CN112806378B - 一种预防冠状病毒的消毒剂组合物 - Google Patents

一种预防冠状病毒的消毒剂组合物 Download PDF

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CN112806378B
CN112806378B CN202110039450.1A CN202110039450A CN112806378B CN 112806378 B CN112806378 B CN 112806378B CN 202110039450 A CN202110039450 A CN 202110039450A CN 112806378 B CN112806378 B CN 112806378B
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alloferon
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曾子晏
胡敏煌
王薛婷
郑炳义
曾才英
曾晓玲
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Xiamen University
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Abstract

本发明公开了一种预防冠状病毒的消毒剂组合物，其有效成分包括含有如SEQ ID NO.01所示的Alloferon、如SEQ ID NO.02所示的Allo‑S2和如SEQ ID NO.03所示的Allo‑S5的混合多肽，其中，Alloferon的浓度为21‑23μM，Allo‑S2的浓度为39‑41μM，Allo‑S5的浓度为12‑14μM。本发明成本低廉，适用范围广，并且能有效预防冠状病毒。

Description

一种预防冠状病毒的消毒剂组合物
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消毒剂组合物。
背景技术
就目前科研成果而言，尚未有特效药能够治疗这些冠状病毒的感染，因此预防病毒的感染仍是最主要的手段。但现有技术中尚未出现满足需求的用来预防冠状病毒感染的消毒剂。
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消毒剂组合物，其有效成分包括含有如SEQ ID NO.01所示的Alloferon、如SEQ ID NO.02所示的Allo-S2和如SEQ ID NO.03所示的Allo-S5的混合多肽，
其中，Alloferon的浓度为21-23μM，Allo-S2的浓度为39-41μM，Allo-S5的浓度为12-14μM。
在本发明的一个优选实施方案中，所述混合多肽由所述Alloferon、Allo-S2和Allo-S5组成。
进一步优选的，所述Alloferon的浓度为21.02μM。
进一步优选的，所述Allo-S2的浓度为39.93μM。
进一步优选的，所述Allo-S5的浓度为12.90μM。
进一步优选的，所述Alloferon的浓度为21.02μM，所述Allo-S2的浓度为39.93μM，所述Allo-S5的浓度为12.90μM。
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。
进一步优选的，所述金银花的含量为15-20％，所述薄荷脑的含量为0.1-1.8％。
更进一步优选的，所述青黛的含量为1-2％，所述硼酸的含量为0.1-0.15％。
再进一步优选的，所述甘油的含量为10-15％。
本发明的有益效果是：本发明成本低廉，适用范围广，并且能有效预防冠状病毒。
附图说明
图1为本发明实施例1中的Alloferon与hACE2的结合能力图。
图2为本发明实施例1中的Allo-S5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Alloferon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、Alloferon及其衍生物(表1所示)、Amine Coupling Kit(GELife100050、PBS、甘氨酸-盐酸缓冲溶液(pH 2.5)。
表1
Figure GDA0002992587360000021
Figure GDA0002992587360000031
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)Alloferon及其衍生物与hACE的亲和力分析
材料：PBS、CM5-hACE2芯片、上述Alloferon及其衍生物、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Alloferon及其衍生物，浓度为3mM，并依次稀释为100μM、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Alloferon与hACE2的结合能力如图1所示，Allo-S5与hACE2的结合能力如图2所示)
表2
Figure GDA0002992587360000032
Figure GDA0002992587360000041
表3
Figure GDA0002992587360000042
实施例2利用免疫共沉淀阐释Alloferon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
仪器：Westernblot电泳仪(BIO-RAD
Figure GDA0002992587360000043
Tetra)。
材料及试剂：hACE2-His纯化蛋白、Alloferon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30μL HA-tag agarose beads置于冰上备用，分装两管等量hACE2-His，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Alloferon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、Alloferon及其衍生物。
具体步骤：分装7管等量HA-tag agarose beads用PBS洗涤后置于冰上备用，而后分装7管等量hACE2-His蛋白，分别加入Alloferon:Allo-S2(21.02μM:39.93μM)、Allo-S2:Allo-S5(39.93μM:12.90μM)、Allo-S8:Allo-S9(29.73μM:16.62μM)、Alloferon:Allo-S5(21.02μM:12.90μM)、Alloferon:Allo-S2:Allo-S5(21.02μM:39.93μM:12.90μM)混匀，将未加多肽的1管中加入PBS作为blank，其余6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未阳性对照组，7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
Figure GDA0002992587360000051
Figure GDA0002992587360000061
实施例3
一种预防冠状病毒的消毒剂组合物，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Alloferon、Allo-S2和Allo-S5组成，Alloferon的浓度为21.02μM，Allo-S2的浓度为39.93μM，Allo-S5的浓度为12.90μM，
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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<213> 人工序列(Artificial Sequence)
<400> 14
Thr Lys Pro Arg Lys His Gly Val Ser Gly His Gly Gln His Gly Val
1 5 10 15
His Gly
<210> 15
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
His Gly Val Ser Gly His Gly Gln His Gly Val His Gly Thr Lys Pro
1 5 10 15
Arg Lys

Claims (5)

1.一种预防冠状病毒的组合物，其特征在于：其有效成分为由SEQ ID NO. 01所示的Alloferon、SEQ ID NO. 02所示的Allo-S2和SEQ ID NO. 03所示的Allo-S5组成的混合多肽，
其中，Alloferon的浓度为21-23μM，Allo-S2的浓度为39-41μM，Allo-S5的浓度为12-14μM。
2.如权利要求1所述的组合物，其特征在于：所述Alloferon的浓度为21.02μM。
3.如权利要求1所述的组合物，其特征在于：所述Allo-S2的浓度为39.93μM。
4.如权利要求1所述的组合物，其特征在于：所述Allo-S5的浓度为12.90μM。
5.如权利要求1所述的组合物，其特征在于：所述Alloferon的浓度为21.02μM，所述Allo-S2的浓度为39.93μM，所述Allo-S5的浓度为12.90μM。
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