WO2022077613A1 - 一种新型冠状病毒 s 蛋白的 b 细胞线性抗原表位、抗体、鉴定方法及应用 - Google Patents

一种新型冠状病毒 s 蛋白的 b 细胞线性抗原表位、抗体、鉴定方法及应用 Download PDF

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WO2022077613A1
WO2022077613A1 PCT/CN2020/126206 CN2020126206W WO2022077613A1 WO 2022077613 A1 WO2022077613 A1 WO 2022077613A1 CN 2020126206 W CN2020126206 W CN 2020126206W WO 2022077613 A1 WO2022077613 A1 WO 2022077613A1
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novel coronavirus
protein
epitope
antibody
seq
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French (fr)
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王晓辉
汪川
张云雯
杨峥嵘
冯铁建
扈庆华
何建凡
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深圳市疾病预防控制中心
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention belongs to the technical field of biology, and in particular relates to a B cell linear antigenic epitope of a novel coronavirus S protein, an identification method and an application.
  • coronavirus coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome corona virus 2
  • SARS-CoV-2 The whole genome of SARS-CoV-2 is about 29.8kb nucleotides in length, encoding four major structural proteins: spike protein S (Spike protein, S protein), membrane protein M (Membrane protein), envelope protein E (Envelope protein) protein) and nucleocapsid protein N (Nucleocapsid).
  • spike protein S spike protein
  • S protein membrane protein M (Membrane protein), envelope protein E (Envelope protein) protein)
  • nucleocapsid protein N Nucleocapsid
  • S1 and S2 subunits are cleaved and dissociated by TMPRSS2. Triggers a conformational change in the S2 subunit, exposing the fusion peptide contained in the S2 subunit.
  • the fusion peptide anchors the target cell membrane and promotes fusion of the virus with the target cell membrane.
  • the S protein is also the main antigenic protein of the virus. Therefore, the SARS-CoV-2 S protein is a popular target antigen for new crown vaccine development.
  • the purpose of the embodiments of the present invention is to provide a B cell linear epitope of the novel coronavirus S protein.
  • a B cell linear epitope of a novel coronavirus S protein the amino acid sequence of the B cell linear epitope of the S protein is as SEQ ID NO.104 or SEQ ID NO. 82 shown.
  • Another object of the embodiment of the present invention is a B cell linear epitope of a novel coronavirus S protein, and the amino acid sequence of the B cell linear epitope of the S protein is such as SEQ ID NO.104, SEQ ID NO. 128 and SEQ ID NO.129.
  • Another object of the embodiment of the present invention is a method for identifying the B cell linear epitope of the novel coronavirus S protein, including:
  • amino acid sequence of the S protein several groups of peptide segments are designed and synthesized, and each of the group of peptide segments includes several segments, and the amino acid sequence of the peptide segments is as shown in SEQ ID NO.1 ⁇ shown in SEQ ID NO.127;
  • novel coronavirus antibody is obtained by coupling an inert protein with the amino acid sequence of the B cell linear epitope according to claim 1, and immunizing animals according to the immunization procedure. resistance serum.
  • Another object of the embodiments of the present invention is a kit for detecting a novel coronavirus, the kit comprising the B cell linear epitope.
  • Another object of the embodiments of the present invention is a method for detecting a novel coronavirus antibody, comprising:
  • the cutoff value was calculated with healthy human serum as a negative control.
  • the average OD value of the serum samples to be tested was greater than the cutoff value, and the antibody reaction was considered positive.
  • Another object of the embodiments of the present invention is a method for identifying a novel coronavirus antibody and an antigen, the identification method comprising using the amino acid sequence of the linear epitope of the B cell as an antigen to detect the novel coronavirus antibody;
  • the novel coronavirus antibody described above detects the novel coronavirus.
  • Another object of the embodiments of the present invention is a novel coronavirus vaccine
  • the novel coronavirus vaccine includes an antigenic epitope
  • the antigenic epitope is the B cell linear epitope of claim 1 .
  • Another object of the embodiments of the present invention is the application of the B cell linear epitope of the S protein of the novel coronavirus in the preparation of antibodies to the novel coronavirus.
  • Another object of the embodiments of the present invention is the application of the B cell linear epitope of the S protein of the novel coronavirus in the preparation of a novel coronavirus vaccine.
  • Another object of the embodiments of the present invention is the application of the B cell linear epitope of the novel coronavirus S protein in identifying novel coronavirus antibodies and antigens.
  • the embodiment of the present invention provides a new B cell linear epitope of the new coronavirus S protein, which can be used to detect specific antibodies in the blood of COVID-19 patients, and can also be used to inoculate healthy people to induce the production of specific antibodies, Thus, it provides important antigen targets for SARS-CoV-2 vaccine design, monoclonal antibody development and antibody detection kit development.
