CN111875709A - 一种融合蛋白及其在构建筛选冠状病毒3cl蛋白酶抑制剂的系统中的应用 - Google Patents
一种融合蛋白及其在构建筛选冠状病毒3cl蛋白酶抑制剂的系统中的应用 Download PDFInfo
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Abstract
本发明公开了一种融合蛋白及其在构建筛选冠状病毒3CL蛋白酶抑制剂的系统中的应用。本发明首先提供了一种融合蛋白,将N端的泛素化蛋白和C端的具有报告功能的蛋白通过3CL蛋白酶酶切位点氨基酸片段融合而成;所述3CL蛋白酶酶切位点氨基酸片段为3CL蛋白酶能够识别并进行酶切的氨基酸片段。利用该融合蛋白、其编码基因、包含其的载体或表达其的重组细胞构建得到了一种筛选冠状病毒3CL蛋白酶抑制剂的系统,该系统方便快捷、操作简单,具有良好的特异性和敏感性;利用该系统能够便捷有效的筛选冠状病毒3CL蛋白酶抑制剂,筛选机理明确,速度快,作用效果直观,且通用性强,重复性好,假阳性率低,在筛选冠状病毒3CL蛋白酶抑制剂中具有良好的应用前景。
Description
技术领域
本发明属于医学病毒学技术领域。更具体地,涉及一种融合蛋白及其在构建筛选冠状病毒3CL蛋白酶抑制剂的系统中的应用。
背景技术
冠状病毒是感染人类呼吸系统的主要病原体之一,导致了严重急性呼吸综合征(SARS)、中东呼吸综合征(MERS),以及2019冠状病毒疾病(COVID-19)。目前,对冠状病毒诱发疾病的机理及发病的分子细胞生物学的机制还缺乏了解,尚没有针对该类疾病的特异性抗病毒药物。
针对冠状病毒药物筛选模型的建立是冠状病毒病致病机理及其药物研究的重要基础,已有的针对冠状病毒的药物筛选模型,主要是基于病毒编码蛋白的体外酶活检测系统,不能充分体现病毒蛋白在细胞内工作的复杂性,因而限制了对病毒复制等机制的深入研究,进而影响冠状病毒致病机理相关知识的拓展,这些知识的缺乏又进一步限制了充分再现病毒蛋白功能的药物筛选模型的开发。近年来,随着冠状病毒一次又一次的爆发,与冠状病毒相关的流行病学、进化学和分子生物学的发展日趋深入,使得冠状病毒筛药模型的建立有了更为准确的理论基础,筛选角度也开始有了多样化发展。
3CL蛋白酶主要针对冠状病毒多聚蛋白进行酶切加工,是调节病毒复制转录的关键蛋白,是抑制病毒复制、实现临床抗病毒治疗效果的重要药物靶点,抑制其酶活性成为了抑制病毒复制和感染的重要策略。目前,已有的靶向3CL蛋白酶的药物筛选手段多基于电脑分析和细胞外系统的细胞外环境(Yu-ChihLiu etal,Screening of drugs by FRETanalysis identifies inhibitors of SARS-CoV 3CLprotease,2005;Pamela Hamilletal,Development of a Red-Shifted Fluorescence-Based Assay for SARS-coronavirus 3CL Protease:Identification of a Novel Class of anti-SARS AgentsFrom the Tropical Marine Sponge Axinella Corrugata,2006;Junwei Zhou etal,Identification of Novel Proteolytically Inactive Mutations in Coronavirus 3C-like Protease Using a Combined Approach,2019),不能真实地再现细胞内3CL蛋白酶工作的状态,因而也限制了筛选高效抑制药物的进程以及对所筛选药物活性的进一步验证,难以获得高效的靶向抑制药物。
因此,提供一种操作简单、方便快捷的基于细胞内环境筛选冠状病毒3CL蛋白酶抑制剂方法,对于筛选治疗冠状病毒相关疾病的药物具有重要意义。
发明内容
本发明要解决的技术问题是克服上述现有技术的缺陷和不足,提供一种融合蛋白及其在构建筛选冠状病毒3CL蛋白酶抑制剂的系统中的应用。
本发明的目的是提供一种融合蛋白。
本发明另一目的是提供编码所述融合蛋白的基因。
本发明又一目的是提供一种单基因双表达病毒载体。
本发明又一目的是提供一种重组细胞。
本发明又一目的是提供所述的融合蛋白、所述的基因、所述的载体或所述的重组细胞在构建筛选冠状病毒3CL蛋白酶抑制剂的系统中的应用。
本发明又一目的是提供一种筛选冠状病毒3CL蛋白酶抑制剂的系统。
本发明又一目的是提供所述系统在筛选冠状病毒3CL蛋白酶抑制剂中的应用。
本发明再一目的是提供一种筛选冠状病毒3CL蛋白酶抑制剂的方法。
本发明上述目的通过以下技术方案实现:
