WO2024055843A1 - 一种基于受体结合检测新冠病毒的方法 - Google Patents

一种基于受体结合检测新冠病毒的方法 Download PDF

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WO2024055843A1
WO2024055843A1 PCT/CN2023/115691 CN2023115691W WO2024055843A1 WO 2024055843 A1 WO2024055843 A1 WO 2024055843A1 CN 2023115691 W CN2023115691 W CN 2023115691W WO 2024055843 A1 WO2024055843 A1 WO 2024055843A1
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new coronavirus
protein
receptor
magnetic microspheres
coronavirus
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孟哲峰
杜玲
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上海市闵行区中心医院
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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  • the invention belongs to the field of novel coronavirus detection, and in particular relates to a method for detecting novel coronavirus based on receptor binding.
  • the new coronavirus (COVID-19) is the pathogen causing the global new crown epidemic. It has infected more than 530 million people and killed more than 6.31 million people. It is a serious public health problem faced by all countries in the world.
  • the detection methods of the new coronavirus are divided into viral gene quantitative PCR detection (viral nucleic acid detection) and viral antigen detection.
  • the former is the currently recognized gold standard for detection, but the new coronavirus nucleic acid detection can only determine whether the new coronavirus infection has occurred and cannot indicate whether the patient has been infected.
  • the purpose of the present invention is to provide a method for detecting the new coronavirus based on receptor binding.
  • the magnetic microspheres are combined with the new coronavirus in a ligand receptor manner to simulate the adsorption of the new coronavirus to the host cell. Learn the behavior and process, and then use quantitative PCR to detect the amount of new coronavirus to determine whether the virus in the patient has receptor binding ability, thereby inferring the infection activity and transmissibility of the new coronavirus in the patient.
  • the present invention provides a method for detecting new coronavirus based on receptor binding, which includes the following steps:
  • receptor protein-coupled agarose magnetic microspheres Preparation of receptor protein-coupled agarose magnetic microspheres: cross-linked agarose is used as the matrix, and agarose magnetic microspheres are obtained after surface modification with carboxyl (-COOH), and are combined with the receptor protein in a covalent coupling manner. , forming receptor protein-coupled agarose magnetic microspheres;
  • Receptor protein-coupled agarose magnetic microspheres capture the new coronavirus:
  • the receptor protein covalently bound to the surface of the receptor protein-coupled agarose magnetic microspheres recognizes the receptor binding region of the outer membrane S protein of the new coronavirus sample. (RBD), the two combine with each other to simulate the binding process between the new coronavirus and the host cell to capture the new coronavirus;
  • the receptor protein is selected from heparin protein (Heparin) or angiotensin-converting enzyme 2 (ACE2) recombinant protein.
  • Heparin heparin protein
  • ACE2 angiotensin-converting enzyme 2
  • the receptor protein is heparin protein derived from porcine small intestinal mucosa.
  • the method for detecting the new coronavirus based on receptor binding includes:
  • Heparinized magnetic microspheres capture the new coronavirus: Heparin covalently bound to the surface of the heparinized magnetic microspheres recognizes the RBD region of the outer membrane S protein of the new coronavirus sample, and the two combine with each other to simulate the binding process of the new coronavirus and host cells. coronavirus;
  • the new coronavirus sample is derived from the in vitro tissue or blood of a person infected with the new coronavirus or a patient who has recovered from the new coronavirus infection and recovered.
  • heparinized magnetic microspheres capture the virus through receptor binding, and then evaluate the infectivity of the new coronavirus through immune binding-fluorescence quantitative PCR combined detection, which can be used to distinguish whether the virus contained in the sample has cell receptor recognition and Combining ability to preliminarily determine whether the virus has infectious activity and transmissibility. It is suitable for evaluating the replication and spread of the virus during the treatment of patients infected with the new coronavirus, as well as the infectivity of the new coronavirus carried in the body of cured patients when they show symptoms of re-positive symptoms.
  • the present invention also provides the application of the above-mentioned method for detecting the new coronavirus based on receptor binding in evaluating the infection activity and transmission ability of the new coronavirus in a subject.
  • the subjects include patients infected with the new coronavirus and patients who become positive after being cured of the new coronavirus infection.
