重组人乙酰胆碱酯酶的表达及其抑制剂的筛选（英文）

Expression of recombinant human acetylcholinesterase and its application in screening its inhibitors

本研究旨在表达重组人乙酰胆碱酯酶(recombinant human acetylcholinesterase,rhAChE)并应用于乙酰胆碱酯酶抑制剂筛选研究。利用质粒pCMV-AChE瞬时转染HEK293细胞,分泌表达rhAChE。考察rhAChE的酶活性:以碘化硫代乙酰胆碱(ATCh)为底物进行酶底物动力学研究获得rhAChE的米氏常数Km,以多奈哌齐(donepezil)为抑制剂进行酶抑制作用动力学研究获得Ki、Ki’等参数。利用rhAChE测定新化合物的IC50值,评价新化合物的抑制活性,同时将研究结果与文献报道的提取大鼠AChE的抑制活性进行比较。结果测得rhAChE的Km为151.9μmol·L-1,多奈哌齐对rhAChE的抑制作用为竞争性和非竞争性结合的混合类型(Ki=16.03 nmol·L-1,Ki’=18.36 nmol·L-1),rhAChE可应用于抑制剂筛选研究。

This study is designed to obtain recombinant human acetylcholinesterase （rhAChE） and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC 50 values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that Km value was 151.9 μmol.L-1, donepezil inhibited rhAChE in a mixed competitive-non- competitive way （Ki= 16.03 nmol.L-1, Ki＇= 18.36 nmol.L-1） and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.