發(fā)布時(shí)間：2021-05-31 21:11

　　親環(huán)素A（Cyclophilin A,Cyp A）是一種廣泛表達(dá)的細(xì)胞蛋白,參與感染、炎癥等多種病理反應(yīng)。Cyp A在多種病毒的復(fù)制過程中都發(fā)揮重要的作用。然而,目前在神經(jīng)壞死病毒復(fù)制中Cyp A發(fā)揮的作用知之甚少。NNV最先是從患病的海洋魚類中分離出來的,目前已在120多種養(yǎng)殖和野生魚類中都有報(bào)道,并給水產(chǎn)養(yǎng)殖業(yè)造成了巨大的經(jīng)濟(jì)損失。最近還報(bào)道發(fā)現(xiàn)在實(shí)驗(yàn)條件下,一些淡水養(yǎng)殖魚類也對NNV敏感。因此,對病毒的預(yù)防以及治療需進(jìn)一步研究。本研究開展了親環(huán)蛋白A在赤點(diǎn)石斑魚神經(jīng)壞死病毒（RGNNV）感染中的功能研究,主要結(jié)果如下:1、斜帶石斑魚（Epinephelus Coioides）親環(huán)蛋白A的克隆和分析。從斜帶石斑魚的鰭條細(xì)胞系（GF-1）中克隆得到石斑魚的Cyp A基因（GF-Cyp A）。通過序列分析得到,GF-Cyp A基因全長為743 bp,開放閱讀框（ORF）全長495 bp,編碼164個(gè)氨基酸,分子量為17.4 k Da。通過生物信息學(xué)分析,并使用軟件phyre²預(yù)測二級結(jié)構(gòu)和三維結(jié)構(gòu),發(fā)現(xiàn)GF-Cyp A存在β折疊、螺旋以及回文結(jié)構(gòu),還預(yù)測了結(jié)合位點(diǎn)... 

【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)】：116 頁

【學(xué)位級別】：博士

【文章目錄】：
Abstract
摘要
Abbreviation
CHAPTER 1 Introduction and Literature review
    1.1. Importance of Grouper fish to Aquaculture
    1.2. Diseases of Grouper fish
    1.3. Viral Nervous Necrosis Disease
    1.4. Nervous Necrosis Virus
    1.5. Genome Composition
    1.6. Occurrence and Geographical distribution
    1.7. Entry to Host and Replication
    1.8. Immune responses and Cytokine secretion during RGNNV infection
    1.9. Cyclophilin A
    1.10. Role of Cyclophilin during Viral replication
    1.11. Cyclosporine A
    1.12. Role of Cyclosporine A during virus replication
    1.13. Fish Cell lines
    1.14. GF-1 cell line
    1.15. Effect of Cyclophilin A on cytokine secretion
    1.16. Aims and objectives:
CHAPTER 2 Molecular Cloning and sequence analysis of Cyp A in Grouper Fish
    2.1. Introduction
    2.2. Material and methods
        2.2.1. Cell Culture
        2.2.2. Plasmid Vector and Escherichia coli
        2.2.3. Reagents and antibodies
        2.2.4. Equipment’s used in current study
        2.2.5. RNA Extraction
        2.2.6. Checking RNA Concentration
        2.2.7. c DNA Synthesis
        2.2.8. Primer designing & Reverse Transcription Polymerase Chain Reaction (RT-PCR)
        2.2.9. Gel Extraction
        2.2.10. Enzyme Digestion
        2.2.11. Ligation with p MD18-T vector
        2.2.12. Media Preparation
        2.2.13. Transformation
    2.3. Results
        2.3.1. Amplification of GF-Cyp A gene
        2.3.2. Sequence analysis of GF-Cyp A gene
        2.3.3. Secondary structure prediction of GF-Cyp A
        2.3.4. 3-D Structure and Phylogenetic analysis of GF-Cyp A
        2.3.5. Secondary structure composition, Binding sites & Solvent accessibility
        2.3.6. Analyzed amino acid composition of GF-Cyp A
        2.3.7. Homology analysis
    2.4. Discussion
CHAPTER 3 Role of Cyclophilin A during RGNNV infection in GF-1 cells
    3.1. Introduction
    3.2. Material and Methods
        3.2.1. Virus and Cell Culture
        3.2.2. Total RNA Isolation and c DNA synthesis
        3.2.3. Recombinant construction of GF-Cyp A plasmid
        3.2.4. Cell infection
        3.2.5. RNA Extraction and Quantitative real-time PCR (q RT-PCR) assay
        3.2.6. Real time Plymerase Chain reaction (RT-PCR)
        3.2.7. Gel Electrophoresis
        3.2.8. Gel extraction
        3.2.9. Enzyme Digestion
        3.2.10. Ligation
        3.2.11. Transformation
        3.2.12. Plasmid DNA Extraction
        3.2.13. Transfection
        3.2.14. Quantitaive Real time PCR
        3.2.15. Hematoxylin & Eosin (HE) Staining
        3.2.16. Titration of Virus
        3.2.17. RNAi Knockdown of GF-Cyp A
        3.2.18. SDS-PAGE & Western blot assay
        3.2.19. Western Blotting
        3.2.20. Immune Fluorescence Assay (IFA) for RGNNV protein expression
        3.2.21. Statistical Analysis
    3.3. Results
        3.3.1. Expression analysis of GF-Cyp A after RGNNV infection
        3.3.2. Effect of GF-Cyp A on RGNNV expression
        3.3.3. Capsid and Rd Rp genes expression after transfection with GF-Cyp A
        3.3.4. RGNNV Capsid Protein Expression after GF-Cyp A transfection
        3.3.5. Effect of Cyp A on RGNNV titration
        3.3.6. Knockdown of GF-Cyp A upregulated the RGNNV level in GF-1 cells
        3.3.7. Effect of Cyp A on cytokines expression during RGNNV infection
    3.4. Discussion
Chapter 4 Cyclosporine A inhibits Red Spotted Grouper Nervous Necrosis Virus (RGNNV) replication at the late step of viral life cycle
    4.1. Introduction
    4.2. Materials and methods
        4.2.1. Virus and Cell culture
        4.2.2. Reagents and Antibodies
        4.2.3. Cell viability assay
        4.2.4. Cell infection
        4.2.5. Total RNA Isolation and c DNA synthesis
        4.2.6. Quantitative real-time PCR (q RT-PCR) assay
        4.2.7. RNAi Knockdown of GF-Cyp A
        4.2.8. Statistical Analysis
    4.3. Results
        4.3.1. Effect of Cyclosporine A (Cs A) on GF-1 cells Viability
        4.3.2. Cs A does not affect the early steps of RGNNV replication
        4.3.3. Cs A negatively effects late stages of RGNNV life cycle
        4.3.4. Silencing Cyp A doesn’t increase RGNNV replication during Cs A treatment
        4.3.5. Effect of Cs A on cytokines expression during RGNNV infection
    4.4. Discussion
CHAPTER 5 Conclusion and Future Perspectives
References
PAPERS PUBLISHED
ACKNOWLEDGMENT


【參考文獻(xiàn)】：
期刊論文
[1]鱖親環(huán)蛋白A在傳染性脾腎壞死病毒增殖中的作用[J]. 胡先勤,付小哲,董星星,涂加鋼,趙麗娟,林強(qiáng),李寧求,林蠡.  水產(chǎn)學(xué)報(bào). 2016(01)



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