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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...

國立高雄大學生物科技研究所

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 = Epitope analysis on a differential protein P25 between Naja naja atra and Bungarus multicinctus venom using phage display technology

紀錄類型:	書目-語言資料,印刷品 : 單行本
並列題名:	Epitope analysis on a differential protein P25 between Naja naja atra and Bungarus multicinctus venom using phage display technology
作者:	林詩婷,
其他團體作者:	國立高雄大學
出版地:	[高雄市]
出版者:	撰者;
出版年:	2009[民98]
面頁冊數:	90面圖、表 : 30公分;
標題:	噬菌體呈現技術
標題:	Naja naja atra
電子資源:	http://handle.ncl.edu.tw/11296/ndltd/95174215971213465997
附註:	指導教授：楊文仁
附註:	參考書目：面
摘要註:	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 secretory protein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。 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 μg 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 2.29×107 to 4.48×108 in ph.D.-C7C library, 8.05×106 to 9.1×108 in ph.D.-7 library, and 7.75×107 to 3.69×109 in 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 C7-20 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 immunoreactivityof NAV from BMV.

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 = Epitope analysis on a differential protein P25 between Naja naja atra and Bungarus multicinctus venom using phage display technology
林, 詩婷

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 = Epitope analysis on a differential protein P25 between Naja naja atra and Bungarus multicinctus venom using phage display technology / 林詩婷撰 - [高雄市] : 撰者, 2009[民98]. - 90面 ; 圖、表 ; 30公分.
指導教授：楊文仁參考書目：面.
噬菌體呈現技術Naja naja atra

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 = Epitope analysis on a differential protein P25 between Naja naja atra and Bungarus multicinctus venom using phage display technology
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330 $a 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 secretory protein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。 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 μg 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 2.29×107 to 4.48×108 in ph.D.-C7C library, 8.05×106 to 9.1×108 in ph.D.-7 library, and 7.75×107 to 3.69×109 in 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 C7-20 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 immunoreactivityof NAV from BMV.
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