WO2023051611A1 - SARS-CoV-2特异性多肽及其应用 - Google Patents

SARS-CoV-2特异性多肽及其应用 Download PDF

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WO2023051611A1
WO2023051611A1 PCT/CN2022/122150 CN2022122150W WO2023051611A1 WO 2023051611 A1 WO2023051611 A1 WO 2023051611A1 CN 2022122150 W CN2022122150 W CN 2022122150W WO 2023051611 A1 WO2023051611 A1 WO 2023051611A1
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seq
polypeptide
cov
sars
amino acid
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PCT/CN2022/122150
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English (en)
French (fr)
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刘军
张�杰
高福
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中国疾病预防控制中心病毒病预防控制所
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Priority claimed from CN202111149547.4A external-priority patent/CN113845577A/zh
Priority claimed from CN202111150064.6A external-priority patent/CN113801208B/zh
Priority claimed from CN202210083824.4A external-priority patent/CN116514930A/zh
Application filed by 中国疾病预防控制中心病毒病预防控制所 filed Critical 中国疾病预防控制中心病毒病预防控制所
Publication of WO2023051611A1 publication Critical patent/WO2023051611A1/zh

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    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • 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
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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

Definitions

  • the invention relates to a SARS-CoV-2 specific polypeptide and application thereof, and belongs to the field of immune detection and vaccines.
  • the continuous spread of the new coronavirus and the continuous emergence of mutant viruses have had a major impact on the health and life of people around the world.
  • the current COVID-19 vaccines mainly include inactivated vaccines, peptide protein vaccines and mRNA vaccines.
  • the mutant strain of the new crown not only changes its pathogenicity and transmissibility, but also affects its immunogenicity, which has an important impact on the effectiveness of currently used detection reagents and vaccines. Therefore, it is imminent to explore more effective detection reagents and vaccines that can deal with mutant strains.
  • HLA human leukocyte antigen
  • the screening of T cell dominant epitopes is based on Enzyme-linked Immunospot Assay (ELISpot), which combines cell culture technology and ELISA technology, and can detect cytokines secreted by a single cell.
  • the principle is: a 96-well plate with a PVDF membrane as the bottom is coated with a specific monoclonal antibody to capture the cytokines secreted by the cells, and the antigen stimulant to be detected and the cells are added to the wells of the culture plate for culture. Under the stimulation of the stimulus, T cells will secrete the corresponding cytokines in the corresponding time period, and the cytokines will be captured by the antibody coated on the membrane at this time.
  • ELISpot Enzyme-linked Immunospot Assay
  • the captured cytokines can be combined with the biotin-labeled secondary antibody, and then the enzyme-labeled avidin can be combined with the biotin for chemical enzyme-linked color development, and circles can be formed locally on the membrane. Each spot corresponds to a cell that secretes cytokines.
  • T cell recognition peptides are limited by individual HLA typing. People in different regions have different characteristics of HLA typing. In the Chinese population, the HLA-A typing that accounts for the majority includes HLA-A2/A11/A24/A3/A1 , HLA-B typing includes B27/B62/B46/B60/B44/B61/B35 and so on. At present, in the identification of new coronavirus T cell epitopes at home and abroad, most of them use software to directly predict short peptides with 8-10 amino terminals of specific HLA types, and use specific HLA types of COVID-19 patients to identify whether they are positive epitopes. bit. The epitopes identified by this method are limited to individual HLA typing, which is not comprehensive enough, and lacks systematic and comprehensive screening of more dominant epitopes applicable to the Chinese population.
  • Enzyme-linked Immunospot Assay combines cell culture technology and ELISA technology to detect cytokines secreted by a single cell.
  • the principle is: a 96-well plate with a PVDF membrane as the bottom is coated with a specific monoclonal antibody to capture the cytokines secreted by the cells, and the antigen stimulant to be detected and the cells are added to the wells of the culture plate for culture. Under the stimulation of the stimulus, T cells will secrete the corresponding cytokines in the corresponding time period, and the cytokines will be captured by the antibody coated on the membrane at this time.
  • the captured cytokines can be combined with the biotin-labeled secondary antibody, and then the enzyme-labeled avidin can be combined with the biotin for chemical enzyme-linked color development, and circles can be formed locally on the membrane. Each spot corresponds to a cell that secretes cytokines.
  • MHC-tetramer technology is a technology that tetramerizes MHC monomer molecules to increase its affinity with TCR on T cells, thereby improving the detection sensitivity.
  • This technology can be applied to the detection of antigen-specific T lymphocytes, direct isolation and cloning of T cells, isolation of specific TCR, in situ staining, etc. It provides an efficient, rapid and sensitive method for studying a series of work related to cellular immune responses. testing method.
  • tetramer technology to evaluate the level of T cell immunity in people who have recovered from SARS-CoV-2 infection or who have been vaccinated.
  • the invention provides a SARS-CoV-2 specific polypeptide, including amino acid sequences such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO. ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO .30, SEQ ID NO.37, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.48 , SEQ ID NO.53, SEQ ID NO.57, SEQ ID NO.59, SEQ ID NO.65, SEQ ID NO.66, SEQ ID NO.72, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.74, SEQ ID NO.
  • the present invention also provides a nucleic acid encoding the above-mentioned SARS-CoV-2 specific polypeptide.
  • the present invention also provides a recombinant vector carrying the above nucleic acid.
  • the present invention also provides host cells carrying the above-mentioned nucleic acid or the above-mentioned recombinant vector.
  • the present invention also provides derivatives of the SARS-CoV-2 specific polypeptide, which are substituted or deleted in the amino acid sequence of the SARS-CoV-2 specific polypeptide, or one or several amino acids are added, and have A polypeptide derivative having the same antigenicity as said polypeptide.
  • the present invention also provides a highly specific and highly sensitive polypeptide-MHC tetramer for SARS-CoV-2 infection or T cells of the population after vaccination.
  • the polypeptide-MHC tetramer is composed of biotinylated MHC- I is combined with the SARS-CoV-2 specific polypeptide, or biotinylated MHC-I is combined with the specific polypeptide derivative.
  • the present invention also provides a preparation method of the polypeptide-MHC tetramer, comprising the following steps: (1) expressing the MHC light chain and the MHC heavy chain with Escherichia coli; (2) diluting and refolding to prepare the polypeptide/MHC complex; (3) preparing biotinylated polypeptide/MHC complexes; (4) reacting biotinylated polypeptide/MHC complexes with labeled streptavidin.
  • the C-terminus of the MHC heavy chain is linked with biotin.
  • an amino acid sequence GGGLNDIFEAQKIEWHE capable of linking biotin is added to the C-terminus of the MHC.
  • the polypeptide/MHC complex binds to D-biotin under the catalysis of BirA enzyme.