本发明提供了一种融合蛋白,将N端的泛素化蛋白和C端的具有报告功能的蛋白通过3CL蛋白酶酶切位点氨基酸片段融合而成;所述3CL蛋白酶酶切位点氨基酸片段为3CL蛋白酶能够识别并进行酶切的氨基酸片段。
本发明将泛素化蛋白和具有报告功能的蛋白通过连接元件(3CL蛋白酶酶切位点氨基酸片段)进行连接,形成融合蛋白,连接元件使得泛素化蛋白和具有报告功能的蛋白的整合不影响其通过泛素-蛋白酶体途径的降解过程;所述连接元件(3CL蛋白酶酶切位点氨基酸片段)可以是多克隆位点所编码的氨基酸序列,其长度没有特别限制,通常为3~10个氨基酸、分子量相对较小、柔性和亲水性强的氨基酸(如A、G、S、T和V)。
优选地,所述3CL蛋白酶酶切位点氨基酸片段的序列为:SEQ ID NO:1所示的氨基酸序列、与SEQ ID NO:1所示的氨基酸序列的同源性大于90%的氨基酸序列或将SEQ IDNO:1所示的氨基酸序列经人工点突变的方式进行改造后得到的氨基酸序列。
更优选地,所述3CL蛋白酶酶切位点氨基酸片段的序列为SEQ ID NO:1所示的氨基酸序列。将所述3CL蛋白酶酶切位点氨基酸片段应用于筛药系统中,能够提高筛药系统中具有报告功能的蛋白的基础表达值,扩大筛药系统的灵敏度。
SEQ ID NO:1:NVATLQSANAT。
优选地,所述泛素化蛋白为人源泛素化蛋白,其氨基酸序列登录号为BAD93019.1,编码基因的碱基序列登录号为AB209782.1。
优选地,所述泛素化蛋白的个数为1~20个。以提高筛药系统中具有报告功能的蛋白的降解效率,扩大筛药系统的灵敏度。
更优选地,所述泛素化蛋白的个数为4个。
优选地,所述3CL蛋白酶酶切位点的个数为1~3。
更优选地,所述3CL蛋白酶酶切位点的个数为3。
优选地,所述具有报告功能的蛋白为荧光素酶、荧光蛋白或报告基因。
更优选地,所述荧光素酶为萤火虫荧光素酶或海鳃荧光素酶。
更优选地,所述荧光蛋白为绿色荧光蛋白、红色荧光蛋白或黄色荧光蛋白。
更优选地,所述报告基因为氯霉素乙酰基转移酶或半乳糖苷酶。
更进一步优选地,所述荧光素酶为萤火虫荧光素酶,其氨基酸序列登录号为QBQ18417.1,编码基因的碱基序列登录号为MK484107.1。
本发明还提供了编码所述融合蛋白的基因,所述基因的碱基序列为通过编码3CL蛋白酶酶切位点的碱基序列将编码泛素化蛋白的基因的碱基序列和编码具有报告功能的蛋白的基因的碱基序列同框连接而成。
本发明通过编码3CL蛋白酶酶切位点的碱基序列将编码泛素化蛋白的基因的碱基序列(N端)和编码具有报告功能的蛋白的基因的碱基序列(C端)同框连接,通过在细胞内表达,形成了以3CL蛋白酶切位点为中心、连接泛素化蛋白与具有报告功能的蛋白的融合蛋白。
优选地,所述编码3CL蛋白酶酶切位点的碱基序列为:SEQ ID NO:2所示的碱基序列、与SEQ ID NO:2所示的碱基序列具有简并性的碱基序列、与SEQ ID NO:2所示的碱基序列的同源性大于60%的碱基序列或将SEQ ID NO:2所示的碱基序列经人工突变点的方式进行改造后得到的碱基序列。
更优选地,所述编码3CL蛋白酶酶切位点的碱基序列为SEQ ID NO:2所示的碱基序列。
SEQ ID NO:2:5’-at gtg gca act tta caa gct gaa aat gta aca-3’。
具体优选地,所述融合蛋白为UB(4)-CScon-Fluc融合蛋白,其氨基酸序列如SEQID NO:3所示,其编码基因的碱基序列如SEQ ID NO:4所示。
本发明还提供了一种单基因双表达病毒载体,包括所述基因的碱基序列。
优选地,所述载体还包括与所述基因操作性相连的调控序列,以便所述载体成功在宿主细胞重组。例如:启动子序列被连接在基因的碱基序列的前端,在宿主细胞内的复制的能力通常由复制起始点控制,用于识别稳定株的筛选基因也可加入载体中。
本发明还提供了一种重组细胞,表达所述的融合蛋白、含有所述的基因、或含有所述的载体。利用该重组细胞,实现了所述融合蛋白和3CL蛋白酶的稳定共表达,确保了两者表达效率的一致性,增加了筛药系统的精确性。
优选地,所述重组细胞为哺乳动物细胞。
更优选地,所述重组细胞为人胚胎肾细胞HEK-293T。
所述的融合蛋白、所述的基因、所述的载体或所述的重组细胞在构建筛选冠状病毒3CL蛋白酶抑制剂的系统(筛药系统)中的应用,也应在本发明的保护范围之内。
本发明还提供了一种筛选冠状病毒3CL蛋白酶抑制剂的系统,包括所述的融合蛋白、所述的基因、所述的载体或所述的重组细胞。
所述筛药系统是一种重组细胞体系,因此,可通过在细胞培养物中加入候选药物(候选冠状病毒3CL蛋白酶抑制剂),观察候选药物是否会在细胞水平上通过改变3CL蛋白酶对融合蛋白的酶切效率而改变具有报告功能的蛋白的总体表达水平,从而筛选潜在的可改变3CL蛋白酶酶切活性的物质;其中,由于融合蛋白中含有便于检测的具有报告功能的蛋白,从而为3CL蛋白酶酶切效率的检测提供了便利途径。
