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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53545

Title: 莫三比克吳郭魚短型顆粒蛋白前體 PGRN1 基因在抗弧菌之分子機制研究
Study on molecular mechanism of short-form progranulin PGRN1 gene from Mozambique tilapia in defense against Vibrio species
Authors: Wu, Sheng-Han
吳聖韓
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 吳郭魚;顆粒蛋白前體;顆粒蛋白;免疫調節;抗菌活性;弧菌
Tilapia;Progranulin;Granulin;Immune modulation;Antibacterial activity;Vibrio species
Date: 2019
Issue Date: 2020-07-03T08:30:19Z
Abstract: 顆粒蛋白前體 (progranulin, PGRN) 是一個多功能性生長因子,具有促進細胞增生和存活、參與癌症生成及轉移、組織修復、發育和發炎反應等功能。莫三比克吳郭魚 (Oreochromis mossambicus) 短型 PGRN 基因 PGRN1 (OmPGRN1) 會表現兩種轉錄本 (transcript),一種可轉譯出由 206 個胺基酸 (amino acid, a.a.) 組成的 PGRN,包含兩個顆粒蛋白 (granulin, GRN) 單元,OmGRN-A 和 OmGRN-B。另一個則為新穎 RNA 選擇性剪接 (RNA alternative splicing) 產生的 mRNA,可轉譯出由 20 個 a.a. 組成的訊號胜肽 (signal peptide) 和 41 個 a.a. 組成的 GRN 單元,命名為 OmGRN-41。OmGRN-41 和 OmPGRN1 主要會表現在免疫相關組織,包含脾臟、頭腎和腸道。而且經創傷弧菌 (Vibrio vulnificus) 感染後 1 及 6 小時後,分別會進一步誘導 OmGRN-41 和 OmPGRN1 在脾臟的表現。本研究首先建立肌肉專一性表現分泌型 OmGRN-41、OmGRNA 和 OmPGRN1 之基因轉殖斑馬魚。結果顯示表現 OmGRN-41、OmGRNA 和 OmPGRN1 皆可提升基因轉殖斑馬魚感染 V. vulnificus 後之相對存活率,分別為 68%、32% 和 36%。而表現分泌型 OmGRN-41 具有顯著性活化基因轉殖斑馬魚之免疫反應相關基因的表現,如 TNFa、TNFb、IL-8、IL-1β、IL-6、IL-26、IL-21、IL-10、complement C3、lysozyme (Lyz) 和肝臟抗微生物胜肽 hepcidin (HAMP) 等。此外 OmGRN-41 亦能在基因轉殖斑馬魚感染 V. vulnificus 初期,進一步提升先天性免疫反應相關基因之表現,如 TNFb、IL-8、IL-1β、IL-6、complement C3、Lyz 和 HAMP 等。接著利用合成胜肽探討 OmGRN-41 對不同的病原性細菌之抗菌活性。結果顯示 OmGRN-41 合成胜肽對多種革蘭氏陰性弧菌屬病原菌具有殺菌活性,包含 V. vulnificus (64 μΜ)、V. alginolyticus (128 μΜ)、V. harveyi (128 μΜ) 和 V. parahaemolyticus (256 μΜ);亦對 V. alginolyticus (64 μΜ 處理後 8 小時內) 和 V. harveyi (64 μΜ 處理後 12 小時內) 和 V. parahaemolyticus (64 μΜ 處理後 8 小時內;128 μM 處理後 12 小時內) 具有抑菌活性。然而對於革蘭氏陰性菌 Aeromonas hydrophila、Edwardsiella tarda 以及革蘭氏陽性菌 Streptococcus agalactiae 和 Streptococcus iniae 皆不具抗菌活性。而由殺菌時間曲線 (time-kill curves) 中發現經 OmGRN-41 合成胜肽處理 2 小時內,即可殺死超過 99% 以上的弧菌屬病原菌,包含 V. vulnificus (64 μΜ 處理 1 小時內)、V. alginolyticus (128 μΜ 處理 1 小時內)、V. harveyi (128 μΜ 處理 2 小時內) 和 V. parahaemolyticus (256 μΜ 處理 2 小時內)。此外 OmGRN-41 經過 pH 2-10 或 40-100℃ 加熱處理 1 小時後,對 V. vulnificus 仍具有 MIC 和 MBC 的活性 (64 μΜ)。進一步利用穿透式電子顯微鏡可觀察到 OmGRN-41 合成胜肽會破壞 V. vulnificus 的外膜,進而導致其菌體破裂,使其胞內物質流出。此外 2-128 μΜ OmGRN-41 合成胜肽對於吳郭魚及綿羊紅血球溶血性低 (<3%)。在 in vivo 實驗也證實 OmGRN-41 合成胜肽能有效提升尼羅吳郭魚 (Oreochromis niloticus) 感染 V. vulnificus 後之存活率,且能降低其肝臟中 V. vulnificus 的菌數。最後利用酵母菌雙雜合系統 (yeast two-hybrid system) 篩選出一些與 OmGRN-41 交互作用的候選分子,如卵黃蛋白原 (vitellogenin)、鈣調蛋白 (calmodulin)、粒線體丙酮酸載體 (mitochondrial pyruvate carrier)、原血紅素結合素 (hemopexin) 等。本研究結果顯示 OmGRN-41 胜肽是一個新穎的殺菌劑,特別是針對弧菌屬病原菌。OmGRN-41 為一個新的宿主防禦胜肽,同時具有免疫調節劑和抗菌胜肽的角色。因此 OmGRN-41 具有潛力應用於治療人類或水產養殖動物之弧菌感染症。
Progranulin (PGRN) is a multi-functional growth factor that mediates cell proliferation, survival, migration, tumorigenesis, wound healing, development, and anti-inflammation activity. A short-form PGRN gene PGRN1 of Mozambique tilapia (Oreochromis mossambicus) expresses two transcripts, OmPGRN1 encoding a 206 a.a. PGRN with two granulin (GRN) units named OmGRN-A and OmGRN-B. And a novel alternatively spliced transcript encoding a novel, secreted GRN peptide composed of 20-a.a. ER signal peptide and 41-a.a. GRN unit named OmGRN-41. OmGRN-41 and OmPGRN1 were not only abundantly expressed in immune-related organs including spleen, head kidney, and intestine of Mozambique tilapia, but also were further induced in the spleen of Mozambique tilapia challenged with