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研究生:鄭智青
論文名稱:核酸酶活性存在於假性狂犬病毒顆粒之製備產物
論文名稱(外文):Nuclease activity associated with the preparation of pseudorabies virus
指導教授:王孟亮
指導教授(外文):Min-Liang Wong
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:65
中文關鍵詞:假性狂犬病毒核酸酶
外文關鍵詞:pseudorabies virusnuclease
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在純化假性狂犬病毒(PRV)的過程中,發現蔗糖梯度離心純化後的假性狂犬病毒溶液攜帶有強烈的DNase活性。此一活性的來源是出自於病毒本身結構成份抑或是病毒顆粒以外的物質?又或者是因假性狂犬病毒的感染而誘發細胞所產生具有某種目的的產物?而如此高產量的DNase活性物質對於病毒本身是否會造成正面或負面的影嚮、其存在的目的為何?目前為止,經由一連串的實驗結果我們發現此一酵素活性物質實為由蛋白質所構成的核酸酶(DNase)。其物理特性上需要二價離子協助活性、受鈣離子保護,而免於結構受到蛋白質酵素的破壞。室溫下穩定,60°C下活性不穩定。此一活性僅存在於假性狂犬病毒溶液及包括MDBK在內的少數幾種細胞培養株的培養液中。在許多與黴漿菌(Mycoplasma)相關的DNase研究中指出黴漿菌具有分泌DNase活性的能力;在利用PCR技術偵測黴漿菌是否存在於細胞株培養液中後發現,在舊MDBK及舊LM TK-有黴漿菌的核酸被偵測到,因此我們認為此一DNase活性的原始來源並非來自於Mycoplasma;但在進一步由細胞中誘導DNase活性產生時發現在MDBK及PK-15細胞皆可受到胎牛血清的誘導而產生DNase的活性,且PK-15細胞也會受到dexamethasone 的誘導而產生DNase活性;但在LM TK-細胞則完全不受誘導產生DNase活性。

A DNA cleavage activity was detected in pseudorabies virion preparation. This activity might come from the structural components of pseudorabies virion or from a product of the infected-cell. Results showed that the DNA cleavage activity was from a nuclease which required Ca2+/Mg2+ to exert its function and was unstable at 60°C . Moreover, in the medium of mock- or pseudorabies-infected MDBK cells, we also found the DNase activity. It is known the foreign body pollution such as mycoplasma spp. may generate a DNase activity in the medium. Using PCR, nucleic acids of mycoplasma were detected in MDBK and LM (TK-) cells. The DNase activity existed in MDBK cells, not in LM (TK-) cells, suggesting that the origin of DNase was not from mycoplama. Our results revealed that the cellular DNase activity could be induced by fetal bovine serum (MDBK, PK-15 cells) or dexamethasone (PK-15); in contrast the LM (TK-) cells exhibited no DNase activity in the presence of fetal bovine serum or dexamethasone.

第一章 緒言…………………………………………………………………1
第二章 文獻探討……………………………………………………………2
2.1 假性狂犬病的歷史背景與病毒特性……………………………2
2.2 核酸酶的歷史背景………………………………………………5
2.3 Dexamethasone誘導DNase活性的影嚮…………………………7
第三章 材枓與方法………………………………………………………8
3.1 細胞培養與病毒增殖…….……………………………………8
3.2 蛋白質成份分析………………………………………………12
3.3 核酸酶活性試驗………………………………………………14
3.4 核酸酶活性物質純化………………………………………………15
3.5 核酸酶活性抑制試驗………………………………………………16
3.6 核酸酶活性來源分析………………………………………………18
第四章 結果………………………………………………………………22
4.1 病毒顆粒與DNase活性物質增殖、純化與特性分析…………22
4.2 病毒溶液中DNase活性物質之DNase活性抑制試驗………………23
4.3 核酸酶活性來源分析………………………………………………25
第五章 討論………………………………………………………………51
參考文獻……………………………………………………………………53

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