點帶石斑之Interleukin-6 receptor (IL-6R) 的基因選殖分析及其與Interleukin-6 (IL-6) 融合蛋白的建構與蛋白表現

Abstract

Interleukin-6 (IL-6) 為一種多效性細胞激素，其在調節免疫反應上佔有非常重要的位置。在先天性及後天性免疫反應中，IL-6 可以誘發急性期蛋白、抗菌胜肽、補體等免疫因子分泌，且促使淋巴球的增生及分化並促進抗體的產生。其作用的機制為在目標細胞上，IL-6 會與其受體 interleukin-6 receptor (IL-6R) 及 glycoprotein 130 (gp130) 進行結合，其刺激訊息會藉由 gp130 傳遞至細胞內，並引起細胞下游免疫反應產生。IL-6 與受體結合在不同的細胞的結合方式有兩種，分別為 classic signaling 及trans-signaling，其不同處在於與不同型態的IL-6R 結合。IL-6 與 membrane-form IL-6R (mIL-6R) 結合後再與gp130聯合稱為classic signaling，而與soluble-form IL-6R (sIL-6R) 結合後再與gp130聯合為trans-signaling。當IL-6 進行trans-signaling時有機會提高 IL-6 產生免疫反應的機率。在過去的研究將sIL-6R 與 IL-6 以一段直鏈蛋白連接，形成一種名為Hyper-IL-6 的融合蛋白。其研究結果顯示，若使用Hyper-IL-6 刺激，在達到同樣的免疫刺激量時，Hyper-IL-6所使用的劑量比IL-6低100-1000倍。因此， Hyper-IL-6 可能是一個在免疫上極具潛力的融合蛋白。本篇研究將從點帶石斑魚體組織內獲取點帶石斑mIL-6R 的基因，藉由生物資訊學的方式預測其信號蛋白(signal peptide)，免疫球蛋白結構域 (immunoglobin domain, Ig domain)，細胞激素結合位 (cytokine-binding domains, CBD, 以IL-6R 來說即為IL-6 binding site)，纖連蛋白III型結構域 (fibronectin type III domain, FN 3 domain)，跨膜結構域 (transmembrane domain), WSXWS motif, cysteine residues 等蛋白結構域，並與其他已知IL-6R 的魚種以及哺乳類的IL-6R 進行比對，以確認獲取之基因為IL-6R。根據以生物資訊預測到的跨膜區，從mIL-6R 中取得sIL-6R 的基因後進行密碼子優化 (codon usage)，再將優化後的sIL-6R 與 IL-6 以基因工程的方式結合，形成點帶石斑之Hyper-IL-6。本研究將 sIL-6R及Hyper-IL-6 進行蛋白表現後，其蛋白純化，功能性分析及與gp130結合性測試將於未來工作中逐一完成，預計融合蛋白Hyper-IL-6 可以提升魚體之免疫效益，可以做為未來免疫促進劑或是疫苗佐劑開發的參考。

Interleukin-6 (IL-6) is a pleiotropic cytokine, which has various effects on innate and acquired immune responses including induce the expression of acute phase proteins (APPs), antimicrobial peptides and complements, promoting antimicrobial activities. When it binds with the specific cognate receptor interleukin-6 receptor (IL-6R) and glycoprotein 130 (gp130), it can activate downstream signaling pathway; there are two different binding type between IL-6, IL-6R and gp130 ‘class-signaling pathway’ was lunched by IL-6 binding the receptor IL-6R and gp130 on the cell membrane of target cell; ‘trans-signaling pathway’ require a soluble IL-6R (sIL-6R) instead of mIL-6R on the target cell surface. In order to increase the effect of IL-6 induced trans-signaling pathway, in previous research, designed a recombinant fusion protein by linking an IL-6, a soluble IL-6R and a flexible peptide linker, and named Hyper-IL-6. It has 100 to 1,000-fold more activity than free form IL-6 and IL-6R. Since Hyper-IL-6’s high immune inducible efficiency, it might be a good candidate protein for immunostimulants or adjuvants of vaccine in teleost. In this study, the Epinephelus coioides (grouper) IL-6R was cloned and IL-6R homologues were identified. Then, the protein and specific domain were defined, including immunoglobulin domain (Ig domain), cytokine-binding domains (CBD, IL-6 binding site), fibronectin type III domain (FN 3 domain), WSXWS motif, cysteine residues. Based on the research in 1997, The combination of the extracellular domain of grouper IL-6R and IL-6 together with a peptide linker was constructed. The functional analyzed and binding assay of Hyper-IL-6 will be confirmed in the future work, and hope that can increasing the immunology effectively enhance in aquaculture.