CN107247143A - 二价金属离子对寨卡病毒非结构蛋白酶活性的影响 - Google Patents
二价金属离子对寨卡病毒非结构蛋白酶活性的影响 Download PDFInfo
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Abstract
利用荧光共振能量转移技术,研究多种二价金属离子对寨卡病毒非结构蛋白的抑制活性的影响,实验结果显示:包括Zn2+、Mg2+、Ca2+、Mn2+金属离子对寨卡病毒非结构蛋白的活性有不同程度的抑制效果。
Description
技术领域
本发明涉及酶学及药学的技术领域,具体说是研究多种二价金属离子对寨卡病毒非结构蛋白酶活性的影响。
背景技术
寨卡病毒属黄病毒科,黄病毒属,单股正链RNA病毒,直径20nm,是一种通过蚊虫进行传播的虫媒病毒,该病毒最早于1947年通过黄热病监测网络偶然在乌干达维多利亚湖畔寨卡丛林的恒河猴身上发现,随后于1952年在乌干达和坦桑尼亚人群中发现。2016年2月9日,我国国家卫生和计划生育委员会专家根据江西赣州1例34岁男性患者的流行病学史、临床表现,以及中国疾病预防控制中心标本复核检测结果,确诊该病例为我国大陆首例输入性寨卡病毒感染病例。由于多例寨卡病毒感染病例的出现,寨卡病毒感染已经引起我国政府与卫生部门的广泛关注。传播媒介伊蚊在我国广泛分布,该病毒传入我国并造成传播的风险极高,但该病毒尚无疫苗和特异性治疗方法,现阶段主要以防控为主。寨卡病毒感染通常症状较轻,无特殊处理。但寨卡病毒感染后潜在的神经系统危害较大,因此找到可以治疗和预防寨卡病毒的药物日益重要。实验证明二价金属离子Zn2+、Mg2+、Ca2+等金属离子对寨卡病毒非结构蛋白酶的活性有不同程度的抑制。
发明内容
本发明要解决的技术问题是提供了多种金属离子对寨卡病毒非结构蛋白NS2B-NS3的影响,并对其影响程度进行分析测定。
本发明为解决公知技术中存在的技术问题所采取的技术方案是:
本发明针对的多种二价金属离子对寨卡病毒非结构蛋白NS2B-NS3 影响,多种二价金属离子包括包括Zn2+、Mg2+、Ca2+、Mn2+。
本发明还可采用以下技术措施:
Zn2+是非竞争性可逆抑制剂。
Mg2+、Ca2+是竞争性可逆抑制剂。
Cu2+、Mn2+对寨卡病毒蛋白活性有抑制活性。
本发明是多种二价金属离子在寨卡病毒非结构蛋白NS2B-NS3抑制剂中的应用。
本发明采用的寨卡病毒非结构蛋白NS2B-NS3蛋白酶的表达、纯化等方法步骤及酶活测定方法参考文献方法(Xia C,Yang K,Chen W,et al. Mechanisms of activationand inhibition of Zika virus NS2B-NS3 protease[J].Cell Research,2016,26(11):1260.)寨卡病毒的非结构蛋白酶的活性测定采用纯度大于95%的荧光底物AC-LKRR-AMC(纯度大于 95%,上海吉尔生化有限公司)作为底物;荧光强度测定的仪器为 FluoraskanAscent酶标仪,激发光和发射光的波长分别为355nm和460 nm,蛋白和化合物震荡25s,孵育10min,用多孔道移液枪将底物加入蛋白中,每10s读一次点,共读100个点;蛋白浓度为100nM,荧光底物选用多个浓度,分别进行测定;
首先测定Zn2+、Mg2+、Ca2+抑制类型,蛋白缓冲液组分为10mM Tris, 20%体积百分数的甘油,pH=9.0,1mM CHAPS;所用体系为40ul蛋白加50ul抑制剂加10ul底物;
测定Zn2+、Mg2+、Ca2+抑制类型,选用蛋白浓度为100nM,在锌离子浓度为0μM、25μM、50μM情况下,寨卡病毒非结构蛋白酶的Km、 Vmax值,底物浓度为150μM、100μM、50μM、25μM、12.5μM、6.25 μM;
利用graphpad prism 6.0处理数据,nonlinear regression中straight line 拟合荧光变化速率slope,以底物浓度为横坐标,slope为纵坐标,利用 graphpad prism 6.0中nonlinear regression中Michaelis-Menten拟合非结构蛋白酶的Km、Vmax值;通过以上数据处理可知在加入不同浓度的 Zn2+后,非结构蛋白酶的Km基本不变,Vmax随锌离子浓度增大而减小,得出锌离子为非竞争性可逆抑制剂的结论,如图1、2;