  • the concentration of the D-biotin is 400-600 ⁇ mol/L.
  • step (4) react with the labeled streptavidin according to the molar ratio (4-6): (0.5-1.5).
  • step (4) react with labeled streptavidin at a molar ratio of 5:1.
  • the preparation method of the polypeptide-MHC tetramer further includes purification.
  • the purification is to purify the prepared polypeptide/MHC complex through molecular sieves.
  • the method specifically includes: using Escherichia coli to express the MHC light chain and the MHC heavy chain connected to the C-terminal biotin, preparing the polypeptide/MHC complex by diluting renaturation, purifying it with superdex200, and then Under the catalysis of BirA enzyme, it combines with D-biotin to form a biotinylated polypeptide/MHC complex, and then reacts with labeled streptavidin at a molar ratio of 5:1 to obtain a polypeptide/MHC complex (polypeptide- MHC tetramer).
  • the invention also provides the application of the polypeptide-MHC tetramer.
  • the application includes: preparing vaccines; as an effective tool for evaluation of T cell immunology, evaluating the T cell immune response of the population after SARS-CoV-2 infection or vaccination; 2 Flow cytometry detection of corresponding immune cells in infected or vaccinated populations, in situ staining of tissue sections, isolation and cloning of specific T cells, isolation of specific TCRs combined with single-cell sequencing technology, and use as T cell activation reagents.
  • the present invention also provides novel coronavirus CD8 + T cell epitope peptides, including epitope peptides with amino acid sequences as shown in any one of SEQ ID NO.272 to SEQ ID NO.286 or a combination of two or more epitope peptides.
  • the present invention also provides nucleic acid encoding the above novel coronavirus CD8 + T cell epitope peptide.
  • the present invention also provides a recombinant vector carrying the above nucleic acid.
  • the recombinant vector includes an adenoviral vector, a lentiviral vector or a prokaryotic expression vector.
  • the present invention also provides host cells carrying the above-mentioned nucleic acid or the above-mentioned recombinant vector.
  • the host cell comprises a mammalian cell or a prokaryotic cell.
  • the present invention also provides a polypeptide vaccine, the active ingredient of which contains the SARS-CoV-2 specific polypeptide, the SARS-CoV-2 specific polypeptide derivative and/or the novel coronavirus CD8 + T cell expression Bit peptide.
  • the present invention also provides a SARS-CoV-2 specific cellular immune detection kit, which contains the SARS-CoV-2 specific polypeptide and/or SARS-CoV-2 specific polypeptide derivatives.
  • the present invention also provides the new coronavirus CD8 + T cell epitope peptide shown in the amino acid sequence of SEQ ID NO.272 to SEQ ID NO.286 or the above-mentioned nucleic acid or the above-mentioned recombinant vector or the above-mentioned host cell in the preparation of the new coronavirus vaccine Application or antibody or detection kit.
  • the novel coronavirus CD8 + T cell epitope peptide with amino acid sequences as shown in SEQ ID NO.272 to SEQ ID NO.286 can bind to MHC-I class molecules, wherein, when the polypeptide binds to MHC- Class I molecules are recognized by CD8 + T cells when bound.
  • the present invention provides a polypeptide composition, and the polypeptide composition is composed of any one or more polypeptide libraries in the S1 polypeptide library, the S2 polypeptide library, the M polypeptide library, and the N polypeptide library.
  • the S1 polypeptide library is composed of polypeptides with amino acid sequences such as SEQ ID NO.87 to SEQ ID NO.178
  • the S2 polypeptide library is composed of amino acid sequences such as SEQ ID NO.179 to SEQ ID NO. 271
  • the M polypeptide library is composed of polypeptides with amino acid sequences such as SEQ ID NO.1 ⁇ SEQ ID NO.29
  • the N polypeptide library is composed of amino acid sequences such as SEQ ID NO.30 ⁇ SEQ ID NO.86.
  • the polypeptide composition has the ability to bind to MHC-I or MHC-II class molecules, wherein when the polypeptide composition binds to MHC, it can be recognized by CD4 + or CD8 + T cells.
  • the present invention also provides the application of the polypeptide composition in the preparation of products for evaluating the cellular immunity level of COVID-19 patients, COVID-19 recoverers or COVID-19 vaccinators.
  • the present invention also provides a kit for assessing the level of cellular immunity of patients with COVID-19, recovering from COVID-19 or vaccine recipients of COVID-19, comprising the above-mentioned polypeptide composition.
  • the kit also contains IFN- ⁇ monoclonal antibody.
  • the polypeptide composition is present in the product in the form of a peptide library stock solution.
  • the kit is also provided with an ELISpot plate, and the ELISpot is pre-coated with monoclonal antibodies that capture cytokines secreted by T cells.
  • the ELISpot plate is pre-coated with a monoclonal antibody capturing IFN- ⁇ .
  • the present invention also provides a detection method for assessing the level of cellular immunity of patients with COVID-19, recovered patients with COVID-19 or vaccine recipients with COVID-19, using the polypeptide composition to stimulate the PBMC cells of patients with COVID-19, recovered patients with COVID-19 or vaccine recipients with COVID-19, and then using Enzyme-linked immunospot technology detects whether spots appear after polypeptide stimulation, that is, the release of IFN- ⁇ .
  • step (2) (4) removing the blocking solution, adding the polypeptide composition and the peripheral blood mononuclear cells of step (2), incubating, washing, and removing the liquid;
  • a negative control is set in the method; the stimulating solution of the positive control is phorbol ester (PMA).
  • PMA phorbol ester
  • the blocking solution is RPMI1640 medium containing 10% fetal bovine serum.
  • the cytokine is IFN- ⁇ .
  • said sample comprises umbilical cord blood, bone marrow, peripheral blood.
  • the screening result of the T cell peptide library is judged by the number of spot-forming cells after the color reaction; if the number of spot-forming cells after the color reaction is twice or more than the number of spot-forming cells in the negative control, the result is Positive.
  • said culturing and expanding said peripheral blood mononuclear cells comprises the following steps:
  • the medium is RPMI1640 medium containing 10% fetal bovine serum; the concentration of IL-7 is 15-25ng/mL; the concentration of IL-2 is 175-225U /mL.
  • the present invention provides amino acid sequences such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.
  • the peptide library covers 9 HLA-A alleles and 17 HLA-B alleles.
  • the peptide library covers HLA-A2, HLA-A11, HLA-A24, HLA-A3, HLA-A1, HLA-A31, HLA-A33, HLA-A30, HLA-A2, HLA- B27, HLA-B62, HLA-B46, HLA-B60, HLA-B44, HLA-B61, HLA-B35, HLA-B51, HLA-B67, HLA-B75, HLA-B55, HLA-B58, HLA-B13, HLA-B54, HLA-B5102, HLA-B52, HLA-B39.
