CN113699212A - 一种筛选新冠病毒主蛋白酶小分子抑制剂的方法及筛选模型 - Google Patents
一种筛选新冠病毒主蛋白酶小分子抑制剂的方法及筛选模型 Download PDFInfo
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Abstract
本发明涉及一种筛选新冠病毒主蛋白酶(main protease,Mpro)小分子抑制剂的方法及筛选模型。该方法及筛选模型基于荧光偏振(fluorescence polarization)原理,以荧光探针FITC‑Substrate‑Biotin作为新冠病毒Mpro的水解底物,再以亲和素(avidin)终止其水解反应,以多功能酶标仪检测实验体系的毫偏值(millipolarization units,mP)。活性化合物在本筛选模型中表现较高的mP值,非活性化合物则表现较低的mP值。
Description
技术领域
本发明属于医药生物技术领域,具体的,涉及一种筛选新冠病毒主蛋白酶小分子抑制剂的方法及筛选 模型。
背景技术
快速研制新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)疫苗、人源化抗 体和抗新冠病毒药物已成为科学界面临的重大科学问题。尽管在研制新冠病毒疫苗方面取得了显著进展, 但对于未接种疫苗的个体或病毒高频基因突变导致疫苗保护力下降等情况,安全有效的广谱抗新冠病毒药 物也是至关重要的(WangZ,et al.Nature,2021)。虽然一些候选抗病毒药物已被用于临床治疗,如瑞德西 韦、法匹拉韦、利巴韦林、替比夫定等,但在大规模临床试验中均显示疗效有限或无效,积极开发新型广 谱抗新冠病毒药物具有重要意义。
目前研究表明,进化高度保守的新冠病毒主蛋白酶(main protease,Mpro)在调控新冠病毒RNA复制 中具有重要的生物学功能,且人体缺乏与其同源的蛋白酶,已成为新型广谱抗冠状病毒药物开发的理想靶 标之一(Morse JS,et al.Chembiochem,2020;Jin Z,et al.Nature,2020;Jin Z,et al.Biochem Biophys Res Commun,2021)。在高选择性新冠病毒Mpro小分子抑制剂的开发过程中,简便、快速、灵敏、经济的药 物高通量筛选模型的建立是其高效筛选与开发的重要基础和关键技术(戚海燕,等.生命的化学,2021)。 目前已报道的Mpro小分子抑制剂筛选方法主要包括虚拟筛选法、荧光共振能量转移法(fluorescence resonance energy transfer,FRET)、荧光素酶报告基因筛选法、绿色荧光蛋白剪切互补法和表型筛选法等(Li Z,et al.Proc Natl Acad Sci USA,2020;Zhu W,etal.ACS Pharmacol Transl Sci,2020;Coelho C,et al.PLoS One,2020;Hung HC,etal.Antimicrob Agents Chemother,2020;马玲,等.药学学报,2020;Rawson J,et al.Viruses,2021;Froggatt HM,et al.J Virol,2020;Rothan HA,et al.Mol Biotechnol,2021;Riva L,et al.Nature, 2020)。但上述筛选方法普遍存在假阳性率高、筛选成本高、操作繁琐、稳定性差、筛选周期长等缺点, 较大地限制了其在大规模高通量筛选中的应用(戚海燕,等.生命的化学,2021)。因此,积极开发简便、 快速、灵敏、经济的新型Mpro小分子抑制剂高通量筛选模型具有重要意义。
发明内容
本发明首先涉及一种用于筛选新冠病毒主蛋白酶小分子抑制剂的方法,所述的方法包括如下步骤:
(1)以大肠杆菌原核表达方法制备高活性Mpro重组蛋白。
(2)添加待筛选化合物与Mpro重组蛋白共同孵育。
优选的,Mpro重组蛋白溶液(Mpro重组蛋白的浓度为100~2000nM)与待筛选化合物(浓度为1~ 5mM)按比例混合后,在室温下共同孵育20~60min。
