黃火鍊臺灣大學：分子與細胞生物學研究所高啟明Kao, Chi-MinChi-MinKao2007-11-252018-07-062007-11-252018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/49906此研究從鯉魚卵巢cDNA基因庫中選殖出一個新型的酵素，此酵素從未在鯉魚身上發表，也缺乏功能性的研究，經過比對，發現它屬於papain family家族的一員，與哺乳類的cathepsin Z相類似。cathepsin Z在分類上屬於cysteine protease。經過比對，此酵素有關於活性調控與形成三級結構的重要區域都與其它cysteine proteases類似。 觀察此酵素在鯉魚卵子最後成熟過程中的變化，發現cathepsin Z的mRNA在鯉魚注射GnRH-analogue 的8-12小時後表現量最多，稍後在排卵時又降到未注射前的水準，推測與卵細胞內卵黃的分解有關，也與先前的研究認定卵細胞內蛋白質分解在成熟過程中8-16小時達到最高峰相吻合。 此外，cathepsin Z存在於成熟大卵的表層顆粒與卵黃細胞質附近。在卵子發生表層反應時，會釋放到圍卵腔中，進而形成受精膜的一部分。最後，實驗表現的重組蛋白具有活性，可以分解gelatin，並且可以切割鯉魚的卵黃前質素(vitellogenin)，證明cathepsin Z具有內切酶的特性。A cDNA encoding cathepsin Z (CTPZ) was cloned from a carp ovarian cDNA library. It is homologous to mammalian CTPZ. The amino acid residues important for protein folding and enzymatic activity of cysteine proteases are conserved in carp CTPZ. During oocyte maturation, cathepsin Z transcripts increased and reached peak at 8 h post-treatment of GnRH analogue by 9-folds compared to that of untreated oocytes, then declined near to the level of untreated oocytes in ovulated eggs. Cathepsin Z was discharged from cortical granules to perivitelline space after cortical reaction and became new component of fertilization envelope. Inside the oocyte, cathepsin Z was stained in the cytoplasm surrounding the yolk granules. The recombinant cathepsin Z has an enzymatic activity toward vitellogenin, and inhibited by PMSF or E-64, a specific inhibitor of thiol protease. The potential roles of cathepsin Z in carp eggs are discussed.中文摘要 ----------------------------------------------------------------------------------------1 Abstract ------------------------------------------------------------------------------------------2 第一章 序----------------------------------------------------------------------------------------3 第一節 卵子的發育----------------------------------------------------------------------3 第二節 卵子發育時細胞內的變化----------------------------------------------------6 第三節 生殖季節中激素、內分泌的變化------------------------------------------10 第四節 卵黃前質素與接受子的基因結構------------------------------------------12 第五節 酵素參與各項的生理反應---------------------------------------------------14 第六節 魚類各種cathepsin proteases 的介紹----------------------------------19 第七節 cathepsin Z的發現-----------------------------------------------------------22 第二章 材料與方法---------------------------------------------------------------------------24 實驗動物與基因庫建構----------------------------------------------------------------24 鯉魚卵巢cysteine protease cDNA之定序----------------------------------------24 5’-RACE (rapid amplification of cDNA ends)-------------------------------------25 重組蛋白cathepsin Z的表現與純化------------------------------------------------27 SDS-膠體電泳---------------------------------------------------------------------------28 蛋白質西方轉印法----------------------------------------------------------------------29 免疫組織化學----------------------------------------------------------------------------30 蛋白質濃度測定-------------------------------------------------------------------------31 重組蛋白之抗血清誘導與製備-------------------------------------------------------31 勝任細胞的製備-------------------------------------------------------------------------32 細胞轉型----------------------------------------------------------------------------------32 卵黃前質素的純化與製備-------------------------------------------------------------33 Northern blotting------------------------------------------------------------------------33 製備DIG標記之DNA探針----------------------------------------------------------34 核酸原位雜交----------------------------------------------------------------------------35 RT-PCR-----------------------------------------------------------------------------------35 統計分析----------------------------------------------------------------------------------36 第三章 結果------------------------------------------------------------------------------------37 鯉魚卵巢中新型酵素cathepsin Z之選殖------------------------------------------37 蛋白質結構分析-------------------------------------------------------------------------37 酵素的表現-------------------------------------------------------------------------------38 酵素的純化-------------------------------------------------------------------------------38 活性測試----------------------------------------------------------------------------------39 cathepsin Z在卵細胞內的分佈位置------------------------------------------------ 40 核酸原位雜交的結果-------------------------------------------------------------------40 組織特異性的探討----------------------------------------------------------------------41 酵素在卵子成熟過程中mRNA表現時間點的探討------------------------------41 第四章 討論------------------------------------------------------------------------------------43 Table 1 實驗用的各項引子(primer)序列------------------------------------------------51 Table 2 鯉魚cathepsin Z與它種生物相似度比較表----------------------------------52 圖表一-------------------------------------------------------------------------------------------53 圖表二-------------------------------------------------------------------------------------------54 圖表三-------------------------------------------------------------------------------------------55 圖表四-------------------------------------------------------------------------------------------56 圖表五-------------------------------------------------------------------------------------------57 圖表六-------------------------------------------------------------------------------------------58 圖表七-------------------------------------------------------------------------------------------59 圖表八-------------------------------------------------------------------------------------------60 參考資料----------------------------------------------------------------------------------------613113423 bytesapplication/pdfen-US鯉魚卵黃前質素硫胱胺酸蛋白酶cathepsin Zvitellogenincarpoocyte maturationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49906/1/ntu-95-D85225006-1.pdf