  • the vaccine comprises an epitope vaccine.
  • the vaccine further includes a carrier, and the carrier includes lipid, heat-activated protein, ovalbumin, bovine serum albumin, and keyhole limpet hemocyanin.
  • the vaccine further includes an adjuvant including Freund's adjuvant and aluminum hydroxide adjuvant.
  • the present invention also provides amino acid sequences such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.30, SEQ ID NO.37, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.53, SEQ ID NO. 57.
  • amino acid sequences such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID
  • SEQ ID NO.59 SEQ ID NO.65, SEQ ID NO.66, SEQ ID NO.72, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.75, SEQ ID NO.77, SEQ ID NO.87, SEQ ID NO.90, SEQ ID NO.91, SEQ ID NO.92, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99, SEQ ID NO.100, SEQ ID NO.101, SEQ ID NO.104, SEQ ID NO.109, SEQ ID NO.110, SEQ ID NO.114, SEQ ID NO.115, SEQ ID NO.116, SEQ ID NO.117, SEQ ID NO. 125.
  • SEQ ID NO.126 SEQ ID NO.137, SEQ ID NO.138, SEQ ID NO.142, SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.161, SEQ ID NO.162, SEQ ID NO.164, SEQ ID NO.169, SEQ ID NO.171, SEQ ID NO.175, SEQ ID NO.185, SEQ ID NO.195, SEQ ID NO.200, SEQ ID NO.201, SEQ ID NO.204, SEQ ID NO.206, SEQ ID NO.214, SEQ ID NO.215, SEQ ID NO.220, SEQ ID NO.222, SEQ ID NO.
  • SEQ ID NO.244 Application of a peptide library composed of any one or more of the polypeptides shown in SEQ ID NO.245, SEQ ID NO.251 or SEQ ID NO.255 in the preparation of a Chinese population HLA-specific novel coronavirus T cell immune detection kit .
  • the peptide library covers 9 HLA-A alleles and 17 HLA-B alleles.
  • the peptide library covers HLA-A2, HLA-A11, HLA-A24, HLA-A3, HLA-A1, HLA-A31, HLA-A33, HLA-A30, HLA-A2, HLA- B27, HLA-B62, HLA-B46, HLA-B60, HLA-B44, HLA-B61, HLA-B35, HLA-B51, HLA-B67, HLA-B75, HLA-B55, HLA-B58, HLA-B13, HLA-B54, HLA-B5102, HLA-B52, HLA-B39.
  • the kit also contains IFN- ⁇ monoclonal antibody.
  • the kit is also provided with an ELISpot reaction plate, and the ELISpot is pre-coated with a monoclonal antibody that captures IFN- ⁇ secreted by T cells.
  • the ELISpot plate is pre-coated with a monoclonal antibody capturing IFN- ⁇ .
  • the present invention also provides amino acid sequences such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.30, SEQ ID NO.37, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.53, SEQ ID NO. 57.
  • amino acid sequences such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID
  • SEQ ID NO.59 SEQ ID NO.65, SEQ ID NO.66, SEQ ID NO.72, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.75, SEQ ID NO.77, SEQ ID NO.87, SEQ ID NO.90, SEQ ID NO.91, SEQ ID NO.92, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99, SEQ ID NO.100, SEQ ID NO.101, SEQ ID NO.104, SEQ ID NO.109, SEQ ID NO.110, SEQ ID NO.114, SEQ ID NO.115, SEQ ID NO.116, SEQ ID NO.117, SEQ ID NO. 125.
  • SEQ ID NO.126 SEQ ID NO.137, SEQ ID NO.138, SEQ ID NO.142, SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.161, SEQ ID NO.162, SEQ ID NO.164, SEQ ID NO.169, SEQ ID NO.171, SEQ ID NO.175, SEQ ID NO.185, SEQ ID NO.195, SEQ ID NO.200, SEQ ID NO.201, SEQ ID NO.204, SEQ ID NO.206, SEQ ID NO.214, SEQ ID NO.215, SEQ ID NO.220, SEQ ID NO.222, SEQ ID NO.
  • SEQ ID NO.244 Application of a peptide library composed of any one or more of the polypeptides shown in SEQ ID NO.245, SEQ ID NO.251 or SEQ ID NO.255 in the preparation of cell therapy or adoptive therapy kits.
  • the present invention has designed 271 15-18-mer SARS-CoV-2 peptides formed by overlapping 10 amino acids, and the amino acid sequences of the 271 SARS-CoV-2 peptides are as SEQ ID NO.1 ⁇ SEQ ID As shown in NO.271, these peptides span the entire S, M and N proteins.
  • the peptides of the S protein are divided into S1 and S2 regions , there are 92 polypeptides of S1 protein (amino acid sequence as shown in SEQ ID NO.87 ⁇ SEQ ID NO.178), and 93 polypeptides of S2 protein (amino acid sequence as shown in SEQ ID NO.179 ⁇ SEQ ID NO.271) shown), there are 29 peptides of M protein (amino acid sequence as shown in SEQ ID NO.1 ⁇ SEQ ID NO.29), and 57 polypeptides of N protein (amino acid sequence as shown in SEQ ID NO.30 ⁇ SEQ ID NO. 86).
  • the present invention first identified the HLA typing of PBMC cells derived from Chinese population, mainly HLA-A2/A11/A24/A3/A1, HLA-B27/B62/B46/B60/B44/B61/B35, etc. 271 polypeptide sequences were verified by ELISpot one by one, and 85 positive epitopes of the new coronavirus covering the main HLA typing of the Chinese population were screened, of which 53 were located in the S protein, 13 were located in the M protein, and 19 were located in the N protein. And identified 35 dominant T cell epitopes, including 17 dominant epitopes located in S protein, 7 dominant epitopes located in M protein, and 11 dominant epitopes located in N protein.
  • the present invention has identified 15 CD8 + T positive epitopes (amino acid sequence as shown in SEQ ID NO.272 to SEQ ID NO.286), these sequences will be used for the development of new coronavirus vaccines, new therapeutic drugs and diagnostic testing in the future.
  • the development of the kit provides the basis.
  • the present invention stimulates the polypeptide antigen and cultures the cells in vitro for 9 days to proliferate the virus-specific T cells and effectively improve the detection sensitivity.
  • the present invention can select the judgment standard according to the specific situation to select the peptide library, and select or combine the ELISpot results obtained from the stimulation samples of different peptide libraries: if it is necessary to improve the detection sensitivity and minimize the missed judgment, you can choose the total peptide library or S1 or Any positive of the M peptide library is judged as the final positive; if it is necessary to reduce misjudgment (excluding non-positive patients) and improve the specificity, the results of the S1 peptide library and the M peptide library can be combined (double positives are judged as the final positive).
