǎn)移酶分類、功能及其研究進展[J]. 王志剛,吳建新.  遺傳. 2009(09)



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