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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 aimed to express recombinant human acetylcholinesterase (rhAChE) and applied it to the screening of acetylcholinesterase inhibitors. HEK293 cells were transiently transfected with plasmid pCMV-AChE and rhAChE was secreted. To investigate the enzyme activity of rhAChE: enzyme substrate kinetics study of thioacetyl choline iodide (ATCh) substrate obtained Kiehmann constant of rhAChE, with donepezil as inhibitor of enzyme inhibition kinetics study Ki , Ki 'and other parameters. The IC50 value of the new compound was measured by rhAChE to evaluate the inhibitory activity of the new compound. The inhibitory activity of AChE was also compared with that reported in the literature. The results showed that the Km of rhAChE was 151.9μmol·L-1, and the inhibitory effect of donepezil on rhAChE was a mixed type with competitive and non-competitive binding (Ki = 16.03 nmol·L-1, Ki '= 18.36 nmol·L-1 ), rhAChE can be used in inhibitor screening studies.