重组人乙酰胆碱酯酶体外表达制备及其相关小分子活性筛选

Expression and preparation of recombinant human acetylcholinesterase in vitro and screening of micromolecule for anti-AchE activity

目的:建立重组人乙酰胆碱酯酶(rhAChE)抑制剂体外筛选模型,并应用该模型对中药单体进行抑制剂的初步筛选。方法:运用pCMV-AChE表达质粒转染HEK293细胞,通过DEAE-Sepharose FF柱纯化酶液,获得更高纯度和活性的纯化酶,并建立其活性检测方法,同时结合SDS-PAGE及Western blot验证模型成功与否;以AChE的可逆性抑制剂多奈哌齐为阳性药,运用该模型探究小檗碱、血根碱和蛇根碱对AChE的影响。结果:纯化液比活力为15.93 U·mg-1,纯化倍数为9.63,酶活回收率为56%;Western blot结果显示在68×103 g·mol^(-1)附近出现明显的rh ACh E的单一条带;测得纯化后rh ACh E的IC50为5.0 nmol·L^(-1),明显低于纯化前(14 nmol·L^(-1));3个不同的化合物对rhAChE分别表现出不同程度的抑制作用,其中小檗碱的IC50接近多奈哌齐。结论:SDS-PAGE、Western blot以及多奈哌齐的阳性结果显示利用pCMV-AChE转染的HEK293细胞成功构建rhAChE抑制剂体外筛选模型,并成功运用于中药单体药物的初筛。

Objectives:To build a cell-based recombinant human acetylcholinesterase(rhAChE) model for screening of micromolecule for anti-AChE activity in vitro. Methods:HEK293 cells were transfected with the reconstructed plasmid pCMV-AChE. DEAE-Sepharose FF affinity chromatography was used to obtain higher purity and higher activity of rhAChE. Detection method of rhAChE was also developed. Meanwhile,the recombinant protein was identified by SDS-PAGE and Western-blot. The established detection system of rhAChE activity was applied to explore the effects of berberine,sanguinarine and serpentine with the AChE’s reversible inhibitor of donepezil as positive drug. Results:Purified enzyme liquid specific activity was 15.93 U·mg-1,the purification ratio was 9.63,the recovery rate was 56%. Western blot analysis showed that rhAChE strip appeared in the vicinity of 68×103 g·mol-1 obviously. The IC50 of purified rhAChE was 5.0 nmol·L-1,which was lower than that of non-purified(14 nmol·L-1). Three kinds of compounds showed diverse degrees for inhibition of rhAChE. The IC50 of berberine was almost equal to that of Donepezil. Conclusion:The rhAChE inhibitor model was verified with SDS-PAGE,Western-Blot and the ability of activation for Donepezil in vitro,which was established by HEK293 cell transfected pCMV-AChE. This model can be used to screen and to evaluate rhAChE natural inhibitor.