抗台灣蛇毒蛋白活性的多株與單株抗體的製備與定性

Preparation and Characterization of Polyclonal and Monoclonal Antibodies against Snake Venom Activity In Taiwan

梁孟慧(Meng-Huei Liang)

指導教授 ： 楊沂淵

共同指導教授 ： 江正榮

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系/碩士(2012年)

https://doi.org/10.6831/TMU.2012.00208

摘要

台灣常見毒蛇有六類，又以龜殼花(Trimeresurus mucrosquamatus；本篇簡稱為MT)與赤尾鮐(Trimeresurus stejnegeri；本篇簡稱為GI)為其中最常見。此兩種蛇類的毒性皆屬於出血性蛇毒，目前仍以馬血清為主要的治療方式，然而製備所需成本相當高。因此，本篇研究選用成本較低的來亨雞為動物模式來進行抗蛇毒抗體的製備。未來，將進一步地發展快速且具專一性的檢驗試劑，甚至改善治療方法。首先，利用疾病管制局所提供的蛇毒蛋白(80 ?慊/次與240 ?慊/次)至少免疫雞隻四次後，從雞蛋蛋黃中快速純化出多株抗體IgY，每顆蛋約可純化50-100 mg的IgY。以西方墨點法和ELISA確認有產生專一性抗體，不過，在高劑量與低劑量免疫雞隻的抗體反應並未有顯著差異。接著利用嗜菌體展現技術(phage display technique)建立了六種重組抗體片段(single chain variable fragment, scFv) 基因庫(library)，分別是有short或long linker的抗龜殼花蛇毒抗體片段基因庫(4×107和1×107)、抗赤尾鮐蛇毒抗體片段基因庫(2.4×107和6.8×107)以及抗龜殼花/赤尾鮐蛇毒的抗體片段基因庫(1.76×107和2.2×107)。我們利用抗龜殼花蛇毒的重組抗體基因庫經四次篩選(panning)後，篩選出18個有表現的抗體片段，將抗體片段基因定序分析，共分成8群具有不同序列的片段。其中有4株scFv經西方墨點法和ELISA證明其對於龜殼花蛇毒具有專一性的結合能力，而不會結合到赤尾鮐蛇毒。間接溶血試驗(Indirect hemolytic assay)結果也顯示，其中三株scFv有抑制龜殼花蛇毒造成溶血的能力。初步的動物實驗結果顯示該四株scFv以及多株抗體IgY能減緩龜殼花蛇毒造成ICR小鼠死亡的時間，而IgY甚至能使部分小鼠存活下來。期望這些具專一性的多株與單株抗體在未來能對檢驗以及治療有重要貢獻，並且有取代馬血清作為治療的機會。

關鍵字

抗蛇毒抗體 ； 免疫球蛋白Y ； 單鏈變異區片段抗體 ； 噬菌體展現抗體技術

並列摘要

There are six major venomous snake species in Taiwan. Two of them are Trimeresurus mucrosquamatus (MT) and Trimeresurus stejnegeri(GI), which both are the majority of snakebite envenomations with necrosis and hemorrhage toxin in Taiwan. Injection the horse-derived T. stejnegeri (MT) and T. mucrosquamatus (GI) bivalent anti-venom horse serum is still the major treatment. However, it costs a lot to generate anti-snake venom serum. Thus, we used the chickens as animal models for immunization with snake venom to get one step ahead to generate rapid and specific dignosis agent and improve the therapy methods. First, we used the snake venom from Centers for Disease Control to immunize chickens. Then, we fast purified the polyclonal chicken IgY from eggs. 50-100 mg IgY could be purified each egg. The specific polyclonal anti-snake venom IgY could recogize snake venom both in western blot and ELISA assay. Otherwise, it was no significant difference between the IgY from immnized higher dose and lower dose chickens. Following, we used the phage display technology to contruct six scFv (single chain variable fragment) antibody library. There were anti-MT scFv antibody library (short linker and long linker, 4×107and 1×107 ), anti-GI scFv antibody library (short linker and long linker, 2.4×107 and 6.8×107) and anti-MT/GI scFv antibody library (short linker and long linker, 1.76×107 and 2.2×107). After four rounds of panning, we selected 18 clones scFv monoclonal antibodies from anti-MT library could express scFv protein. Then, we alignmented these 18 scFv clone sequences and classed them among eight groups. In addition, there were four monclonal scFv antibody could specifictly recognize MT snake venom but not to GI snake venom protein in western blot and ELISA assay. Next, we used the four scFv to do indirect hemolytic assay in vitro. According the results, three of these scFv could inhibt the hemolysis induced by MT venom. We hoped these polyclonal and monoclonal antibodies colud be used to detect and treat the venom envenomations in the future.

並列關鍵字

anti-snake venom antibodies ； IgY ； single chain variable fragment, scFv ； phage display technology

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