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研究生:林文福
研究生(外文):Wen-Fu Lin
論文名稱:尼羅吳郭魚短型顆粒蛋白前體2(PGRN2)調節先天性免疫相關基因抵抗鏈球菌之研究
論文名稱(外文):Study on Short-form Progranulin 2 (PGRN2) of Nile Tilapia Modulates Innate Immune-related Genes against Streptococcus
指導教授:龔紘毅
指導教授(外文):Hong-Yi Gong
口試委員:陳志毅胡紹揚邱品文龔紘毅
口試委員(外文):Jyh-Yih ChenShao-Yang HuPinwen Peter ChiouHong-Yi Gong
口試日期:2016-07-18
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:86
中文關鍵詞:尼羅吳郭魚基因轉殖斑馬魚顆粒蛋白前體顆粒蛋白先天免疫鏈球菌
外文關鍵詞:Nile TilapiaTransgenic ZebrafishProgranulinGranulinInnate ImmunityStreptococcus
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  • 被引用被引用:0
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  • 下載下載:21
  • 收藏至我的研究室書目清單書目收藏:1
顆粒蛋白前體 (Progranulin, PGRN) 為一多功能生長因子,具有促進細胞增生、抑制細胞凋亡、參與癌細胞的形成與轉移、抗發炎反應、發育與組織修復等功能。當個體受傷或感染時, PGRN 可經蛋白酶剪切成顆粒蛋白 (Granulins, GRN) 胜肽單位,促進先天性免疫反應。本實驗室於吳郭魚發現多種短型 PGRN 基因,如 PGRN1 、 PGRN2 等,分別由 206 和 199 個胺基酸組成,均具有 2 個 GRN 胜肽單位。本研究從尼羅吳郭魚 (Oreochromis niloticus) 基因體選殖出 OnPGRN2 ,發現 OnPGRN2 基因於公母成魚中皆大量表現於脾臟、頭腎兩個免疫相關組織。進一步使用海豚鏈球菌 (Streptococcus iniae) 以腹腔注射感染尼羅吳郭魚後,萃取組織分析發現 OnPGRN2 基因表現量被誘導提升。利用 Tol2 轉位子系統建立肌肉專一性啟動子表現不同程度分泌型 OnPGRN2 (OnPGRN2-S 、 OnPGRN2-W) 蛋白質兩種品系之基因轉殖斑馬魚,探討其參與調節先天性免疫之分子機制。在無細菌感染情況下, OnPGRN2 基因轉殖斑馬魚與螢光斑馬魚控制組於先天性免疫相關基因表現上並無顯著差異。使用無乳鏈球菌 (S. agalactiae) 以腹腔注射基因轉殖斑馬魚, OnPGRN2-S 相對於螢光斑馬魚控制組有較高的存活率,相對存活百分率為 40% 。經無乳鏈球菌感染後,發現 OnPGRN2 基因轉殖斑馬魚促發炎反應基因表現量會顯著提升。 OnPGRN2-S 於 24 hpi 顯著活化 TNFa 、 IL-6 、 COX-2a 等促發炎反應因子,來達到抵抗細菌性病原之效果。本研究使用抗病的 NT1 與對鏈球菌敏感的 NT2 兩種品系之尼羅吳郭魚,進一步分析兩品系的 OnPGRN2 基因 mRNA 表現量與 2.6 kb 上游序列。研究結果指出 NT1 之 OnPGRN2 表現量顯著高於 NT2 ,且發現兩品系之間在 5 個先天性免疫相關轉錄因子結合位點上具有序列變異。未來需進一步驗證 OnPGRN2 啟動子序列與基因表現量之關係,將可作為尼羅吳郭魚全新抗病之分子標誌,輔助抗鏈球菌尼羅吳郭魚品系建立。
Progranulins (PGRN) is a multifunctional growth factor that regulate cell proliferation, cell survival, tumorigenesis, inflammation, embryonic development, and tissue repair. When organisms are injured or infected, PGRN can be digested into granulin units (GRN) by elastases. GRN units have the function of innate immunity regulation. We had isolated several types of tilapia PGRN genes, PGRN1 and PGRN2, which encode 206 and 199 amino acids composed of two GRN units, respectively. In this study, we isolated one OnPGRN2 gene from Nile tilapia (Oreochromis niloticus). OnPGRN2 was abundantly expressed in spleen and head kidney of male and female adult Nile tilapia. Intraperitoneal injection with S. iniae can induce OnPGRN2 mRNA expression in Nile tilapia. We used Tol2 transposon system to establish two transgenic zebrafish lines expressing