以即時定量聚合酶鏈鎖反應為平台分析虹彩病毒感染後點帶石斑之基因表現

Abstract

虹彩病毒是一可感染點帶石斑之生活史各階段，並會造成嚴重死亡之病毒，其為一遺傳物質全長140 kb之雙股DNA病毒，其主要感染石斑魚之免疫器官，如脾臟、腎臟等部位。截至今日為止，雖然虹彩病毒之基因序列已全部解序，其各別基因也已有不少研究成果發表，但對於病毒入侵後石斑魚體內基因表現量之調控研究仍尚缺乏。本實驗室於2008年以點帶石斑魚的腎臟製作成的功能性基因體微陣列晶片以研究經注射過虹彩病毒、lipopolysaccharide（LPS）、poly I:C之點帶石斑腎臟於不同時間點之基因表現量的變化，篩選出18個2倍正調控與23個2倍負調控基因群。 本實驗重新以GeneSpring GX 11.5軟體檢視微陣列晶片，排除背景值後篩選出39個兩倍正調控與48個兩倍負調控之基因群。本實驗進一步以即時定量聚合酶鏈鎖反應來分析這些差異性表現的基因群。實驗中共分析24個微陣列晶片篩選出的基因以及6個免疫相關基因，其中RNA helicase DHX58 homolog、CD9 antigen、interferon stimulated gene 15、HECT E3 ubiquitin ligase、reverse transcriptase-like protein、vig-1、very large inducible GTPase-1、urokinase plasminogen activator surface receptor、26S proteasome non-ATPase regulatory subunit 1 homolog、interleukin-1β、interleukin-8、interferon-induced GTP-binding protein MxI、tumor-necrosis factor α和cyclooxygenase-2等15個基因，其注射虹彩病毒處理組之頭腎組織基因表現量比無注射之控制組高20倍以上。而UDP-glucose 6-dehydrogenase在病毒處理中的基因表現量則較控制組低了近100倍。本實驗分析出虹彩病毒感染石斑魚之頭腎基因差異表現之基因群將有助於了解病毒與宿主間的交互作用。

Grouper iridovirus (GIV), a double-strand DNA virus, can infect with all life stages of orange-spotted grouper (Epinephelus coioides), and causes groupers serious death. It mainly attacks immune system of host, such as spleen and kidney. Until now, the total 140 kb genome size of GIV had been sequenced and the research of several virus genes expression upon infection had been studied. However, investigation of host gene expression during GIV infection is still know very little. The grouper gene expression profiles after different kinds of challenges including GIV, lipopolysaccharide, and poly I:C using GIV-infected grouper kidney cDNA microarray had been established in our lab previously. We reanalyzed the previous array data of GIV-challenged only, and the newly 39 up-regulated and 48 down-regulated genes under two-fold thresholud were selected. To validate the result of microarray analysis, the quantitative real-time RT-PCR was performed. The kidney parts of non-injected control groupers and GIV-, lipopolysaccharide-, poly I:C-treated of groupers were collected at 1, 3, 5 day post-injection and the total RNAs were extracted for further RT-qPCR. Twenty four candidate genes from microarray analysis and another 6 immune-related genes were estimated. Fifteen genes including RNA helicase DHX58 homolog, CD9 antigen, interferon stimulated gene 15, HECT E3 ubiquitin ligase, reverse transcriptase-like protein, vig-1, very large inducible GTPase-1, urokinase plasminogen activator surface receptor, 26S proteasome non-ATPase regulatory subunit 1 homolog, interleukin-1β, interleukin-8, interferon-induced GTP-binding protein MxI, tumor-necrosis factor α and cyclooxygenase-2 are significant expression up to 20 fold compared to non-injected control candidate gene, but UDP-glucose 6-dehydrogenase is suppressed down to around 100 fold after GIV-injected treatment. Based on this experiment, we believe that those genes with significant expressed or suppressed may participate in the host-viral interaction.