神經(jīng)壞死病毒(NNV)可感染多種重要魚類,對全球魚類養(yǎng)殖業(yè)造成了巨大的經(jīng)濟(jì)損失。谷氨酸胺是三羧酸循環(huán)途徑中一個重要的成分,并且己經(jīng)被病毒利用用來進(jìn)行高效復(fù)制。然而,人們對NNV的復(fù)制和谷氨酰胺之間的關(guān)系了解很少。本論文采用赤點(diǎn)石斑魚神經(jīng)壞死病毒(RGNNV)感染石斑魚鰭條(GF-1)和條紋月鱧細(xì)胞(SSN-1),研究了谷氨酰胺對RGNNV復(fù)制的影響。用焚光定量方法檢測病毒mRNA的表達(dá),用Western blotting方法檢測病毒的蛋白表達(dá)。得出了以下主要結(jié)果:缺乏谷氨酰胺對細(xì)胞活性幾乎沒有影響,但顯著限制RGNNV的復(fù)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的復(fù)制有一定程度的恢復(fù)。此外,添加谷氨酰胺酶抑制劑BPTES后可以抑制病毒復(fù)制;然而,當(dāng)添加a-KG后,RGNNV病毒的復(fù)制也可部分恢復(fù)。我們在mRNA的轉(zhuǎn)錄水平檢測了不同時間點(diǎn)病毒RNA聚合酶基因和衣殼蛋白基因mRNA的變化,發(fā)現(xiàn)在使用缺乏谷氨酰胺時,病毒復(fù)制都受到顯著抑制。綜上所述,這些研究結(jié)果表明谷氨酰胺可以通過三羧酸循環(huán)調(diào)節(jié)RGNNV復(fù)制,對RGNNV的復(fù)制是必要的。這些結(jié)...

【文章頁數(shù)】：55 頁

【學(xué)位級別】：碩士

【文章目錄】：
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



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