亲环蛋白A在赤点石斑鱼神经坏死病毒（RGNNV）感染中的功能研究
发布时间：2021-05-31 21:11
亲环素A（Cyclophilin A,Cyp A）是一种广泛表达的细胞蛋白,参与感染、炎症等多种病理反应。Cyp A在多种病毒的复制过程中都发挥重要的作用。然而,目前在神经坏死病毒复制中Cyp A发挥的作用知之甚少。NNV最先是从患病的海洋鱼类中分离出来的,目前已在120多种养殖和野生鱼类中都有报道,并给水产养殖业造成了巨大的经济损失。最近还报道发现在实验条件下,一些淡水养殖鱼类也对NNV敏感。因此,对病毒的预防以及治疗需进一步研究。本研究开展了亲环蛋白A在赤点石斑鱼神经坏死病毒（RGNNV）感染中的功能研究,主要结果如下:1、斜带石斑鱼（Epinephelus Coioides）亲环蛋白A的克隆和分析。从斜带石斑鱼的鳍条细胞系（GF-1）中克隆得到石斑鱼的Cyp A基因（GF-Cyp A）。通过序列分析得到,GF-Cyp A基因全长为743 bp,开放阅读框（ORF）全长495 bp,编码164个氨基酸,分子量为17.4 k Da。通过生物信息学分析,并使用软件phyre2预测二级结构和三维结构,发现GF-Cyp A存在β折叠、螺旋以及回文结构,还预测了结合位点...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：116 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]鳜亲环蛋白A在传染性脾肾坏死病毒增殖中的作用[J]. 胡先勤,付小哲,董星星,涂加钢,赵丽娟,林强,李宁求,林蠡. 水产学报. 2016(01)
本文编号：3208982
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：116 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]鳜亲环蛋白A在传染性脾肾坏死病毒增殖中的作用[J]. 胡先勤,付小哲,董星星,涂加钢,赵丽娟,林强,李宁求,林蠡. 水产学报. 2016(01)
本文编号：3208982
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