- 學位論文
草原蝰(Vipera ursinii renardi)蛇毒促凝蛋白酶的基因選殖 與特性
cDNA cloning and protein characterization of the pro-coagulant proteases from Vipera ursinii renardi venom
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摘要
本篇研究目的在於發現並了解Vipera ursinii renardi蛇毒內的促凝蛋白，Vipera ursinii renardi的蛇毒先前較少被研究，因此仍然存在著許多未知。利用前人已發表的類似蛋白的cDNA序列可設計出特殊的primer，並可經由PCR放大所需的基因，在本研究中已利用此技術成功放大了Vipera ursinii renardi蛇囊內編碼Factor X activator subunits與Factor V activator的基因，其中在絲胺酸蛋白酶的部分，除了FV activator (本文稱之為Vur-FVA)還發現了chymotrypsin-like protease，前者的氨基酸序列與Vipera lebetina FV activator的具有93%的高度相似性，而後者與V. lebetina的chymotrypsin-like protease也具有90%的相似性，Vur-FVA可經由凝膠層析(gel filtration)與陽離子交換(cation exchanger )管柱而被純化出來，並可經由SDS-PAGE觀察到Vur-FVA具有將FV活化的能力。除了Vipera ursinii renardi 蛇毒活化FX的能力外，本研究中亦測量了其他許多Vipernae的蛇毒，儘管V. u. renard蛇毒活化FX的能力比Russell`s viper弱了上百倍，然而其蛇毒確實具有FX activator的活性，V. u. renardi FX activator在本研究中被命名為Vur-FXA，經過凝膠層析(gel filtration)管柱的初步純化後我們觀察到Vur-FXA的分子量大概為95 kDa，且經由SDS-PAGE 的分析可得知Vur-FXA具有多個subunit。研究中還經由基因選殖的技術，發現了Vur-FXA的CTLL light chains與Vipera lebetina 和Daboia russelli的CTLL light chains具有高度的相似性，而針對Vur-FXA heavy chain的部分，我們得到了一個pIII金屬蛋白酶，然而卻發現其缺少了負責與light chains形成雙硫鍵的Cysteine。由以上結果可發現V. u. renardi蛇毒中具有多種促凝成份，這些成份可能會與前人在V. u. renardi蛇毒中所發現的抗凝成份具有協同作用，共同造成凝血症狀或獵物的死亡，因此未來可針對此一現象做更深入的研究。
關鍵字
草原蝰促凝蛋白酶並列摘要
The aim of this study is to identify and characterize the pro-coagulant proteins in the venom of a less studied Viperinae species, Vipera ursinii renardi. Using the specific primers designed based on previously published cDNA sequences of the pro-coagulant proteases from the other two Viperinae species, we have cloned the cDNA encoding all the subunits or proteins of Factor X and Factor V activators from the venom glands of V. u. renardi (Vur). Two distinct serine proteases have been cloned from Vur cDNAs. One encodes a FV activator, and the other encodes a chymotrypsin-like protease. The deduced amino acid sequence of the former enzyme revealed 93% identity with Vipera lebetina FV activator and was designated as Vur-FVA. Another was 90% identical to a chymotrypsin-like protease of V. lebetina. Vur-FVA was purified from the crude venom by gel filtration and cation exchanger columns; its FV activating activity was confirmed by SDS-PAGE analysis of the produced FVa. FX activating activities of many Vipernae venoms were compared by assay the FXa generated. Although the activity is 2 order of magnitude weaker than Russell`s viper venom, V. u. renardi venom do contain the activity. The FX activator (designated as Vur-FXA) was partially purified by gel filtration and shown the molecular weight is about 95 kDa. We also could find Vur-FXA was a oligomer by comparing the fractions containing Vur-FXA on reducing and non-reducing SDS-PAGE gel. The CTLL light chains of Vur-FXA were cloned and the deduced amino acid sequences are highly similar to those of V. lebetina and Daboia russelli. The amino acid sequence of a PIII metalloprotease was also cloned. However, the deduced protease sequence lacks the Cys residue which involved in disulfide linking to the light chain in RVV-X. These results characterized Vur venom pro-coagulant components. It has been reported anticoagulant components exist in Vur venom. The anticoagulant components and procoagulant components may act synergistically, leading to coagulopathy symptom or the death of prey.
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