點帶石斑魚CD9基因之選殖、特性鑑定及感染虹彩病毒之表現分析

Abstract

點帶石斑魚(Epinephelus coioides)是目前水產養殖生物中具有高度經濟價值的魚種。在台灣，石斑魚產業的發展受限於許多因素，其中以病毒的威脅最為嚴重。GIV (grouper iridovirus)為一種二十面體的DNA病毒，可以造成從稚魚到成魚階段極高的致死率。本實驗室在2011年以GIV病毒液感染活體點帶石斑魚，發現到點帶石斑魚的腎臟與胰臟細胞中CD9的表現量有大幅上升的趨勢存在，而使用poly I:C的刺激下亦有大量的誘導表現，但是對於LPS並無任何反應，因此本研究想要進一步了解點帶石斑魚CD9的特性，並探討其與與虹彩病毒的交互作用。CD9是一種屬於四穿膜蛋白家族的醣蛋白。四穿膜蛋白家族成員間及相關的附屬蛋白會有形成四穿膜蛋白網絡的特性，這個特性使四穿膜蛋白具有訊息傳導的功能。目前已知許多疾病致病機轉中可以看到四穿膜蛋白家族成員的參與，如瘧疾以及病毒的感染。 本實驗設計了三種點帶石斑魚CD9基因的專一性引子進而以5’RACE得到CD9 cDNA 5’端的序列，結合先前本實驗室的點帶石斑魚cDNA資料庫中已知不完整3’端片段的資訊，我們得到點帶石斑魚的開放閱讀框架全長為720鹼基對。分析所轉譯出的239胺基酸序列具有典型的四穿膜區及兩個膜外環區，而具功能的大膜外環區也會有保守的CCG motif，進一步和不同物種CD9進行多重序列比對及親緣演化樹，分析顯示點帶石斑魚的CD9基因在演化上的保守性。此外，我們利用大腸桿菌生產石斑魚CD9重組蛋白，並以此為抗原，成功的製備出抗CD9的多株抗體，並且將CD9全長序列送進真核細胞中表現，配合免疫螢光染色，我們觀察到點帶石斑魚的CD9融合蛋白確實表現在核膜及細胞膜上。最後我們把GK細胞株以GIV病毒液進行感染試驗，結果顯示GK細胞在GIV不同的感染時程中會有不同程度的CD9表現量。

Orange-spotted grouper (Epinephelus coioides) is one of the high valued target species in aquaculture. The fiercest menace to the grouper industry is the breakout of virus disease. GIV (grouper iridovirus) is an icosahedron DNA virus and could cause highly lethal rate from fry to adult stage. In 2011, our laboratory found that the orange-spotted grouper kidney and spleen CD9 gene expression level could increase obviously after GIV intraperitoneal injection. Also, the tendency of CD9 gene expression was in accordance with GIV as we injected poly I:C. But there was no significance increasing phenomenon while we used LPS as artificial stimulator. This research engages in revealing the characteristic of orange-spotted grouper CD9 gene and discusses the interplay between GIV and CD9. CD9 , a surface glycoprotein, belongs to the tetraspanin family. The members of this family appear to form large integrated signaling complexes or tetraspanin-enriched microdomains (TEM) by their associated molecules. Tetraspanins are also known to play roles in pathology of infectious disease such as malara and numerous viral infections. We designed three CD9 gene specific primers to sequence the 5‘ end of orange-spotted grouper CD9 gene. Base on the partial sequence form the orange-spotted kidney cDNA library databank, we obtained the complete sequence and deduced the open reading frame length of orange-spotted grouper CD9 gene to 720 bp. We analyzed the 239 amino acid sequence which spans the plasma membrane four times, producing two extracellular loops, and processes a conserved CCG motif. We compared multiple sequence alignmernt of orange-spotted grouper CD9 to that of other vertebrate, and plotted the phylogenic tree to realize the conservation of orange-spotted grouper CD9 gene. Moreover, we produced the CD9 recombinant protein to generate polyclonal mouse anti-CD9 antibody. Meanwhile, we also tansfected the CD9 expression vectors into Hela and GK cells to explore CD9 subcellular localization. The immunochemistry observation showed that the CD9 fusion protein is mainly expressed on the nucleus and plasma membrane. Finally ,we challenged GK cell with GIV (moi=1) in 12 hrs time course and semi-quantated the RNA expression level of CD9 by RT-PCR. The result gives the sign of the fact that GK could alter the CD9 transcription magnitude according to the GIV infection elapsed time.