石斑魚虹彩病毒極早期基因030L之分子特性表現及其功能之分析

石斑魚(Epinephelus spp.)是具有高度經濟價值的養殖魚種，在台灣，除了東南亞及中國大陸的養殖興起，造成的經濟競爭外，其受到虹彩病毒的感染，造成魚隻死亡，更是各國都面臨的棘手問題，因此研究虹彩病毒為當前的重要課題。本實驗室藉由cDNA微陣列晶片，分析石斑魚虹彩病毒(grouper iridovirus, GIV)基因表現次序，推測其具有二十一個極早期基因(immediate early gene, IE)，這些基因能藉由調控病毒表現及改變宿主的生理狀態，使病毒得以進行複製增殖。而石斑魚虹彩病毒030L就被偵測為極早期基因，其全長為696個鹼基對，由231個胺基酸組成，蛋白質分子量為25.9 kDa，再經由NCBI資料庫分析GIV030L開放閱讀框架的結果顯示，其為腫瘤壞死因子受體(tumor necrosis factor receptor, TNFR)超級家族的一員。此TNFR蛋白具有膜外TNF配體結合區和穿膜區，卻缺少了膜內功能區，推測此TNFR-like基因可能是虹彩病毒為了對抗寄主TNF誘發系統，所演化出來的圈套。　　本實驗建構了真核表現載體pcDNA3CF-030L，轉染於子宮頸部腫瘤(HeLa)細胞中，經紫外光照射後，藉由免疫細胞化學染色及DNA斷裂端標記試驗(TUNEL assay)，發現過量表現的GIV030L無法阻擋紫外光所造成的細胞凋亡，紫外光照射所誘發的細胞凋亡是經由內部路徑(intrinsic pathway)所造成。因此本實驗進一步想藉由TNF-α細胞凋亡外部路徑(extrinsic pathway)來誘發石斑魚腎臟(GK)細胞的細胞凋亡。接著本實驗建構石斑魚TNF-α基因於原核表現載體pET-43.1a(+)-Factor Xa-TNF-α，並成功地在大腸桿菌BL-21(DE3) plysS以IPTG誘導表現出可溶性的重組蛋白，藉由親和性層析管柱，以不同濃度之咪唑(imidazole)沖提出目標蛋白，再由Factor Xa蛋白質切割酶切割後，得到重組的TNF-α蛋白。以不同濃度之TNF-α蛋白，與相同劑量之Actinomycin D處理於石斑魚腎臟細胞中，在顯微鏡下進行觀察，得知隨著TNF-α重組蛋白濃度的增加，GK細胞凋亡的情形也逐步惡化。

關鍵字

石斑魚虹彩病毒；極早期基因

並列摘要

Grouper (Epinephelus spp.) are highly economic value farmed fish in Taiwan. In addition to the farming competition from China and Southeast Asia countries, the high mortality caused by iridovirus infection became one of the thorniest issues in grouper aquaculture. Therefore, the study of mechanism involving in the iridovirus infection is the most important current events. Using cDNA microarray assay, our laboratory had identified 21 immediate early genes from grouper iridovirus including GIV030L. The GIV030L contains 696 nucleotides and is composed of 231 amino acids which encode a 25.9 kDa protein. According NCBI database comparative sequence analysis, GIV030L represents a tumor necrosis factor receptor (TNFR) homolog with a TNF ligand binding extracellular domain and a transmembrane domain, but lacking a intracellular domain. This incomplete TNFR-like viral protein may serve as a decoy and play a role to help iridovirus against host TNF attacking system. In this study, GIV030L was constructed into pcDNA3CF eukaryotic expression vector and was transfected into HeLa cells. However, the overexpressed GIV030L can not prevent the HeLa cells from apoptosis trigged by UV-irradiation mediated intrinsic apoptosis pathway in the immunocytochemistry and Terminal deoxynucleotidyl Transferase mediated dUTP Nick End Labeling (TUNEL) assays. To establish the grouper kidney (GK) cells apoptosis through TNF and TNFR extrinsic pathway, the TNF-α gene was cloned from orange-spotted grouper and was constructed into pET-43.1a(+)-Factor Xa prokaryotic expression vector. The E. coli expressed recombinant TNF-α contains a NusA protein at N-terminal to help TNF-α under correct folding. After Ni2+ affinity column purification and Factor Xa protease digestion, the soluble recombinant grouper TNF-α was subjected to apoptosis experiment. With no harmful concentration of Actinomycin D, the GK cells were gradually deteriorated fallowing the increase of recombinant TNF-α addition.

並列關鍵字

GIV ； iridovirus

參考文獻

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石斑魚虹彩病毒極早期基因030L之分子特性表現及其功能之分析

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