  • the positive rate of T cell reaction of patients with new coronary pneumonia stimulated by the total peptide library was 93.42% after 6 months of recovery; the positive rate of T cell reaction after 12 months of recovery was 91.78%.
  • using the method of compatibility evaluation of S1 peptide library and M peptide library can obtain higher sensitivity and specificity (the gold standard is virus nucleic acid detection): sensitivity 71.43%, specificity 96.43%.
  • Figure 3 is: HLA-A*1101 and epitope polypeptide superdex200 molecular sieve column diagram
  • Figure 4 is a column diagram of superdex200 molecular sieve after biotinylation of the MHC complex formed by HLA-A*1101 and epitope polypeptide;
  • Figure 5 is: M23-Tetramer stained SARS-CoV-2 infection recovery T cell flow cytometry results representative figure
  • Figure 6 is a representative diagram of the results of flow cytometry analysis of T cells from recovered patients infected with SARS-CoV-2 infected with N25-Tetramer.
  • the present invention will be further described below in conjunction with specific examples, but the examples do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • step 6 Transfer all the liquid in step 5 to the adsorption column that has been loaded into the collection tube. If there is more liquid, it can be added in multiple times. Centrifuge at 12000rpm for 1min, discard the waste liquid in the collection tube, and put the adsorption column back.
  • the extracted blood DNA of the research subjects was sent to Beijing Mantaili Biotechnology Co., Ltd., and the company used the flow cytometric SSO typing technology of ONE LAMBDA company in the United States to determine the HLA typing. Both HLA-A and HLA-B gene loci were sequenced for each sample.
  • SARS-CoV-2 peptides overlapped by 10 amino acids were designed by PeptGen software, and the amino acid sequences of 271 SARS-CoV-2 peptides are shown as SEQ ID NO.1 ⁇ SEQ ID NO As shown in .271, these peptides cover the entire S, M and N proteins.
  • the polypeptides of the S protein are divided into S1 and S2, and the polypeptides of the S1 protein are 92 (amino acid sequence as shown in SEQ ID NO.87 ⁇ SEQ ID NO.178), there are 93 peptides of S2 protein (amino acid sequence as shown in SEQ ID NO.179 ⁇ SEQ ID NO.271), M protein There are 29 polypeptides (amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.29), and 57 peptides of N protein (amino acid sequences shown in SEQ ID NO.30 to SEQ ID NO.86). 271 peptides were mixed to form peptide libraries, namely S1, S2, M and N peptide libraries, which were used as antigenic stimuli for stimulating culture of recovered PBMCs.
  • the S1 peptide library contains 92 polypeptides of the S1 protein (the amino acid sequence is shown in SEQ ID NO.87 ⁇ SEQ ID NO.178), similarly, the S2 peptide library Contains 93 peptides of S2 protein (amino acid sequence shown in SEQ ID NO.179 ⁇ SEQ ID NO.271), M peptide library contains 29 polypeptides of M protein (amino acid sequence shown in SEQ ID NO.1 ⁇ SEQ ID NO. 29), the N peptide library contains 57 polypeptide amino acid sequences of N proteins as shown in SEQ ID NO.30 ⁇ SEQ ID NO.86). Dissolve the synthesized peptides with DMSO, the target concentration is 20 ⁇ g/ ⁇ l, mix well, take 1 ⁇ l of each polypeptide and mix it into a peptide library.
  • the peptide library prepared in Example 1 was used to stimulate PBMC cells, and the positive rate of T cell reaction was detected by ELISpot.
  • 10ml anticoagulant tube and coagulant tube (BD), 10ml pipette, lymphocyte separation tube (Cat#DKW-LST-24015SK), 1640 medium, DMSO, 15ml centrifuge tube, 50ml centrifuge tube, cell freezing box, Cell cryopreservation tube (Thermo), domestic cell cryopreservation tube (Jiangsu Haimen, yellow cap/blue cap), liquid nitrogen cryopreservation box, electric pipette, isopropanol, calf serum (FBS), pasteurization Suction tubes with lifting acceleration Horizontal centrifuge equipped with 15mL centrifuge tube bucket.
  • Pretreatment take out the lymphocyte separation tube, and centrifuge at 800 g at 20° C. for 1 min. Centrifuge the test tube stored with anticoagulant blood at 400g for 5 minutes (or draw it directly after a long period of storage), absorb 2ml of the upper layer of plasma, and divide it into 2 tubes (blue-capped domestic cell cryopreservation tubes) for cryopreservation.
  • PBMC Absorb PBMC: After centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation fluid, and the uppermost layer is plasma. The middle layer between the plasma and the separation fluid is a dense white membrane containing mononuclear cells (including lymphocytes and monocytes).
  • a Pasteur pipette to suck up the white cell layer in the middle, transfer it to a new 15ml centrifuge tube, and add RPMI 1640 culture medium to 12ml. Centrifuge at 400g for 10min.
  • PBMC cleaning After centrifugation, pour off the supernatant in the cell operation platform, shake the centrifuge tube, and use the remaining liquid of about 200 microliters to resuspend the cells. Add 10ml of RPMI 1640 medium, 400g, and wash the cells for 10min.
  • 15ml centrifuge tube 1.5ml ep tube, 1ml pipette, 10ul pipette, cell counting plate, cell counter, 24-well cell culture plate, RPMI 1640 medium, interleukin-2 (IL-2), interleukin-7 (IL-7), fetal bovine serum (FBS), penicillin and streptomycin, trypan blue staining solution, water bath, horizontal centrifuge equipped with 15mL centrifuge tube bucket, cell culture incubator.
  • IL-2 interleukin-2
  • IL-7 interleukin-7
  • FBS fetal bovine serum
  • penicillin and streptomycin trypan blue staining solution
  • water bath horizontal centrifuge equipped with 15mL centrifuge tube bucket, cell culture incubator.
  • PBMCs cell recovery recover the cells and mix them in 10ml of complete medium (10% FBS, RPMI 1640 medium), mix well and centrifuge at 1500r for 10min, discard the supernatant, add x ml of complete medium to resuspend.
  • Cell counting add 10 ⁇ l of trypan blue to the ep tube, 10 ⁇ l of mixed cells, and read the cell number y in the two nine-square grids.
  • PBMC stimulation put the 24-well plate into the incubator and let it settle for 3-5 hours, then stimulate it with the peptide library in Example 1 for 45 minutes (peptide library: number of strips ⁇ 0.2 ⁇ l + 100 ⁇ l medium/well), and then add 900 ⁇ l of complete medium containing IL-7 (20 ug/ml).
  • Cell culture Cells were observed every day and cultured for 9 days. Change the medium in half every two days (aspirate 1mL of the supernatant and discard it, and slowly add fresh medium by circling around the wall). The medium to be replaced was a complete medium containing 200 U/ml IL-2.
