石斑魚虹彩病毒027L基因之分子特性, 表現及其功能之分析

Abstract

石斑魚為台灣重要的經濟魚種，然而養殖石斑魚卻經常受到虹彩病毒(grouper iridovirus, GIV)的感染而造成魚隻大量死亡，重創南台灣的養殖產業。本實驗室已將石斑魚虹彩病毒基因組序列解出，並用微陣列基因座方式找出21個極早期基因，其中包含石斑魚虹彩病毒027L開放讀架。 石斑魚虹彩病毒027L開放讀架由91個胺基酸組成，可轉錄出一個分子量為10,505 dalton 的蛋白質。此蛋白質包含一個硫胱氨酸蛋白酶聚集功能區(caspase recruitment domain, CARD) ，序列比對結果顯示其胺基酸序列與人類的CARD-only 蛋白質ICEBERG及pseudo-ICE有高度相似性。反轉錄聚合酶連鎖反應偵測到在石斑魚虹彩病毒感染石斑魚腎臟細胞株早期即轉錄出GIV027L的 RNA，此表現時間點的偵測結果和其在轉譯抑制劑cycloheximide的處理下，仍可大量表現，證實石斑魚虹彩病毒GIV027L確實為一極早期基因。利用大腸桿菌表現系統pET20b(+)表現出GIV027L重組蛋白質,並接種至紐西蘭大白兔,製造出抗GIV027L的多株抗體。將pEGFP-N/GIV027L質體轉染進GK及HeLa細胞株中並配合胞器染色，顯示EGFP/GIV027L融合蛋白質會分布在細胞質與細胞核中。最後，利用免疫螢光染色法實驗，發現有轉染進pcDNA3CF/GIV027L的HeLa細胞株皆不會產生由UV引起之細胞凋亡，證實GIV027L具有抗細胞凋亡的能力。

Groupers are vital fish in Taiwan, while grouper iridovirus (GIV) has caused mass mortality, which economically impacts on culture of marine fish. Iridovirus infections have led to serious economic loss in the aquaculture industry in Southern Taiwan. Our laboratory had determined the complete DNA sequence of GIV and identified 21 immediate-early genes from GIV by using the microarray method, which include GIV 027L open reading frame (ORF). This GIV027L ORF is a caspase recruitment domain (CARD) domain containing gene which encodes a protein of 91 amino acid with predicted molecular mass of 10,505 Da and shows high similarity to human CARD-only protein, ICEBERG and pseudo-ICE. RT-PCR result revealed that GIV027L transcript at an early stage (1 h) of GIV infection. Cycloheximide and aphidicolin further confirmed that GIV027L was an immediate-early gene. By using E. coli pET20b(+) expression system, the recombinant GIV027L protein can be used to immunize New Zealand rabbits to produce rabbit anti-GIV027L polyclonal antibody. The subcellular localization of pEGFP-N/GIV027L fusion protein was in the nucleus as well as cytoplasm. By immunocytochemical staining, it was proven that GIV027L-expressing cells can inhibit apoptosis induced by UV irridiation.