  • Fig. 1 is the epitope peptide screening flow chart provided in the embodiment of the present invention.
  • Figure 2 is a schematic diagram of identification of SARS-CoV-2 S protein B cell epitopes provided in the embodiment of the present invention
  • FIG. 3 is a schematic diagram of the positive reaction rates of epitope peptides P82 and P104 provided in the embodiment of the present invention, as well as existing epitope peptides S21P2 and S14P5 and the convalescent serum of COVID-19 patients;
  • FIG. 4 is a schematic diagram of the comparison of antibody S/CO values and the correlation analysis of antibody levels of the epitope peptides P82 and P104 provided in the embodiment of the present invention, and the existing epitope peptides S21P2 and S14P5.
  • Such amino acid sequence-specific single peptides can be used to detect specific antibodies in the blood of COVID-19 patients, and can also be used to inoculate healthy people to induce specific antibodies, which are SARS-CoV.
  • -2Vaccine design, development of monoclonal antibodies and development of antibody detection kits provide important antigen targets.
  • amino acid sequence of the B cell linear epitope of the novel coronavirus S protein is as shown in SEQ ID NO.104 (P104) or SEQ ID NO.82 (P82).
  • the amino acid sequence of the B cell linear epitope of the novel coronavirus S protein is as SEQ ID NO.104 (P104), SEQ ID NO.128 (S14P5) and SEQ ID NO. 129 (S21P2).
  • SEQ ID NO: 129 S21P2 PSKPSKRSFIEDLLFNKV.
  • Example 1 Identification method of B cell linear epitope of novel coronavirus S protein
  • indirect ELISA is as follows: ELISA 96-well plate (Corning, #42592) for antigen coating, adding 100 ⁇ L of peptide (2.5 ⁇ g/mL per peptide) or 0.2 ⁇ g/mL RBD (CUSABIO, #CSB-MP3324GMY1b1) to each well ) at 4°C overnight. Wash the plate 6 times with 300 ⁇ L of wash buffer (PBS containing 0.05% Twynn-20). Add 300 ⁇ L of blocking solution (PBS containing 0.1% Tween-20 and 5% nonfat dry milk) to each well to block for 2 hours.
  • wash buffer PBS containing 0.05% Twynn-20
  • blocking solution PBS containing 0.1% Tween-20 and 5% nonfat dry milk
  • the reaction was stopped by adding 100 ⁇ L of Stop Solution (Multi Science, #EK0011) to each well. Absorbance was measured at 450nm and 630nm according to the instructions, and OD450nm-630nm was calculated. The average OD of the negative control serum + 3 times the standard deviation (SD) was used as the cutoff value, and the average OD value of the sample to be tested was greater than the cutoff value, and the reaction was considered positive.
  • Stop Solution Multi Science, #EK0011
  • each peptide group (G1-G13) was coated on a 96-well plate, and 20 convalescent sera from patients discharged from COVID-19 were randomly selected for indirect ELISA detection, and the peptide groups with positive reactions were screened. Then, each peptide contained in the positive peptide group was coated on a 96-well plate, and the indirect ELISA detection with the serum was carried out one by one to determine the positive peptides ( Figure 1).
  • peptide groups G9 and G11 had positive response values (Fig. 2a, convalescent serum was tested with each peptide group by indirect ELISA, and positive peptide groups (G9 and G11) were screened). Then each peptide in G9 and G11 was tested with convalescent serum one by one, and finally two linear epitopes of B cells recognized by convalescent serum IgG were identified, P82 ( 811 KPSKRSFIEDLLFNK 825 ) and P104 ( 103 1ECVLGQSKRVDFCGK 1045 ) (Fig.
  • convalescent serum was subjected to indirect ELISA with each peptide segment included in the screened positive peptide groups G9 and G11, and positive peptides (P82 and P104) were screened.
  • the circles represent epitopes and recovery The mean OD values of the sera responses during the period.
  • the squares represent the cutoff values of the epitope responses with healthy human sera). Further, it is found that P82 is completely contained in the B cell epitope S21P2, while the P104 identified by the present invention is a new 15 amino acid SARS-CoV-2 B cell epitope.
  • Fig. 3a shows the positive reaction rates of peptides S21P2, S14P5, P82 and P104 in the convalescent sera of all COVID-19 patients and asymptomatic patients
  • Figure 3b shows the positive reaction rates of different peptide combinations analyzed.S14P4, S21P2 + S14P5 and S21P2 + S14P5 + P104 were the best combination with the highest overall positive rate among single combination, paired combination and triple triple combination, respectively.
  • Figure 3c represents the detailed clinical types of patients detected by different peptide combinations).