  • PVDF membrane-bottomed 96-well plate 1.5mlep tube, 1ml pipette, 10ul pipette, cell counting plate, cell counter, liquid separation tank, coated antibody (purified anti-IFN- ⁇ ), 1XPBS, RPMI 1640 Culture medium, fetal bovine serum (FBS), DMSO, phorbol ester (PMA), deionized water, Tween, detection antibody (Biotinylated anti-IFN- ⁇ ), avidin-HRP conjugate (streptavidin-HRP), AEC substrate solution (AEC substrate), AEC dye (AEC chromogen), cell culture incubator, refrigerator, ELISpot Reader System (CTL-Immunospot S5Versa), etc.
  • FBS fetal bovine serum
  • PMA phorbol ester
  • detection antibody Biotinylated anti-IFN- ⁇
  • avidin-HRP conjugate streptavidin-HRP
  • AEC substrate solution AEC substrate
  • AEC dye A
  • Coating antibody prepare a PVDF membrane-bottomed 96-well plate (100 ⁇ l/well), and mix the coating antibody (purified anti-IFN- ⁇ ) with 1 ⁇ PBS at a ratio of 1:200.
  • Blocking Pour out the solution, wash 2 times with 1640 medium (180 ⁇ l/well), add 180 ⁇ l complete medium (RPMI 1640 medium + 10% calf serum + 1-2% penicillin and streptomycin) per well closed.
  • Polypeptide Peptide library stimulation: The final concentration of single peptide is 2 ⁇ g/ml, 0.4 ⁇ g per well.
  • Example 1 Add the peptide library in Example 1 (0.02*number of strips ⁇ l+100 ⁇ l complete medium/well), mock (100 ⁇ l complete medium/well), cells (1 ⁇ 10 5 /well), PMA (10 ⁇ l PMA+90 ⁇ l Complete medium/well, powdered PMA dissolved in 500 ⁇ l RPMI 1640 medium or PBS per bottle).
  • Antibody incubation Dilute the detection antibody (Biotinylated anti-IFN- ⁇ ) 1:250 into PBS containing 10% FBS and mix well. 100 ⁇ l per well, incubate at room temperature for 2 hours. Remove the original liquid, wash 3 times with deionized water at room temperature, 5 minutes each time, wash 3 times with PBST, 2 minutes apart each time, and blot dry on absorbent paper. Avidin-HRP conjugate (streptavidin-HRP) was diluted 1:100 into PBS containing 10% serum and mixed well. 100 ⁇ l per well, incubate at room temperature for 1 h. Remove the original liquid, wash 3 times with PBST, and 2 times with PBS, with an interval of 2 minutes between each time, and blot dry on absorbent paper.
  • detection antibody Biotinylated anti-IFN- ⁇
  • Color development system: 100 ⁇ l/well AEC substrate solution (AEC substrate) + 1 drop/ml AEC dye (AEC chromogen) and mix well in the dark, add 100 ⁇ l to each well.
  • the positive rate of T cell reaction of patients with new coronary pneumonia stimulated by the total peptide library after 6 months of recovery is 93.42%; the positive rate of T cell reaction after 12 months of recovery is 91.78%.
  • using the method of compatibility evaluation of S1 peptide library and M peptide library can obtain higher sensitivity and specificity (the gold standard is virus nucleic acid detection): sensitivity 71.43%, specificity 96.43%.
  • 10mL anticoagulant tube and coagulant tube (BD), 10mL pipette, lymphocyte separation tube (Cat#DKW-LST-24015SK), 1640 medium, DMSO, 15mL centrifuge tube, 50mL centrifuge tube, cell freezing box, Cell cryopreservation tube (Thermo), domestic cell cryopreservation tube (Jiangsu Haimen, yellow cap/blue cap), liquid nitrogen cryopreservation box, electric pipette, isopropanol, fetal bovine serum (FBS), Pasteur pipette , a horizontal centrifuge with lifting acceleration equipped with a 15mL centrifuge tube bucket.
  • BD anticoagulant tube and coagulant tube
  • 10mL pipette lymphocyte separation tube
  • 1640 medium 1640 medium
  • DMSO 15mL centrifuge tube
  • 50mL centrifuge tube cell freezing box
  • Cell cryopreservation tube Thermo
  • domestic cell cryopreservation tube Juangsu Haimen
  • Pretreatment take out the lymphocyte separation tube, and centrifuge at 800 g at 20° C. for 1 min. Centrifuge the test tube stored with anticoagulant blood at 400g for 5 minutes, absorb 2 mL of the upper layer of plasma, and divide it into 2 tubes (blue-capped domestic cell cryopreservation tubes) for frozen storage, or directly absorb after a long period of storage.
  • PBMCs After centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation fluid, and the uppermost layer is plasma.
  • the middle layer between the plasma and the separation fluid is a dense white membrane containing mononuclear cells (including lymphocytes and monocytes).
  • a Pasteur pipette suck the white cell layer in the middle with a Pasteur pipette, transfer it to a new 15mL centrifuge tube, and add 1640 culture medium to 12mL. Centrifuge at 400g for 10min.
  • Growth medium 1640 medium containing 10% FBS and 1% penicillin and streptomycin.
  • PBMCs recovery mix the recovered cells in 10mL 1640 medium, centrifuge at 1500r for 10min, discard the liquid and add x mL of growth medium to resuspend.
  • Stimulation of PBMCs Put the 24-well plate into the incubator and let it settle for 3-5 hours, then absorb 1 mL of the supernatant, and then add the peptide library in Example 1 (peptide library: number of strips ⁇ 0.2 ⁇ L + 100 ⁇ L medium /well), stimulated for 45 min, and then added 900 ⁇ L of growth medium containing interleukin 7 (IL-7, 20 ⁇ g/mL).
  • IL-7 interleukin 7
  • Cell culture Cells were observed every day and cultured for 9 days. Change the medium in half every two days (aspirate 1mL of the supernatant and discard it, and slowly add fresh medium by circling around the wall). The replacement ratio is: 1 mL/well of 1640 medium (containing 10% FBS), containing a final concentration of 200 U/mL interleukin 2 (IL-2), and 1% penicillin and streptomycin.
  • IL-2 interleukin 2
  • PVDF membrane-bottomed 96-well plate 1.5mL centrifuge tube, 1mL pipette gun, 10 ⁇ L pipette gun, cell counting plate, cell counter, liquid separation tank, coated antibody (purified anti-IFN- ⁇ ), 1 ⁇ PBS , 1640 medium, fetal bovine serum (FBS), DMSO, PMA, deionized water, Tween 20, detection antibody (Biotinylated anti-IFN- ⁇ ), avidin-HRP conjugate (streptavidin-HRP), AEC substrate Substrate solution (AEC substrate), AEC dye (AEC chromogen), cell culture incubator, refrigerator, ELISpot Reader System (CTL-Immunospot S5 Versa), etc.
