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標題: | 鯉魚卵巢Cystatin基因之選殖與表現之研究 Molecular Cloning and Expression of Carp Ovarian Cystatin |
作者: | 李肇昌 |
出版年 : | 1995 |
學位: | 碩士 |
摘要: | Cystatin family是由一群結構相似的蛋白質所組成,能強烈抑制cysteine proteinase的活性。它們約含115個氨基酸,且都有兩個雙硫鍵。比較它們的氨基酸序列可發現,它們除了形成雙硫鍵的四個cysteine及其所在的位置保持不變外,還有三個高度保留區域,這三個區域與其抑制活性有關。 由本實驗室的鯉魚卵巢cDNA基因庫中分離出的三個clones,其cDNA長度分別為0.55、0.6及1.3kb,其核?酸序列與其他脊椎動物的cystatin family成員有很高的相似度,但它們彼此之間的序列並不盡相同。進一步定序後發現,1.3 kb長之cDNA與另外兩個cDNA的相似部份在其5'端,而其3'端卻額外多出約770個鹼基。推導的氨基酸序列顯示它們是不相同的蛋白質,但同樣都包含129個氨基酸,同時顯示1.3kb長之cDNA所多出的鹼基是屬於3'端非轉譯區(3'UTR)。而它們N端的前18-20個氨基酸極可能是一段訊號序列。此外,它們也俱備了四個位置不變的cysteine及三個高度保留區域,顯示它們確實是屬於cystatin family的蛋白質。 本篇論文除了分析了這三個cDNA的核?酸序列外,還以之為探針,瞭解此等cystatin mRNA在鯉魚各組織的表現情形。另外還將這些cDNA之片段接至表現型質體,在E.coli表現出鯉魚cystatin蛋白質,用來引發抗體,再用Western blotting來探討cystatin蛋白質在鯉魚組織或體液的分佈狀況。實驗結果發現,不管是mRNA或是蛋白質的表現都具有明顯的組織特異性,即只在卵巢中有表現。至於此等蛋白質在鯉魚卵巢的生理功能為何?則有待進一步探討。 The cystatin family comprises a group of structurally related proteins with strong inhibitory activity on cysteine proteinase. They are composed of about 115 amino acid residues with two intrachain disulfide bonds. In addition to four invariable cysteine, they contain three highly conserved regions which may participate in inhibitory activity. Three clones containing the inserts of 0.55、0.6 and 1.3 kb respectively, were isolated from a carp ovarian cDNA library. Nucleotide sequence analysis revealed that they are all homologous to the members of cystatin family of other vertebrates although they differ from each other. The open reading frames of all these cDNAs encode a protein of 129 amino acids which include a putative signal peptide of 18-20 amino acid residues. In addition, they also contain four invariable cysteine residues and three highly conserved regions found in cystatin family. The above data suggest that all the three cDNAs encode a polypeptide which belongs to the cystatin family of cysteine proteinase inhibitor. In this paper, the three cDNAs were also used as probes to detect the mRNAs expressed in carp tissues. In addititon, they were constructed to an expression vector and expressed in E.coli. The expressed proteins were used as antigens to induce antibodies in rabbits, with which the distribution of these proteins in carp tissues or body fluids were investigated. The results indicate that both cystatin mRNAs and proteins are tissue-specifically expressed only in the ovary. The physiological roles of these proteins in carp remain to be investigated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76162 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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