虹彩病毒感染點帶石斑魚腎臟基因表現之研究

Abstract

石斑魚虹彩病毒為一雙股DNA 病毒，在140 Kb 的基因體序列中，分析得到139 個開放讀架。虹彩病毒主要攻擊宿主的免疫器官如脾臟與腎臟，感染虹彩病毒後病毒基因表現之研究已經大致完成，然而對於在感染期間宿主基因之表現仍尚未被廣泛研究，為了進一步了解宿主在感染期的免疫反應，首先我們先建構虹彩病毒感染的石斑魚腎臟組織λ 噬菌體cDNA基因庫，轉換為質體載體基因庫後，純化出6000 個候選質體來製作cDNA 微陣列晶片。製作完成的晶片再分別以注射過虹彩病毒、lipopolysaccharide (LPS) 或polyriboinosinic:polyribocytidylic acid (Poly I:C)之石斑幼苗萃取出的腎臟組織total RNA 所製作的螢光探針進行雜合反應。利用共軛焦雷射激發所得影像經分析後，挑選出在虹彩病毒感 染後任一天數中有2.5 倍以上或以下表現之候選基因進行核酸序列分析。所得基因核酸序列轉譯為胺基酸序列後與NCBI 蛋白質資料庫進行比對後，共分析出53 個不重複之基因。這些基因中22 個為2.5 倍以上正調控、31 個為2.5 倍以下負調控。依照基因功能可分類為:細胞凋亡、泛素/類泛素所調控的蛋白質降解系統、免疫反應、細胞組成骨架/細胞構形、紅血球生成、轉錄、轉譯、粒線體呼吸鏈、氫離子幫浦、代謝以及未知功能等11 個基因群。本實驗所篩選出來的基因有助於我們了解虹彩病毒感染魚體後，為達成病毒增殖以及宿主為抵抗病毒侵入時所進行的分子防禦策略，同時也證實利用質體當作cDNA 微陣列雜合方法在搜索宿主基因差異性表現具可行性。

Grouper iridovirus (GIV), a double strand DNA virus, encoded 139 open reading frames (ORFs) in its 140 kb genome size. Iridovirus mainly attack immune system such as spleen and kidney of host. The research of virus gene expression profile upon infection had been revealed. However, investigation of host gene expression during virus infection is still unknown. In order to understand virus infection mechanism and gene expressing profile of host, we have constructed GIV-infected grouper kidney λcDNA library. After transfering the vector from phage to plasmid, total 6000 candidate plasmids were purified and spotted on to cDNA microarray chip. Resulting chip was hybridized with different time-coursed fluorsence-labeled cDNA probes made from GIV, polyriboinosinic polyribocytidylic (Poly I:C) or lipopolysaccharide (LPS)-injected grouper larvae kidney total RNA. Images obtained from dual-laser scanner were analyzed and genes were selected by the criteria of at least 2.5-fold up or down-regulation of expression level in any of the experiment days. Candidate genes were sequenced, translated into protein sequence and blasted in NCBI protein database. Total 53 non-redundant genes were obtained, in which 22 of them were up-regulated and the rest 31 genes were down-regulated. Functional categories referred these genes into 11 groups, which were apoptosis, ubiquitin/protein degradation, respiratory chain, immune, transcription, translation, structure protein, erythropoiesis, proton pump, metabolism and genes with unknown function. Selected genes from this experiment help us elucidating the strategies of host genes in response to virus propagation and host defense during virus infection. Meanwhile, the plasmid DNA chip is proof of use in searching host differential expressed genes.