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研究生:何宗澧
研究生(外文):Chung-Li Ho
論文名稱:臺灣龜殼花多半胱胺酸毒蛋白特性之研究
論文名稱(外文):Characterization of a Cysteine-rich Venom Protein from Trimeresurus mucrosquamatus (Taiwan habu)
指導教授:郭耀文郭耀文引用關係
指導教授(外文):Yaw-Wen Guo
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:多半胱胺酸臺灣龜殼花基寧蛋白原降血壓降體溫
外文關鍵詞:Cysteine-rich secretory proteinsCystein-rich venom protein (CRVP)hypotensionkininogenkallikrein-like activitybradykinin
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多半胱胺酸蛇毒蛋白 (cysteine-rich venom protein, CRVP) 基因是由台灣龜殼花 (Trimeresurus mucrosquamatus) 毒腺基因庫所篩選出。CRVP蛇毒蛋白屬於多半胱胺酸分泌蛋白 (cysteine-rich secretory proteins, CRISPs) 家族的一員。其共同特徵在於蛋白質胺基酸序列的C端具有十六個半胱胺酸,能形成含有八條雙硫鍵的結構。屬於CRISP的蛇毒蛋白也陸續被發現,如ablomin、pseudechetoxin、和 tigrin等,其作用皆和抑制離子通道的活性有關。CRVP蛇毒蛋白注射小鼠會造成小鼠產生動作遲緩、後肢麻痺及低體溫的現象和已知之墨西哥毒蜥蜴helothermine毒蛋白的藥理活性相似,推測其可能具有抑制鈣離子通道的功能以及影響血壓的作用。本實驗室在後續的大鼠動脈插管血壓試驗中,發現CRVP蛇毒蛋白會使大鼠血壓由原先的95mmHg驟降至55mmHg並一直維持此血壓長達六小時以上,而心跳卻沒有因而改變的結果,非常適合做為降血壓藥物的研發。為了探討其可能的作用機轉,本論文中比較股靜脈插管給藥及腦室給藥實驗發現,CRVP蛇毒蛋白只能在股靜脈給藥實驗發揮作用,產生使實驗動物降體溫及降血壓的作用,而以離體主動脈環張力試驗探究其可能作用機制的測試中,得知CRVP蛇毒蛋白並不具有抑制平滑肌鈣離子通道的功能,但是在體外生化試驗卻發現 CRVP 具有分解基寧蛋白原 (kininogen) 的活性,因此推測 CRVP 蛇毒蛋白血壓調控的作用機制可能和蛇毒蛋白中的絲胺酸蛋白酶類同。本實驗成功的將 CRVP蛇毒蛋白由原核之大腸桿菌表現系統轉換至酵母菌真核系統,且表現之CRVP蛇毒蛋白於動物活體試驗中具有降體溫及降血壓的生物活性,並發現降血壓的可能路徑是經由分解血漿中基寧蛋白原而產生的作用。
The cDNA encoding cysteine-rich venom protein (CRVP) was isolated from a cDNA library of venom gland of Trimeresurus mucrosquamatus. CRVP was belonged to the family of cysteine-rich secretory proteins, CRISPs. The CRISPs have sixteen consensus cysteines in their C terminus which can be bridged to form eight disulfide bonds. There were several snake venom proteins belonging to the CRISPs been isolated, such as ablomin, pseudechetoxin, tigrin and so on. CRISPs displayed a specific commom activity in the inhibition of ion channels. When recombinant CRVP was given to mice, a Ca2+ channel blocker resemblance effects of lethargy, paralysis, hypothermia, and death were observed. In addition, we also found that CRVP decreased the blood pressure of the rats without disturbing their heart beats. Therefore, CRVP may be ideally for the development of hypotensive drugs. In this study, CRVP could led into hypothermia and htpotension following intravenous administration in vivo. However, there were no similar effects in vivo in intracereventricular administration. In the search of possible biological mechanisms, we have found that CRVP could degrade kininogen to produce bradykinin in vitro. It is similar to some snake venom serine protease that produces it’s hypotensive effect by kallikrain-like protease activities. In this study, we successfully produced a functional CRVP from a yeast expression system and found that the hypotensive effect of CRVP might be mediated through kininogenase activity.
頁次
目錄……………………………………………………………………… I
圖次目錄………………………………………………………………… IV
中文摘要………………………………………………………………… V
英文摘要………………………………………………………………… VI
第一章 緒言……………………………………………………………… 1
第一節 毒蛇簡介………………………………………………………… 1
第二節 蛇毒蛋白的藥理作用…………………………………………… 1
第三節 多半胱胺酸分泌蛋白 (cysteine-rich secretory proteins, CRISPs)…………………………………………………………………… 8
第四節 臺灣龜殼花毒腺多半胱胺酸蛋白介紹………………………… 12
第五節 血壓和蛇毒蛋白………………………………………………… 14
第六節 實驗動機及目的………………………………………………… 15
第二章 實驗材料與方法………………………………………………… 17
第一節 實驗材料………………………………………………………… 17
第二節 實驗方法………………………………………………………… 23
第三章 結果……………………………………………………………… 40
第一節 pPICZαB 表現載體的確認……………………………………… 40
第二節 CRVP 蛇毒蛋白的表現及西方轉印法分析…………………… 40
第三節 CRVP 蛇毒蛋白的純化………………………………………… 41
第四節 CRVP 蛇毒蛋白去醣基化測試………………………………… 41
第五節 小鼠(BALC/c)降體溫活性測試…………………………… 41
第六節 大鼠(SD rat)股靜脈給藥及腦室給藥降體溫測試……… 42
第七節 大鼠(SD rat)股靜脈給藥及腦室給藥降血壓測試……… 42
第八節 大鼠(SD rat)離體主動脈環張力活性測試……………… 43
第九節 基寧蛋白原(kininogen)分解活性測試.………………… 43
第四章 討論…………………………………………………………… 58
第一節 利用真核表現系統大量表現微量蛇毒蛋白………………… 58
第二節 CRVP 蛇毒蛋白之西方轉印法分析與去醣基化測試 ……… 58
第三節 小鼠(BALC/c)降體溫活性測試分析……………………… 59
第四節 大鼠(SD rat)股靜脈給藥及腦室給藥降體溫血壓分析 60
第五節 大鼠(SD rat)離體主動脈環張力活性測試分析………… 61
第六節 基寧蛋白原(kininogen)分解活性測試分析…………… 61
第七節 未來展望…………………………………………………… 62
第五章 結論………………………………………………………… 63
第六章 參考文獻……………………………………………………… 64
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