加入不同浓度Mg2+和Ca2+后,非结构蛋白酶的Km值随离子浓度增大而增大,得出Mg2 +和Ca2+为竞争性可逆抑制剂的结论,如图3和4,图5和6;
测定锌离子浓度为0μM、25μM、50μM、100μM、200μM下寨卡病毒非结构蛋白的Km、Vmax的值;
测定三组独立实验,每组独立实验包括三个副孔,并计算三组实验间的标准偏差和相对标准偏差;
抑制剂浓度为横坐标,以slope为纵坐标得到一条直线,当y=0时对应的x值为-Ki;以抑制剂浓度为横坐标,以1/Vmax为纵坐标得到一条直线,当y=0时对应的x值为-αKi,因此得到Ki=126μM,α=0.85,IC50= 113μM,如图7和8;
测定Mg2+、Ca2+的IC50,寨卡病毒非结构蛋白酶的浓度为100nM,金属离子浓度为5000μM、2500μM、1250μM、625μM、312.5μM、156.25 μM、78.125μM、39.0625μM、19.53125μM、0μM;底物浓度为50μM,所用体系为40μl蛋白加50μl抑制剂加10μl底物;
以时间为横坐标,荧光值为纵坐标,利用graphpad prism 6.0中 nonlinearregression中straight line拟合荧光变化速率slope,计算各抑制剂浓度下的抑制活性,以及抑制剂浓度的Log值Log[I],以Log[I]为横坐标,抑制活性为纵坐标,通过variable-slope拟合IC50,如图9和10;
蛋白缓冲液组分为10mM Tris,20%v0/v的甘油,pH=7.2,1mM CHAPS;所用体系为40ul蛋白加50ul抑制剂加10ul底物;
定性测验了锰离子对寨卡病毒蛋白酶的抑制活性,利用梯度稀释检测分别带有306.25μM Mn2+蛋白酶和无抑制剂蛋白酶的活性,加入上述二价离子的蛋白酶活性降低Zn2+是非竞争性可逆抑制剂。Mg2+、Ca2+是竞争性可逆抑制剂。
Zn2+抑制寨卡病毒蛋白酶活性的IC50值为113μM。
Ca2+抑制寨卡病毒蛋白酶活性的IC50值为400μM。
Mg2+抑制寨卡病毒蛋白酶活性的IC50值为807μM。
Mn2+对寨卡病毒蛋白活性有抑制活性。
本发明具有的优点和积极效果是:
本发明的针对多种二价金属离子对寨卡病毒非结构蛋白NS2B-NS3 影响的研究中,突破传统的化学治疗药物,利用锌离子等二价金属离子研究寨卡病毒的治疗药物的开发,为开发以锌离子为辅助药物做出了一定的贡献。本发明是多种二价金属离子在寨卡病毒非结构蛋白 NS2B-NS3抑制剂中的应用。
附图说明
图1、2是Zn2+的抑制类型测试图,图1为Michaelis-Menten图,图2为其双倒数图;
图3、4是Ca2+的抑制类型测试图,图3为Michaelis-Menten图,图4为其双倒数图;
图5、6是Mg2+的抑制类型测试图,图5为Michaelis-Menten图,图6为其双倒数图;
图7、8是Zn2+的抑制效果图
图9、10是Ca2+、Mg2+的IC50图;
图11是Mn2+的抑制效果对比图;
具体实施方式
下面将详细描述本发明的具体实施方式,这仅用于解释本发明,而不能解释为对本发明的限制。
本发明针对的多种二价金属离子对寨卡病毒非结构蛋白NS2B-NS3 影响,多种二价金属离子包括包括Zn2+、Mg2+、Ca2+、Mn2+。
Zn2+是非竞争性可逆抑制剂。
Mg2+、Ca2+是竞争性可逆抑制剂。
Mn2+对寨卡病毒蛋白活性有抑制活性。
本发明是多种二价金属离子在寨卡病毒非结构蛋白NS2B-NS3抑制剂中的应用。
本发明采用的寨卡病毒非结构蛋白NS2B-NS3蛋白酶的表达、纯化等方法步骤及酶活测定方法参考文献方法(Xia C,Yang K,Chen W,et al. Mechanisms of activationand inhibition of Zika virus NS2B-NS3 protease[J].Cell Research,2016,26(11):1260.)寨卡病毒的非结构蛋白酶的活性测定采用纯度大于95%的荧光底物AC-LKRR-AMC(纯度大于 95%,上海吉尔生化有限公司)作为底物;荧光强度测定的仪器为 FluoraskanAscent酶标仪,激发光和发射光的波长分别为355nm和460 nm,蛋白和化合物震荡25s,孵育10min,用多孔道移液枪将底物加入蛋白中,每10s读一次点,共读100个点;蛋白浓度为100nM,荧光底物选用多个浓度,分别进行测定;