更优选的,Mpro重组蛋白溶液(Mpro重组蛋白的浓度为400nM)与待筛选化合物(浓度为3mM) 按照1:29(v/v)的比例混合后,在室温下共同孵育40min。
(3)以异硫氰酸荧光素(fluorescein isothiocyanate,FITC)和Biotin标记的FITC-Substrate-Biotin多肽 (FITC-S-Biotin:FITC-AVLQSGFRKK-Biotin)作为Mpro水解底物,在室温下共同孵育5~60min。
优选的,FITC-S-Biotin浓度为20~100nM,加入的FITC-S-Biotin溶液的体积为步骤(2)的体系的 1/2~1/1(v/v)。加入FITC-S-Biotin后,室温孵育5~40min。
更优选的,FITC-S-Biotin浓度为60nM,加入的FITC-S-Biotin溶液的体积为步骤(2)的体系的2/3 (v/v)。加入FITC-S-Biotin后,室温孵育20min。
(4)以亲和素终止水解反应,在室温下共同孵育1~30min,以多功能酶标仪检测mP值。
优选的,亲和素浓度为10~500nM,加入的亲和素溶液的体积为步骤(2)的体系的1/6~1/1(v/v)。 加入亲和素后,室温孵育1~10min,以多功能酶标仪检测mP值。
更优选的,亲和素浓度为300nM,加入的亲和素溶液的体积为步骤(2)的体系的1/3(v/v)。加入亲 和素后,室温孵育5min,以多功能酶标仪检测mP值。
(5)绘制目标化合物的抑制曲线,计算目标化合物对新冠病毒主蛋白酶的IC50值。
步骤(1)所述的大肠杆菌原核表达方法制备高活性Mpro重组蛋白的方法为:
①将密码子优化的Mpro基因连接到pET-21a(+)表达载体中,构建重组质粒Mpro-pET-21a;
②再将重组质粒转化到E.coli Rosetta(DE3)感受态细胞中,进行Mpro可溶表达,以HisTrap亲和层 析柱分离纯化新冠病毒Mpro重组蛋白。
步骤(5)所述的绘制目标化合物抑制曲线的步骤中,其抑制率的计算公式如下:
本发明还涉及一种用于筛选新冠病毒主蛋白酶小分子抑制剂的荧光偏振筛选试剂盒,所述的试剂盒中 包含:
(1)检测有效量的新冠病毒Mpro重组蛋白、荧光探针FITC-S-Biotin、亲和素;
(2)必要的溶剂与试剂。
本发明还涉及所述的用于筛选新冠病毒主蛋白酶小分子抑制剂的荧光偏振筛选试剂盒在筛选新冠病 毒主蛋白酶小分子抑制剂中的应用。
本发明的有益效果如下:
本发明公开了一种用于新型冠状病毒主蛋白酶小分子抑制剂筛选的荧光偏振高通量筛选模型及其使 用方法,属于医药生物技术领域。该高通量筛选模型基于荧光偏振原理,以荧光探针FITC-S-Biotin作为新 冠病毒Mpro的水解底物,再以亲和素终止其水解反应,以多功能酶标仪检测实验体系的mP值。活性化 合物在本筛选模型中表现较高的mP值,非活性化合物则表现较低的mP值。具体地包括:
(1)本发明建立的荧光偏振高通量筛选模型适用于以主蛋白酶为靶标的抗新冠病毒药物的快速筛选、 发现与活性评价;
(2)本发明建立的荧光偏振高通量筛选模型具有均相反应、操作简便、检测灵敏、成本低廉等优点;
(3)本发明建立的荧光偏振高通量筛选模型不仅仅局限于新冠病毒主蛋白酶小分子抑制剂的高通量 筛选,其他致病性病毒中调控病毒复制的关键蛋白酶小分子抑制剂同样可以应用或改进本发明进行快速筛 选与发现。
附图说明
图1、本发明实施例1中新冠病毒Mpro重组蛋白的密码子优化序列与氨基酸序列。
图2、本发明实施例1与实施例2中新冠病毒Mpro重组蛋白原核表达、分离纯化与生物学活性鉴定。 2A,新冠病毒Mpro重组蛋白的原核表达与分离纯化:泳道M:蛋白质标准分子量;泳道1:菌体蛋白质;泳 道2:裂解上清液;泳道3:粗提液;泳道4:纯化的Mpro重组蛋白。2B:MCA标准品荧光强度标准曲线; 2C:不同浓度的Mpro重组蛋白水解MCA底物后的荧光强度曲线。
图3、本发明实施例3中新冠病毒Mpro重组蛋白水解FITC-S-Biotin底物的反应曲线。
图4、本发明实施例4中GC-376在荧光偏振高通量筛选模型中的量效曲线与IC50值。
图5、本发明实施例5中荧光偏振高通量筛选模型的Z因子值。
图6、本发明实施例6中漆树酸与银杏酚酸在荧光偏振高通量筛选模型中的量效曲线与IC50值。