另外,所述系统在筛选冠状病毒3CL蛋白酶抑制剂中的应用,也应在本发明的保护范围之内。
本发明还提供了一种筛选冠状病毒3CL蛋白酶抑制剂的方法,将候选药物与所述系统进行接触,检测候选药物对系统中融合蛋白的具有报告功能的蛋白总体表达水平的影响,若候选药物能够使得细胞水平上融合蛋白的具有报告功能的蛋白总体表达水平降低1.5倍以上,则该候选药物为冠状病毒3CL蛋白酶抑制剂。
具体地,本发明构建了一种细胞水平上基于3CL蛋白酶酶切作用的萤火虫荧光素酶检测系统,并用于候选冠状病毒3CL蛋白酶抑制剂的高通量筛选,泛素化蛋白通过3CL蛋白酶的酶切位点氨基酸片段与萤火虫荧光素酶连接,介导萤火虫荧光素酶的降解,而3CL蛋白酶作用于酶切序列的酶切活性,可以阻断这一降解过程,并且这一酶切活性可以通过萤火虫荧光素酶的活性总体表达水平来体现,对3CL蛋白酶活性具有抑制功能的药物(冠状病毒3CL蛋白酶抑制剂)可降低萤火虫荧光素酶的表达水平,从而可被快速筛选出来。
本发明中,可以采用各种常规仪器和技术手段检测融合蛋白的具有报告功能的蛋白总体表达水平,如酶标仪等。该方法还可以包括进一步进行其他细胞水平或者动物水平实验,以更准确筛选出冠状病毒3CL蛋白酶抑制剂。
本发明具有以下有益效果:
(1)本发明首先提供了一种融合蛋白、其编码基因、包含其的载体或表达其的重组细胞,利用3CL蛋白酶酶切机理及酶切位点的特征,该融合蛋白既保留了泛素化蛋白以及具有报告功能的蛋白的结构功能特性,又能够方便的检测因酶切位点被3CL蛋白酶识别酶切而导致的融合蛋白中泛素化蛋白与具有报告功能的蛋白分离,最终影响具有报告功能的蛋白降解水平,从而为筛选针对3CL蛋白酶活性的抗冠状病毒感染的物质提供了良好的途径。
(2)本发明还提供了一种筛选冠状病毒3CL蛋白酶抑制剂的系统,该系统基于细胞内的固有机制,利用具有报告功能的蛋白的活性重复再现3CL蛋白酶在细胞内的工作状态,能够将3CL蛋白酶酶切效率转化为可视化的具有报告功能的蛋白总体表达水平的检测值,且该系统方便快捷、操作简单,具有良好的特异性和敏感性,适合于高通量筛选抗冠状病毒感染的药物。
(3)另外,本发明还提供了一种筛选冠状病毒3CL蛋白酶抑制剂的方法,该方法能够作为便捷有效的检测筛选抗冠状病毒感染的物质的实验手段,可同时进行高通量的化合物筛选,筛选机理明确,速度快,作用效果直观;且该方法通用性强,重复性好,假阳性率低;因此,该方法不仅可以应用于实验室对抗冠状病毒感染的理论基础研究,还可应用获取针对新型冠状病毒治疗的先导药物、挖掘具有抗新冠病毒活性的已有临床药物,在筛选冠状病毒3CL蛋白酶抑制剂中具有良好的应用前景。
附图说明
图1是同时含有UB(4)-CScon-Fluc基因和3CL蛋白酶基因的单基因双表达病毒载体的图谱。
图2是UB-CS-Fluc融合蛋白与3CL蛋白酶在筛药系统中工作模式图;其中,UB为泛素化蛋白,3CL为3CL蛋白酶,Fluc为萤火虫荧光素酶。
图3是伊马替尼作为阳性对照药物证实筛药系统工作有效性结果图。
图4是UB(4)-Fluc融合蛋白在细胞水平上降解情况结果图。
图5是融合蛋白中酶切位点的插入对荧光素酶降解情况的影响结果图。
图6是在3CL蛋白酶作用下,融合蛋白中酶切位点的插入对荧光素酶降解情况的影响结果图。
图7是分析3CL蛋白酶所识别酶切位点的氨基酸序列结果图。
图8是融合蛋白中泛素化蛋白个数对荧光素酶降解情况的影响结果图。
图9是融合蛋白中酶切位点个数对荧光素酶降解情况的影响结果图。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
以下实施例所用实验材料、仪器及试剂:
单基因双表达病毒载体、菌株E.coli DH5α/Stable3、人胚胎肾细胞HEK-293T均为本实验室保存。
恒温金属浴购自林贝尔公司;电泳仪购自Biorad公司;PCR仪购自Biorad公司;细菌培养箱购自安捷来公司;恒温摇床购自Thermo公司、ND-2000超微量核酸蛋白测定仪及细胞培养箱购自NanoDrop;凝胶成像系统购自天能公司;酶标仪购自BioTek公司。
引物由广州擎科生物技术有限公司合成;限制性内切酶Hind III、Nhe I和BamH I以及T4连接酶、Dpn I酶、Quick CIP酶购自NEB有限公司;pfu聚合酶购自Stratagene技术有限公司;dNTP购自GeneStar公司;DNA胶回收产物纯化试剂盒购自Thermo Scientific公司;质粒提取试剂盒购自天根生化科技有限公司;DMEM培养基、胎牛血清、0.05%胰酶、PBS购自Gibco公司;其它生化试剂主要购自广州擎科生物技术有限公司、生工生物工程(上海)股份有限公司;双荧光素酶报告系统试剂盒购自Promege公司;
