CN112741893B - 一种预防冠状病毒的多肽消毒剂组合物 - Google Patents
一种预防冠状病毒的多肽消毒剂组合物 Download PDFInfo
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Abstract
本发明公开了一种预防冠状病毒的多肽消毒剂组合物,其有效成分包括由如SEQ ID NO.01所示的第一多肽、如SEQ ID NO.02所示的第二多肽和如SEQ ID NO.03所示的第三多肽组成的混合多肽。本发明成本低廉,适用范围广,并且能有效预防冠状病毒。本发明的混合多肽的组分均是由六个氨基酸残基组成的小肽,易于生产合成,成本低廉,适用范围广,并且能有效预防冠状病毒。
Description
技术领域
本发明属于病毒消毒剂技术领域,具体涉及一种预防冠状病毒的多肽消毒剂组合物。
背景技术
冠状病毒属的病毒是具备囊膜、基因组为线性单股正链的RNA病毒,是自然界广泛存在的一大类病毒。到目前为止已知有其中能够感染人类的冠状病毒,分别是HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV以及2019-nCOV,其中SARS-CoV、MERS-CoV以及2019-nCoV属于β-冠状病毒,具有极强的感染能力,对人类健康具有极大的危害并且造成极严重的经济损失,同时,根据已有冠状病毒的出现规律,在往后几十年内将交替出现不同类别的冠状病毒,从而严重影响人类社会的发展。并且鉴于目前该类别冠状病毒并无特效药,因此探索更有效的预防手段无疑是应对冠状病毒的最佳手段。
发明内容
本发明的目的在于克服现有技术缺陷,提供一种预防冠状病毒的多肽消毒剂组合物。
本发明的技术方案如下:
一种预防冠状病毒的多肽消毒剂组合物,其有效成分包括由如SEQ ID NO.01所示的第一多肽、如SEQ ID NO.02所示的第二多肽和如SEQ ID NO.03所示的第三多肽组成的混合多肽,
其中,第一多肽的浓度为21-22μM,第二多肽的浓度为25-26μM,第三多肽的浓度为36-37μM。
在本发明的一个优选实施方案中,所述第一多肽的浓度为21.38μM。
在本发明的一个优选实施方案中,所述第二多肽的浓度为25.30μM。
在本发明的一个优选实施方案中,所述第三多肽的浓度为36.82μM。
在本发明的一个优选实施方案中,还包括金银花、薄荷脑、青黛、硼酸、甘油和水。
本发明的另一技术方案如下:
一种预防冠状病毒的多肽消毒剂组合物,其有效成分包括由如SEQ ID NO.01所示的第一多肽和如SEQ ID NO.03所示的第三多肽组成的混合多肽,
其中,第一多肽的浓度为21-22μM,第三多肽的浓度为36-37μM。
在本发明的一个优选实施方案中,所述第一多肽的浓度为21.38μM。
在本发明的一个优选实施方案中,所述第三多肽的浓度为36.82μM。
在本发明的一个优选实施方案中,所述第一多肽的浓度为21.38μM,所述第三多肽的浓度为36.82μM。
在本发明的一个优选实施方案中,还包括金银花、薄荷脑、青黛、硼酸、甘油和水。
本发明的有益效果是:
1、本发明的混合多肽的组分均是由六个氨基酸残基组成的小肽,易于生产合成,成本低廉,适用范围广,并且能有效预防冠状病毒。
2、本发明的辅料来源简单易得,使得本发明具有高效的预防作用以及良好的推广使用价值。
附图说明
图1为本发明实施例1中的Peptide1与hACE2的结合能力图。
图2为本发明实施例1中的Peptide2与hACE2的结合能力图。
图3为本发明实施例2的hACE2-His与S1-HA纯化蛋白体外结合实验的结果图。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1基于Biacore分子结合系统检测Peptide1&2及其衍生物与hACE2的结合能力
仪器:GE Biacore T200。
材料及试剂:Series S Sensor Chip CM5(GE Life100530)、hACE2纯化蛋白、peptide1&2其衍生物(表1所示)、Amine Coupling Kit(GELife100050、PBS、甘氨酸-盐酸缓冲溶液(pH 2.5)。
表1
实验方法:
(1)捕获表面的制备和结合实验
材料:EDC、NHS、氨基乙醇(氨基偶联试剂盒)、PBS、hACE2、Series S Sensor ChipCM5。
