臺灣鋏蠓化學感應蛋白之高親和性配體之研究

A study on high-affinity ligands of chemosensory protein of Forcipomyia taiwana

魏大鈞(Da-jiun Wei)

指導教授 ： 呂兆倉

中臺科技大學/健康科學院/生命科學研究所/碩士(2012年)

摘要

臺灣鋏蠓 (Forcipomyia taiwana) 為日行性昆蟲，體長約 1.4 mm，雌蟲嗜吸人血，由於體型極小不易被人察覺，在繁殖密度高的區域經常群體叮咬人體裸露的皮膚，體質敏感者會產生劇烈的搔癢與紅腫，為一種騷擾性害蟲。昆蟲個體與外界環境的聯繫通常是透過化學溝通進行調控，包含嗅覺與味覺的感知。在先前研究中發現到臺灣鋏蠓身上有 CSP，初步推測臺灣鋏蠓 CSP 可能參與嗅覺感應的功能，本研究以大腸桿菌表現臺灣鋏蠓 CSP ，純化後進行結合試驗分析其結合性質，以嘗試藉由干擾 CSP 的作用影響臺灣鋏蠓的嗅覺進而達到臺灣鋏蠓的防制。本研究將臺灣鋏蠓 CSP 表現基因轉殖到大腸桿菌進行表現，再進一步將 CSP 加以純化，以純化後的 CSP 用螢光光譜儀進行配體結合試驗篩選出合適的螢光探針進行競爭結合試驗，透過競爭結合試驗篩選出具有高親和性的配體，將具有高親和性之配體進行 Y 型管誘引與忌避實驗篩選具有高親和性且具忌避性質之配體，之後進一步進行忌避劑之試驗。競爭結合試驗指出 2-methoxycinnamaldehyde、α-amylcinnamaldehyde 及 α-hexylcinnamaldehyde 三種醛類化合物與 CSP 具有高度的親和性。Y 型管誘引與忌避實驗結果 α-hexylcinnamaldehyde 忌避趨勢最為明顯。忌避劑試驗最終指出 2-Methoxycinnamaldehyde 及 α-hexylcinnamaldehyde 做為忌避劑有相似於相同濃度的敵避 (N,N-Diethyl-meta-toluamide) 的效果。

關鍵字

忌避劑 ； Y 型管嗅覺計 ； 臺灣鋏蠓 ； 化學感應蛋白

並列摘要

Forcipomyia taiwana is a diurnal insects, about 1.4 mm body length. The female insects are addicted to suck human blood. Because of minimum size, can''t easily be detected. In the region of high insect density, they often bite in group skin of human. The ones susceptible to F.taiwana often resulted in through biting.Interaction between the insects and environment is usually regulated by chemical communication, either through olfaction or taste. Previous studies found chemosensory proteins (CSPs) in F.taiwana, suggested that CSPs are involved in the olfactory response. The CSP gene has been cloned in the expression vector Escherichia coli. In this study, CSP proteins were expressed and purified. The purified CSP was used to study it characteristics, and screen its binding ligands for interfering prevention of F.taiwana. CSP of F. taiwana expressed and purified from cloned E. coli. The expressed CSP was used to screen ligands by binding assay through fluorescence spectrometry. The repellent response of high-affinity ligand was conducted using Y-tube olfactometer, then for repellents test. Three kinds of aldehyde compounds ( 2-methoxycinnamaldehyde, α-amylcinnamaldehyde, α-hexylcinnamaldehyde ) revealed high affinity for CSP through competitive binding. α-hexylcinnamaldehyde showed the most significant repellent trend for F. taiwana by Y-tube olfactometer. Repellents test showed that the repellent effect 2-Methoxycinnamaldehyde and α-hexylcinnamaldehyde were similar to DEET (N, N-Diethyl-meta-toluamide).

並列關鍵字

Repellents ； Y-tube olfactometer ； Chemosensory proteins ； Forcipomyia taiwana

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