实施例1UB(4)-CScon-Fluc融合蛋白与3CL蛋白酶的单基因双表达病毒载体构建
本实施例利用酶切克隆方法实现3CL蛋白酶酶切位点氨基酸序列对应的小核酸片段CScon定向插入到已构建好的表达UB(4)-Fluc融合蛋白的基因中,得到UB(4)-CScon-Fluc基因;再利用无缝克隆技术将构建好的UB(4)-CScon-Fluc基因与单基因双表达病毒载体进行重组,得到含有UB(4)-CScon-Fluc基因的单基因双表达病毒载体;最后再将编码3CL蛋白酶的基因与单基因双表达病毒载体进行重组,得到同时含有UB(4)-CScon-Fluc基因和3CL蛋白酶基因的单基因双表达病毒载体。具体步骤如下:
1、UB(4)-CScon-Fluc基因的克隆
Insert-1(CScon)引物由广州擎科生物技术有限公司合成,正义和反义DNA链退火反应条件:95℃金属浴5min,自然缓慢退火至室温,获得Insert-1(CScon)DNA片段,4℃保存。
Vector-1(UB(4)-Fluc)利用限制性内切酶Hind III(NEB有限公司)酶切条件:37℃气浴2h,对酶切完成的载体进行1.5%琼脂糖凝胶电泳分析。
电泳结果显示:扩增出了与预期大小相符的条带,利用CIP酶(NEB有限公司)处理载体5’末端,减少自连;再按照购买的DNA胶回收纯化试剂盒(ThermoScientific公司)说明书对目的条带进行纯化。
然后,分别将Insert-1(CScon)DNA片段与Vector-1酶切产物按比例混合,4℃连接过夜;连接产物取2.5μL转化DH5α感受态细胞(天根生化(TIANGEN)),在含有卡那抗生素的LB平板上进行筛选,挑取阳性菌落克隆PCR鉴定后,将初步鉴定成功的阳性克隆摇菌提取质粒,送广州擎科生物技术有限公司测序。
测序结果说明:所得重组质粒序列完全正确,并能够在细胞水平中正常表达,表明已成功构建了UB(4)-CScon-Fluc基因。
2、含有UB(4)-CScon-Fluc基因的单基因双表达病毒载体的构建
利用pfu聚合酶(Stratagene公司)PCR反应体系扩展Insert-2(UB(4)-CScon-Fluc),扩增反应条件:95℃预变性2min;95℃变性20s,55℃退火20s,72℃延伸3min;30个循环。72℃,3min,4℃保存。PCR扩增反应完成后,进行1.5%琼脂糖凝胶电泳分析。
电泳结果显示:扩增出了与预期大小相符的条带,Insert-2 PCR扩增产物按照购买的DNA胶回收纯化试剂盒说明书对目的条带进行纯化,PCR扩增产物利用DpnI酶(NEB有限公司)处理除去背景。
利用Nhe I和BamH I(NEB有限公司)对Vector-2(单基因双表达病毒载体)酶切条件:37℃气浴2h。对酶切完成的载体进行1.5%琼脂糖凝胶电泳分析。
电泳结果显示:扩增出了与预期大小相符的条带,再按照购买的DNA胶回收纯化试剂盒说明书对目的条带进行纯化。
然后,按照购买的博迈德重组酶说明书,针对已获得的Insert-2 PCR扩增产物与酶切完成的Vector-2载体进行重组连接:50℃保温15min。连接产物取2.5μL转化Stable3感受态细胞,在含有氨苄抗生素的LB平板上进行筛选,挑取阳性菌落克隆PCR鉴定后,将初步鉴定成功的阳性克隆摇菌提取质粒,送广州擎科生物技术有限公司测序。
测序结果说明:所得重组质粒序列完全正确,表明已成功构建了含有UB(4)-Cscon-Fluc基因的单基因双表达病毒载体。
3、同时含有UB(4)-CScon-Fluc基因和3CL蛋白酶基因的单基因双表达病毒载体的构建
Insert-3(3CL蛋白酶基因)PCR反应条件:98℃预变性2min;98℃变性10s,56℃退火15s,72℃延伸20s;30个循环。72℃,5min,4℃保存。PCR扩增完成后,进行1.5%琼脂糖凝胶电泳分析。
电泳结果显示:扩增出了与预期大小相符的条带,Insert-3 PCR扩增产物按照购买的GeneJET回收纯化试剂盒说明书进行纯化,PCR扩增产物利用DpnI酶处理除去背景。
Vector-3(含有UB(4)-CScon-Fluc基因的单基因双表达病毒载体)酶切条件:37℃气浴2h。对酶切完的载体进行1.5%琼脂糖凝胶电泳分析。
电泳结果显示:扩增出了与预期大小相符的条带,再按照购买的DNA胶回收纯化试剂盒说明书对目的条带进行纯化。
然后,按照购买的博迈德重组酶说明,针对已获得的Insert-3 PCR扩增产物与Vector-3载体进行重组连接:50℃保温15min。连接产物取2.5μL转化Stable3感受态细胞,在含有氨苄抗生素的LB平板上进行筛选,挑取阳性菌落克隆PCR鉴定后,将初步鉴定成功的阳性克隆摇菌提取质粒,送广州擎科生物技术有限公司测序。