  • the receptor protein is covalently bound to the surface of the agarose magnetic microsphere and recognizes the RBD region of the outer membrane S protein of the new coronavirus sample. They combine with each other to capture the new coronavirus. After magnetic microsphere purification, elution collection and virus lysis, the new coronavirus gene with cell binding ability is obtained, and then the new coronavirus infection activity and infectivity of the test sample are evaluated through immunoconjugation-fluorescence quantitative PCR joint detection. It is particularly suitable for patients who have recovered from COVID-19 infection and has important practical value.
  • Figure 1 is a schematic flow chart of the method for detecting new coronavirus based on receptor binding in the embodiment.
  • Figure 2 shows the results of the inhibitory activity of heparin protein and ACE2 recombinant protein against new coronavirus infection in the examples.
  • Figure 3 shows the absorbance of heparin protein and ACE2 recombinant protein inhibiting the binding of S protein to A549 human lung epithelial cells in the example, where the absorbance value is the standardized 450nm OD value.
  • Figure 4 is an inhibitory effect curve of different concentrations of heparin protein inhibiting the binding of S protein to A549 human lung epithelial cells in the Example.
  • This example uses heparin protein derived from pig small intestinal mucosa as the receptor protein to prepare heparinized magnetic microspheres, detect the new coronavirus through immunoconjugation-fluorescence quantitative PCR, and evaluate its infectious activity and transmissibility (Figure 1).
  • the method is: : Using cross-linked agarose as the matrix, through carboxyl surface modification, it is combined with heparin proteins from different sources (heparin derived from pig small intestine, heparin derived from bovine intestinal mucosa and heparin recombinant protein) in a covalent coupling manner to form a heparinized magnet. Bead microspheres are used to capture the new coronavirus.
  • the heparin protein covalently bound to the surface of the heparinized magnetic bead microspheres binds to the RBD region of the outer membrane S protein of the new coronavirus, capturing the virus and simulating the infection process of its recognition receptor. It is obtained through magnetic bead cleaning, purification and virus elution.
  • the new coronavirus particles with receptor binding ability are detected through immunoconjugation-fluorescence quantitative PCR, and their infective activity and transmissibility are evaluated.
  • Block with PBS-T detect the coupling efficiency of heparin-agarose magnetic beads, and store it at 4°C for later use: fluorescently label the unreacted binding sites on the surface of the magnetic beads coupled to the protein to obtain the sample to be tested; after activation, All binding sites on the surface of the magnetic beads are fluorescently labeled to obtain a positive control substance; blank magnetic beads, activated magnetic beads or protein-coupled magnetic beads are used as the blank control substance; measure the test sample, positive control substance, and blank The fluorescence value of the reference substance was used to calculate the protein coupling efficiency of the magnetic beads according to the formula.
  • Heparin-coupled magnetic beads perform an immune binding reaction with the new coronavirus from positive patient samples
  • the coupled magnetic beads that have completed the binding reaction are added to the virus lysis solution for lysis to obtain the viral genome, and the magnetic beads are removed by centrifugation;
  • This example verifies the competitive neutralization of virus infection by heparin-coupled magnetic beads through pseudovirus infection in vitro.
  • the method is: using heparinized magnetic beads to neutralize the infective activity of the new coronavirus pseudovirus on host cells, thereby verifying the heparin protein-coupled magnetic beads.
  • the specific operations are as follows:
  • Figure 2 shows the activity of heparin protein (derived from porcine small intestinal mucosa) in inhibiting the infection of the new coronavirus. It can be seen that heparin significantly inhibits the activity of vero cells infected by the new coronavirus pseudovirus.
  • ACE2 is used as a positive control, and the heparin protein IC50: 24.6 ⁇ g/mL ( Figure 2 ).

