CN111896504A - 一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒及其应用 - Google Patents
一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒及其应用，包括Mpro蛋白、荧光多肽底物以及反应试剂缓冲液，多肽底物两端分别含有酶解荧光标记，所述多肽底物的序列为SEQ ID NO:1所示的多肽底物。本发明开发出试剂盒可以快速筛选出可能是Mpro蛋白酶的靶标化合物的药物，并且可通过计算Mpro的米氏常数Km值及催化效率Kcat值，定量判定靶标化合物的抑制能力。
Description
技术领域
本发明涉及生物化学领域，具体地说，是涉及一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒及应用。
背景技术
人感染了COVID-19病毒后多会出现有呼吸道症状、发热、咳嗽、气促和呼吸困难等症状。严重者会发生肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。
COVID-19疫苗的研制还在研制过程中，筛选靶向COVID-19病毒的药物对于疾病的治疗至关重要。在中草药、海洋生物、陆地微生物及产物等自然界中萃取、寻找抗COVID-19病毒药物将为是新结构、新机制抗COVID-19病毒药物提供重要来源，对于抗COVID-19病毒药物的研究具有重要意义。但COVID-19病毒致病性和传染性非常强，相关病毒培养实验仅能在生物安全等级三级以上实验室才可进行，严重制约了药物筛选的速度和效率。
COVID-19的生存和复制依赖于其主蛋白酶(Mpro)活性，因此抑制Mpro酶活的药物有很大几率对COVID-19病毒感染具有治疗效果因此需要一种能有效解决上述问题的新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒及应用。
发明内容
本发明的目的在于提供一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒及应用.
为实现上述目的，本发明采用的技术方案如下：
本发明包括以下步骤：
本发明包括Mpro蛋白、荧光多肽底物以及反应试剂缓冲液，多肽底物两端分别含有酶解荧光标记，所述多肽底物的序列为SEQ ID NO:1所示的多肽底物；。
进一步地，所述多肽底物的N端连接标记基团，C端连接荧光淬灭基团。
进一步地，所述酶解荧光标记的标记基团和荧光淬灭基团可以为Dabcyl和Edans、Mca和lys(dnp)、Abz和Tyr(3-NO2)、6－TMARA和FITC中的一种。进一步地，所述试剂盒的酶活反应20μl反应体系如下：
Mpro蛋白2μl
多肽底物5μl
反应buffer 13μl
一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒在筛选靶点药物检测其抑制活性的应用。
一种靶点药物筛选酶活测定定量分析的方法，Mpro蛋白通过酶活反应水解所述多肽底物使得所述多肽底物的酶解荧光标记的基团分离后在336nm波长光激发后检测显色。
进一步地，通过计算Mpro的米氏常数Km值及催化效率Kcat值，定量判定靶标化合物的抑制能力。
与现有技术相比，本发明具有以下有益效果：
本发明开发出试剂盒可以快速筛选出可能是Mpro蛋白酶的靶标化合物的药物，并且可通过计算Mpro的米氏常数Km值及催化效率Kcat值，定量判定靶标化合物的抑制能力。
附图说明
图1为实施例子中的蛋白酶抑制剂M5对Mpro蛋白降解底物TE peptide的酶活抑制曲线结果图；
图2为实施例子中的蛋白酶抑制剂M5对Mpro蛋白对不同浓度底物TE peptide的酶活曲线结果图以及相应Kcat及Km值。
具体实施方式
下面根据实施例对本发明作进一步说明，本发明的方式包括但不仅限于以下实施例。
在本实施例子中,采用基因工程的方法构建COVID-19病毒Mpro蛋白基因的原核表达质粒，使用大肠杆菌Bl21菌株进行蛋白的表达和纯化。蛋白序列如下：
SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIRKSNH NFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNGSPSGVYQCAMRP NFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTI TVNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASL KELLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQ
1.2：荧光多肽底物
通过检测了Mpro蛋白酶对5条十肽底物的酶活，筛选出稳定性好、可有效被Mpro蛋白酶降解的多肽(表1)。这一底物是基于COVID-19上orf1ab polyprotein蛋白序列设计。酶解位点为谷氨酰胺与丙氨酸之间的肽键。多肽的 N端连接Dabcyl基团，C端连接Edans基团。Dabcyl和Edans两基团靠近时发生荧光催灭，不显示荧光。当多肽被降解后，两基团分离，在336nm波长光激发下，可在490nm处检测到荧光。