Vibrio vulnificus at 1 h post infection (hpi) and 6 hpi, respectively. In this study, we established three transgenic zebrafish lines expressing the secreted OmGRN-41, OmGRN-A and OmPGRN1 specifically in muscle. The relative percent of survival (RPS) was enhanced in adult transgenic zebrafish expressing OmGRN-41 (68%), OmGRN-A (32%) and OmPGRN1 (36%) compared with control transgenic zebrafish expressing AcGFP after challenge with V. vulnificus. And the secreted OmGRN-41 can induce the expression of innate immune response-related genes, such as TNFa, TNFb, IL-8, IL-1β, IL-6, IL-26, IL-21, IL-10, complement C3, lysozyme (Lyz) and the hepatic antimicrobial peptide hepcidin (HAMP), in adult transgenic zebrafish without V. vulnificus infection. In addition, the OmGRN-41 can enhance the innate immune response by further elevating TNFb, IL-1β, IL-8, IL-6, and HAMP expression in early responsive time to the V. vulnificus challenge in transgenic zebrafish. Furthermore, synthetic OmGRN-41 peptide was used to investigate its anti-bacterial activities against various bacterial pathogens. The results showed that synthetic OmGRN-41 had bactericidal activity against gram-negative Vibrio species including V. vulnificus (64 μM), V. alginolyticus (128 μM), V. harveyi (128 μM), V. parahaemolyticus (256 μΜ); and exhibited bacteriostatic activities to V. alginolyticus (64 μM in 8 hours), and V. harveyi (64 μM in 12 hours), and V. parahaemolyticus (64 μM in 8 hours, 128 μM in 12 hours). However, no anti-bacterial activities of synthetic OmGRN-41 was observed for gram-negative Aeromonas hydrophila, Edwardsiella tarda, and gram-positive Streptococcus agalactiae, and Streptococcus iniae. Time-kill curves showed that the Vibrio species was eradicated over 99% of bacteria by synthetic OmGRN-41 treatment for 2 hours, including V. vulnificus (64 μM in 1 hour), V. alginolyticus (128 μM in 1 hour), V. harveyi (128 μM in 2 hours) and V. parahaemolyticus (256 μM in 2 hours). Synthetic OmGRN-41 exerted the antimicrobial activity to V. vulnificus with MIC and MBC at 64 μM after treated in pH 2 to pH 10 or after heated for 1 hour at high temperature from 40℃ to 100℃. The TEM observed that the outer membrane of V. vulnificus was disrupted by synthetic OmGRN-41 leading to morphology rupture and loss of cytoplasmic contents. Additionally, little hemolytic activity (<3%) for the tilapia and sheep erythrocytes were detected after treated with 2 to 128 μM synthetic OmGRN-41. The in vivo experiments also confirmed that synthetic OmGRN-41 can effectively enhance the survival of Nile tilapia (Oreochromis niloticus) infected by V. vulnificus, and decreased the number of V. vulnificus in the liver of infected Nile tilapia. Finally, some candidate molecules interacting with OmGRN-41 were identified by the yeast two-hybrid system, such as vitellogenin, calmodulin, mitochondrial pyruvate carrier, hemopexin and so on. Our results suggest that the OmGRN-41 peptide is a novel bactericidal agent, especially for Vibrio species. OmGRN-41 as a new host defense peptide is not only an immune modulator but also an antimicrobial peptide. It indicates that OmGRN-41 can be applied to therapy for vibriosis in human or aquaculture animals.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0020133004.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53545
Appears in Collections:[水產養殖學系] 博碩士論文

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