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Abstract

一种基于受体结合检测新冠病毒的方法，包括以交联琼脂糖为基质，通过羧基表面修饰后得到琼脂糖磁性微球，与受体蛋白以共价偶联方式结合形成受体蛋白偶联琼脂糖磁性微球，其表面共价结合的受体蛋白识别新冠病毒样本外膜S蛋白的受体结合区域，两者相互结合模拟新冠病毒与宿主细胞的结合过程捕获新冠病毒，依次经过磁性微球清洗、纯化和洗脱得到具备细胞结合能力的新冠病毒，通过免疫结合-荧光定量PCR联合检测并评估其感染活性和传播能力，适用于评价新冠病毒感染者治疗过程中病毒复制和传播情况，以及治愈患者出现复阳症状时体内携带新冠病毒的传染性。

Description

一种基于受体结合检测新冠病毒的方法 技术领域
本发明属于新冠病毒检测领域，尤其涉及一种基于受体结合检测新冠病毒的方法。
背景技术
新冠病毒(COVID-19)是造成全球新冠疫情的病原体，累计感染5.3亿多人，其中631万多人死亡，是世界各国共同面临的严重公共卫生问题。新冠病毒的检测方法分为病毒基因定量PCR检测(病毒核酸检测)和病毒抗原检测，前者为当前公认的检测金标准，但新冠病毒核酸检测只能定性是否发生新冠病毒感染，不能提示该患者是否具有传播性，尤其对于新冠病毒感染恢复后发生复阳的患者群体(即出院后的患者再次出现新冠病毒阳性)，因这部分患者体内可能存在抗病毒的中和抗体，传统的定量PCR检测能发现其体内病毒，但无法判断病毒是否具备感染活性和传播能力。
发明内容
针对现有技术中的上述问题，本发明的目的是提供一种基于受体结合检测新冠病毒的方法，通过磁性微球与新冠病毒以配受体方式结合，模拟新冠病毒吸附在宿主细胞的病毒学行为及过程，然后定量PCR检测新冠病毒量，判断患者体内病毒是否具备受体结合能力，从而推断患者体内新冠病毒的感染活性和传播能力。
为实现上述目的，本发明采用如下技术方案：
本发明提供一种基于受体结合检测新冠病毒的方法，包括以下步骤：
(1)制备受体蛋白偶联琼脂糖磁性微球：以交联琼脂糖为基质，通过羧基(-COOH)表面修饰后得到琼脂糖磁性微球，与受体蛋白以共价偶联方式结合，形成受体蛋白偶联琼脂糖磁性微球；
(2)受体蛋白偶联琼脂糖磁性微球捕获新冠病毒：所述受体蛋白偶联琼脂糖磁性微球表面共价结合的受体蛋白识别新冠病毒样本外膜S蛋白的受体结合区域(RBD)，两者相互结合模拟新冠病毒与宿主细胞的结合过程捕获新冠病毒；
(3)受体蛋白偶联琼脂糖磁性微球捕获新冠病毒后，依次经过磁性微球清洗、纯化和洗脱，得到具备细胞结合能力的新冠病毒；
(4)通过免疫结合-荧光定量PCR联合检测上述具备受体结合能力的新冠病毒，评估其感染活性和传播能力。
作为优选，所述受体蛋白选自肝素蛋白(Heparin)或血管紧张素转化酶2(ACE2)重组蛋白。
作为优选，所述受体蛋白为来源于猪小肠粘膜的肝素蛋白。
作为更优选，所述基于受体结合检测新冠病毒的方法包括：
(a)制备肝素化磁性微球：以交联琼脂糖为基质，通过羧基表面修饰后得到琼脂糖磁性微球，与肝素蛋白以共价偶联方式结合，形成肝素化磁性微球；
(b)肝素化磁性微球捕获新冠病毒：所述肝素化磁性微球表面共价结合的肝素识别新冠病毒样本外膜S蛋白RBD区域，两者相互结合模拟新冠病毒与宿主细胞的结合过程捕获新冠病毒；
(c)所述肝素化磁性微球捕获新冠病毒后，依次经过磁性微球清洗、纯化 和洗脱，得到具备细胞结合能力的新冠病毒；
(d)通过免疫结合-荧光定量PCR联合检测上述具备受体结合能力的新冠病毒，并评估其感染活性和传播能力。
作为优选，所述新冠病毒样本来源于新冠病毒感染者或新冠病毒感染治愈后复阳患者的体外组织或血液。
本发明的上述方法中，肝素化磁性微球通过受体结合捕获病毒，再通过免疫结合-荧光定量PCR联合检测评价新冠病毒感染性，可用于区分样本中所含病毒是否具备细胞受体识别和结合能力，从而初步判断该病毒是否具备感染活性和传播能力，适用于评价新冠病毒感染者治疗过程中病毒复制和传播情况，以及治愈患者出现复阳症状时体内携带新冠病毒的传染性。