  • coated antibody purified anti-IFN- ⁇
  • 1 ⁇ PBS 1640 medium, fetal bovine serum (FBS), DMSO, PMA, deionized water, Tween 20, detection antibody (Biotinylated anti-IFN- ⁇ ), avidin-HRP
  • Coating antibody prepare the coating antibody (purified anti-IFN- ⁇ ) mixed with 1 ⁇ PBS at a ratio of 1:200, and add to a 96-well plate with PVDF membrane as the bottom (100 ⁇ L/well).
  • Blocking decant the solution, wash 2 times with 1640 medium (180 ⁇ L/well), add 180 ⁇ L of growth medium (1640 medium + 10% fetal bovine serum + 1% penicillin) to block each well.
  • Polypeptides 271 peptides designed in Chemical Synthesis Example 1, 125 ⁇ L DMSO was added to 2.5 mg of synthetic polypeptides to dissolve (20 ⁇ g/ ⁇ L); single peptide stimulation: the final concentration of a single polypeptide added to each well was 10 ⁇ g/mL, 2 ⁇ g per well;
  • wash the plate remove cells, wash with deionized water at room temperature 3 times, 5 minutes each time, wash 3 times with PBST, 2 minutes each time, shake off the cleaning solution vigorously after each wash, and then blot dry on absorbent paper.
  • Antibody incubation Dilute the detection antibody (Biotinylated anti-IFN- ⁇ ) 1:250 into PBS containing 10% FBS and mix well. 100 ⁇ L per well, incubate at room temperature for 2 h. Remove the original liquid, wash 3 times with deionized water at room temperature, 5 minutes each time, wash 3 times with PBST, 2 minutes apart each time, and blot dry on absorbent paper. Avidin-HRP conjugate (streptavidin-HRP) was diluted 1:100 into PBS containing 10% FBS and mixed thoroughly. 100 ⁇ L per well, incubate at room temperature for 1 h. Remove the original liquid, wash 3 times with PBST, and 2 times with PBS, with an interval of 2 minutes between each time, and blot dry on absorbent paper.
  • detection antibody Biotinylated anti-IFN- ⁇
  • Color development system: 100 ⁇ L/well AEC substrate solution (AEC substrate) + 1 drop/mL AEC dye (AEC chromogen) and mix well in the dark, add 100 ⁇ L to each well.
  • the CD8 + T epitope was predicted according to the HLA typing of the positive recovered individual.
  • %Rank and Affinity are usually used as the indicators for determining the prediction results of the NetMHC website.
  • Affinity ⁇ 50 and %Rank ⁇ 0.5 are defined as high affinity
  • Affinity ⁇ 500 and %Rank ⁇ 2 are defined as weak affinity.
  • step 2) Cultivate the Escherichia coli BL21 carrying the plasmid in step 1) at 37°C and add 1mmol/L IPTG to induce protein expression, collect the bacteria, ultrasonically disrupt the bacteria, and dissolve the precipitate after high-speed centrifugation (12000rpm, 10min)
  • dissolution buffer 6mol/L guanidine hydrochloride, 10% glycerol, 50mmol/L Tris pH8.0, 100mmol/L NaCl, 10mmol/L EDTA
  • heavy chain HLA-A*1101 and light chain B2m were obtained;
  • polypeptide whose sequence is shown in SEQ ID NO.279 and the heavy chain and light chain are diluted and refolded in renatured buffer (100mmol/L Tris pH 8.0, 400mmol/L arginine, 2mmol/L EDTA) Simultaneous renaturation in the medium, so that it forms an MHC complex; adopt the same method as above to prepare the MHC complex with the polypeptide shown in SEQ ID NO.278, the heavy chain, and the light chain;
  • renatured buffer 100mmol/L Tris pH 8.0, 400mmol/L arginine, 2mmol/L EDTA
  • step (1) Collect the molecular sieve-purified polypeptide/MHC complex protein sample obtained in step (1) in an ultrafiltration concentrator tube, concentrate to about 300 ⁇ L, react with D-biotin under the catalysis of BirA enzyme, and incubate at 4°C Overnight, biotinylated protein samples were obtained.
  • the biotinylated MHC can combine with Streptavidin to form a macromolecule, which makes its band lag in SDS-PAGE.
  • the effect of biotinylation can be judged by comparing the ratio of (C-B)/C MHC content. Proportional MHC is better biotinylated, and the biotinylation effect of the technical solution of the present invention is about 70%.
  • biotinylated MHC molecules are concentrated, and the biotinylated MHC molecules are tetramerized according to the molar ratio of streptavidin to polypeptide/MHC complex of 1:5. Streptavidin is fluorescently labeled streptavidin and prime, and incubated overnight at 4°C to prepare M23 tetramers.
  • the N25 tetramer was prepared following the same procedure as above.
  • the sequence corresponding to the novel coronavirus positive epitope polypeptide provided in Example 3 or Example 4 is used to synthesize the polypeptide, or the expression of the nucleic acid sequence to synthesize RNA or DNA fragments in vivo or in vitro to provide an immunogen for the new crown,
  • the polypeptide is linked to a certain vaccine carrier (lipid, heat-activated protein, ovalbumin, bovine serum albumin or keyhole limpet hemocyanin) to obtain a vaccine, and the polypeptide can also be coupled to the carrier protein through a linking peptide.
  • the vaccine is prepared as an injection, and an adjuvant is also included in the vaccine, and the adjuvant is selected from Freund's incomplete adjuvant, Freund's complete adjuvant, Bacillus pertussis adjuvant, lipopolysaccharide, MF59 (oil-in-water emulsion containing squalene ), AS03 (containing squalene, vitamin E and Tween80), containing monophosphoryl lipid A (monophos-phoryl lipid A, MPL) AS01 and AS02 adjuvants, cytosine guanine oligodeoxynucleotide (CpG- ODN), aluminum hydroxide, alum, MONTANIDE ISA 51 VG or MONTANIDE ISA 720 VG.
  • the adjuvant is selected from Freund's incomplete adjuvant, Freund's complete adjuvant, Bacillus pertussis adjuvant, lipopolysaccharide, MF59 (oil-in-water emulsion
  • the vaccine can be preserved in the form of solid powder before being mixed with an adjuvant to immunize the body.
  • the solid powder can be prepared into a liquid, and an equal volume of adjuvant can be added to form an injection for immunization.
  • the present invention selects 6-8 week-old healthy HLA transgenic mice for intramuscular injection/peritoneal injection/subcutaneous injection of the vaccine, and isolates mouse splenocytes 1-2 weeks after the last immunization to detect IFN- ⁇ by ELISpot to measure memory T cell response , the results showed that after the vaccine was immunized, a strong memory T cell response could be detected in the mice.
