以噬菌體呈現技術探討眼鏡蛇與雨傘節 蛇毒差異性
蛋白質P25之抗原決定位
指導教授：楊文仁 博士 國立高雄大學生物科技研究所 學生：林詩婷 國立高雄大學生物科技研究所 摘要 毒蛇咬傷是一全球性重要課題，在被咬的當下，許多患者無法看清楚或確定其被咬 的毒蛇種類，因此迅速診斷出何種蛇毒咬傷，將能減少誤用抗蛇毒血清及降低血清 病發生之機率。本實驗期藉由噬菌體呈現技術來分析不同蛇種間差異性蛋白質之抗 原決定位， 以利於相關檢驗試劑的開發。從蛋白質電泳分析，發現眼鏡蛇和雨傘節 蛇毒在分子量大約25 kDa 處有明顯之差異性蛋白質存在，將其命名為P25。以馬的 抗眼鏡蛇蛇毒血清進行西方點墨法分析，會辨識眼鏡蛇蛇毒P25，在雨傘節蛇毒中未 出現P25 蛋白，因此以P25 來探討具有鑑辨別眼鏡蛇與雨傘節蛇毒之抗原決定位。 首先從蛋白質電泳膠體片中回收P25 蛋白，用0.25%戊乙醯醛進行去毒性處理。以 100 μg 之去毒性P25 免疫兔子製備抗血清，經西方墨點法證實血清抗體呈陽性反 應。以protein A 純化抗血清獲得抗P25 抗體，經活性測試後，分別與三種不同噬菌 體胜肽庫進行三次生物親和性篩選， 在每個胜肽庫進行三回合篩選後，ph.D.-C7C 噬菌體胜肽庫沖提數目由2.29×107 上升至4.48×108，ph.D.-7 噬菌體胜肽庫沖提數目 由8.05×106 上升至9.1×108 和ph.D.-12 噬菌體胜肽庫沖提數目由7.75×107 上升至 3.69×109，證實經過三回合生物親和性篩選，確實篩選出有較強親和性之噬菌體族 群。隨機挑選單一噬菌體定序分析，將胺基酸進行排列後發現有七種一致的序列存 在。比對NCBI 資料庫中已知蛇毒序列，可以比對到平顏海蛇(Lapernis hardwickii) 的 磷 脂 酶 A2 蛋 白 (Accession No. AF144349.1) 和 眼 鏡 蛇 的 cysteine rich secretoryprotein 蛋白(Accession No. Q7T1K6)。以純化之抗P25 IgG 與篩選出的噬菌體進行墨 點法，結果顯示訊號強度大於陽性對照組且雨傘節蛇毒抗血清為陰性反應的噬菌體 為編號C7-20 和D12-9。用競爭型ELISA 分析，發現D12-9 能和P25 競爭P25 抗體， 與P25 抗體結合，其序列可以對應到磷脂酶A2 活性催化位上的Arg49 及Tyr52。推
測篩選出D12-9 所帶的外源胜肽序列具有開發為鑑別眼鏡蛇與雨傘節毒蛇檢驗試劑 之潛力。
Epitope analysis on a differential protein P25 between
Naja naja atra and Bungarus multicinctus venom
using phage display technology
Advisor: Dr. Wen-Jen Yang Institute of Biotechnology National University of Kaohsiung
Student: Shih-Ting Lin Institute of Biotechnology National University of Kaohsiung
ABSTRACT
Poisonous snake bite is a global important issue. Several victims could not see or identify clearly what kind of the snake bit them. Therefore, it could reduce the misuse of antivenin and lower the occurrence of serum sickness through the rapid identification what kind of the snake bit them. This study analyzed epitopes on a differential protein that existed in different kinds of snake using phage display technology to facilitate the development of diagnostic reagent. Based on the analysis of protein electrophoresis, a differential 25-kDa protein, named P25, was found existing in Taiwan cobra Naja naja atra venom (NAV) but not in Bungarus
multicinctus venom (BMV). Western blot analysis showed that P25 could be
recognized by horse-derived anti-NAV antivenin. Therefore, P25 protein was used to study the differential epitopes between NAV and BMV. First of all, we recovered the P25 from protein electrophoresis gel and detoxified it with the treatment of 0.25% glutaraldehyde. Antiserum was prepared by rabbit immunized with 100 g of detoxified P25 and the antiserum activity against P25 was verified by Western blot analysis. Anti-P25 IgG was purified through protein A, its activity verified again, and was used for biopanning with three different phage display peptide libraries. After three rounds of biopanning for each library, the eluted phage numbers (pfu/ml) increased from 2.29×107to 4.48×108 in ph.D.-C7C library, 8.05×106to 9.1×108in ph.D.-7 library, and 7.75×107to 3.69×109in ph.D-12 library, respectively. It indicated that the high affinity phage clones were selected through three rounds of biopanning. Phage clones were randomly selected and sequenced, the deduced amino acid
sequences were aligned and seven consensus sequences were found between them. Comparison with the sequences of known snake venoms in NCBI database, several sequences matched on the phospholipase A2 of Lapernis hardwickii (Accession No.
AF144349.1) and cysteine-rich secretory protein (CRISP) of Naja naja atra (Accession No. Q7T1K6). Selected phage clones were further analyzed by dot blot assay using purified anti-P25 IgG. The results showed that the clones which has dot blot signal higher than positive control and no cross reaction with anti-BMV antiserum were phage C7-2 and D12-9. Competitive ELISA analysis revealed that the D12-9 clone could compete with P25 bound to anti-P25 IgG. The D12-9 sequence matched the amino acid residues Arg49 and Tyr52 on the active site of phospholipase A2 of Lapernis hardwickii. It indicated that the sequence of D12-9 has diagnostic
potential in distinguishing the antivenin immunoreactivity of NAV from BMV.