本发明还提供上述基于受体结合检测新冠病毒的方法在评价受试者体内新冠病毒感染活性和传播能力方面的应用。
作为优选，所述受试者包括新冠病毒感染者和新冠病毒感染治愈后复阳患者。
与现有技术相比，本发明基于受体结合检测新冠病毒的方法中，受体蛋白偶联琼脂糖磁性微球表面共价结合的受体蛋白识别新冠病毒样本外膜S蛋白RBD区域，两者相互结合捕获新冠病毒，经过磁性微球纯化、洗脱收集和病毒裂解，获得具备细胞结合能力的新冠病毒基因，然后通过免疫结合-荧光定量PCR联合检测评估检测样本的新冠病毒感染活性和传染能力，特别适用于新冠病毒感染治愈后的复阳患者，具有重要的实用价值。
附图说明
图1为实施例中基于受体结合检测新冠病毒的方法流程示意图。
图2为实施例中肝素蛋白和ACE2重组蛋白抑制新冠病毒感染活性的结果。
图3为实施例中肝素蛋白和ACE2重组蛋白抑制S蛋白与A549人肺上皮细胞结合的吸光度，其中吸光度值为标准化的450nm OD值。
图4为实施例中不同浓度的肝素蛋白抑制S蛋白与A549人肺上皮细胞结合的抑制效应曲线。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1
本实施例以来源于猪小肠粘膜的肝素蛋白作为受体蛋白制备肝素化磁性微球，通过免疫结合-荧光定量PCR联合检测新冠病毒，并评估其感染活性和传播能力(图1)，方法为：以交联琼脂糖为基质，通过羧基表面修饰后，与不同来源的肝素蛋白(猪小肠来源肝素、牛肠粘膜来源肝素和肝素重组蛋白)以共价偶联的方式结合，形成肝素化磁珠微球，用来捕获新冠病毒。肝素化磁珠微球表面共价结合的肝素蛋白与新冠病毒外膜S蛋白RBD区域相互结合，捕获病毒并模拟其识别受体的感染过程，依次经过磁珠清洗、纯化和病毒洗脱得到具备细胞结合能力的新冠病毒，通过免疫结合-荧光定量PCR联合检测该具备受体结合能力的新冠病毒颗粒，并评估其感染活性和传播能力。
具体操作如下：
1)配制15mM、pH6.0 2-吗啉乙磺酸缓冲液(MES)，以5-20mM碳二亚胺和10-50mM n-羟基硫代琥珀酰亚胺，在MES溶液中进行肝素与羧基化琼脂糖磁珠偶联反应；
2)加入200mM Tris缓冲液终止偶联反应；
3)PBS-T进行封闭，检测肝素-琼脂糖磁珠偶联效率，保存于4℃备用：对偶联蛋白的磁珠表面未反应的结合位点进行荧光标记，得到待测样品；对活化后的磁珠表面的全部结合位点进行荧光标记，得到阳性对照品；以空白磁珠、活化后的磁珠或者偶联蛋白的磁珠作为空白对照品；测定待测样品、阳性对照品、空白对照品的荧光值，按公式计算磁珠的蛋白偶联效率。
4)肝素偶联磁珠与阳性患者样本新冠病毒进行免疫结合反应；
5)通过清洗去除未结合的游离病毒后，完成结合反应的偶联磁珠加入病毒裂解液中进行裂解，获得病毒基因组，离心去除磁珠；
6)提取病毒基因组，进行荧光定量PCR检测，并与原样本中所检测病毒Ct值进行比较。
实施例2
本实施例通过体外假病毒感染验证肝素偶联磁珠竞争性中和病毒感染，方法为：利用肝素化磁珠中和新冠病毒假病毒对宿主细胞的感染活性，从而验证肝素蛋白偶联磁珠捕获新冠病毒的能力，及初步评价肝素蛋白偶联磁珠—荧光定量PCR检测新冠病毒感染活性的准确性和可行性，具体操作如下：
1)利用HIV慢病毒包装系统制备新冠病毒假病毒并感染vero细胞，具体步骤如下：
①构建VSV-G表达质粒以及新冠病毒S蛋白表达质粒，根据美国NIH网站GenBank数据库下载新冠病毒奥密克戎株(BA.2.2)的S基因全长DNA序 列，在S蛋白表达载体上合成；
②提取S蛋白表达质粒和pNL4-3.luc.R-.E-骨架质粒，采用jetPRIME转染试剂将其共转染293T细胞，48-72h后收获培养上清，过滤后获得新冠病毒假病毒悬液；
