臺灣博碩士論文加值系統

本實驗室蔡元彰學長 (1995) 從鯉魚卵巢 cDNA 庫篩選出一長度為
1228bp 的 cDNA( 附 1)，其轉譯的 331 個胺基酸序列與其它已知的
Cysteine protease 有相當程度的相似性，因此該 cDNA 可能載錄鯉魚
Cysteine protease。本論文即以此 cDNA為探針，進行此基因在鯉魚卵巢
表現之研究。並將此基因載入 pQE30 質體，然後以此重組蛋白為抗原誘
發抗體，再以抗體進行組織分佈之研究； 此外，本論文也嚐試自卵巢組
織純化 Cysteine protease。
原位雜交結果顯示 Cysteine protease mRNA 出現在卵母細胞， 且在卵
黃堆積前之卵母細胞 (previtellogen oocytes) 已開始轉錄，此外，卵
母細胞外圍之濾泡細胞亦發現有 Cysteine protease mRNA 存在。 免疫
組織化學之觀察則發現此 Cysteineprotease 主要分佈於卵細胞之表層
囊泡 (cortical alveoli) 以及濾泡細胞，且在發育早期之卵母細胞外圍
之濾泡細胞即已出現 Cysteine protease。 而位於表層囊泡之Cysteine
protease 在卵細胞進行表層反應 (cortical reaction) 時會釋放到圍卵
腔。西方轉印分析結果顯示鯉魚卵巢內 PBS 可溶部分Cysteine protease
主要有26.8kDa與 30.2kDa二種形式，而在 PBS 不可溶部分，除了26.8
kDa 與30.2kDa 外，尚有許多可能與其它物質接合在一起的高分子量之型
式。 當以 ovaprim 誘發鯉魚排
卵時， 鯉魚卵巢 Cysteine protease mRNA(0.98 kb)之含量在 ovaprim
注射四小時後有明顯增加，然後逐漸減少。而西方轉印分析顯示在鯉魚卵
成熟及排卵過程， 卵巢內 PBS 可溶部分之 26.8kDa Cysteine protease
含量逐漸增加，而 30.2kDa 之 Cysteine protease 含量則沒有變化。相
反地，在 PBS 不可溶部分，23.8kDa 及 30.2kDa 之 Cysteine protease
均隨著 ovaprim 之處理時間增長而增加。
本實驗又以 TSK-DEAE 650 陰離子交換樹脂與 Cystatin 親和性膠體進
行鯉魚卵巢 Cysteine protease 的初步純化， 結果獲得之主要產物為
30.2kDa 之 Cysteineprotease，但經分析並不表現酵素之活性，如何獲
得有活性之 Cysteine protease 則仍待進一步的探討。

In this study, a 1228bp cDNA previously isolate from a
carp ovariancDNA library and encoding a 331 residue
polypeptide homologous toCysteine protease was used as a
probe to study its gene expression inovary. In situ
hybridization showed that Cysteine protease mRNA arepresent
in oocytes and follicle cells. Transcription of Cysteine
proteasegene starts very early during oogensis. Antibody
against the recombinantCysteine protease was used to
investigate the tissue distribution andbiochemical nature
of c arp ovarian Cysteine protease.
Byimmunohistological study, the Cysteine protease was found
majorly incortical alveoli and minorly in zona pellucida of
oocytes, and also thefollicle cells surrounding developing
oocytes. During the corticalreaction of egg, the Cysteine
protease in the cortical alveoli wasreleased to the
perivitelline space. From western blot analysis, theCysteine
protease in the PBS-soluble fraction of carp ovary and two
form,26/8kDa and 30.2kDa,while those in the PBS-insoluble
fractio n are morethan two form,26.8kDa and 30.2kDa and those of
larger molecular weights. To study the gene expression
during oocyte maturation and ovulation,ovapim was injected to
mature female carp. Northern blot analysis showedthat ovarian
Cysteine protease mRNA(0.98KB) was significantly increased4hr
after ovaprim injection and decreased thereafter. In
addition, the26.8kDa Cysteine protease in the PBS-soluble
fraction was increasedgradually during maturation and
ovulation while the content of 30.2kDaform was not changed.
However, both the 26.8kDa and 30.2kDa Cysteineprotease in the
PBS -insoluble fraction were gradually increased afterovaprim
injection. They were decreased after 7hr. postinjection.
In order to purify carp ovarian Cysteine protease, TSK-DEAE 650
anionexchanged chromatography and Cystatin-affinity
Agarose columnchromatography were employed. A partially
purified Cysteine protease of30.2kDa without protease activity
were obtained. Further experiment needto be done for the
purification of the biologically active Cysteineprotease.