具体步骤:
a、启动Biacore T200,将Series S Sensor Chip CM5嵌入Biacore T200中,在实验过程中全程使用除气的PBS缓冲液;
b、按照系统要求将NHS、EDC、乙醇胺和hACE2(1mg/mL)按照系统提示的体积,用EP管足量添加后置于Biacore T200的进样板上;
c、以5μL/min的流速进样,依次完成Series S Sensor Chip CM5活化、hACE2的氨基偶联以及用乙醇胺饱和未偶联上hACE2的活化羧基,获得hACE2的RUs值为14826.0;
d、关闭流动液,关闭命令窗口,保存报告,获得CM5-hACE2芯片;
(2)表1的多肽与hACE的亲和力分析
材料:PBS、CM5-hACE2芯片、上述表1中的多肽、甘氨酸-盐酸缓冲液(pH2.5)。
具体步骤:
a、制备样本检测用稀释液:用PBS溶解待测的表1中的多肽,浓度为3mM,并依次稀释为I00μM、60μM、30μM、10μM、3μM、1μM、0.3μM、0的不同稀释液,每一份均保证体积为400μL及以上,分装于EP管上,将样品置于BiacoreT200的检测板上,将该检测板卡入检测槽中;
b、载入CM5-hACE2芯片,设定检测程序为样品进样流速30μL/min,进样120s,解离时间设定为300s,而后用甘氨酸-盐酸缓冲液按照30μL/min流速进样30s再生芯片,按照相同程序依次对样品不同浓度进行检测,根据不同浓度获得的RU值进行拟合,获得亲和力常数KD值。(如表2和3所示,其中Peptide1与hACE2的结合能力如图1所示,Peptide2与hACE2的结合能力如图2所示)
表2
表3
实施例2利用免疫共沉淀阐释Peptide1&2及其衍生物以及多肽间不同配比下抑制S1蛋白结合hACE2的机制
材料及试剂:hACE2-His纯化蛋白、Peptide1&2及其衍生物、S1-HA纯化蛋白、PBS、5%BSA封闭液、1.5MTris(pH8.8)、1.0M Tris(pH6.8)、10%SDS、10%过硫酸铵、抗体洗脱液、DAB辣根过氧化物酶显色试剂盒(beyotime P0202)、SDS-Gly电泳液、电转液、PVDF膜(Beyotime,FFP28)、anti-6XHis(Abcam,ab18184)、anti-HA tag(Abcam,ab9110)、HA-tagagarose beads(Engibody,AT0079-1mL)、SDS-PAGE蛋白上样缓冲液(Beyotime,P0286-2mL)。
实验方法:
(1)hACE2-His与S1-HA纯化蛋白体外结合实验
材料:PBS、hACE2-His、S1-HA、HA-tag agarose beads、SDS-PAGE蛋白上样缓冲液。
具体步骤:分装两管30μLHAagarosebeads置于冰上备用,分装两管等量hACE2,而后在一管中加入等量S1-HA蛋白,另一管加入与S1-HA等量体积的PBS,分别混匀后在水浴锅37℃水浴加热30min,而后取出十分之一样本冻存在-80℃作为Input,将剩余组分分别与两管Beads混匀,补足体积到450μL,而后在4℃进行IP过夜,于次日用PBS洗涤beads外游离蛋白,加入100μL PBS以及蛋白上样缓冲液,连同Input样本加热变性后进行Westernblot检测,分别利用anti-His以及anti-HA检测Input以IP样本中对照组以及实验组的hACE2-His以及S1-HA含量,如图3。
(2)检测在上述IP体系中加入Peptide1&2及其衍生物后,hACE2-His以及S1-HA的结合情况
材料:PBS、hACE2-His、S1-HA、HA-tag agarose beads、SDS-PAGE蛋白上样缓冲液、Peptide1&2及其衍生物。
