槲寄生乙型核醣體失活蛋白基因之選殖及其重組蛋白之製備與活性探討

Abstract

乙型核醣體失活蛋白由一具毒性之A鏈與一具凝集素活性之B鏈所組成，此類分子擁有多樣的生物活性，包括細胞毒性與免疫調節活性。多種此類分子展現應用於臨床治療的潛能，其中最令人矚目者為槲寄生凝集素，其分離自白果槲寄生植物並且為槲寄生萃取液之有效成分，此槲寄生萃取液廣泛在歐洲地區應用於癌症治療。令人好奇的是，在實驗動物模型中發現此槲寄生凝集素主要是藉者有效調控免疫反應來達到抗腫瘤的效果。高單位的槲寄生凝集素往往因為細胞毒性的副作用，反而失去對抗腫瘤的效果。目前已知槲寄生凝集素需要B鏈之凝集素活性才能調節免疫細胞的活性，然而，單獨B鏈是否具備獨立刺激免疫細胞活性之能力仍有待進一步實驗測試。台灣擁有數十種的槲寄生植物分布全島，也使用這類植物於中醫藥臨床治療。我的論文報告了在其中二種槲寄生植物鑑定出的新型槲寄生乙型核醣體失活蛋白基因，分別是椆櫟槲寄生的椆櫟槲寄生凝集素基因與台灣槲寄生的台灣槲寄生凝集素基因，並且發現此類槲寄生凝集素基因獨特的多樣性。此多樣性的獨特性值得未來進一步收集或栽培此類相對少量的槲寄生植物以用作分離可能存在的獨特槲寄生凝集素來應用於醫藥研發。我們同時也選殖了椆櫟槲寄生凝集素A鏈與B鏈用以大量生產具有實質應用活性之重組蛋白。這過程中，我們開發了一種低離子強度溶液來穩定貯存中易於聚集而變性的椆櫟槲寄生凝集素B鏈蛋白，這個技術是研究與應用此重組蛋白或其它乙型核醣體失活B鏈蛋白的重要關鍵。最後我們證明了我們所製備的A鏈具備相當的核醣體失活活性以及此無醣化B鏈之活化免疫細胞的潛能。這樣的成果讓我們可以繼續發展它們未來在生技醫療的相關應用，例如 A鏈可作為提供高毒性來源的分子，B鏈則可作為低毒性的免疫活化分子。

Type-2 ribosome-inactivating proteins (RIP), composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. One of the most noticeable for their potential therapeutic uses might be mistletoe lectin I (ML-I), which was isolated from Viscum album and has been identified as the main active component of mistletoe extracts, a widely applied herbal medicine in Europe for complementary treatment of cancer patients. Interestingly, experiments in animal models suggest that ML-I-mediated inhibition of tumor growth is primarily associated with immunomodulatory efficacy. High-dose ML-I may fail because of its cytotoxicity. Although the B-chain binding activity was proved necessary for immunostimulating effects induced by ML-I, whether a B-chain fragment is an active immunomodulator still remains questionable. In Taiwan, there are dozens of various mistletoe plants, which have also been used for alternative medicine. In this dissertation, we reported identification of two new mistletoe type-2 RIP genes, articulatin and alniformosanin genes, from V. articulatum and V. alniformosanae, respectively. We showed certain diversity existing among these mistletoe type-2 RIP genes and suggested further collection or planting of these scarce mistletoe plants for isolation and characterization of novel mistletoe type-2 RIPs be warranted. We also cloned articulatin A- and B-chain (rATA and rATB) to produce large quantity of active recombinant proteins for potential practical use. We have developed a low ionic strength solution to stabilize the aggregation-prone rATB during storage, which was very critical to studies on rATB and other type-2 RIP B-chain. Finally, we in vitro demonstrated the ribosome-inactivating activity of rATA and immunostimulating potential of such nonglycosylated rATB, and discussed their possible biotechnological uses as a toxin or an immunomodulator without unwanted cytotoxicity.