首页|SARS-CoV-2 Mpro抑制剂筛选体系的构建及临床常用抗病毒中药制剂的初步筛选

SARS-CoV-2 Mpro抑制剂筛选体系的构建及临床常用抗病毒中药制剂的初步筛选

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目的:利用荧光共振能量转移(Fluorescence resonance energy transfer，FRET)技术构建新冠病毒主蛋白酶(SARS-CoV-2 Mpro)中药抑制剂的体外筛选方法。方法:构建Mpro及筛选探针原核表达的工程质粒pGEX-4T-1-Mpro、pET-28a(+)-Ls-mK。使用荧光探针Ls-mK检测Mpro的生物活性及酶动力学参数，并对筛选条件进行优化。采用阳性药物GC-376及阴性对照PLpro(Pa-pain-like protease)和TEV酶(Tobacco Etch Virus Proteinase)评价荧光探针Ls-mK的特异性及检测能力与商业化探针的差异。根据所构建的筛选体系考察五种中药制剂筛选热毒宁注射液、血必净注射液、抗病毒口服液和蒲地蓝口服液对Mpro的抑制能力。结果:本研究成功构建原核表达的工程质粒并获得纯度为90％左右的重组蛋白荧光探针Ls-mK及Mpro。荧光探针Ls-mK可成功产生FRET现象并具有良好的特异性。Mpro具有良好的生物活性。本研究所构建的筛选体系具有与商业化探针相同的检测能力，对五种中药制剂考察发现仅喜炎平注射液对Mpro具有良好的抑制活性，其IC50值为(3。32±0。03)mg/mL。结论:本研究成功构建基于荧光蛋白SARS-CoV-2 Mpro的简便、灵敏和低成本的筛选体系，为新冠病毒主蛋白酶抑制剂的筛选与发现奠定了实验基础。
Construction of Screening System for SARS-CoV-2 Mpro Inhibitors and Preliminary Screening of Antiviral Chinese Medicine Preparations
Objective:To construct an in vitro screening system of Chinese medicine inhibitors of SARS-CoV-2 Mpro by fluorescence resonance en-ergy transfer(FRET)technology.Methods:The engineering plasmids pGEX-4T-1-Mpro and pET-28a(+)-Ls-mK were constructed.The bi-ological activity and enzyme kinetic parameters of Mpro were determined by the fluorescent probe Ls-mK,and the screening conditions were optimized.The differences between fluorescent probe Ls-mK and commercial probe in specificity and detection ability were evaluated by the positive drug GC-376 and the negative control papain-like protease(PLpro)and tobacco etch virus(TEV)protease.According to the con-structed screening system,the inhibitory ability of five Chinese medicine preparations against Mpro was investigated,including Reduning(热毒宁)Injection,Xuebijing(血必净)Injection,Antiviral Oral Liquid and Pudilan(蒲地蓝)Oral Liquid.Results:The engineering plasmid expressed in prokaryotes was successfully constructed,and the recombinant protein fluorescent probes Ls-mK and Mpro with a purity of about 90％were obtained.The fluorescent probe Ls-mK produced FRET phenomenon and presented good specificity.Mpro had good biological ac-tivity.The screening system constructed in this paper specifically had the same detection ability as commercial probes.It was found that only Xiyanping Injection inhibited Mpro,with an IC50 value of 3.32±0.03 mg/mL.Conclusion:This paper successfully constructed a simple,sen-sitive and low-cost screening system based on SARS-CoV-2 Mpro,which laid an experimental foundation for the screening and discovery of SARS-CoV-2 Mpro inhibitors.

SARS-CoV-2Main protease inhibitorFluorescence resonance energy transferAntiviral Chinese medicine preparationsBiologi-cal probe

韩冰、张岱、陈小菲、李伟霞、吴娅丽、王晓艳、张辉、汪彬、蒋雪松、贾金浩、纪秋如、孟高全、孟伟亭、王炜、唐进法

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河南中医药大学药学院,郑州 450046

河南中医药大学第一附属医院检验科,郑州 450046

河南中医药大学第一附属医院河南省中药临床应用、评价与转化工程研究中心河南省中药安全评价与风险防控工程研究中心河南省中药临床药学中医药重点实验室,郑州 450000

河南中医药大学呼吸疾病中医药防治省部共建协同创新中心,郑州 450046

河南中医药大学第一附属医院感染控制科,郑州 450000

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新冠病毒 主蛋白酶抑制剂 荧光共振能量转移 抗病毒中药制剂 生物探针

河南省中医药科学研究专项课题河南省高校科技创新团队2022财政专项-中医药传承与创新人才工程(仲景工程)2023年财政专项-中药创新能力提升项目

2023ZY204223IRTSTHN026

2024

中药药理与临床
中国药理学会 四川省中医药科学院

中药药理与临床

北大核心
影响因子：0.996
ISSN：1001-859X
年,卷(期)：2024.40(6)
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