具体步骤:分装10管等量HA-tag agarose beads用PBS洗涤后置于冰上备用,而后分装10管等量hACE2-His蛋白,分别加入Peptide1∶Peptide2(50.60μM∶94.97μM)、Peptide1∶Peptide12(50.60μM∶21.38μM)、Peptide12∶Peptide13(21.38μM∶25.30μM)、Peptide1∶Peptide11(50.60μM∶60.70μM)、Peptide1∶Peptide21(50.60μM∶36.82μM)、Peptide12∶Peptide21(21.38μM∶36.82μM)、Peptide11∶Peptide22+Peptide2(60.70μM∶58.37μM∶94.97μM)、Peptide12∶Peptide13∶Peptide21(21.38μM∶25.30μM∶36.82μM)混匀,将未加多肽的1管中加入PBS作为blank,其余9管分别加入S1-HA蛋白水浴37℃加热30min,其中未加多肽的实验组为阳性对照组,10管混合物中分别取出十分之一作为Input冻存在-80℃,其余组分与Beads混匀后补足450μL在4℃过夜进行免疫共沉淀,次日进行Westernblot检测,检测各组Input与IP样本中hACE2-His以及S1-HA的含量,将阳性对照及实验组的hACE2-His灰度值减去Blank组的相应灰度值,而后得到的实验组灰度值比去阳性对照组灰度值,最后得出各实验组中多肽组合后抑制S1-HA与hACE2-His结合的能力,如表4。
表4
实施例3
一种预防冠状病毒的多肽消毒剂组合物,由混合多肽、金银花、薄荷脑、青黛、硼酸、甘油和注射用水组成,其中,
混合多肽由第一多肽Peptide12、第二多肽Peptide13和第三多肽Peptide21组成,第一多肽Peptide12的浓度为21.38μM,第二多肽Peptide13的浓度为25.30μM,第三多肽Peptide21的浓度为36.82μM,
金银花的含量为17%,所述薄荷脑的含量为1.0%,青黛的含量为2%,硼酸的含量为0.15%,甘油的含量为15%,余量为水。
该多肽消毒剂组合物的制备方法为:先将注射用水溶解金银花、薄荷脑、青黛、硼酸、甘油充分溶解后配置成辅料母液,之后用辅料母液溶解上述多肽,最后以注射用水补足,即成。
实施例4
一种预防冠状病毒的多肽消毒剂组合物,由混合多肽、金银花、薄荷脑、青黛、硼酸、甘油和注射用水组成,其中,
混合多肽由第一多肽Peptide12和第三多肽Peptide21组成,第一多肽Peptide12的浓度为21.38μM,第三多肽Peptide21的浓度为36.82μM,
金银花的含量为17%,所述薄荷脑的含量为1.0%,青黛的含量为2%,硼酸的含量为0.15%,甘油的含量为15%,余量为水。
该多肽消毒剂组合物的制备方法为:先将注射用水溶解金银花、薄荷脑、青黛、硼酸、甘油充分溶解后配置成辅料母液,之后用辅料母液溶解上述多肽,最后以注射用水补足,即成。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
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Claims (10)
1.一种预防冠状病毒的多肽消毒剂组合物,其特征在于:其有效成分为由如SEQ IDNO. 01所示的第一多肽、如SEQ ID NO. 02所示的第二多肽和如SEQ ID NO. 03所示的第三多肽组成的混合多肽,
其中,第一多肽的浓度为21-22μM,第二多肽的浓度为25-26μM,第三多肽的浓度为36-37μM。
2.如权利要求1所述的多肽消毒剂组合物,其特征在于:所述第一多肽的浓度为21.38μM。
3.如权利要求1所述的多肽消毒剂组合物,其特征在于:所述第二多肽的浓度为25.30μM。
4.如权利要求1所述的多肽消毒剂组合物,其特征在于:所述第三多肽的浓度为36.82μM。
5.如权利要求1至4中任一权利要求所述的多肽消毒剂组合物,其特征在于:还包括金银花、薄荷脑、青黛、硼酸、甘油和水。
6.一种预防冠状病毒的多肽消毒剂组合物,其特征在于:其有效成分为由如SEQ IDNO. 01所示的第一多肽和如SEQ ID NO. 03所示的第三多肽组成的混合多肽,
其中,第一多肽的浓度为21-22μM,第三多肽的浓度为36-37μM。
7.如权利要求6所述的多肽消毒剂组合物,其特征在于:所述第一多肽的浓度为21.38μM。
8.如权利要求6所述的多肽消毒剂组合物,其特征在于:所述第三多肽的浓度为36.82μM。
9.如权利要求6所述的多肽消毒剂组合物,其特征在于:所述第一多肽的浓度为21.38μM,所述第三多肽的浓度为36.82μM。
10.如权利要求6至9中任一权利要求所述的多肽消毒剂组合物,其特征在于:还包括金银花、薄荷脑、青黛、硼酸、甘油和水。
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