  • Localization of P82 and P104 in the three-dimensional structure of the S protein showed that both P82 and P104 were located in the S protein S2 subunit (Fig. 2d).
  • P82 is located on the surface of the protein
  • P104 is located in the groove on the surface of the protein
  • FIG. 2d localization of the two epitope peptides in the S protein
  • the 3D structure of the SARS-CoV-2 S protein was obtained from the NCBI website (PDB ID: 6VXX); this image was processed through the NCBI website.
  • the three monomers of the S protein are indicated in different colors).
  • P104 epitope was screened by synthesizing the 127 polypeptides in Table 1, establishing an indirect enzyme-linked immune reaction (ELISA) method, and then testing 165 COVID-19 patients' convalescent sera. selected from the technical route.
  • ELISA enzyme-linked immune reaction
  • Example 2 Detection of novel coronavirus antibodies with B-cell linear epitope, embodiment of detection of novel coronavirus antibodies comprising B-cell linear epitope P104
  • ELISA 96-well plate (Corning, #42592) is used for antigen coating, and 100 ⁇ L of peptide (2.5 ⁇ g/mL for each peptide) or 0.2 ⁇ g/mL RBD (CUSABIO, #CSB) is added to each well.
  • -MP3324GMY1b1) at 4°C overnight. Wash the plate 6 times with 300 ⁇ L of wash buffer (PBS containing 0.05% Twynn-20). Add 300 ⁇ L of blocking solution (PBS containing 0.1% Tween-20 and 5% nonfat dry milk) to each well to block for 2 hours.
  • the reaction was stopped by adding 100 ⁇ L of Stop Solution (Multi Science, #EK0011) to each well. Absorbance was measured at 450nm and 630nm according to the instructions, and OD450nm-630nm was calculated. The average OD of the negative control serum + 3 times the standard deviation (SD) was used as the cutoff value, and the average OD value of the sample to be tested was greater than the cutoff value, and the reaction was considered positive.
  • Stop Solution Multi Science, #EK0011
  • the convalescent sera of 165 COVID-19 patients were tested by indirect ELISA with 4 epitope peptides S14P5, S21P2, P82 and P104, respectively, and the overall positive rates were 77.0%, 73.9%, 61.2% and 30.3%, respectively.
  • the epitope peptides S14P5, S21P2 and P82 showed a higher positive rate of reaction, and the difference from the positive rate of P104 was statistically significant ( P ⁇ 0.001).
  • the four epitope peptides were combined in different pairs, and their combined detection rates were analyzed.
  • S12P2+S14P5 is the pairwise combination with the highest detection rate (88.5%)
  • S12P2+S14P5+P104 is the highest detection rate in patients (92.7%) of all three combinations, and the results of combined detection with four epitope peptides same.
  • the combined use of epitope peptides could significantly improve the patient detection rate ( P ⁇ 0.001, Fig. 3b).
  • Figures 4a-b represent the S/CO values of the convalescent sera reacting with epitope peptides in all patients (a) or patients with different clinical types (b); the median (interquartile range) for each group of data ) indicates; each point represents the antibody detection result of each serum.
  • * represents P ⁇ 0.05, ** represents P ⁇ 0.01, and *** represents P ⁇ 0.001.
  • the overall detection rate of S14P5+S21P2+P104 was the highest (92.7%), which was the same as the combined detection rate of the four epitope peptides, indicating that the rational combination of multiple epitope peptides could achieve the best detection effect.
  • serological testing has high specificity. Especially in the screening of asymptomatic infections, serological testing is used as an auxiliary means, which is faster and more convenient, and can reduce the missed detection rate.
  • amino acid sequence of the antigenic epitope P104 is a linear epitope of SARS-CoV-2, and its amino acid sequence has not been reported; while the amino acid sequence of the antigenic epitope P82 is included in S21P2, it can at least show that , S21P2 lacks the P82 linear epitope formed by 3 amino acids in the head and tail, and still has a certain ability of antibody binding.
  • the positive rate was 88.5%, and the combined detection of the three epitopes S14P5, S21P2 and P104, the positive rate increased to 92.7%, which shows that the new discovery of P104
  • the epitope has the effect of improving the positive rate of detection.
  • the antigenic epitope P104 is used for the preparation of the new coronavirus vaccine, that is, the antigenic epitope P104 polypeptide is used to immunize humans or animals, and the vaccinated humans or animals will produce corresponding antibodies, and the production of antibodies is the main purpose of vaccination.
  • the antigenic epitope P104 polypeptide is used to immunize humans or animals, and the vaccinated humans or animals will produce corresponding antibodies, and the production of antibodies is the main purpose of vaccination.
  • the embodiment of the present invention provides a new B cell linear epitope of coronavirus S protein, which can be used to detect specific antibodies in the blood of COVID-19 patients, and can also be used to inoculate healthy people to induce specific antibodies.
