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標題: | 鯉魚腎泌分解?及其相關基因之研究 Carp Head Kidney Nephrosin and Embryonic Hatching Enzymes Are Members of the Astacin Family. |
作者: | Hon-Ren Huang 黃宏仁 |
出版年 : | 1996 |
學位: | 碩士 |
摘要: | 鯉魚的頭腎包含免疫、造血、內分泌及神經四種組織,我們實驗室已經從鯉魚頭腎純化到一個耦合金屬蛋白分解?,利用蛋白質電泳(SDS-PAGE)估計分子大小約為25 kD,經由西方轉印法分析得知,它分佈在頭腎、腎臟和脾臟,這三個器官在魚類都具有免疫的功能,我們將這個耦合金屬蛋白分解?命名為腎泌分解?(Nephrosin)。我以純化得到的腎泌分解?在天竺鼠生產抗體,然後利用免疫篩選法從鯉魚頭腎cDNA庫選殖到一個cDNA株,經由定序後發現它是腎泌分解?的基因,從一級結構的比對,我們得知腎泌分解?是屬於astacin家族;這是一個在九零年代才浮現的耦合金屬蛋白分解?家族,目前已知的成員有二十多個,分佈於許多種生物的不同系統。腎泌分解?的一級結構和日本大肚魚medaka胚胎的孵化蛋白?最相似,它是用來水解卵膜,幫助仔魚進行孵化。為了進一步瞭解腎泌分解?和孵化蛋白?的關係,我們用西方轉印法和北方轉印法分析鯉魚胚胎,並沒有偵測到腎泌分解?的蛋白質和mRNA。另一方面,我們利用腎泌分解?和medaka孵化蛋白?胺基酸的相似區域設計引子(primers),以RT-PCR的方式在鯉魚胚胎找到一些DNA片段,也是屬於astacin家族,之後我們挑選兩個差異較大的片段,以RACE的方法找出整個cDNA的序列,分別稱為CCE-1和CCE-2。Medaka孵化蛋白?的一級結構比較像CCE-1和CCE-2,與腎泌分解?的相似度則較低。從這些結果來看,腎泌分解?不是鯉魚的孵化蛋白?,而是astacin家族的一個新成員。 We have recently purified a metalloproteinase from the carp head kidney, an organ containing immune, hematopoietic, endocrine, and nervous tissues. Its molecular size was 25 kD, as estimated from SDS-PAGE. It was a secreted proteinase and present in the head kidney, kidney, and spleen, all of which are responsible for the immune and hematopoietic function in fish. In this study, we have cloned the cDNA encoding this metalloproteinase through antibody screening and named it nephrosin. Its transcript is present in many tissues, and is more abundant in the head kidney, kidney, spleen, and heart. It belongs to a new proteinase family, astacin family, which contains more than 20 members up to date. All of them have a signature domain, HEXXHXXGFXHEXXRXDR, and need one zinc ion as a cofactor for their activity. The first member discovered in this family is astacin, purified from the crayfish Astacus astacus. The only known members in fish are medaka, Oryzias latipes, hatching enzymes. The primary structure of nephrosin is similar to the hatching enzymes, HCE-l, HCE-2, and LCE, which are secreted to digest the egg envelope when embryos are hatching. The expression level of nephrosin in 2-days embryo is not detectable by northern and western blot in contrast to adult head kidney. In order to understand the relationship between nephrosin and hatching enzymes, we isolated two cDNA clones, CCE-l and CCE-2, from carp embryo via RT-PCR and RACE. From the infered amino acid sequences, CCE-l and CCE-2 are more similar to the hatching enzymes of medaka than nepbrosin. Therefore, nepbrosin is structurally and functionally different from hatching enzymes and is a new member of astacin family. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76184 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
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