Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96409
標題: 人造油體系統應用於家蠅的重組乙醯膽鹼酯酶之純化
Purification of the recombinant acetylcholinesterase from Musca domestica via an artificial oil body system
作者: 陳靖茹
Ching-Ju Chen
關鍵字: 乙醯膽鹼酯酶;農藥殘留檢測;家蠅;人造油體;內含肽;acetylcholinesterase;pesticide residue testing;Musca domestica;artificial oil body;intein
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摘要: 
乙醯膽鹼酯酶(acetylcholinesterase,AChE)是一種透過水解活性使乙醯膽鹼降解為膽鹼與醋酸之酵素,可應用於農藥檢測。有機磷及氨基甲酸鹽類殺蟲劑即是藉由抑制昆蟲體內的乙醯膽鹼酯酶活性造成乙醯膽鹼大量累積,進而影響昆蟲正常的神經傳導而致死,此兩類殺蟲劑為目前國內登記農藥之大宗,對於人體亦具有毒性。隨著近年來食安問題逐漸受到重視,各類農產品的農藥殘留量均有嚴格規定,而傳統的生化法農藥殘留快速檢驗技術需從無受外界汙染之家蠅中萃取乙醯膽鹼酯酶,生產成本較高,因此利用人造油體作為純化重組乙醯膽鹼酯酶是本研究之目標。將前人研究之敏感家蠅乙醯膽鹼酯酶基因構築於表現載體並融合油體膜蛋白(oleosin),經誘導大量表現後,不溶性之包涵體(inclusion body)蛋白以人造油體(artificial oil body,AOB)系統純化,發現因乳化形成之白濁狀影響呈色法之活性檢測,但若在低溫誘導表達3天後取水溶性部分,則可得到具有活性之重組乙醯膽鹼酯酶,推測其可能以微膠粒(micelle)型式存在。為了改善因人造油體純化包涵體所造成檢測之不便,本研究構築了內含肽(intein)於油體蛋白與乙醯膽鹼酯酶之間,期望內含肽透過溫度、酸鹼值改變或是加入DTT發生自我剪切(self-splicing)的作用,從人造油體上分離出具活性之乙醯膽鹼酯酶。由Coomassie blue染色以及西方墨點法分析結果顯示,目前測試的剪切條件下,乙醯膽鹼酯酶仍無法由人造油體中剪切分離出來。

Acetylcholinesterase (AChE, EC 3.1.1.7) hydrolyses the neurotransmitter acetylcholine to choline and acetic acid. Insect AChE is known to be a primary target of organophosphorus and carbamate insecticides which are the majority of registered pesticides in Taiwan. Both kinds of pesticides are also highly acutely toxic to human beings. Recently, agricultural products meet stringent pesticide safety standards for potential health concern. The traditional biochemical technique for pesticide residues detection utilizes AChE extracted from the housefly which is breeding in an isolated environment and thus the production cost stays high. In order to improve the AChE production, generation of recombinant AChE is the aim of this project. In this study, a housefly AChE gene fused with oleosin sequence was constructed and then overexpressed by T7-RNA polymerase dependent system. The inclusion body was purified via artificial oil body (AOB) system. However, it was difficult to determine the activity using Ellman's method due to the formation of white turbidity with AOB. Surprisingly, the soluble fraction of crude extract showed higher AChE activity suggesting that the micelles were formed after expression at low temperature for three days. Furthermore, in order to determine the AChE activity from AOB, the self-splicing ability of intein in pH/temperature shift was employed. So far, coomassie blue staining and western blot analysis showed that the AChE cannot be recovered from AOB using current methods.
URI: http://hdl.handle.net/11455/96409
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