近幾年在亞洲地區流行的腸病毒71型(EV71)是一種生長於腸道的RNA病毒，是一種手足口症(hand-foot-mouth disease，HFMD)，這類感染病出現神經系統的病徵如抽蓄、肢體麻痺、腦膜炎及呼吸困難等症狀。有些研究已經指出EV71的受體P-selectin glycoprotein ligand-1 (PSGL-1)一旦硫化之後確實和EV71的結合能力會增加，但是在PSGL-1的序列當中，能夠被硫酸化和EV71結合的酪氨酸(Tyrosine, Y)有三個(酪氨酸-46,48,51)，目前推測此三個位置可能在硫酸化後和EV71的反應能力會有所不同。為了探討這三個酪氨酸對外鞘膜蛋白VP1的結合能力，本研究將使用苯丙胺酸(Phenylanine, F)取代酪氨酸。本研究使用原子力顯微鏡(Atomic Force Microscopy , AFM)作為量測EV71的外鞘膜蛋白VP1和PSGL-1之間解離力的工具，使用自組裝單分子層製程，VP1和突變的PSGL-1蛋白便可以分別固定於AFM探針以及矽基板之上，在使用原子力顯微鏡在緩衝液中量測兩種蛋白質的解離力。不論是抗體還是VP1的量測結果可知硫酸化的PSGL-1 (46, 48F)的解離力明顯大於硫酸化PSGL-1 (48, 51F)以及硫酸化PSGL-1 (46, 51F)。此結果指出當酪胺酸-51被苯丙胺酸取代而無法硫酸化時會使得硫酸化PSGL-1和VP1的反應性明顯下降，證實酪氨酸-51是最重要的因為只要能組止它被硫酸化，便可以減少PSGL-1和腸病毒71的反應性。原子力顯微鏡能夠利用力學曲線可以分辨突變PSGL-1的反應性，所以適合量測這種與EV71相關的研究。

Enteroviruses 71 (EV71) are a genus of positive-sense single-stranded RNA viruses which are notable as ones of the major causative agents associated with hand, foot and mouth diseases (hand-foot-mouth disease, HFMD) and also associated with severe central nervous system diseases like twitch, paralysis, meningitis and difficulty breathing, etc. It has been indicated that once the receptor of EV71, P-selectin glycoprotein ligand-1 (PSGL-1) is sulfated, the binding ability with EV71 tends to be increased. There are three tyrosines (tyrosine-46, 48, and 51) in PSGL-1, while three different positions of tyrosines with varied binding abilities to EV71 as PSGL-1 is sulfated. In order to study the different binding abilities of three tyrosines with outer sheath protein Viral Protein 1 (VP1) of EV71, three substitute tyrosines replaced by phenylalanine are studied. Atomic force microscopy (AFM) was utilized as force apparatus to measure the unbinding force between VP1 of and PSGL-1. At first, VP1 and mutated PSGL-1 proteins were immobilized on AFM tip and Si substrate, respectively. The unbinding forces between these two proteins were then measured in a buffer condition. Whether reacted with antibody or VP1, the unbinding forces of sulfated PSGL-1 (46, 48F) were significantly larger than sulfated PSGL-1 (48, 51F) as well as sulfated PSGL-1 (46, 51F). The results indicate that the reactivity between sulfated PSGL-1 and VP1 obviously decreases as tyrosine-51 is replaced by phenylalanine. As a consequence, tyrosine-51 exhibits the major importance because preventing it from being sulfated will reduce the reactivity of PSGL-1 with EV71. In this study, AFM study is competent to tell the difference of reactivity of PSGL-1 mutations through AFM force curves and is therefore promising forEV71 related studies based on the mutated PSGL-1 and VP1.

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