具体实施方式
若未特别指明,实施中所用的技术手段为本领域技术人员所熟知的常规生化方法,所用试剂与材料均 可从商业途径获得。
实施例1.新冠病毒Mpro重组蛋白的原核表达与分离纯化
利用大肠杆菌原核表达技术,将密码子优化的Mpro基因(图1)连接到pET-21a(+)表达载体中,构建重 组质粒Mpro-pET-21a。再将重组质粒转化到E.coli Rosetta(DE3)感受态细胞中,以氨苄西林抗性筛选重组 子。将重组子接种到1升LB液体培养基(含100μg/mL氨苄西林)中,37℃培养7h,加入0.2mM IPTG,30℃ 诱导8h。菌体以超声波法破碎后,裂解上清液再以HisTrap亲和层析柱分离纯化,纯化的Mpro表观分子量 为34kDa,其氨基端仅残留一个甲酰蛋氨酸,羧基端融合有多聚组氨酸标签,电泳纯度大于90%,浓度为3 mg/mL(图1、图2A)。
密码子优化的Mpro基因序列如SEQ ID NO.1所示,使用的酶切位点为Nde Ⅰ(CATATG)与Xho Ⅰ (CTCGAG)。Mpro重组蛋白的氨基端含有一个甲酰蛋氨酸(fMet),羧基端含有多聚组氨酸标签。
SEQ ID NO.1:
CATATGAGTGGCTTTCGTAAAATGGCCTTTCCGAGCGGCAAAGTTGAAGGTTGTATGGTGCAGGTGACCTGCGGTACCACCACCCTGAATGGTCTGTGGCTGGATGATGTGGTTTATTGCCCGCGTCATGTGATTTG TACCAGTGAAGATATGCTGAATCCGAATTATGAAGATCTGCTGATTCGCAAAAGCAATCATAATTTTCT GGTGCAGGCCGGCAATGTTCAGCTGCGCGTGATTGGCCATAGTATGCAGAATTGCGTTCTGAAACTGA AAGTGGATACCGCAAATCCGAAAACCCCGAAATATAAATTTGTTCGCATTCAGCCGGGTCAGACCTTT AGCGTGCTGGCATGTTATAATGGTAGTCCGAGCGGTGTGTATCAGTGCGCAATGCGTCCGAATTTTACC ATTAAGGGCAGTTTTCTGAATGGTAGCTGCGGCAGCGTTGGTTTTAATATTGATTATGATTGCGTGAGT TTCTGCTATATGCATCACATGGAACTGCCGACCGGTGTGCATGCAGGCACCGATCTGGAAGGTAATTTT TATGGCCCGTTTGTGGATCGCCAGACCGCACAGGCAGCCGGTACCGATACCACCATTACCGTTAATGT TCTGGCATGGCTGTATGCAGCCGTTATTAATGGTGACCGTTGGTTTCTGAATCGTTTTACCACCACCTT AAATGATTTTAATCTGGTTGCCATGAAGTATAATTACGAACCGCTGACCCAGGATCATGTGGATATTCT GGGCCCGCTGAGTGCCCAGACCGGTATTGCAGTTCTGGATATGTGTGCAAGCCTGAAAGAACTGCTG CAGAATGGTATGAATGGTCGTACCATTCTGGGTAGTGCACTGCTGGAAGATGAATTCACTCCGTTTGAT GTTGTGCGCCAGTGTAGCGGTGTGACCTTTCAGCTCGAG
其编码的氨基酸序列如SEQ ID NO.2所示,
SEQ ID NO.2:
MSGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIRKSNH NFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNGSPSGVYQCAMRP NFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTIT VNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKE LLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQLEHHHHHH
实施例2.新冠病毒Mpro重组蛋白的生物学活性鉴定
1、将10μM MCA(7-甲氧基香豆素,7-methoxycoumarin-4-acetic acid,MCA)在TBS溶液(50mM Tris, 150mM NaCl pH8.0)中以2倍倍比法稀释5个浓度梯度,以只含TBS溶液孔为阴性对照孔。上述MCA稀释 液以50μL/孔加入到384孔板中,各孔中MCA总量分别为0、31.25、62.5、125、250、500pmol,设置自动 增益模式,以多功能酶标仪检测相对荧光强度值(relative fluorescence unit,RFU)。根据各孔MCA总量和 ΔRFU值(ΔRFU=RFUMCA-RFU0)拟合回归方程,绘制MCA荧光强度标准曲线(图2B)。
2、将2mM MCA-Substrate(MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2)以TBS溶液稀释至10μM,加入Mpro 使其终浓度分别为0、0.25、0.5、1μM。上述反应液以50μL/孔加入到384孔板中,设置增益值为58,检测 温度为25℃,激发光为320nm,发射光为405nm,检测间隔为1s,检测总时间为3min,以多功能酶标仪 检测RFU值。在30s水解反应时间内,第T1和T2时间点的RFU值分别记录为RFUT1和RFUT2,计算 ΔRFU=RFUT2-RFUT1,通过图2B的标准曲线计算在ΔT=T2-T1时间内,Mpro水解MCA-Substrate产生的产物 MCA-AVLQ总量(pmol)。活力单位(U)定义:在25℃和pH 8.0条件下的水解反应中,Mpro每分钟水解 MCA-Substrate生成1pmolMCA-AVLQ(product,P)所需的酶量。
利用式2,计算纯化的Mpro比活力不低于50 000U/mg(图2C)。
实施例3.新冠病毒Mpro重组蛋白对FITC-S-Biotin底物的水解作用
将2mM FITC-S-Biotin以荧光偏振反应液(10mM Tris,50mM NaCl,1mM DTTpH8.0)稀释至60nM, 以20μL/孔加入到384孔板中,再依次加入0、25、50、100、150、175、200、300、400、500、600、700nM Mpro,每孔30μL,每组设置3组复孔,室温孵育20min,再以10μL/孔加入300nM亲和素反应液,室温避 光孵育5min,以多功能酶标仪检测mP值。
Mpro水解反应曲线说明,200nM Mpro在室温孵育20min条件下,可充分水解FITC-S-Biotin底物为产 物FITC-AVLQ(图3)。
实施例4.GC-376在荧光偏振高通量筛选模型中的抑制活性
GC-376是目前已报道的活性俱佳的新冠病毒Mpro小分子抑制剂(图4A)。将10mMGC-376以400nM Mpro进行2倍倍比稀释(起始浓度2μM,共稀释8个浓度梯度),加入到384孔板中,每孔30μL,每组设置3 组复孔,室温孵育40min。再将60nM FITC-S-Biotin依次加入到384孔板中,每孔20μL,室温避光孵育20min。 再以10μL/孔继续加入300nM亲和素反应液,室温继续孵育5min,以多功酶标仪检测mP值。
阴性孔设置为Mpro(400nM,29μL/孔)+DMSO(1μL/孔)+FITC-S-Biotin(60nM,20μL/孔)+亲和 素(300nM,10μL/孔),阳性孔设置为荧光偏振反应液(29μL/孔)+DMSO(1μL/孔)+FITC-S-Biotin(60 nM,20μL/孔)+亲和素(300nM,10μL/孔),拟合GC-376的抑制曲线,计算其IC50值。
利用式3,计算GC-376在荧光偏振筛选模型中的IC50值为0.127μM(图4B),与其在FRET筛选模型中 已报道的IC50值基本一致(Vuong W,et al.Nat Commun,2020),这说明所建立的荧光偏振高通量筛选模型 具有良好的特异性。
实施例5.荧光偏振高通量筛选模型的Z因子值
Z因子是评价药物高通量筛选模型稳定性的核心参数。利用多功能酶标仪,计算本荧光偏振高通量筛 选模型的Z因子值为0.85,满足了高通量筛选模型中Z因子大于0.5的基本要求(图5)。