  • Example 3 separate the PBMCs of a single patient, stimulate the cells with the new coronavirus positive epitope polypeptide provided in Example 3 and Example 4 in the culture medium, and detect the T cells against the new coronavirus after the induction is completed
  • the specific killing ability of T cells can be obtained by sorting, which can be used for the treatment of patients with new crowns.

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Abstract

SARS-CoV-2特异性多肽及其应用。设计了271条由10个氨基酸重叠而成的15-18聚体SARS-CoV-2肽段，氨基酸序列如SEQ ID NO.1～SEQ ID NO.271所示，这些肽段跨越了整个S、M和N蛋白。鉴定了中国人群来源的PBMC细胞的HLA分型，将271条多肽序列一一进行ELISpot验证，筛选得到85条覆盖中国人群主要HLA分型的新冠病毒阳性表位，其中位于S蛋白53条，位于M蛋白的13条，位于N蛋白的19条。还鉴定得到15条氨基酸序列如SEQ ID NO.272～SEQ ID NO.286所示的CD8 +T阳性表位。提供的SARS-CoV-2特异性多肽、新冠病毒阳性表位以及CD8 +T阳性表位为今后新型冠状病毒疫苗研发、新型治疗药物研发和诊断检测试剂盒的的开发提供了基础。

Description

SARS-CoV-2特异性多肽及其应用 技术领域
本发明涉及SARS-CoV-2特异性多肽及其应用，属于免疫检测和疫苗领域。
背景技术
新型冠状病毒的持续传播及突变病毒的不断出现，对全球人民的健康及生活产生重大影响。当前的新冠疫苗主要推广接种的有灭活疫苗、多肽蛋白疫苗及mRNA疫苗等。新冠突变株不仅改变其致病性、传播力，同时也影响到其免疫原性，对目前应用的检测试剂和疫苗的效果造成重要影响。因而探索更有效的能够应对变异株的检测试剂和疫苗迫在眉睫。在抗病毒免疫反应中，细胞免疫和体液免疫同样发挥重要的作用，而在细胞免疫中，表位多肽激发的细胞免疫反应受到人群白细胞抗原(HLA)限制性的影响，基于这一现象，可筛选适用于中国人群的免疫优势的T细胞表位，以用于T细胞免疫检测技术和相关疫苗的研发。
T细胞优势表位的筛选基于酶联免疫斑点检测(Enzyme-linked Immunospot Assay，ELISpot)，该技术结合了细胞培养技术与酶联免疫吸附技术，能够检测到单个细胞分泌的细胞因子情况。其原理是：用PVDF膜为底的96孔板包被上特异性的单克隆抗体以捕获细胞分泌的细胞因子，在培养板的孔内加入待检测的抗原刺激物和细胞进行培养。在刺激物的刺激下，T细胞就会在对应的时间段分泌对应的细胞因子，此时细胞因子就被包被在膜上的抗体所捕获。洗去细胞后，被捕获的细胞因子可以与生物素标记的第二抗体结合，然后用酶标亲和素与生物素结合，进行化学酶联显色，就可以在膜的局部形成一个个圆形的斑点，每一个斑点就对应了当初一个分泌细胞因子的细胞。
T细胞识别多肽受到个体HLA分型的限制，不同地区的人群具有不同的HLA分型特点，在中国人群中，占主要比例的HLA-A分型包括HLA-A2/A11/A24/A3/A1，HLA-B分型包括B27/B62/B46/B60/B44/B61/B35等。当前，国内外在新冠病毒T细胞表位鉴定中，多数采用软件直接预测特定HLA分型的8-10个氨基端的短肽，利用特定HLA分型的COVID-19康复者来鉴定是否是阳性表位。这种方法鉴定的表位局限于个别HLA分型，不够全面，且缺乏适用于中国人群的较为优势表位系统而全面的筛选。
酶联免疫斑点检测(Enzyme-linked Immunospot Assay，ELISpot)结合了细胞培养技术与酶联免疫吸附技术，能够检测到单个细胞分泌的细胞因子情况。其原理是：用PVDF膜为底的96孔板包被上特异性的单克隆抗体以捕获细胞分泌的细胞因子，在培养板的孔内加入待检测的抗原刺激物和细胞进行培养。在刺激物的刺激下，T细胞就会在对应的时间段分泌对应的细胞因子，此时细胞因子就被包被在膜上的抗体所捕获。洗去细胞后，被捕获的细胞因子可以与生物素标记的第二抗体结合，然后用酶标亲和素与生物素结合，进行化学酶联显色，就可以在膜的局部形成一个个圆形的斑点，每一个斑点就对应了当初一个分泌细胞因子的细胞。
MHC-四聚体技术是将MHC单体分子四聚体化，提高其与T细胞上的TCR的亲和力，进而提高检测的灵敏度的技术。该技术可应用于检测抗原特异性T淋巴细胞、T细胞的直接分离与克隆、分离特异性TCR、原位染色等，为研究与细胞免疫反应有关的一系列工作提供了高效、快速、敏感的检测手段。然而目前还没有用四聚体技术评价SARS-CoV-2感染康复人群或疫苗接种人群T细胞免疫水平的报道。
发明内容
本发明提供了一种SARS-CoV-2特异性多肽，包括氨基酸序列如SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.30、SEQ ID NO.37、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.53、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.77、SEQ ID NO.87、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92、SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.104、SEQ ID NO.109、SEQ ID NO.110、SEQ ID NO.114、SEQ ID NO.115、SEQ ID NO.116、SEQ ID NO.117、SEQ ID NO.125、SEQ ID NO.126、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.151、SEQ ID NO.152、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.164、SEQ ID NO.169、SEQ ID NO.171、SEQ ID NO.175、SEQ ID NO.185、SEQ ID NO.195、SEQ ID  NO.200、SEQ ID NO.201、SEQ ID NO.204、SEQ ID NO.206、SEQ ID NO.214、SEQ ID NO.215、SEQ ID NO.220、SEQ ID NO.222、SEQ ID NO.224、SEQ ID NO.229、SEQ ID NO.234、SEQ ID NO.235、SEQ ID NO.236、SEQ ID NO.238、SEQ ID NO.242、SEQ ID NO.243、SEQ ID NO.244、SEQ ID NO.245、SEQ ID NO.251或SEQ ID NO.255任一所示的多肽或两种多肽以上的组合。