首先测定Zn2+、Mg2+、Ca2+抑制类型,蛋白缓冲液组分为10mM Tris, 20%体积百分数的甘油,pH=9.0,1mM CHAPS;所用体系为40ul蛋白加50ul抑制剂加10ul底物;
测定Zn2+、Mg2+、Ca2+抑制类型,选用蛋白浓度为100nM,在锌离子浓度为0μM、25μM、50μM情况下,寨卡病毒非结构蛋白酶的Km、 Vmax值,底物浓度为150μM、100μM、50μM、25μM、12.5μM、6.25 μM;
利用graphpad prism 6.0处理数据,nonlinear regression中straight line 拟合荧光变化速率slope,以底物浓度为横坐标,slope为纵坐标,利用 graphpad prism 6.0中nonlinear regression中Michaelis-Menten拟合非结构蛋白酶的Km、Vmax值;
通过以上数据处理可知在加入不同浓度的Zn2+后,非结构蛋白酶的 Km基本不变,Vmax随锌离子浓度增大而减小,得出锌离子为非竞争性可逆抑制剂的结论,如图1、2;
加入不同浓度Mg2+和Ca2+后,非结构蛋白酶的Km值随离子浓度增大而增大,得出Mg2 +和Ca2+为竞争性可逆抑制剂的结论,如图3和4,图5和6;
测定锌离子浓度为0μM、25μM、50μM、100μM、200μM下寨卡病毒非结构蛋白的Km、Vmax的值;
测定三组独立实验,每组独立实验包括三个副孔,并计算三组实验间的标准偏差和相对标准偏差;
抑制剂浓度为横坐标,以slope为纵坐标得到一条直线,当y=0时对应的x值为-Ki;以抑制剂浓度为横坐标,以1/Vmax为纵坐标得到一条直线,当y=0时对应的x值为-αKi,因此得到Ki=126μM,α=0.85,IC50= 113μM,如图7和8;
测定Mg2+、Ca2+的IC50,寨卡病毒非结构蛋白酶的浓度为100nM,金属离子浓度为5000μM、2500μM、1250μM、625μM、312.5μM、156.25 μM、78.125μM、39.0625μM、19.53125μM、0μM,底物浓度为50μM,所用体系为40μl蛋白加50μl抑制剂加10μl底物;
以时间为横坐标,荧光值为纵坐标,利用graphpad prism 6.0中 nonlinearregression中straight line拟合荧光变化速率slope,计算各抑制剂浓度下的抑制活性,以及抑制剂浓度的Log值Log[I],以Log[I]为横坐标,抑制活性为纵坐标,通过variable-slope拟合IC50,如图9和10;
蛋白缓冲液组分为10mM Tris,20%v0/v的甘油,pH=7.2,1mM CHAPS;所用体系为40ul蛋白加50ul抑制剂加10ul底物;
定性测验了锰离子对寨卡病毒蛋白酶的抑制活性,利用梯度稀释检测分别带有306.25μM Mn2+蛋白酶和无抑制剂蛋白酶的活性,加入上述二价离子的蛋白酶活性降低,如图11;
本文中所涉及的各种实验用品(包括但不限于:化学试剂、生物制品、细胞、生物体、仪器等)之中,对于那些特殊的或者不宜获得的,文中均已注明了制造商、参考文献或详细的制备方法;未经特别说明的,均为常规实验用品,在本发明申请日之前,可以通过各种方式(例如购买、自行制备等)很方便地获得。在不偏离本发明的精神和范围的情况下,本领域的普通技术人员可以在形式和细节上对其做出各种改变和改进,而这些均被认为落入了本发明的保护范围。
Claims (4)
1.多种二价金属离子对寨卡病毒非结构蛋白的活性的影响,其特征在于:金属离子包括Zn2+、Mg2+、Ca2+、Mn2+。
2.根据权利要求1所述的多种离子对寨卡病毒非结构蛋白影响,其特征在于Zn2+、Mg2+、Ca2+、Mn2+都对寨卡病毒非结构蛋白酶活有抑制效果,如表1。
3.根据权利要求书2所述的Zn2+对寨卡病毒非结构蛋白酶活有抑制效果,其特征在于Zn2+是非竞争性抑制剂。
4.根据权利要求书2所述的Mg2+、Ca2+对寨卡病毒非结构蛋白酶活有抑制效果,其特征在于Mg2+、Ca2+是竞争性抑制剂;
表1
*-为抑制影响,+为促进影响
注:因为在pH=9.0的情况下,Mn2+不能以二价离子形式存在,所以将缓冲液的pH调整为7.2,定性的测定了其抑制效果。
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