鯉魚卵巢Cysteine Protease 之生合成、組織分佈與純化

Abstract

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個胺基酸序列與其他已知的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Cysteine Protease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26.8kDa與30.2kDa外，尚有許多可能與其他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 (0.98kb)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Cysteine protease，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 ovarian cDNA library and encoding a 331 residue polypeptide homologous to Cysteine protease was used as a probe to study its gene expression in ovary. In situ hybridization showed that Cysteine protease mRNA are present in oocytes and follicle cells. Transcription of Cysteine protease gene starts very early during oogensis. Antibody against the recombinant Cysteine protease was used to investigate the tissue distribution and biochemical nature of carp ovarian Cysteine protease. By immunohistological study, the Cysteine protease was found majorly in cortical alveoli and minorly in zona pellucida of oocytes, and also the follicle cells surrounding developing oocytes. During the cortical reaction of egg, the Cysteine protease in the cortical alveoli was released to the perivitelline space. From western blot analysis, the Cysteine protease in the PBS-soluble fraction of carp ovary and two form, 26.8kDa and 30.2kDa,while those in the PBS-insoluble fraction are more than 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 showed that ovarian Cysteine protease mRNA (0.98KB) was significantly increased 4hr after ovaprim injection and decreased thereafter. In addition, the 26.8kDa Cysteine protease in the PBS-soluble fraction was increased gradually during maturation and ovulation while the content of 30.2kDa form was not changed. However, both the 26.8kDa and 30.2kDa Cysteine protease in the PBS-insoluble fraction were gradually increased after ovaprim injection. They were decreased after 7hr. postinjection. In order to purify carp ovarian Cysteine protease, TSK-DEAE 650 anion exchanged chromatography and Cystatin-affinity Agarose column n chromatography were employed. A partially purified Cysteine protease of 30.2kDa without protease activity were obtained. Further experiment need to be done for the purification of the biologically active Cysteine protease.