  • Antibodies thus providing important antigen targets for SARS-CoV-2 vaccine design, monoclonal antibody development and antibody detection kit development.

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Abstract

适用于生物学技术领域，提供了一种新型冠状病毒S蛋白的B细胞线性抗原表位、抗体、鉴定方法及应用，其中，所述S蛋白的B细胞线性抗原表位的氨基酸序列为如SEQ ID NO.104或SEQ ID NO.82所示，提供了全新的新型冠状病毒S蛋白的B细胞线性抗原表位，可以用于检测COVID-19患者血液中的特定抗体，也可以用于接种健康人群从而诱导产生特异性的抗体，从而为SARS-CoV-2疫苗设计、单克隆抗体研发以及抗体检测试剂盒的研发提供重要的抗原靶点。

Description

一种新型冠状病毒S蛋白的B细胞线性抗原表位、抗体、鉴定方法及应用 技术领域
本发明属于生物学技术领域，尤其涉及一种新型冠状病毒S蛋白的B细胞线性抗原表位、鉴定方法及应用。
 背景技术
2019年12月暴发的新型冠状病毒肺炎（coronavirus disease 2019，COVID-19），病原体为SARS-CoV-2（severe acute respiratory syndrome corona virus 2）。COVID-19已经成为全球性大流行病，疫情已经发展至200多个国家且形势严峻。
SARS-CoV-2全基因组长度大约为29.8kb核苷酸，编码四种主要的结构蛋白：棘突蛋白S（Spike protein，S蛋白）、膜蛋白M（Membrane protein）、包膜蛋白E（Envelope protein）和核衣壳蛋白N（Nucleocapsid）。其中，SARS-CoV-2通过S蛋白上的受体结合域（receptor binding domain，RBD）介导病毒与细胞受体结合后，S1与S2亚基被TMPRSS2切割解离。触发S2亚基发生构象改变，使S2亚基包含的融合肽暴露。融合肽锚定靶细胞膜，促进病毒与靶细胞膜融合。同时，S蛋白也是病毒的主要抗原蛋白。因此， SARS-CoV-2 S蛋白是新冠疫苗研发的热门靶抗原。
 技术问题
最近的报道鉴定出了部分SARS-CoV-2 S蛋白B细胞线性表位，提供了一部分SARS-CoV-2的B细胞线性表位分布信息，但截至目前，SARS-CoV-2抗原表位研究资料还很有限，多数报道仅基于软件预测，准确率低。
 技术解决方案
本发明实施例的目的在于提供一种新型冠状病毒S蛋白的B细胞线性抗原表位。
本发明实施例是这样实现的，一种新型冠状病毒S蛋白的B细胞线性抗原表位，所述S蛋白的B细胞线性抗原表位的氨基酸序列为如SEQ ID NO.104或SEQ ID NO.82所示。
本发明实施例的另一目的在于一种新型冠状病毒S蛋白的B细胞线性抗原表位，所述S蛋白的B细胞线性抗原表位的氨基酸序列为如SEQ ID NO.104、SEQ ID NO.128和SEQ ID NO.129所示。
本发明实施例的另一目的在于一种新型冠状病毒S蛋白的B细胞线性抗原表位的鉴定方法，包括：
根据S蛋白的氨基酸序列，设计合成若干肽段组，每个所述肽段组包括若干肽段，所述肽段的氨基酸序列如SEQ ID NO.1~ SEQ ID NO.127所示；
采集恢复期血清或血浆样本，灭活备用；
将所述每个肽段组分别包被ELISA 96孔板，并与所述恢复期血清或血浆样本进行间接酶联免疫反应检测，确定阳性肽段组；
将所述阳性肽段组内的每个肽段分别包被ELISA 96孔板，继续与所述恢复期血清或血浆样本逐一进行间接酶联免疫反应检测，确定阳性反应肽段，所述阳性反应肽段的氨基酸序列为权利要求1所述的SEQ ID NO.104和SEQ ID NO.82。
本发明实施例的另一目的在于一种新型冠状病毒抗体，所述新型冠状病毒抗体是采用权利要求1所述的B细胞线性抗原表位的氨基酸序列偶联惰性蛋白，按照免疫程序免疫动物获得的抗性血清。
本发明实施例的另一目的在于一种检测新型冠状病毒的试剂盒，所述试剂盒包括所述的B细胞线性抗原表位。
本发明实施例的另一目的在于一种新型冠状病毒抗体的检测方法，包括：
将所述的B细胞线性抗原表位包被96孔板，与待测血清样本逐一进行间接ELISA检测，重复测定两次；
