Functional Genomics Study of Teleost Progranulin Genes Involved in Regulation of Innate Immunity and Growth

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Project title

Code/計畫編號

NSC102-2313-B019-004-MY3

Translated Name/計畫中文名

魚類Progranulin基因參與先天免疫調控及生長之功能性基因體研究

Project Coordinator/計畫主持人

Hong-Yi Gong

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Aquaculture

Website

https://www.grb.gov.tw/search/planDetail?id=11274258

Year

2015

Start date/計畫起

01-08-2015

Expected Completion/計畫迄

31-07-2016

Bugetid/研究經費

1578千元

ResearchField/研究領域

生物技術(農)
漁業

"顆粒蛋白前體 (Progranulin; PGRN)是一個多功能的生長因子，具有促進細胞增生、存活、遷移，參與癌症生成、組織修復、發育及抗發炎反應等功能。PGRN 會被嗜中性球所產生的絲氨酸蛋白質酶裂解為GRN 胜肽來誘導促發炎反應的細胞激素IL-8 表現。本研究利用基因體南方墨點雜交分析及PCR 選殖發現莫三比克吳郭魚的基因體中具有多個由5 個exons 及4 個introns 組成的PGRN 家族基因，均可產生兩個56 或57 個胺基酸的不同GRN 單元。依據其前端GRN 的變異可將吳郭魚PGRN 基因分為三類。我們首先發現一個使用79 個核苷酸intron 2 序列之選擇性剪接mRNA 產物，造成氨基酸轉譯序列提前終止而產生C 端縮短成為由20 個氨基酸signal peptide 及41 個氨基酸成熟胜肽組成的新穎分泌型GRN 胜肽，將之命名為GRN-41。此GRN-41 胜肽具有與PGRN 前端GRN 相同的N 端30 個氨基酸及變異的C 端11 個氨基酸。吳郭魚脾臟及小腸第一類與第三類PGRN 基因大量表現206 個胺基酸的 PGRN，而肌肉偏好表現GRN-41。特別的是第二類PGRN 基因在所有組織中只表現GRN-41。本研究利用肌肉專一性啟動子結合雙向表現TetOff 載體系統，已建立表現吳郭魚分泌型GRN-41、GRN-A 及PGRN-1 之基因轉殖斑馬魚。結果發現新穎吳郭魚GRN-41 具有活化及調節IL-8、IL-1β、TNF及c3b 等先天免疫相關基因的作用。本計畫將以功能性基因體方法利用基因轉殖斑馬魚及重組蛋白處理，以表現微晶片(microarray)或數位基因表現(DGE)分析研究吳郭魚GRN-41、GRN 胜肽、吳郭魚與龍膽石斑之短型PGRN 及長型PGRN 其調控魚類先天免疫作用及生長之分子機制。進一步建立以大腸桿菌及酵母菌表現系統來生產高活性GRN-41、GRN 胜肽、短型PGRN 及長型PGRN 等重組蛋白，以應用於重要養殖魚種如台灣鯛及石斑魚等之抗病及促進生長之功能性飼料添加物。" "Progranulin (PGRN) is a multi-functional growth factor regulating cell proliferation, survival, migration, tumorgenesis, would healing, development and anti-inflammation. PGRN undergoes proteolysis by neutrophil serine protease into GRN peptides to induce pro-inflammatory cytokine IL-8. In this study, multiple PGRN family genes composed of five exons and four introns encoding PGRNs with two variant GRN units (56 or 57 a. a.) in tilapia (Oreochromis mossambicus) genome were identified by genomic Southern hybridization and PCR cloning. Three groups of tilapia PGRN genes were classified according to variations in anterior GRN units. We identified a novel tilapia PGRN alternative splicing transcript using 79-nucleotide intron 2 sequence resulting in in-frame stop codon to generate a C-terminal truncated secretory GRN. The novel tilapia secretory GRN named as GRN-41 is composed of 20-a. a. signal peptide and 41-a. a. mature peptide which share identical N-terminal 30 a. a. with anterior GRN but a variant 11-a.a. C-terminal. Tilapia spleen and intestine abundantly express normal PGRN with 206 a. a. whereas muscle prefers to express GRN-41 of group I and III PGRN genes. Especially tilapia group II PGRN gene abundantly expresses GRN-41 in all tested tissues. We had established secretory GRN-41, GRN-A, and PGRN-1 transgenic zebrafish lines by muscle-specific promoter and bi-directional TetOff regulatory system. The novel tilapia secretory GRB-41 can activate and modulate innate immunity genes such as IL-8, IL-1β, TNFc3b expression in transgenic zebrafish. In this project, transgenic zebrafish, tilapia and giant grouper treated with GRN or PGRN recombinant protein will be analyzed by using functional genomics approaches as expression microarray and digital gene expression (DGE) to study function and molecular mechanism of tilapia GRN-41, tilapia and giant grouper GRN peptides, short PGRN and long PGRN involved in regulation of innate immunity and growth in teleots. We will use E. coli and yeast expression system to produce potent recombinant GRN and PGRN proteins as food functional supplements for disease-resistant and growth promotion to apply in the administration of the important aquaculture fish species such as Taiwan tilapia, grouper etc."

Keyword(s)

顆粒蛋白前體
顆粒蛋白
吳郭魚
免疫
生長
Progranulin
Granulin
Tilapia
Immunity
Growth

DSpace CRIS

Functional Genomics Study of Teleost Progranulin Genes Involved in Regulation of Innate Immunity and Growth

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