Dabcyl基团和Edans基团对的肽键水解后产生的荧光可衡量纳摩尔级浓度的酶活性。当FRET肽是完整的，表现出的是内部的荧光猝灭，但当Dabcyl基团和Edans基团对的任何肽键断裂就会释放出荧光，此荧光可被连续检测，从而可对酶的活性进行定量分析。
表1荧光多肽底物
方法步骤：蛋白酶活力测定实验
酶活反应20μl反应体系如下：
Mpro蛋白2μl(约0.5μM)
多肽底物5μl(约10μM)
反应buffer 13μl
将Mpro蛋白及底物加入到酶活反应体系后，混匀并立即倒入玻璃比色皿中，在336nm波长激发下，获得其在490nm处的发射波谱随时间扫描的曲线。在上述反应体系中加入不同浓度的待检测化合物，并重复以上步骤操作，获得化合物存在时的酶活曲线，确定化合物对Mpro蛋白酶的酶活性的影响。
多肽的N端连接Dabcyl基团，C端连接Edans基团。个供体基团(EDANS) 和接受基因(DABCYL)匀被连接到一蛋白酶的天然底物上，当该底物未被切断时，Dabcyl和Edans两基团靠近时发生荧光淬灭，从而检测不到荧光。当多肽被逐渐被Mpro蛋白酶降解后，两基团分离，当该底物被Mpro蛋白酶切断后，EDANS 不再被DABCYL淬灭，随即可检测到EDANS荧光。蛋白酶抑制剂的有效性可凭借 EDANS荧光强度的变化进行监测。
荧光共振能量转移是在供体基团的激发状态下由一对偶极子介导的能量从供体(染料1)向受体(染料2)转移的过程。通常，供体(Donor)荧光基团的发射光谱要与受体(Acceptor)基团的吸收光谱有一定的重叠。当这两个荧光基团间的距离合适时
)，就可观察到荧光能量由供体向受体转移的现象。能量转移发生方式依赖于受体的化学结构：
在336nm波长光激发下，可在490nm处检测到荧光，并且随着降解的加速荧光逐渐变强直至反应完全。通过检测到的酶活曲线，在阳性试剂、阴性试剂对照下，通过对比米氏常数Km值及催化效率Kcat值，评价受试样品是否通过抑制Mpro蛋白酶活性。
实验结果
取0.5μM Mpro蛋白加入含10μM多肽底物的反应体系中，37度反应 10-60分钟，测定荧光强度，绘制酶活曲线。根据以上实验方法，分别检测有无 5μM抑制剂M5存在时的酶活曲线，如图1所示,为蛋白酶抑制剂M5对Mpro蛋白降解底物TE peptide的酶活抑制结果图。如图2所示为实施例子中的蛋白酶抑制剂M5对Mpro蛋白对不同浓度底物TE peptide的酶活曲线结果图以及相应 Kcat及Km值。
申请人声明，本发明通过上述实施例来说明本发明的一种新型冠状病毒 Mpro蛋白酶靶点药物筛选试剂盒，但本发明并不局限于上述实施例，即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
上述实施例仅为本发明的优选实施方式之一，不应当用于限制本发明的保护范围，但凡在本发明的主体设计思想和精神上作出的毫无实质意义的改动或润色，其所解决的技术问题仍然与本发明一致的，均应当包含在本发明的保护范围之内。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> 一种新型冠状病毒 Mpro蛋白酶靶点药物筛选试剂盒及其应用
<141> 2020-06-16
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 新型冠状病毒(COVID-19病毒)
<400> 1
Thr Val Arg Leu Gln Ala Gly Asn Ala Thr
1 5 10
Claims (7)
1.一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒，其特征在于，包括Mpro蛋白、荧光多肽底物以及反应试剂缓冲液，多肽底物两端分别含有酶解荧光标记，所述多肽底物的序列为SEQ ID NO:1所示的多肽底物。
2.根据权利要求1所述的一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒，其特征在于，所述多肽底物的N端连接标记基团，C端连接荧光淬灭基团。
3.根据权利要求1所述的一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒，其特征在于，所述酶解荧光标记的标记基团和荧光淬灭基团可以为Dabcyl和Edans、Mca和lys(dnp)、Abz和Tyr(3-NO2)、6－TMARA和FITC中的一种。
4.根据权利要求1所述的一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒，其特征在于，所述试剂盒的酶活反应20μl反应体系如下：
Mpro蛋白2μl
多肽底物5μl
反应buffer 13μl 。
5.根据权利要求1所述的一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒在筛选靶点药物检测其抑制活性的应用。
6.一种靶点药物筛选酶活测定定量分析的方法，其特征在于，Mpro蛋白通过酶活反应水解所述多肽底物使得所述多肽底物的酶解荧光标记的基团分离后在336nm波长光激发后检测显色。
7.根据权利要求6所述的一种新型冠状病毒Mpro蛋白酶靶点药物筛选试剂盒，其特征在于，通过计算Mpro的米氏常数Km值及催化效率Kcat值，定量判定靶标化合物的抑制能力。
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