以健康人血清作为阴性对照计算cutoff值，待测血清样本的平均OD值大于cutoff值认为抗体反应阳性。
本发明实施例的另一目的在于一种新型冠状病毒抗体和抗原的鉴别方法，所述鉴别方法包括采用所述的B细胞线性抗原表位的氨基酸序列作为抗原去检测新型冠状病毒抗体；采用所述的新型冠状病毒抗体检测新型冠状病毒。
本发明实施例的另一目的在于一种新型冠状病毒疫苗，所述新型冠状病毒疫苗包括抗原表位，所述抗原表位为权利要求1所述的B细胞线性抗原表位。
本发明实施例的另一目的在于一种所述的新型冠状病毒S蛋白的B细胞线性抗原表位在制备新型冠状病毒抗体中的应用。
本发明实施例的另一目的在于一种所述的新型冠状病毒S蛋白的B细胞线性抗原表位在制备新型冠状病毒疫苗中的应用。
本发明实施例的另一目的在于一种所述的新型冠状病毒S蛋白的B细胞线性抗原表位在鉴别新型冠状病毒抗体和抗原的应用。
 有益效果
本发明实施例提供了全新的新型冠状病毒S蛋白的B细胞线性抗原表位，可以用于检测COVID-19患者血液中的特定抗体，也可以用于接种健康人群从而诱导产生特异性的抗体，从而为SARS-CoV-2疫苗设计、单克隆抗体研发以及抗体检测试剂盒的研发提供重要的抗原靶点。
 附图说明
图1是本发明实施例提供的表位肽筛选流程图；
图2是本发明实施例提供的SARS-CoV-2 S 蛋白B细胞表位的鉴定示意图；
图3是本发明实施例提供的表位肽P82、P104，以及现有表位肽S21P2、S14P5与COVID-19患者恢复期血清的阳性反应率的示意图；
图4是本发明实施例提供的表位肽P82、P104，以及现有表位肽S21P2、S14P5的抗体S / CO值比较和抗体水平的相关性分析示意图。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
S蛋白的B细胞线性抗原表位诱导的特异性抗体对抑制病毒入侵机体有着重要作用。B细胞表位又称抗原决定簇，它是病原体蛋白（也称为抗原）上特定序列的氨基酸组合（也称为多肽），可以与B细胞抗原受体及特定的抗体发生特异性结合。B细胞表位包括B细胞表面抗原受体或B细胞分泌的特异性抗体所识别的线性表位和构象表位。本发明所指的是S蛋白的B细胞线性表位，即具有15个氨基酸序列特异性的多肽，其可以与COVID-19患者血液中的特异性抗体产生特异性结合。这类氨基酸序列特异性的单条多肽（或与其它多肽的组合）可以用于检测COVID-19患者血液中的特定抗体，也可以用于接种健康人群从而诱导产生特异性的抗体，为SARS-CoV-2疫苗设计、单克隆抗体研发以及抗体检测试剂盒的研发提供重要的抗原靶点。
在本发明实施例中，所述新型冠状病毒S蛋白的B细胞线性抗原表位的氨基酸序列为如SEQ ID NO.104（P104）或SEQ ID NO.82（P82）所示。
在本发明一个优选实施例中，所述新型冠状病毒S蛋白的B细胞线性抗原表位的氨基酸序列为如SEQ ID NO.104（P104）、SEQ ID NO.128（S14P5）和SEQ ID NO.129（S21P2）所示。
SEQ ID NO:104（P104）  KPSKRSFIEDLLFNK，
SEQ ID NO:82（P82）  ECVLGQSKRVDFCGK，
SEQ ID NO:128（S14P5）  TESNKKFLPFQQFGRDIA，
SEQ ID NO:129（S21P2）  PSKPSKRSFIEDLLFNKV。
下面结合具体实施例对本发明的新型冠状病毒S蛋白的B细胞抗原表位、鉴定方法及应用的技术效果做进一步的说明，但这些实施例所提及的具体实施方法只是对本发明的技术方案进行的列举解释，并非限制本发明的实施范围。
实施例1：新型冠状病毒S蛋白的B细胞线性抗原表位的鉴定方法
1、肽段库的建立
根据SARS-CoV-2 S蛋白的氨基酸序列（access number :YP_009724390.1）设计合成多肽。按照氨基酸排列顺序合成长度为15个氨基酸的肽段，相邻肽段重叠5个氨基酸。共获得127个肽段（P1～P127），覆盖S蛋白全长。肽段按氨基酸位置顺序分为13个组（G1～G13），每个组包含10或7个连续肽段（表1）。
表1   SARS-CoV-2重叠肽段库序列（肽库）
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Figure 321375dest_path_image005
Figure 282378dest_path_image006
Figure 789582dest_path_image007
2、COVID-19患者恢复期血清收集
