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研究生:張凱貿
研究生(外文):Kai-Mao Chang
論文名稱:鯉魚GranzymeB之純化及特性
論文名稱(外文):Purification and Characterization of Granzyme B from Carp Gill
指導教授:張震東張震東引用關係
指導教授(外文):Green-Dong Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:57
中文關鍵詞:granzyme B硬骨魚
外文關鍵詞:granzyme Bteleost fishgill
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Granzyme B是serine proteinase的一員,主要存在於cytotoxic淋巴細胞的分泌顆粒內。此分泌顆粒中含有穿孔素(perforin)、granzymes和其它蛋白分解。當cytotoxic淋巴細胞和受病原感染的細胞接觸時,分泌顆粒便會釋放內涵物,granzymes和perforin就會進入目標細胞內,引發細胞自殺機制使細胞死亡。在本篇報告中,我們以肝制凝素(Heparin)親和力管柱從鯉魚鰓組織純化到一個還原後分子量約26 kDa的蛋白,經N端15個基酸序列比對後,和mature human granzyme B的N端15個基酸序列有七成的相似度。此酵素活性在pH 7.5-8.5間最大,由受質專一性判斷為Asp-ase。因此,確認為鯉魚granzyme B;它是granzymes在硬骨魚類被發現的首例。實驗結果顯示,鯉魚granzyme B的活性會被高濃度鋅離子(1 mM, 0.5 mM)抑制住。鎘、鎳、錳、鈷離子在1 mM濃度下也具有抑制效果,但作用較鋅離子弱。此外,也發現肝制凝素和Dermatan sulfate(DS)會增強鯉魚granzyme B的活性,但是無法恢復受到鋅離子抑制的鯉魚granzyme B活性。組織分佈調查的結果顯示,鯉魚granzyme B主要出現在頭腎、腎臟和鰓,表示鯉魚granzyme B可能參與硬骨魚的免疫反應。由上述結果,我們可以推論,granzyme B的活性會受到高濃度鋅離子及proteoglycans的調控。

Mammalian granzyme B is a neutral serine proteinase found, together with other granzymes and perforin, in the exocytotic granules of the cytotoxic lymphocytes. The cytotoxic proteins are released to initiate typical programmed cell death in the target cells when encountered by the cytotoxic lymphocytes.
We reported in this thesis the purification and characterization of granzyme B from carp gill. The protein is around 26 kDa as determined by SDS gel electrophoresis and binds strongly in the heparin affinity column chromatography. N-terminal amino acid sequence was determined from the purified protein for up to 15 residues and was identical to human granzyme B at 11 corresponding positions. In addition, the carp proteinase exhibits optimal enzyme activity at pH 7.5 to 8.5 and prefers cutting scissile bonds right after Asp residues. Therefore, we believe that the purified protein is a fish granzyme B and this is the first report on the purification of a teleost granzyme B.
In this thesis, we also examined the effects of metal ions and glycosaminoglycans (GAGs) on the activity of gill granzyme B. Zinc ion at 0.5 mM and higher concentrations greatly reduces the enzyme activity of carp granzyme B. Other divalent metal ions such as Cd2+, Ni2+, Mn2+ and Co2+ may also inhibit granzyme B, but to lesser extents. On the contrary, heparin (0.1-100 μg/ml) and dermatan sulfate (DS, 1-1,000 μg/ml) can enhance the enzyme activity of carp granzyme B. However, heparin and DS cannot reactivate the activity of carp granzyme B suppressed by 1 mM Zn2+. The presence of carp granzyme B in different tissues was examined by immunoblotting with a polyclonal antiserum against the purified protein. The carp granzyme B is present mostly in the head kidney, kidney and gill, all of which are immune organs. Therefore, the data suggest that carp granzyme B may be involved in the function of fish immune system. Furthermore, regulation of carp granzyme B involves both proteoglycans and zinc ion.

目錄…………………………………………………………………………Ⅰ
中文摘要……………………………………………………………………Ⅲ
英文摘要……………………………………………………………………Ⅳ
第一章 前言…………………………………….….…………….………1
第一節 Granzymes…………………………………..………….……..…1
第二節 Granzyme B……………………………………..………….……3
第三節 Proteoglycans…………………………………..………….…..…4
第四節 Granzymes和Proteoglycans的交互作用………….……….....….6
第五節 鋅離子………………………………………………..…….……7
第六節 硬骨魚類的鰓………………………………...……..…..………8
第二章 材料與方法…………………………………….……..………..10
第一節 實驗動物…………………………………………….…..……..10
第二節 實驗藥品………………………………………….……………10
第三節 實驗儀器…………………………………………….…………10
第四節 SDS聚丙烯醯胺凝膠電泳(SDS-PAGE)…………...…....…..11
第五節 蛋白質膠片染色….....…………………………….….…....…..11
第六節 Alcian Blue Stain………………………………..…………..…..12
第七節 銀染色(Silver-staining)……………………….………….....12
第八節 西方轉印法……………………………………….……....……12
第九節 蛋白電泳分布圖 (Proteolytic zymography)………..….….....13
第十節 鯉魚Granzyme B之純化…………………………..…….…......13
第十一節 RCM(Reduced Carboxymethylated)-BSA之製備…….....…15
第十二節 鯉魚 Granzyme B 酵素活性測定…………………..……...…15
第十三節 蛋白質定量法……………………………………………...…15
第十四節 製備鯉魚Granzyme B 之多株抗體……………………….….16
第十五節 鯉魚組織萃取液之製備………………………………..…….17
第三章 結果……………………………………………………...…..…18
第一節 鯉魚 Granzyme B的純化與確認…………………….…....…...18
第二節 鯉魚 Granzyme B的活性取決於酸鹼值…………………....…19
第三節 高濃度鋅離子抑制鯉魚Granzyme B活性…………….……....20
第四節 抑制鯉魚 Granzyme B的金屬離子種類……………….......….21
第五節 鈣、鎂離子不能活化鋅離子抑制之鯉魚 Granzyme B
活性……………………………………………………..…........…..21
第六節 肝制凝素增強鯉魚 Granzyme B活性………………..….….....21
第七節 調控鯉魚 Granzyme B活性的多醣種類…………….………..22
第八節 肝制凝素、多醣不能活化鋅離子抑制之鯉魚Granzyme B
活性…...…...………………………………………..……..….........22
第九節 鯉魚 Granzyme B的抗體製備和組織調查………..……..…...23
第十節 鯉魚鰓proteoglycans的純化和確認…………………..…....…23
第四章 討論…...…...……………………………………….….…........25
第一節 鯉魚Granzyme B的純化……………………………….…...…25
第二節 鯉魚Granzyme B的活性取決於環境酸鹼度………………....25
第三節 高濃度鋅離子抑制鯉魚Granzyme B活性…………….…...…26
第四節 鈣、鎂離子無法活化鋅離子抑制之鯉魚granzyme B
活性……………………………………………………….....…….27
第五節 肝制凝素、多醣活化鯉魚Granzyme B活性…………..…….28
第六節 肝制凝素、多醣無法再活化鋅離子抑制之鯉granzyme B
活性…...…...……………………………………….….…….....…29
第七節 鯉魚Granzyme B的組織分佈………………………………...29
第八節 結語………………………………………………....…......….30
參考資料………………………………………………………...….…...32
圖及表………………………………………………………….…….….39

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