测序结果说明:所得重组质粒序列完全正确,表明已成功构建了同时含有UB(4)-CScon-Fluc基因和3CL蛋白酶基因的单基因双表达病毒载体。
同时含有UB(4)-CScon-Fluc基因和3CL蛋白酶基因的单基因双表达病毒载体的图谱如图1所示。
实施例2利用哺乳动物细胞构建稳定表达细胞株
将实施例1构建得到的同时含有UB(4)-CScon-Fluc基因和3CL蛋白酶基因的单基因双表达病毒载体用于稳定表达细胞株的构建,利用病毒转染的方法将其整合到宿主细胞(哺乳动物细胞)染色体上,使宿主细胞可长期表达UB(4)-CScon-Fluc融合蛋白和3CL蛋白酶,构建筛药系统。具体步骤如下:
1、病毒包装
准备状态良好的HEK-293T细胞,在转染前24h进行细胞铺板(根据实验需求选择直径为6厘米的细胞培养皿),取处于对数生长期的HEK-293T细胞进行病毒包装,细胞密度控制在80%左右,取去除内毒素的高质量质粒(包括病毒包装质粒和病毒载体)进行转染,转染完毕放入培养箱中,7h后将培养皿中的培养液移弃,加入新鲜的适量的培养液。每隔24h收集一次病毒颗粒上清,收集3次,于12000rpm离心10分钟去除细胞碎片,得到的病毒颗粒上清放置于-80℃保存待用。
2、细胞感染
准备状态良好的HEK-293T细胞,在感染前21h进行细胞铺板,控制感染时细胞密度在80%左右,加入适量步骤1中所获得的含有病毒的细胞上清及polybrene(10ug/mL)进行病毒对靶细胞的感染,培养24h后,用新鲜培养基替换含有病毒的培养基继续培养。
3、药物筛选
待已感染完毕的细胞生长状态稳定后,利用嘌呤霉素对成功整合目的基因(UB(4)-CScon-Fluc融合基因和3CL蛋白酶基因)的细胞进行筛选,得到符合实验要求的稳定高表达目的蛋白(UB(4)-CScon-Fluc融合蛋白和3CL蛋白酶)的细胞株,扩大培养并冻存。
得到了稳定高表达目的蛋白的细胞株后,筛药系统模型已基本成立完毕,可直接用于抗冠状病毒感染药物的高通量筛选。
4、鉴定稳定表达细胞株
利用PCR和Western blot技术对筛选得到的稳定高表达目的蛋白的细胞株在核酸水平以及蛋白水平进行检测。
实施例3抗冠状病毒感染药物的高通量筛选
1、药物处理
准备状态良好的实施例2得到的稳定高表达目的蛋白的细胞株,在药物处理前21h进行细胞铺板(根据实验需求选择细胞培养皿),根据药物溶解性等特质分别用适当浓度的所得候选药物对细胞进行处理12h。
2、荧光素酶检测
弃去原培养基,用PBS溶液轻轻洗去残留的培养基,按照双荧光素酶报告基因检测试剂盒说明书,检测细胞水平上荧光素酶的总体表达水平,采用伊马替尼(Imatinib)作为阳性对照,与阴性对照(不添加所述候选药物的细胞)进行数据分析对比,筛选能够使得荧光素酶总体表达水平下调的可能抗冠状病毒感染的药物。
UB-CS-Fluc融合蛋白与3CL蛋白酶在筛药系统中工作模式图如图2所示。
伊马替尼作为阳性对照药物证实筛药系统工作有效性结果如图3所示,可以看出,伊马替尼作为候选药物具有抑制3CL蛋白酶酶切活性的作用。
以上结果说明:本发明构建得到的抗冠状病毒感染药物的高通量筛选系统是可行的。
实施例4单基因双表达病毒载体构建条件的优化
1、实验方法
利用实施例2同样的实验方法,构建单独稳定高表达UB(4)-Fluc的细胞株,在药物处理前21h进行细胞铺板(根据实验需求选择细胞培养皿),利用MG132(终浓度0.5μM)对细胞进行处理12h,检测细胞水平上荧光素酶的降解情况。
利用实施例2同样的实验方法,构建单独稳定高表达UB(4)-CS-Fluc的细胞株,与上述构建的单独稳定高表达UB(4)-Fluc的细胞株分别进行细胞铺板(根据实验需求选择细胞培养皿),检测比较细胞水平上荧光素酶的总体表达水平。
利用实施例2同样的实验方法,构建稳定高表达UB(4)–Fluc与3CL蛋白酶的细胞株,与上述构建的稳定高表达UB(4)-CS-Fluc与3CL蛋白酶的细胞株分别进行细胞铺板(根据实验需求选择细胞培养皿),检测比较细胞水平上荧光素酶的总体表达水平。
利用实施例2同样的实验方法,构建稳定高表达UB(1)-CS-Fluc或UB(2)-CS-Fluc与3CL蛋白酶的细胞株,与上述构建的稳定高表达UB(4)-CS-Fluc与3CL蛋白酶的细胞株分别进行细胞铺板(根据实验需求选择细胞培养皿),检测比较细胞水平上荧光素酶的总体表达水平。
利用实施例2同样的实验方法,构建稳定高表达UB(4)-CS(2)-Fluc或UB(4)–CS(3)-Fluc与3CL蛋白酶的细胞株,与上述构建的稳定高表达UB(4)-CS-Fluc(即为UB(4)-CS(1)-Fluc)与3CL蛋白酶的细胞株及稳定高表达UB(4)–Fluc(即为UB(4)-CS(0)-Fluc)与3CL蛋白酶的细胞株分别进行细胞铺板(根据实验需求选择细胞培养皿),检测比较细胞水平上荧光素酶的总体表达水平。