实施例6.荧光偏振高通量筛选模型的使用方法
化合物库的初筛浓度为1mg/mL,取化合物1μL与400nM Mpro(29μL/孔)加入到384孔板中,室温孵 育40min后,继续加入60nM FITC-S-Biotin,每孔20μL,室温孵育20min,再加入300nM亲合素(10μL/ 孔),室温孵育5min。同时设置阴性对照组(DMSO孔)和阳性对照组(GC-376孔,1μM),以多功能酶标 仪检测mP值。利用已建立的荧光偏振高通量筛选模型对天然产物化合物库进行高通量筛选,成功筛选到漆 树酸(anacardic acid,AA)与银杏酚酸(ginkgolic acid,GA)具有良好的抑制活性,利用式1,计算其IC50值分别为9.62、7.15μM(图6A,6B)。目前,已有研究证实漆树酸与银杏酚酸是新冠病毒Mpro的小分子抑 制剂,并对新冠病毒具有良好的抗病毒活性(Xiong Y,et al.Fitoterapia,2021;Chen Z,et al.CellBiosci, 2021),这也说明了本荧光偏振高通量筛选模型具有良好的实用性与推广性。
虽然,上文中已用一般性说明及其具体实施方案对本发明做了详尽的描述,但在本发明的基础上,本 领域技术人员仍可对之做进一步的改进或修改,这是显而易见的。因此,在本发明所述的荧光偏振原理基 础上所做的任何修改或改进或其他致病性病毒中调控病毒复制的关键蛋白酶小分子抑制剂的荧光偏振高 通量筛选模型的建立与使用,均属于本发明的保护范围。本发明中所应用的FITC-S-Biotin底物不仅仅局限 于应用实施方案中所述的多肽序列进行FITC与Biotin双标记,凡是含有Mpro保守切割序列(L-Q-S/A)及 其他荧光分子标记或以其他氨基酸修饰技术合成的Mpro底物多肽,也均属于本发明的保护范围。此外,本 发明中所应用的新冠病毒Mpro不仅仅局限于应用大肠杆菌原核表达技术制备,利用酵母细胞、CHO细胞、 昆虫细胞、植物细胞等其他真核生物表达系统制备Mpro同样是可行的,但高活性的Mpro应是本文所叙述 的结构,氨基端仅残留一个甲酰蛋氨酸或为自由氨基端,羧基端的多聚组氨酸标签可有可无,其对Mpro 生物学活性影响较小,Mpro此种构建策略也属于本发明的保护范围。
SEQUENCE LISTING
<110> 中国医学科学院医药生物技术研究所
<120> 一种筛选新冠病毒主蛋白酶小分子抑制剂的方法及筛选模型
<130> CP121020635C
<160> 2
<170> PatentIn version 3.3
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<213> 人工序列
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catatgagtg gctttcgtaa aatggccttt ccgagcggca aagttgaagg ttgtatggtg 60
caggtgacct gcggtaccac caccctgaat ggtctgtggc tggatgatgt ggtttattgc 120
ccgcgtcatg tgatttgtac cagtgaagat atgctgaatc cgaattatga agatctgctg 180
attcgcaaaa gcaatcataa ttttctggtg caggccggca atgttcagct gcgcgtgatt 240
ggccatagta tgcagaattg cgttctgaaa ctgaaagtgg ataccgcaaa tccgaaaacc 300
ccgaaatata aatttgttcg cattcagccg ggtcagacct ttagcgtgct ggcatgttat 360
aatggtagtc cgagcggtgt gtatcagtgc gcaatgcgtc cgaattttac cattaagggc 420
agttttctga atggtagctg cggcagcgtt ggttttaata ttgattatga ttgcgtgagt 480