2020年3月5日至2020年5月12日，对包括无症状感染者在内的165名深圳市COVID-19出院患者进行血清采集，冻存于-80℃冰柜备用，使用前血清置于56℃30min灭活可能存在的残余病毒再进行正式试验。
3、B细胞线性表位鉴定方法——间接酶联免疫反应（ELISA）
间接ELISA主要操作如下：ELISA 96孔板（Corning，＃42592）进行抗原包被，每孔加入100 µL肽段（每种肽2.5μg/ mL）或0.2μg/ mL RBD（CUSABIO，＃CSB-MP3324GMY1b1），4℃过夜。300μL洗涤缓冲液（含0.05％Twenn-20的PBS）洗板6次。每孔加入300μL封闭液（含0.1％Tween-20和5％脱脂奶粉的PBS）封闭2小时。洗板后，将经过热灭活的COVID-19患者恢复期血清或作为阴性对照的健康人血清以1:80稀释度，每孔加入100 µL。在37℃下孵育1小时。每个样品重复两个孔。孵育后洗板，以100μL/孔加入1：130000稀释的兔抗人IgG HRP（abcam，＃6759）二抗缓冲液，37℃孵育1小时。孵育后洗板，每孔加入100 µL TMB（Multi Science，＃EK0011）。将板在黑暗中于室温孵育25分钟。每孔加入100 µL终止溶液（Multi Science，＃EK0011）终止反应。根据说明书于450nm和630nm处测定吸光度，计算OD450nm-630nm。以阴性对照血清OD的平均值+ 3倍标准差（SD）作为cutoff值，待测样品的平均OD值大于cutoff值认为反应阳性。
4、B细胞线性表位筛选流程
首先将每个肽段组（G1～G13）分别包被96孔板，随机选取20份COVID-19出院患者恢复期血清进行间接ELISA检测，筛选具有阳性反应的肽段组。再将阳性肽段组包含的每个肽段分别包被96孔板，继续与血清逐一进行间接ELISA检测，确定阳性反应肽段（图1）。
鉴定结果：
将S蛋白的重叠肽段库与COVID-19患者恢复期血清进行间接ELISA检测。在第一轮筛选中，肽段组G9和G11反应值呈阳性（图2a，通过间接ELISA将恢复期血清与每个肽段组进行检测，筛选出阳性肽组（G9和G11））。再逐一将G9和G11中的每个肽段与恢复期血清进行检测，最后鉴定出了2个恢复期血清IgG可识别的B细胞线性表位P82（ 811KPSKRSFIEDLLFNK 825）和P104（ 1031ECVLGQSKRVDFCGK 1045）（图2b,c, 表2，恢复期血清与筛选出的阳性肽组G9和G11中包含的每个肽段进行间接ELISA，筛选出阳性肽（P82和P104）。圆点代表表位与恢复期血清反应的平均OD值。方块代表表位与健康人血清反应的cutoff值）。进一步，比较发现P82完全包含于B细胞表位S21P2中，而本发明鉴定的P104是一个新的15个氨基酸的SARS-CoV-2 B细胞表位。P82虽然被B细胞表位S21P2包围，但S21P2是18个氨基酸多肽，而P82是15个氨基酸的多肽，两者与165份血清反应的阳性率和反应强度也不一样（图3，其中，图3a表示在全部COVID-19患者和无症状患者的恢复期血清中，肽S21P2，S14P5，P82和P104的阳性反应率；图3b表示分析不同肽组合的阳性反应率。 S14P4，S21P2 + S14P5和S21P2 + S14P5 + P104分别是单组合，成对组合和三三组合中具有最高总体阳性率的最佳组合。图3c表示通过不同肽段组合检测的详细患者临床类型）。将P82和P104在S蛋白三维结构（PDB ID：6VXX）中定位，显示P82和P104都位于S蛋白S2亚基（图2d）。就空间位置来看，P82位于蛋白表面，P104位于蛋白表面的凹槽内（图2d，S蛋白中两个表位肽的定位；SARS-CoV-2S蛋白的3D结构从NCBI网站（PDB ID：6VXX）获得；该图像是通过NCBI网站处理的。S蛋白的三个单体分别以不同颜色示意）。
表2  表位肽序列及氨基酸位置    
Figure 595864dest_path_image008
值得注意的是，P104抗原表位是通过合成表1的127个多肽，建立间接酶联免疫反应（ELISA）方法，然后进行165份COVID-19患者恢复期血清的检测才筛选出来，是通过独特的技术路线筛选出来的。
实施例2：以B细胞线性表位检测新型冠状病毒抗体、包含B细胞线性表位P104的检测新型冠状病毒抗体的实施方案