different levels of secretory OnPGRN2 (OnPGRN2-S, OnPGRN2-W) by muscle-specific promoter. Expressions of innate immune-related genes had no significant difference in OnPGRN2 transgenic zebrafish lines. Upon intraperitoneal injection with S. agalactiae, the survival rate was enhanced in OnPGRN2-S transgenic zebrafish line, and the relative survival rate was 40%. Pro-inflammatory genes of OnPGRN2 transgenic zebrafish lines were highly activated after challenge with S. agalactiae. It suggests OnPGRN2-S can activate pro-inflammatory factors including TNFa, IL-6, COX-2a at 24 hpi to enhance innate immunity against bacterial pathogen. We detected mRNA expressions of OnPGRN2 and 2.6 kb upstream sequences in two Nile tilapia strains, NT1 and NT2. OnPGRN2 mRNA expression in NT1 which is resistant strain was higher than NT2 which is sensitive strain to Streptococcus, and five immune-related transcription factor binding sites were identified to bear sequence variations between two strains. The relationship between OnPGRN2 promoter sequence and expression will be further investigated. OnPGRN2 gene will be a novel molecular marker for marker-assisted selection of disease-resistant Nile tilapia.
謝辭 I
摘要 II
Abstract III
目錄 IV
圖目錄 VI
表目錄 VIII
附錄 IX
縮寫表 X
壹、前言 1
1.1 哺乳類顆粒蛋白前體 (Progranulin, PGRN) 研究 1
1.2 硬骨魚類 PGRN 研究 2
1.3 硬骨魚類先天性免疫系統 (Innate immune system of teleost fish) 3
1.4 基因轉殖技術 (Transgenic technology) 6
1.5 Tol2 轉位子系統 (Tol2 transposon system) 6
1.6 tTA 系統 (Tetracycline-controlled transactivator system) 7
1.7 研究目的 7
貳、實驗材料 9
2.1 實驗動物 9
2.2 實驗載體 (Vector) 9
2.3 實驗菌株 9
2.4 培養基 10
2.5 引子 (Primer) 與探針 (Probe) 10
2.6 市售生物反應試劑套組 (Kit) 10
2.7 生物及化學藥品 11
2.8 儀器設備 11
參、實驗方法 13
3.1 選殖尼羅吳郭魚 OnPGRN2 之 cDNA 13
3.2 選殖尼羅吳郭魚 OnPGRN2 之 Genomic DNA (gDNA) 16
3.3 尼羅吳郭魚 OnPGRN2 之組織分佈 (OnPGRN2 mRNA tissue distribution) 17
3.4 使用海豚鏈球菌 (Streptococcus iniae) 感染尼羅吳郭魚之 OnPGRN2 基因表現分析 19
3.5 基因轉殖表現載體構築 19
3.6 建立 OnPGRN2 基因轉殖斑馬魚品系 19
3.7 OnPGRN2 基因轉殖斑馬魚之基因表現分析 22
3.8 使用無乳鏈球菌 (Streptococcus agalactiae) 感染 OnPGRN2 基因轉殖斑馬魚之存活率 22
3.9 使用無乳鏈球菌 (Streptococcus agalactiae) 感染 OnPGRN2 基因轉殖斑馬魚之基因表現分析 22
肆、實驗結果 23
4.1 選殖尼羅吳郭魚之 OnPGRN2 基因序列 23
4.2 尼羅吳郭魚 OnPGRN2 基因組織分佈 23
4.3 尼羅吳郭魚經海豚鏈球菌 (Streptococcus iniae) 感染 OnPGRN2 基因表現量變化 23
4.4 建立 OnPGRN2 基因轉殖斑馬魚 24
4.5 OnPGRN2 基因轉殖斑馬魚之先天性免疫相關基因表現 24
4.6 OnPGRN2 基因轉殖斑馬魚經無乳鏈球菌 (Streptococcus agalactiae) 感染之存活率 25
4.7 OnPGRN2 基因轉殖斑馬魚經無乳鏈球菌 (Streptococcus agalactiae) 感染之先天性免疫相關基因表現量變化 25
4.8 尼羅吳郭魚品系間 OnPGRN2 基因表現量差異 26
伍、討論 27
陸、參考文獻 31
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