ttctgctata tgcatcacat ggaactgccg accggtgtgc atgcaggcac cgatctggaa 540
ggtaattttt atggcccgtt tgtggatcgc cagaccgcac aggcagccgg taccgatacc 600
accattaccg ttaatgttct ggcatggctg tatgcagccg ttattaatgg tgaccgttgg 660
tttctgaatc gttttaccac caccttaaat gattttaatc tggttgccat gaagtataat 720
tacgaaccgc tgacccagga tcatgtggat attctgggcc cgctgagtgc ccagaccggt 780
attgcagttc tggatatgtg tgcaagcctg aaagaactgc tgcagaatgg tatgaatggt 840
cgtaccattc tgggtagtgc actgctggaa gatgaattca ctccgtttga tgttgtgcgc 900
cagtgtagcg gtgtgacctt tcagctcgag 930
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<212> PRT
<213> 人工序列
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Met Ser Gly Phe Arg Lys Met Ala Phe Pro Ser Gly Lys Val Glu Gly
1 5 10 15
Cys Met Val Gln Val Thr Cys Gly Thr Thr Thr Leu Asn Gly Leu Trp
20 25 30
Leu Asp Asp Val Val Tyr Cys Pro Arg His Val Ile Cys Thr Ser Glu
35 40 45
Asp Met Leu Asn Pro Asn Tyr Glu Asp Leu Leu Ile Arg Lys Ser Asn
50 55 60
His Asn Phe Leu Val Gln Ala Gly Asn Val Gln Leu Arg Val Ile Gly
65 70 75 80
His Ser Met Gln Asn Cys Val Leu Lys Leu Lys Val Asp Thr Ala Asn
85 90 95
Pro Lys Thr Pro Lys Tyr Lys Phe Val Arg Ile Gln Pro Gly Gln Thr
100 105 110
Phe Ser Val Leu Ala Cys Tyr Asn Gly Ser Pro Ser Gly Val Tyr Gln
115 120 125
Cys Ala Met Arg Pro Asn Phe Thr Ile Lys Gly Ser Phe Leu Asn Gly
130 135 140
Ser Cys Gly Ser Val Gly Phe Asn Ile Asp Tyr Asp Cys Val Ser Phe
145 150 155 160
Cys Tyr Met His His Met Glu Leu Pro Thr Gly Val His Ala Gly Thr
165 170 175
Asp Leu Glu Gly Asn Phe Tyr Gly Pro Phe Val Asp Arg Gln Thr Ala
180 185 190
Gln Ala Ala Gly Thr Asp Thr Thr Ile Thr Val Asn Val Leu Ala Trp
195 200 205
Leu Tyr Ala Ala Val Ile Asn Gly Asp Arg Trp Phe Leu Asn Arg Phe
210 215 220
Thr Thr Thr Leu Asn Asp Phe Asn Leu Val Ala Met Lys Tyr Asn Tyr
225 230 235 240
