- 學位論文
點帶石斑之介白素-6、介白素-6受體與醣蛋白130間蛋白質交互作用探討及介白素-6受體異構型選殖
Protein-Protein Interactions between Interleukin-6, Interleukin-6 Receptor and Glycoprotein 130 as well as Cloning of IL-6R Isoform of Epinephelus coioides
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摘要
介白素-6 (IL-6)是一種多功能性細胞激素，作為細胞間負責聯絡功能的蛋白質，影響細胞活化、增殖與分化等反應。在哺乳類IL-6機制研究當中，IL-6與介白素-6受體(IL-6R)及傳訊分子醣蛋白130 (gp130)組成六聚體複合物以調控生化反應，然而IL-6可依據與膜上型或游離型IL-6R結合而分為古典路徑及替代路徑，藉此發揮其眾多功能。魚類IL-6研究方面目前則多偏向分子選殖與功能分析，先前研究證實魚類IL-6具抑制病原感染擴散與促進抗體保護的效果，也確實能誘發魚體免疫反應如抗菌胜肽表現並降低魚隻遭受細菌及病毒感染的死亡率，未來有機會應用於水生生物疾病防治並作為疫苗佐劑或免疫促進劑。 本篇研究為探討魚類IL-6作用機制與功能，先透過載體重構、訊號胜肽移除、重新篩選單一菌落、基因序列點突變方式突破先前點帶石斑IL-6R與gp130膜外全長蛋白表現與純化之困境，接著透過原核蛋白表現系統、親和性管柱純化法及磁珠純化法取得點帶石斑IL-6、IL-6R、gp130膜外全長及Hyper IL-6純化蛋白，接著透過遠端西方墨點法、親和性層析法與蛋白拉下實驗來測試蛋白質間交互作用，實驗結果顯示點帶石斑IL-6、IL-6R與gp130膜外全長間具有蛋白質交互作用結合關係，此外也從石斑組織中選殖出IL-6R異構型，並進一步探討其可能形成原因為mRNA替代性剪接，另已製備石斑IL-6R及其異構型血清抗體，可用於未來探討及解釋更多石斑IL-6R異構型之生物功能。 本研究為第一篇提出魚類IL-6、IL-6R與gp130蛋白質間交互作用及IL-6R異構型的論文，此份結果可串連先前研究中點帶石斑之IL-6、IL-6R及gp130基因選殖、訊息傳遞及免疫功能分析等研究成果，並作為點帶石斑IL-6作用機制探討、水生生物疾病預防及現場應用的參考基礎，期許未來可應用於石斑水產養殖產業，成功降低細菌及病毒感染所帶來的肆虐，有效提升經濟價值。
並列摘要
Interleukin-6 (IL-6) is a multifunctional cytokine. As a protein responsible for communication between cells, it affects responses such as cell activation, proliferation, and differentiation. In the mechanism study of IL-6 in mammals, IL-6 forms a hexameric complex with its receptor IL-6R and the signaling molecule glycoprotein 130 (gp130) to regulate biochemical reactions. This complex performs many functions by activating different signaling mechanisms and biochemical functions such as classical pathway and alternative pathway. However, few studies have explored the biochemical function of fish IL-6. Previous studies have confirmed that fish IL-6 has the effect of inhibiting the spread of infection and promoting antibody protection. IL-6 also induce immune responses such as antimicrobial peptides and reduce the mortality of fish from bacterial and viral infections. Fish IL-6 research will have the opportunity to be practically applied for disease control and become an adjuvant or immune booster in the future. In this study, targeted protein expression and purification were solved using vector reconstruction, signal peptide removal, re-screening of single colonies, point mutation, and then the targeted protein were expressed through the prokaryotic protein expression system, and then the affinity column was used to obtain the purified proteins of grouper IL-6, IL-6R, gp130 and Hyper IL-6. Then, the interaction between proteins were tested by far western blotting, affinity chromatography and protein pull-down assay. The experimental results show that there are protein interactions between IL-6, IL-6R and gp130 of grouper. In addition, IL-6R isoform was also cloned from grouper tissue, and the possible cause of this formation was further explored as mRNA alternative splicing. Serum antibodies of grouper IL-6R and its isoform have been prepared, which can be used to explore and explain more biological functions of grouper IL-6R isoforms in the future. This is the first thesis to propose the protein-protein interactions between IL-6, IL-6R and gp130 and discovery of IL-6R isoform in fish. This result can be linked to previous research of grouper IL-6, IL-6R and gp130 in our laboratory including gene cloning, signal pathway transduction, immune function analysis and other related research results. The result also be a reference basis for the mechanism study of grouper IL-6, disease prevention and field application. It is expected that it can be applied to the grouper aquaculture industry to reduce the spread of diseases that caused by pathogens infection and effectively enhance economic value in the future.
參考文獻
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