2、实验结果
UB(4)-Fluc融合蛋白在细胞水平上降解情况结果如图4所示,融合蛋白中酶切位点的插入对荧光素酶降解情况的影响结果如图5所示,结果表明:细胞水平上,融合蛋白中荧光素酶通过泛素化蛋白被蛋白酶解体降解,3CL蛋白酶酶切位点的插入对融合蛋白的表达水平没有显著影响。
在3CL蛋白酶作用下,融合蛋白中酶切位点的插入对荧光素酶降解情况的影响结果如图6所示,结果表明:在3CL蛋白酶的作用下,融合蛋白中有无酶切位点的插入直接影响荧光素酶降解情况,3CL蛋白酶通过酶切融合蛋白中的酶切位点,降低融合蛋白泛素化降解情况。
分析3CL蛋白酶所识别酶切位点的氨基酸序列结果如图7所示,根据3CL蛋白酶所识别酶切位点的氨基酸序列,统计分析得到了可能使其酶切效率达到最优的CScon的氨基酸序列:NVATLQSANAT,其编码基因的碱基序列为aat gtg gca act tta caa gct gaa aatgta aca。
融合蛋白中泛素化蛋白个数对荧光素酶降解情况的影响结果如图8所示,结果表明:在3CL蛋白酶的作用下,融合蛋白中泛素化蛋白个数直接影响荧光素酶降解情况,泛素化蛋白融合个数与融合蛋白的降解水平呈正相关。
融合蛋白中酶切位点个数对荧光素酶降解情况的影响结果如图9所示,结果表明:在3CL蛋白酶的作用下,融合蛋白中酶切位点个数直接影响3CL蛋白酶对融合蛋白的酶切效率,酶切位点个数与3CL蛋白酶对融合蛋白的酶切效率呈正相关。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 中山大学
<120> 一种融合蛋白及其在构建筛选冠状病毒3CL 蛋白酶抑制剂的系统中的应用
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Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe
690 695 700
Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val
705 710 715 720
Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly
725 730 735
Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly
740 745 750
Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe
755 760 765
Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln
770 775 780
Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile
785 790 795 800
Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu
805 810 815
Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys
820 825 830
Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu
835 840 845
Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly
850 855 860
Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys
865 870 875 880
Gly Gly Lys Ile
<210> 4
<211> 2652
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgcagatct tcgtgaagac tctgactggt aagaccatca ccctcgaggt tgagcccagt 60
gacaccattg agaatgtcaa ggcaaagatc caagataagg aaggcatccc tcctgaccag 120
cagaggctga tctttgctgg aaaacagctg gaagatgggc gcaccctgtc tgactacaac 180
atccagaaag agtccaccct gcacctggta ctccgtctca gaggtgtggt gactagattc 240