③假病毒液感染293T-ACE2细胞，采用荧光素酶试剂盒检测获得假病毒TCID50。
2)肝素化磁珠进行假病毒感染中和试验，具体步骤如下：
①接种vero细胞到细胞培养板中，生长成单层；
②新冠病毒假病毒悬液与不同量肝素化磁珠进行结合反应；
③去除磁珠后病毒液加入vero细胞层上；37℃培养，去除培养液；
④加新培养液，37℃，培养3-6天，检测细胞内荧光素酶表达；
⑤判定肝素化磁珠吸附新冠病毒假病毒并抑制假病毒感染细胞程度。
结果
(1)图2为肝素蛋白(猪小肠粘膜来源)抑制新冠病毒感染活性，可见肝素显著抑制新冠病毒假病毒感染vero细胞活性，ACE2作为阳性对照，其中肝素蛋白IC50：24.6μg/mL(图2)。
(2)ACE2重组蛋白和肝素蛋白对S蛋白与A549人肺上皮细胞结合的影响，吸光度值为标准化的450nm OD值，可见肝素蛋白显著抑制S蛋白与A549细胞结合(图3)。
(3)利用生物素化人ACE2蛋白包备96孔板后，进行竞争性ELISA分析测量不同浓度的肝素蛋白对S蛋白结合的抑制效应，可见肝素蛋白呈浓度依赖性抑制结合效应(图4)。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于 此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应所述以权利要求的保护范围为准。

Claims (7)

  1. 一种基于受体结合检测新冠病毒的方法，其特征在于，包括以下步骤：
    (1)制备受体蛋白偶联琼脂糖磁性微球：以交联琼脂糖为基质，通过羧基表面修饰后得到琼脂糖磁性微球，与受体蛋白以共价偶联方式结合形成受体蛋白偶联琼脂糖磁性微球；
    (2)受体蛋白偶联琼脂糖磁性微球捕获新冠病毒：所述受体蛋白偶联琼脂糖磁性微球表面共价结合的受体蛋白识别新冠病毒样本外膜S蛋白RBD区域，两者相互结合模拟新冠病毒与宿主细胞的结合过程捕获新冠病毒；
    (3)受体蛋白偶联琼脂糖磁性微球捕获新冠病毒后，依次经过磁性微球清洗、纯化和洗脱，得到具备细胞结合能力的新冠病毒；
    (4)通过免疫结合-荧光定量PCR联合检测上述具备受体结合能力的新冠病毒，评估其感染活性和传播能力。
  2. 根据权利要求1所述基于受体结合检测新冠病毒的方法，其特征在于，所述受体蛋白选自肝素蛋白或ACE2重组蛋白。
  3. 根据权利要求2所述基于受体结合检测新冠病毒的方法，其特征在于，所述受体蛋白为来源于猪小肠粘膜的肝素蛋白。
  4. 根据权利要求3所述基于受体结合检测新冠病毒的方法，其特征在于，所述基于受体结合检测新冠病毒的方法包括：
    (a)制备肝素化磁性微球：以交联琼脂糖为基质，通过羧基表面修饰后得到琼脂糖磁性微球，与所述肝素蛋白以共价偶联方式结合形成肝素化磁性微球；
    (b)肝素化磁性微球捕获新冠病毒：所述肝素化磁性微球表面共价结合的肝素识别新冠病毒样本外膜S蛋白RBD区域，两者相互结合模拟新冠病毒与宿主细胞的结合过程捕获新冠病毒；
    (c)所述肝素化磁性微球捕获新冠病毒后，依次经过磁性微球清洗、纯化和洗脱，得到具备细胞结合能力的新冠病毒；
    (d)通过免疫结合-荧光定量PCR联合检测上述具备受体结合能力的新冠病毒，并评估其感染活性和传播能力。
  5. 根据权利要求1所述基于受体结合检测新冠病毒的方法，其特征在于，所述新冠病毒样本来源于新冠病毒感染者或新冠病毒感染治愈后复阳患者的体外组织或血液。
  6. 权利要求1–5任一项所述基于受体结合检测新冠病毒的方法在评价受试者体内新冠病毒感染活性和传播能力方面的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述受试者包括新冠病毒感染者和新冠病毒感染治愈后复阳患者。
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