将4个B细胞线性表位（S21P2、S14P5、P82、P104）分别包被96孔板，与165份COVID-19患者恢复期血清逐一进行间接ELISA检测。每个样本重复测定两次。以健康人血清作为阴性对照计算cutoff值，然后计算S/CO值（样品平均OD450nm-630nm值/cutoff值）。
其中，间接ELISA主要操作如下：ELISA 96孔板（Corning，＃42592）进行抗原包被，每孔加入100 µL肽段（每种肽2.5μg/ mL）或0.2μg/ mL RBD（CUSABIO，＃CSB-MP3324GMY1b1），4℃过夜。300μL洗涤缓冲液（含0.05％Twenn-20的PBS）洗板6次。每孔加入300μL封闭液（含0.1％Tween-20和5％脱脂奶粉的PBS）封闭2小时。洗板后，将经过热灭活的COVID-19患者恢复期血清或作为阴性对照的健康人血清以1:80稀释度，每孔加入100 µL。在37℃下孵育1小时。每个样品重复两个孔。孵育后洗板，以100μL/孔加入1：130000稀释的兔抗人IgG HRP（abcam，＃6759）二抗缓冲液，37℃孵育1小时。孵育后洗板，每孔加入100 µL TMB（Multi Science，＃EK0011）。将板在黑暗中于室温孵育25分钟。每孔加入100 µL终止溶液（Multi Science，＃EK0011）终止反应。根据说明书于450nm和630nm处测定吸光度，计算OD450nm-630nm。以阴性对照血清OD的平均值+ 3倍标准差（SD）作为cutoff值，待测样品的平均OD值大于cutoff值认为反应阳性。
检测结果：
（1）表位肽S14P5、S21P2、P82和P104与患者恢复期血清的反应阳性率
165名COVID-19患者恢复期血清与4个表位肽S14P5、S21P2、P82和P104分别进行间接ELISA检测，总体反应阳性率分别为77.0%、73.9%、61.2%和30.3%。表位肽S14P5、S21P2和P82显示出较高的反应阳性率，与P104阳性率的差异有统计学意义（ P<0.001）。30名无症状感染者恢复期血清与4个表位肽反应阳性率分别为63.3%、53.3%、46.7%和36.7%（图3a, P=0.209）。将4个表位肽进行不同的配对组合，分析它们的联合检出率。S12P2+S14P5是检出率最高的两两组合（88.5%），S12P2+S14P5+P104是所有三个组合中患者检出率最高的（92.7%），且与四个表位肽联合检测的结果相同。与单个表位肽检测相比，表位肽的联合使用可以明显提高患者检出率（ P<0.001，图3b）。对于无症状感染者，虽然不同表位肽的联合检测阳性率差异无统计学意义，但S12P2+S14P5+P104的联合检测将阳性率由63.3%提高至86.7%（ P=0.084）。表位肽的联合使用能明显提高COVID-19所有类型患者的检出率（图3c）。鉴于S12P2+S14P5+P104的组合能与92.7%的患者恢复期血清发生阳性反应，认为该组合是最有效的包含B细胞线性表位P104的检测新型冠状病毒抗体的实施方案。
（2）表位肽S14P5、S21P2、P82和P104的血清反应性及抗体相关性分析
165份COVID-19患者恢复期血清与表位肽S14P5、S21P2、P82和P104抗原抗体反应的S/CO值中位数（IQR）分别为1.4339（1.2299）、1.5564（4.2269）、1.2237（2.7857）和0.6917（0.6294）。P104的S/CO值明显低于其它3个表位肽（P<0.001）。在普通型与轻型患者中，P104的S/CO值也均低于其他3个表位肽（图4a,b）。其中，图4a-b表示所有患者（a）或具有不同临床类型的患者（b）的恢复期血清与表位肽反应的S / CO值；每组数据均用中位数（四分位间距）表示；每个点代表每个血清的抗体检测结果。*代表 P <0.05，**代表 P<0.01，***代表 P<0.001。
综上，本发明提供的抗原表位P104在COVID-19患者中的反应阳性率虽然较低，但经研究发现，P104与无症状感染者的反应阳性率（36.7%）高于其与有症状患者的反应阳性率（28.9%， P=0.388），而另3个表位肽与无症状感染者的阳性率均比有症状患者低。虽然S14P5和S21P2在感染人群中的检出率较高，但由于个体免疫应答差异等原因，单一表位肽检测存在较高的漏检率，但多个表位肽组合后能显著提高患者检出率。其中，S14P5+S21P2+P104的总体检出率最高（92.7%），与4个表位肽的联合检出率相同，说明多个表位肽的合理搭配可以达到最优检测效果。对于疑似病例的确证，血清学检测具有较高的特异性。尤其是无症状感染者的筛查，将血清学检测作为辅助手段，更加快捷、方便，且可以降低漏检率。