accatgcaga tcttcgtgaa gactctgact ggtaagacca tcaccctcga ggttgagccc 300
agtgacacca ttgagaatgt caaggcaaag atccaagata aggaaggcat ccctcctgac 360
cagcagaggc tgatctttgc tggaaaacag ctggaagatg ggcgcaccct gtctgactac 420
aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgt ggtgactaga 480
ttcaccatgc agatcttcgt gaagactctg actggtaaga ccatcaccct cgaggttgag 540
cccagtgaca ccattgagaa tgtcaaggca aagatccaag ataaggaagg catccctcct 600
gaccagcaga ggctgatctt tgctggaaaa cagctggaag atgggcgcac cctgtctgac 660
tacaacatcc agaaagagtc caccctgcac ctggtactcc gtctcagagg tgtggtgact 720
agattcacca tgcagatctt cgtgaagact ctgactggta agaccatcac cctcgaggtt 780
gagcccagtg acaccattga gaatgtcaag gcaaagatcc aagataagga aggcatccct 840
cctgaccagc agaggctgat ctttgctgga aaacagctgg aagatgggcg caccctgtct 900
gactacaaca tccagaaaga gtccaccctg cacctggtac tccgtctcag aggtgtggtg 960
actagtaagc ttaatgtggc aactttacag agtgctaatg ctactaagct tgaagacgcc 1020
aaaaacataa agaaaggccc ggcgccattc tatccgctgg aagatggaac cgctggagag 1080
caactgcata aggctatgaa gagatacgcc ctggttcctg gaacaattgc ttttacagat 1140
gcacatatcg aggtggacat cacttacgct gagtacttcg aaatgtccgt tcggttggca 1200
gaagctatga aacgatatgg gctgaataca aatcacagaa tcgtcgtatg cagtgaaaac 1260
tctcttcaat tctttatgcc ggtgttgggc gcgttattta tcggagttgc agttgcgccc 1320
gcgaacgaca tttataatga acgtgaattg ctcaacagta tgggcatttc gcagcctacc 1380
gtggtgttcg tttccaaaaa ggggttgcaa aaaattttga acgtgcaaaa aaagctccca 1440
atcatccaaa aaattattat catggattct aaaacggatt accagggatt tcagtcgatg 1500
tacacgttcg tcacatctca tctacctccc ggttttaatg aatacgattt tgtgccagag 1560
tccttcgata gggacaagac aattgcactg atcatgaact cctctggatc tactggtctg 1620
cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga gattctcgca tgccagagat 1680
cctatttttg gcaatcaaat cattccggat actgcgattt taagtgttgt tccattccat 1740
cacggttttg gaatgtttac tacactcgga tatttgatat gtggatttcg agtcgtctta 1800
atgtatagat ttgaagaaga gctgtttctg aggagccttc aggattacaa gattcaaagt 1860
gcgctgctgg tgccaaccct attctccttc ttcgccaaaa gcactctgat tgacaaatac 1920
gatttatcta atttacacga aattgcttct ggtggcgctc ccctctctaa ggaagtcggg 1980