Glu Pro Leu Thr Gln Asp His Val Asp Ile Leu Gly Pro Leu Ser Ala
245 250 255
Gln Thr Gly Ile Ala Val Leu Asp Met Cys Ala Ser Leu Lys Glu Leu
260 265 270
Leu Gln Asn Gly Met Asn Gly Arg Thr Ile Leu Gly Ser Ala Leu Leu
275 280 285
Glu Asp Glu Phe Thr Pro Phe Asp Val Val Arg Gln Cys Ser Gly Val
290 295 300
Thr Phe Gln Leu Glu His His His His His His
305 310 315
Claims (8)
1.一种用于筛选新冠病毒主蛋白酶(main protease,Mpro)小分子抑制剂的方法,所述的方法包括如下步骤:
(1)以大肠杆菌原核表达方法制备高活性Mpro重组蛋白;
(2)添加待筛选化合物与Mpro重组蛋白共同孵育;
优选的,步骤(2)中,Mpro重组蛋白溶液(Mpro重组蛋白的浓度为100~2000nM)与待筛选化合物(浓度为1~5mM)按比例混合后,在室温下共同孵育20~60min;
(3)以异硫氰酸荧光素(fluorescein isothiocyanate,FITC)和Biotin标记的FITC-Substrate-Biotin多肽(FITC-S-Biotin:FITC-AVLQSGFRKK-Biotin)作为Mpro水解底物,在室温下共同孵育5~60min;
优选的,步骤(3)中,FITC-S-Biotin浓度为20~100nM,加入的FITC-S-Biotin溶液的体积为步骤(2)的体系的1/2~1/1(v/v)。加入FITC-S-Biotin后,室温孵育5~40min;
(4)以亲和素终止水解反应,在室温下共同孵育1~30min,以多功能酶标仪检测mP值;
优选的,步骤(4)中,亲和素浓度为10~500nM,加入的亲和素溶液的体积为步骤(2)的体系的1/6~1/1(v/v)。加入亲和素后,室温孵育1~10min,以多功能酶标仪检测mP值;
(5)绘制目标化合物的抑制曲线,计算目标化合物对新冠病毒主蛋白酶的IC50值;
所述的新冠病毒主蛋白酶的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的方法,其特征在于,
步骤(2)中:将Mpro重组蛋白溶液(Mpro重组蛋白的浓度为400nM)与待筛选化合物(浓度为3mM)按照1:29(v/v)的比例混合后,在室温下共同孵育40min。
3.根据权利要求1所述的方法,其特征在于,
步骤(3)中:FITC-S-Biotin浓度为60nM,加入的FITC-S-Biotin溶液的体积为步骤(2)体系的2/3(v/v)。加入FITC-S-Biotin后,室温孵育20min。
4.根据权利要求1所述的方法,其特征在于,
步骤(4)中:亲和素浓度为300nM,加入的亲和素溶液的体积为步骤(2)体系的1/3(v/v)。加入亲和素后,室温孵育5min,以多功能酶标仪检测mP值。
5.根据权利要求1所述的方法,其特征在于,
步骤(1)所述的大肠杆菌原核表达方法制备高活性Mpro重组蛋白的方法为:
①将核苷酸序列如SEQ ID NO.1所示的Mpro基因片段连接到pET-21a(+)表达载体中,构建重组质粒Mpro-pET-21a;
②再将重组质粒转化到E.coli Rosetta(DE3)感受态细胞中,进行Mpro可溶表达,以HisTrap亲和层析柱分离纯化新冠病毒Mpro重组蛋白。
7.一种用于筛选新冠病毒主蛋白酶小分子抑制剂的荧光偏振筛选试剂盒,所述的试剂盒中包含:
(1)检测有效量的新冠病毒Mpro重组蛋白、荧光探针FITC-S-Biotin、亲和素;
(2)必要的溶剂与试剂。
8.权利要求7所述的用于筛选新冠病毒主蛋白酶小分子抑制剂的荧光偏振筛选试剂盒在筛选新冠病毒主蛋白酶小分子抑制剂中的应用。
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