虹彩病毒感染點帶石斑魚腎臟基因表現之研究

• Main Authors: 黃聖翔, Huang, Hseng-Hsiang
• Other Authors: 張繼堯, 臺灣大學:漁業科學研究所
• Language: Chinese; English
• Published: 2008
• Subjects: 虹彩病毒;微陣列; iridovirus;microarray; Andenes
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/181687; http://ntur.lib.ntu.edu.tw/bitstream/246246/181687/1/ntu-97-R95B45014-1.pdf

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 framesORFs) in its 140 kb genome size. Iridovirus mainly attack immune system such as spleen andidney of host. The research of virus gene expression profile upon infection had been revealed.owever, investigation of host gene expression during virus infection is still unknown. In order tonderstand virus infection mechanism and gene expressing profile of host, we have constructedIV-infected grouper kidney λcDNA library. After transfering the vector from phage to plasmid,otal 6000 candidate plasmids were purified and spotted on to cDNA microarray chip.esulting chip was hybridized with different time-coursed fluorsence-labeled cDNA probesade from GIV, polyriboinosinic polyribocytidylic (Poly I:C) or lipopolysaccharide (LPS)-injectedrouper larvae kidney total RNA. Images obtained from dual-laser scanner were analyzed andenes were selected by the criteria of at least 2.5-fold up or down-regulation of expression level inny of the experiment days. Candidate genes were sequenced, translated into protein sequence andlasted in NCBI protein database. Total 53 non-redundant genes were obtained, in which 22 ofhem were up-regulated and the rest 31 genes were down-regulated. Functional categories referredhese genes into 11 groups, which were apoptosis, ubiquitin/protein degradation, respiratory chain,mmune, transcription, translation, structure protein, erythropoiesis, proton pump, metabolism andenes with unknown function.elected genes from this experiment help us elucidating the strategies of host genes inesponse to virus propagation and host defense during virus infection. Meanwhile, the plasmidNA chip is proof of use in searching host differential expressed genes. 摘要 . Ibstract . II錄 . III目次 . VI目次 . VII一章 前言 . 1.1 虹彩病毒 . 1.1.1 魚類虹彩病毒感染症之發現 . 1.1.2 虹彩病毒之特性 . 3.1.3 虹彩病毒之分類 . 4.2 石斑魚之簡介與台灣養殖概況 . 5.3 病毒與免疫系統 . 6.3.1 魚類免疫系統 . 6.3.2 虹彩病毒感染魚類相關之研究 . 7二章 材料與方法 . 9.1 虹彩病毒之增殖與純化 . 9.1.1 細胞繼代培養 . 10.1.2 虹彩病毒之增殖 . 11.1.3 虹彩病毒之純化 . 11.1.4 病毒力價之測定 . 12.1.5 病毒蛋白質之電泳分析 . 12.2 石斑魚受虹彩病毒感染腎臟組織λ 噬菌體cDNA 基因庫之構築 . 13.2.1 石斑魚腎臟組織檢體之準備 . 15.2.2 石斑魚腎臟組織mRNA 之萃取及RNA 電泳 . 16.2.3 cDNA 之合成與修飾 . 17.2.4 分離不同大小之cDNA 片段 . 18.2.5 cDNA 嵌入片段與載體之接合反應 . 19.2.6 λ 噬菌體之包裝 . 19.2.7 宿主細胞之製備 . 19.2.8 腎臟組織cDNA 基因庫力價之測定 . 20.2.9 轉換噬菌體載體為質體載體 . 20.2.10 噬菌質體力價測定、候選基因藍白篩選和質體純化 . 21.3 cDNA 微陣列之雜合反應. 22.3.1 石斑魚幼魚樣品探針之製備 . 24.3.2 Total RNA 之萃取、純化與RNA 電泳 . 24.3.3 基因微陣列晶片之前準備 . 25.3.4 cDNA 合成與標定 . 25.3.5 cDNA 微陣列之雜合反應 . 26.3.6 候選基因定序及序列比對分析 . 27三章 結果 . 28.1 石斑魚腎臟組織λ 噬菌體cDNA 基因庫力價之測定 . 28.2 噬菌質體(phagemids)基因庫力價之測定 . 28.3 cDNA 微陣列之雜合反應. 28.4 cDNA 微陣列數據之分析. 28.5 差異表現 cDNA 之序列分析 . 29四章 討論 . 31.1 粒線體呼吸鏈相關基因 . 31.2 CCAAT enhancer binding protein beta . 32.3 IGF binging protein 5 . 32.4 泛素/類泛素相關基因 . 33.5 Very large inducible GTPase (VLIG) . 33.6 Serum lectin precusor . 34.7 Butyrophilin、CD 9 protein 和thymosin beta . 34五章 參考文獻 . 36目次igure 1. Complementary DNA microarray images of grouper kidney infected with grouper iridovirus . 44igure 2. Complementary DNA microarray images of grouper kidney infected with polyriboinosinic:olyribocytidylic acid . 45igure 3. Complementary DNA microarray images of grouper kidney infected with lipopolisaccharide . 46igure 4. Gene expression profile of GIV-infected grouper kidney . 47igure 5. Gene expression profile of poly I:C-injected grouper kidney . 48igure 6. Gene expression profile of LPS-injected grouper kidney . 49igure 7. Core reference set for 2 and 2.5 fold up- or down-regulated genes . 50igure 8. Clustering gene tree of 2.5-fold differentially expressed genes . 51igure 9. Line graph of 2.5-fold up-regulated genes by hierarchical clustering . 52igure 10. Line graph of 2.5-fold down-regulated genes by hierarchical clustering . 53igure 11. Functional categories of differentially expressed genes . 54目次able 1. Up-regulated genes for at least 2.5-fold change of expression level underwent either GIV、oly I:C or LPS treatment. . 55able 2. Down-regulated genes for at least 2.5-fold change of expression level underwent either GIV、oly I:C or LPS treatment. . 57