gaagcggttg ccaagaggtt ccatctgcca ggtatcaggc aaggatatgg gctcactgag 2040
actacatcag ctattctgat tacacccgag ggggatgata aaccgggcgc ggtcggtaaa 2100
gttgttccat tttttgaagc gaaggttgtg gatctggata ccgggaaaac gctgggcgtt 2160
aatcaaagag gcgaactgtg tgtgagaggt cctatgatta tgtccggtta tgtaaacaat 2220
ccggaagcga ccaacgcctt gattgacaag gatggatggc tacattctgg agacatagct 2280
tactgggacg aagacgaaca cttcttcatc gttgaccgcc tgaagtctct gattaagtac 2340
aaaggctatc aggtggctcc cgctgaattg gaatccatct tgctccaaca ccccaacatc 2400
ttcgacgcag gtgtcgcagg tcttcccgac gatgacgccg gtgaacttcc cgccgccgtt 2460
gttgttttgg agcacggaaa gacgatgacg gaaaaagaga tcgtggatta cgtcgccagt 2520
caagtaacaa ccgcgaaaaa gttgcgcgga ggagttgtgt ttgtggacga agtaccgaaa 2580
ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga tcctcataaa ggccaagaag 2640
ggcggaaaga tc 2652
Claims (13)
1.一种融合蛋白,其特征在于,将N端的泛素化蛋白和C端的具有报告功能的蛋白通过3CL蛋白酶酶切位点氨基酸片段融合而成;所述3CL蛋白酶酶切位点氨基酸片段为3CL蛋白酶能够识别并进行酶切的氨基酸片段。
2.根据权利要求1所述的融合蛋白,其特征在于,所述3CL蛋白酶酶切位点氨基酸片段的序列为:SEQ ID NO:1所示的氨基酸序列、与SEQ ID NO:1所示的氨基酸序列的同源性大于90%的氨基酸序列或将SEQ ID NO:1所示的氨基酸序列经人工点突变的方式进行改造后得到的氨基酸序列。
3.根据权利要求1所述的融合蛋白,其特征在于,所述泛素化蛋白为人源泛素化蛋白;所述泛素化蛋白的个数为1~20。
4.根据权利要求1所述的融合蛋白,其特征在于,所述3CL蛋白酶酶切位点的个数为1~3。
5.根据权利要求1~4任一所述的融合蛋白,其特征在于,所述融合蛋白为UB(4)-CScon-Fluc融合蛋白,其氨基酸序列如SEQ ID NO:3所示,其编码基因的碱基序列如SEQ IDNO:4所示。
6.编码权利要求1~5任一所述融合蛋白的基因,其特征在于,所述基因的碱基序列为通过编码3CL蛋白酶酶切位点的碱基序列将编码泛素化蛋白的基因的碱基序列和编码具有报告功能的蛋白的基因的碱基序列同框连接而成。
7.根据权利要求6所述的基因,其特征在于,所述编码3CL蛋白酶酶切位点的碱基序列为:SEQ ID NO:2所示的碱基序列、与SEQ ID NO:2所示的碱基序列具有简并性的碱基序列、与SEQ ID NO:2所示的碱基序列的同源性大于60%的碱基序列或将SEQ ID NO:2所示的碱基序列经人工突变点的方式进行改造后得到的碱基序列。
8.一种单基因双表达病毒载体,其特征在于,包括权利要求6或7所述基因的碱基序列。
9.一种重组细胞,其特征在于,表达权利要求1~5任一所述的融合蛋白、含有权利要求6或7所述的基因、或含有权利要求8所述的载体。
10.权利要求1~5任一所述的融合蛋白、权利要求6或7所述的基因、权利要求8所述的载体或权利要求9所述的重组细胞在构建筛选冠状病毒3CL蛋白酶抑制剂的系统中的应用。
11.一种筛选冠状病毒3CL蛋白酶抑制剂的系统,其特征在于,包括权利要求1~5任一所述的融合蛋白、权利要求6或7所述的基因、权利要求8所述的载体或权利要求9所述的重组细胞。
12.权利要求11所述系统在筛选冠状病毒3CL蛋白酶抑制剂中的应用。
13.一种筛选冠状病毒3CL蛋白酶抑制剂的方法,其特征在于,将候选药物与所述系统进行接触,检测候选药物对系统中融合蛋白的具有报告功能的蛋白总体表达水平的影响,若候选药物能够使得细胞水平上融合蛋白的具有报告功能的蛋白总体表达水平降低1.5倍以上,则该候选药物为冠状病毒3CL蛋白酶抑制剂。
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