石斑魚虹彩病毒108L極早期基因之特性鑑定

Abstract

石斑魚(Epinephelus spp.)為台灣重要的經濟養殖魚種之一，卻長期飽受虹彩病毒威脅，使得石斑魚養殖業面臨重大的經濟損害，因此研究虹彩病毒為刻不容緩的課題。石斑魚虹彩病毒(grouper iridovirus, GIV)的基因依據轉錄的先後順序可分為三大類：極早期基因( immediate early gene )、早期基因( early gene )和晚期基因( late gene )；其中，極早期基因能藉由調控病毒基因表現或改變宿主細胞的生理狀態，例如影響宿主細胞生長週期、細胞凋亡以及免疫防禦系統等，促進病毒進行複製增殖，因此極早期基因對於病毒感染宿主具有重要功能。本實驗室分析石斑魚虹彩病毒基因表現次序，推測其具有二十一個極早期基因，其中包含了ORF108L。108L基因全長為1,149個鹼基對，由382個胺基酸組成，蛋白質分子量 44.1 kDa，經由序列分析比對顯示其與 ICP46蛋白相似，且在虹彩病毒科中具有高度的保留性，因此認為108L對於病毒感染應扮演十分重要的角色；然而，目前尚未發現 ICP46具有已知有意義的功能區，所以它的功能仍是未知的。 本實驗建構了原核表現載體pET-28a-CBP-Factor Xa-108L，並成功地在大腸桿菌BL-21(DE3)以IPTG誘導表現出可溶性的重組蛋白。經由反轉錄聚合酶鏈鎖反應(RT-PCR)偵測石斑魚虹彩病毒感染石斑魚腎臟(GK)細胞病毒的基因表現，顯示108L於感染後2小時即開始轉錄，此外以轉錄抑制劑 (cycloheximide) 處理後仍可大量表現，證實108L基因確實屬於病毒極早期基因。藉由免疫細胞化學染色發現無論在GK或HeLa細胞內，108L蛋白表現位置主要為細胞核；此外本實驗基於同源性重組(homologous recombination)概念建構108L基因剔除(108L gene knockout )的重組病毒，並比較野生型石斑魚虹彩病毒與108L基因剔除重組病毒感染石斑魚腎臟細胞的差異性，顯示重組病毒出現效價(titer)下降與病毒斑(plaque)變小之現象。本實驗結果顯示108L可能於細胞核內作用並參與石斑魚虹彩病毒增殖與複製調控。

Grouper (Epinephelus spp.) is an important aquaculture fish species in Taiwan, but it is highly susceptible to iridovirus which often cause significant economic losses to grouper aquaculture. Accordingly, it is imperative to investigate the mechanisms of iridovirus infection and pathogenesis. The grouper iridovirus (GIV) genes can be classed into immediate early (IE), early (E) and late (L) genes according to their temporal synthesis upon infection. IE genes are regard as major roles in virus life cycle, because the transcripts of viral IE genes manipulate essential functions to benefit viral replication including controlling itself gene expression and altering host cell physiological status, such as cell cycle control, apoptosis and immune response. ORF108L is one of the immediate-early genes which our laboratory had identified from GIV. It contains 1,149 nucleotide and is composed of 382 amino acids which encode a 44.1 kDa protein. By comparative sequence analysis, 108L encodes infected cell polypeptide (ICP) 46 homolog which is highly conserved among the Iridoviridae family. However, there are no putative conserved domains have been found in ICP46 protein, so its actual function remains unknown. The prokaryotic expression plasmid, pET-28a-CBP-Factor Xa-108L, was constructed and transformed into the E.coli strain BL-21 (DE3) for expression. Besides, the best conditions of expression soluble 108L-his recombinant protein and purification by Ni2+ affinity column are well-established. The RT-PCR data confirmed that GIV108L is an immediate early gene of GIV, because the transcript of GIV108L was firstly detected at 2 hours post infection and still expressed after cycloheximide treatment. By immunocytochemistry assay, GIV108L protein was predominantly distributed at the nucleus both in GK and HeLa cells. Finally, the 108L gene knockout virus is generated by homologous recombination. Comparing wild-type virus with recombinant virus, the virus titeration is lower and the presence of cytopathic effect (CPE) obscured by infecting GK cells with recombinant virus than wild-type virus. In summary, this study demonstrated that GIV108L may function in nucleus and involve in the propagation and replication of grouper iridovirus.