谷氨酰胺对赤点石斑鱼神经坏死病毒复制影响的研究
发布时间：2024-06-02 05:36
神经坏死病毒(NNV)可感染多种重要鱼类,对全球鱼类养殖业造成了巨大的经济损失。谷氨酸胺是三羧酸循环途径中一个重要的成分,并且己经被病毒利用用来进行高效复制。然而,人们对NNV的复制和谷氨酰胺之间的关系了解很少。本论文采用赤点石斑鱼神经坏死病毒(RGNNV)感染石斑鱼鳍条(GF-1)和条纹月鳢细胞(SSN-1),研究了谷氨酰胺对RGNNV复制的影响。用焚光定量方法检测病毒mRNA的表达,用Western blotting方法检测病毒的蛋白表达。得出了以下主要结果:缺乏谷氨酰胺对细胞活性几乎没有影响,但显著限制RGNNV的复制。此外,添加丙酮酸,草酰乙酸(OAA),和二甲基α-酮戊二酸(a-KG)后,RGNNV的复制有一定程度的恢复。此外,添加谷氨酰胺酶抑制剂BPTES后可以抑制病毒复制;然而,当添加a-KG后,RGNNV病毒的复制也可部分恢复。我们在mRNA的转录水平检测了不同时间点病毒RNA聚合酶基因和衣壳蛋白基因mRNA的变化,发现在使用缺乏谷氨酰胺时,病毒复制都受到显著抑制。综上所述,这些研究结果表明谷氨酰胺可以通过三羧酸循环调节RGNNV复制,对RGNNV的复制是必要的。这些结...
【文章页数】：55 页
【学位级别】：硕士
【文章目录】：
Abstract
摘要
Abbreviations
Chapter Ⅰ Introduction
1.1. Viral-induced changes in carbon metabolism
1.1.2. Warburg Effect
1.1.3. Glucose and Glutamine alteration by Viral Infections
1.1.4. SSN-1 and GF-1 cell lines
1.2. Nervous Necrosis Virus
1.2.1. Etiology, Clinical Signs and pathology
1.2.2. Genome Composition
1.2.3. Entry to Host and Replication
1.2.4. Immune responses against Nervous Necrosis Virus infection
1.3. Hypothesis
1.4. Aims and Objectives of proposed work
1.5. Experimental Design
Chapter Ⅱ Materials and Methods
2.2. Virus and Cell Culture
2.3. Reagents and antibodies
2.4. Equipments
2.5. Cell viability assay
2.6. Virus titration
2.7. Cell infection
2.8. Reagents standardization
2.9. Nutrient starvation and the addition of metabolic intermediates of TCA cycle
2.10.Transcription studies of RGNNV at different time points
2.11. RNA Extraction and cDNA Library construction
2.12. Primer Designing and RT-PCR
2.13. Quantitative Real time PCR Assay (qRT-PCR Assay)
2.14. SDS-PAGE and Western blotting assay for the expression of RGNNV
2.15. Immune Fluorescence Assay (IFA) for RGNNV protein expression
2.16. Statistical Analysis
Chapter Ⅲ Results
3.1. Glucose but not glutamine was essential for cell proliferation
3.2. Glutamine was essential for SSN-1 or GF-1 cells infected with RGNNV
3.3. Additions of intermediates of TCA cycle could rescue the replication of RGNNV
3.4. Blocking glutaminolysis could inhibit the replication of RGNNV
3.5. Glutamine regulated the transcription of capsid gene of RGNNV:
3.6. Glutamine effect the transcription of RdRp gene of RGNNV
3.7. Glutamine affected RGNNV protein synthesis
Chapter Ⅳ Discussion
Conclusion
References
Acknowledgements
本文编号：3986931
【文章页数】：55 页
【学位级别】：硕士
【文章目录】：
Abstract
摘要
Abbreviations
Chapter Ⅰ Introduction
1.1. Viral-induced changes in carbon metabolism
1.1.2. Warburg Effect
1.1.3. Glucose and Glutamine alteration by Viral Infections
1.1.4. SSN-1 and GF-1 cell lines
1.2. Nervous Necrosis Virus
1.2.1. Etiology, Clinical Signs and pathology
1.2.2. Genome Composition
1.2.3. Entry to Host and Replication
1.2.4. Immune responses against Nervous Necrosis Virus infection
1.3. Hypothesis
1.4. Aims and Objectives of proposed work
1.5. Experimental Design
Chapter Ⅱ Materials and Methods
2.2. Virus and Cell Culture
2.3. Reagents and antibodies
2.4. Equipments
2.5. Cell viability assay
2.6. Virus titration
2.7. Cell infection
2.8. Reagents standardization
2.9. Nutrient starvation and the addition of metabolic intermediates of TCA cycle
2.10.Transcription studies of RGNNV at different time points
2.11. RNA Extraction and cDNA Library construction
2.12. Primer Designing and RT-PCR
2.13. Quantitative Real time PCR Assay (qRT-PCR Assay)
2.14. SDS-PAGE and Western blotting assay for the expression of RGNNV
2.15. Immune Fluorescence Assay (IFA) for RGNNV protein expression
2.16. Statistical Analysis
Chapter Ⅲ Results
3.1. Glucose but not glutamine was essential for cell proliferation
3.2. Glutamine was essential for SSN-1 or GF-1 cells infected with RGNNV
3.3. Additions of intermediates of TCA cycle could rescue the replication of RGNNV
3.4. Blocking glutaminolysis could inhibit the replication of RGNNV
3.5. Glutamine regulated the transcription of capsid gene of RGNNV:
3.6. Glutamine effect the transcription of RdRp gene of RGNNV
3.7. Glutamine affected RGNNV protein synthesis
Chapter Ⅳ Discussion
Conclusion
References
Acknowledgements
本文编号：3986931
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