值得注意的是，抗原表位P104的氨基酸序列作为SARS-CoV-2的线性表位，其氨基酸序列是没有人报道过的；而抗原表位P82氨基酸序列虽然包含在S21P2中，但至少可以表明，S21P2少了头尾3个氨基酸所形成的P82线性表位，依然具有一定抗体结合的能力。另外，通过使用两个线性表位S14P5和S21P2联合进行165份血清检测阳性率为88.5%，S14P5、S21P2和P104三个表位的联合检测，阳性率提高到92.7%，这说明P104这个新发现的表位具有提高检测阳性率的作用。
实施例3：新型冠状病毒疫苗
将抗原表位P104用于新型冠状病毒疫苗的制备，即用抗原表位P104多肽免疫人或动物，接种后的人或动物会产生相对应的抗体，而产生抗体是疫苗接种的主要目的。具体实现方式可参照现有技术，在此不再做具体赘述。
实施例4：采用P104多肽制备的新型冠状病毒抗体检测新型冠状病毒
通过用抗原表位P104多肽偶连惰性蛋白免疫人或动物，使人或动物产生与该抗原表位P104多肽结合的抗体，提取纯化抗体，再用该抗体去检测新型冠状病毒。具体实现方式可参照现有技术，在此不再做具体赘述。
综上，本发明实施例提供了全新的冠状病毒S蛋白的B细胞线性抗原表位，可以用于检测COVID-19患者血液中的特定抗体，也可以用于接种健康人群从而诱导产生特异性的抗体，从而为SARS-CoV-2疫苗设计、单克隆抗体研发以及抗体检测试剂盒的研发提供重要的抗原靶点。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (1)

  1. 一种新型冠状病毒S蛋白的B细胞线性抗原表位，其特征在于，所述S蛋白的B细胞线性抗原表位的氨基酸序列为如SEQ ID NO.104或SEQ ID NO.82所示。
    2.一种新型冠状病毒S蛋白的B细胞线性抗原表位，其特征在于，所述S蛋白的B细胞线性抗原表位的氨基酸序列为如SEQ ID NO.104、SEQ ID NO.128和SEQ ID NO.129所示。
    3.一种新型冠状病毒S蛋白的B细胞线性抗原表位的鉴定方法，其特征在于，包括：
    根据S蛋白的氨基酸序列，设计合成若干肽段组，每个所述肽段组包括若干肽段，所述肽段的氨基酸序列如SEQ ID NO.1~ SEQ ID NO.127所示；
    采集恢复期血清或血浆样本，灭活备用；
    将所述每个肽段组分别包被ELISA 96孔板，并与所述恢复期血清或血浆样本进行间接酶联免疫反应检测，确定阳性肽段组；
    将所述阳性肽段组内的每个肽段分别包被ELISA 96孔板，继续与所述恢复期血清或血浆样本逐一进行间接酶联免疫反应检测，确定阳性反应肽段，所述阳性反应肽段的氨基酸序列为权利要求1所述的SEQ ID NO.104和SEQ ID NO.82。
    4.一种新型冠状病毒抗体，其特征在于，所述新型冠状病毒抗体是采用权利要求1所述的B细胞线性抗原表位的氨基酸序列偶联惰性蛋白，按照免疫程序免疫动物获得的抗性血清。
    5.一种检测新型冠状病毒的试剂盒，其特征在于，所述试剂盒包括权利要求1所述的B细胞线性抗原表位。
    6.一种新型冠状病毒抗体的检测方法，其特征在于，包括：
    将权利要求1所述的B细胞线性抗原表位包被96孔板，与待测血清样本逐一进行间接ELISA检测，重复测定两次；
    以健康人血清作为阴性对照计算cutoff值，待测血清样本的平均OD值大于cutoff值认为抗体反应阳性。
    7.一种新型冠状病毒抗体和抗原的鉴别方法，其特征在于，所述鉴别方法包括采用权利要求1所述的B细胞线性抗原表位的氨基酸序列作为抗原去检测新型冠状病毒抗体；采用权利要求5所述的新型冠状病毒抗体检测新型冠状病毒。
    8.一种新型冠状病毒疫苗，其特征在于，所述新型冠状病毒疫苗包括抗原表位，所述抗原表位为权利要求1所述的B细胞线性抗原表位。
    9.如权利要求1所述的新型冠状病毒S蛋白的B细胞线性抗原表位在制备新型冠状病毒抗体中的应用或在制备新型冠状病毒疫苗中的应用。
    10.如权利要求1所述的新型冠状病毒S蛋白的B细胞线性抗原表位在鉴别新型冠状病毒抗体和抗原的应用。
PCT/CN2020/126206 2020-10-15 2020-11-03 一种新型冠状病毒 s 蛋白的 b 细胞线性抗原表位、抗体、鉴定方法及应用 WO2022077613A1 (zh)

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