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

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rhAChE的单一条带；测得纯化后rhAChE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 nonpurified (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 SDSPAGE, 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.

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