斑点叉尾鮰病毒感染及鲶科鱼（斑点叉尾鮰和黄颡鱼）的免疫特性

发布时间：2023-02-12 08:39

　　鲶科鱼类广泛分布在除南极洲以外的其他大陆上,在世界各地被养殖了好几个世纪。鲶科鱼类作为世界鱼类产业的重要物种,具有较高的市场价值。鲶科鱼类在养殖过程中饱受病毒和细菌疾病的侵害,造成了严重的经济损失,影响其产量。学者们报道了几种能大规模造成鲶科鱼类死亡和发表的病毒和细菌性疾病。目前认为鱼类通过建立物理和生化免疫系统来应对入侵的微生物。然而,免疫系统组成成分十分复杂,相关酶和信号通路会有效的保护鱼体。已从鲶科鱼类中分离出多种免疫相关蛋白和酶,它们的免疫活性在包括黏膜层和分泌物在内的各种器官中被阐明。本研究报道了斑点叉尾鮰在受到斑点叉尾鮰病毒(CCV)感染后的免疫应答反应,和重组激活基因2 (RAG2)的发现及在黄颡鱼中的抗菌作用。此外,还在黄颡鱼中发现了一种炎性因子CYPA并研究了其在粘膜免疫的作用。斑点叉尾鮰病毒(CCV)可以对斑点叉尾鮰幼鱼造成致死性感染,从而导致巨大的经济损失。CCV的基因组已经完成测序,其患病率是有据可查的。然而,CCV致病的分子机理却很少有报道。在此,斑点叉尾鮰卵巢细胞(CCO)感染CCV后,利用RNA测序技术分析转录组草图。总的来说,从73231128个测序读取...

【文章页数】：110 页

【学位级别】：博士

【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER 1 INTRODUCTION AND REVIEW OF LITERATURE
    1. CATFISH CULTURE
    2. DISEASES OF CATFISH
    3. OVERVIEW OF FISH IMMUNITY
    4. IMMUNE-RELEVANT PROTEIN IN FISH
        4.1. RAG2
        4.2. CYCLOPHILIN A
        4.3. MECHANISM OF CYPA CHEMOTAXIS AND LEUKOCYTE ACTIVATION
        4.4. CYPA SERVES AS A CO-FACTOR IN LEUKOCYTE CHEMOTAXIS
        4.5. CYPA INDUCED LEUKOCYTE CHEMOTAXIS
        4.6. INHIBITION OF THE CHEMOTACTIC ACTIVITY OF CYPA
    5. OBJECTIVES
CHAPTER 2 CHANNEL CATFISH VIRUS INFECTION IN CHANNEL CATFISHOVARY CELLS
    1. INTRODUCTION
    2. MATERIALS AND METHODS
        2.1. CELL CULTURE, CCV INFECTION AND SAMPLE COLLECTION
        2.2. EXTRACTION OF TOTAL RNA, PURIFICATION OF MRNA AND SEQUENCING OF CDNA
        2.3. CLEANING OF INITIAL (RAW) DATA,CONSTRUCTION OF THE DE-NOVO ASSEMBLY ANDANNOTATION OF THE UNIGENE
        2.4. EVALUATION OF THE EXPRESSION LEVEL OF GENES
        2.5. VERIFICATION OF THE EXPRESSION LEVEL OF GENES BY QRT-PCR
        2.6. APOPTOSIS EVALUATION IN CCO CELLS USING STAINING
        2.7. CASPASES ASSAY
        2.8. Statistics
    3. RESULTS
        3.1. CONSTRUCTION AND ANALYSIS OF THE TRANSCRIPTOMIC SEQUENCES ASSEMBLY
        3.2. EVALUATION OF THE FUNCTIONALLY ANNOTATED DEGS
        3.3. EVALUATION OF THE UP AND DOWN REGULATED UNIGENES
        3.4. CONFIRMATION OF THE TRANSCRIPTOMIC GENES VIA QRT-PCR
        3.5. THE APOPTOSIS INDUCED BY CCV INFECTION IN CCO CELLS
        3.6. CCV INFECTION INDUCED APOPTOSIS VIA EXTRINSIC PATHWAY IN CCO CELLS
    4. DISCUSSION
CHAPTER 3 ANALYSIS OF RAG2 IN CHINESE YELLOW CATFISH
    1. INTRODUCTION
    2. MATERIAL AND METHODS
        2.1. FISH
        2.2. ISOLATION (CLONING AND SEQUENCING) OF RAG2
        2.3. SEQUENCE CHARACTERIZATION AND PHYLOGENETIC ANALYSIS
        2.4. PRODUCTION OF YC-RAG2 POLYCLONAL ANTIBODY
        2.5. ANALYSIS OF YELLOW CATFISH RAG2 TRANSCRIPT BY QRT-PCR
        2.6. EXAMINATION OF YELLOW CATFISH RAG2 PROTEINS BY WESTERN BLOT
        2.7. ANALYSIS OF THE TRANSCRIPT EXPRESSION LEVEL OF RAG2,IL-1B AND TNF-A INFISH TISSUES AFTER E. ICTALURI INFECTION
        2.8. EXAMINATION OF YC-RAG2 PROTEIN DISTRIBUTION AFTER E.ICTALURI INFECTION
        2.9. Statistics
    3. RESULTS
        3.1. CLONING, CLASSIFICATION AND PHYLOGENETIC EVALUATION OF RAG2 SEQUENCE
        3.2. ANTIBODY SPECIFICITY AND EXPRESSION LEVEL OF YC-RAG2 IN VARIOUS TISSUES
        3.3. THE TRANSCRIPT EXPRESSION OF YC-RAG2 IN E. ICTALURI CHALLENGED FISH
        3.4. THE LEVEL OF EXPRESSIONS OF IL-1 B AND TNF-A IN LYMPHOID TISSUE OF YELLOWCATFISH AFTER BACTERIAL INFECTION
    4. DISCUSSION
CHAPTER 4 FUNCTIONAL ANALYSIS OF CYCLOPHILIN A
    1. INTRODUCTION
    2. MATERIALS AND METHODS
        2.1. ANIMALS AND SAMPLE COLLECTION
        2.2. MASS SPECTROMETRIC ANALYSIS
        2.3. WESTERN BLOT ANALYSIS
        2.4. SAMPLE PREPARATION, PLASMIDS CONSTRUCTION AND PROTEIN EXPRESSION
        2.5. LEUKOCYTES SEPARATION
        2.6. CHEMOTAXIS ASSAY OF SKIN MUCUS
        2.7. CHEMOTAXIS ASSAY OF CYPA RECOMBINANT PROTEIN
        2.8. STATISTICS
    3. RESULTS
        3.1. MASS SPECTROMETRY AND WESTERN BLOT ANALYSIS
        3.2. CHEMOTAXIS ASSAY OF SKIN MUCUS
        3.3. CHEMOTACTIC ACTIVITY OF MUTANT CYPA
    4. DISCUSSION
CONCLUSIONS
REFERENCES
LIST OF PUBLICATIONS
Submitted papers
ACKNOWLEDGEMENTS



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