鯰科魚類廣泛分布在除南極洲以外的其他大陸上,在世界各地被養(yǎng)殖了好幾個世紀(jì)。鯰科魚類作為世界魚類產(chǎn)業(yè)的重要物種,具有較高的市場價值。鯰科魚類在養(yǎng)殖過程中飽受病毒和細(xì)菌疾病的侵害,造成了嚴(yán)重的經(jīng)濟(jì)損失,影響其產(chǎn)量。學(xué)者們報道了幾種能大規(guī)模造成鯰科魚類死亡和發(fā)表的病毒和細(xì)菌性疾病。目前認(rèn)為魚類通過建立物理和生化免疫系統(tǒng)來應(yīng)對入侵的微生物。然而,免疫系統(tǒng)組成成分十分復(fù)雜,相關(guān)酶和信號通路會有效的保護(hù)魚體。已從鯰科魚類中分離出多種免疫相關(guān)蛋白和酶,它們的免疫活性在包括黏膜層和分泌物在內(nèi)的各種器官中被闡明。本研究報道了斑點(diǎn)叉尾鮰在受到斑點(diǎn)叉尾鮰病毒(CCV)感染后的免疫應(yīng)答反應(yīng),和重組激活基因2 (RAG2)的發(fā)現(xiàn)及在黃顙魚中的抗菌作用。此外,還在黃顙魚中發(fā)現(xiàn)了一種炎性因子CYPA并研究了其在粘膜免疫的作用。斑點(diǎn)叉尾鮰病毒(CCV)可以對斑點(diǎn)叉尾鮰幼魚造成致死性感染,從而導(dǎo)致巨大的經(jīng)濟(jì)損失。CCV的基因組已經(jīng)完成測序,其患病率是有據(jù)可查的。然而,CCV致病的分子機(jī)理卻很少有報道。在此,斑點(diǎn)叉尾鮰卵巢細(xì)胞(CCO)感染CCV后,利用RNA測序技術(shù)分析轉(zhuǎn)錄組草圖�？偟膩碚f,從73231128個測序讀取...

【文章頁數(shù)】：110 頁

【學(xué)位級別】：博士

【文章目錄】：
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



本文編號：3740760