本发明还提供了编码上述SARS-CoV-2特异性多肽的核酸。
本发明还提供了携带上述核酸的重组载体。
本发明还提供了携带上述核酸或上述重组载体的宿主细胞。
本发明还提供了所述SARS-CoV-2特异性多肽的衍生物，是在所述SARS-CoV-2特异性多肽的氨基酸序列上取代、或缺失，或添加一个或几个氨基酸，且具有与所述多肽相同抗原性的多肽衍生物。
本发明还提供了对SARS-CoV-2感染或疫苗接种后人群的T细胞高度特异性和高度灵敏性的多肽-MHC四聚体，所述多肽-MHC四聚体由生物素化的MHC-I与所述SARS-CoV-2特异性多肽，或，由生物素化的MHC-I与所述特异性多肽衍生物结合。
本发明还提供了所述多肽-MHC四聚体的制备方法，包括如下步骤：(1)用大肠杆菌表达MHC轻链和MHC重链；(2)稀释复性，制备多肽/MHC复合物；(3)制备生物素化的多肽/MHC复合物；(4)生物素化的多肽/MHC复合物与带标记的链亲和素反应。
在一种实施方式中，所述MHC重链C端连接生物素。
在一种实施方式中，所述MHC中的C端添加能够连接生物素的氨基酸序列GGGLNDIFEAQKIEWHE。
在一种实施方式中，所述步骤(3)中，在BirA酶的催化下多肽/MHC复合物与D-biotin结合。
在一种实施方式中，所述步骤(3)中，所述D-biotin的浓度为400～600μmol/L。
在一种实施方式中，所述步骤(4)中，按照摩尔比(4～6)：(0.5～1.5)的比例与带标记的链亲和素反应。
在一种实施方式中，所述步骤(4)中，按照摩尔比5：1的比例与带标记的链亲和素反应。
在一种实施方式中，所述多肽-MHC四聚体的制备方法还包括纯化。
在一种实施方式中，所述纯化为将制备得到的多肽/MHC复合物经分子筛纯化。
在一种实施方式中，所述方法具体是：利用大肠杆菌表达MHC轻链及C端连接生物素的MHC重链，利用稀释复性的方法制备多肽/MHC复合物，用superdex200纯化，然后再BirA酶的催化下与D-biotin结合，形成生物素化的多肽/MHC复合物，再与带标记的链亲和素按照摩尔比5：1的比例反应，得到多肽/MHC复合物(多肽-MHC四聚体)。
本发明还提供所述多肽-MHC四聚体的应用。
在一种实施方式中，所述应用包括：制备疫苗；作为T细胞免疫学评价的有效工具，对SARS-CoV-2感染或疫苗接种后人群的T细胞免疫应答进行评估；对SARS-CoV-2感染或疫苗接种人群相应免疫细胞的流式细胞术检测、组织切片的原位染色、特异性T细胞的分离与克隆、结合单细胞测序技术分离特异性TCR、作为T细胞激活试剂。
本发明还提供了新型冠状病毒CD8 +T细胞表位肽，包括氨基酸序列如SEQ ID NO.272～SEQ ID NO.286任一所示的表位肽或两条表位肽以上的组合。
本发明还提供了编码上述新型冠状病毒CD8 +T细胞表位肽的核酸。
本发明还提供了携带上述核酸的重组载体。
在一种实施方式中，所述重组载体包括腺病毒载体、慢病毒载体或原核表达载体。
本发明还提供了携带上述核酸或上述重组载体的宿主细胞。
在一种实施方式中，所述宿主细胞包括哺乳动物细胞或原核细胞。
本发明还提供了一种多肽疫苗，其活性成分含有所述SARS-CoV-2特异性多肽、所述SARS-CoV-2特异性多肽衍生物和/或所述新型冠状病毒CD8 +T细胞表位肽。
本发明还提供了SARS-CoV-2特异性细胞免疫检测试剂盒，所述试剂盒含有所述SARS-CoV-2特异性多肽和/或SARS-CoV-2特异性多肽衍生物。
本发明还提供了氨基酸序列SEQ ID NO.272～SEQ ID NO.286所示的新型冠状病毒CD8 +T细胞表位肽或上述核酸或上述重组载体或上述宿主细胞在制备新型冠状病毒疫苗中的应用或抗体或检测试剂盒。
在一种实施方式中，氨基酸序列如SEQ ID NO.272～SEQ ID NO.286所示的新型冠状病毒CD8 +T细胞表位肽能够与MHC-I类分子结合，其中，当多肽与MHC-I类分子结合时能被CD8 +T细胞识别。
本发明提供了一种多肽组合物，所述多肽组合物由S1多肽库、S2多肽库、M多肽库、N多肽库中的任意一个以上的多肽库组成。
在一种实施方式中，所述S1多肽库由氨基酸序列如SEQ ID NO.87～SEQ ID NO.178所示的多肽组成，S2多肽库由氨基酸序列如SEQ ID NO.179～SEQ ID NO.271所示的多肽组成，M多肽库由氨基酸序列如SEQ ID NO.1～SEQ ID NO.29所示的多肽组成，N多肽库由氨基酸序列如SEQ ID NO.30～SEQ ID NO.86所示的多肽组成。
在一种实施方式中，所述多肽组合物由能力与MHC-I或MHC-II类分子结合，其中，当多肽组合物与MHC结合时能被CD4 +或CD8 +T细胞识别。
本发明还提供了所述多肽组合物在制备用于评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的产品中的应用。
本发明还提供了一种评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的试剂盒，含有上述多肽组合物。
在一种实施方式中，所述试剂盒中还含有IFN-γ单克隆抗体。
在一种实施方式中，所述多肽组合物以肽库储液的形式存在于产品中。
在一种实施方式中，所述试剂盒中还设置有ELISpot板，ELISpot上预包被捕获T细胞分泌的细胞因子的单克隆抗体。
在一种实施方式中，所述ELISpot板上预包被有捕获IFN-γ的单克隆抗体。
本发明还提供了一种评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的检测方法，使用所述多肽组合物刺激新冠患者、新冠康复者或新冠疫苗接种者的PBMC细胞，然后使用酶联免疫斑点技术检测多肽刺激后是否出现斑点，即IFN-γ的释放。
在一种实施方式中，所述使用方法具体步骤如下：
(1)分离样品中的外周血单个核细胞；
(2)培养扩增步骤(1)的外周血单个核细胞；
(3)取IFN-γ捕获抗体进行包被，孵育，加入封闭液封闭；
(4)去除封闭液，加入多肽组合物和步骤(2)的外周血单个核细胞，孵育，洗涤，去除液体；
(5)加入IFN-γ单克隆抗体，孵育，去除上清，洗涤；
(6)加入亲和素-HRP结合物，孵育，去除上清，洗涤；
(7)检测并读取斑点。
在一种实施方式中，所述方法中设置有阴性对照；所述阳性对照的刺激溶液为佛波酯(PMA)。
在一种实施方式中，所述封闭液为含10％胎牛血清的RPMI1640培养基。
在一种实施方式中，所述细胞因子为IFN-γ。
在一种实施方式中，所述样品包括脐带血、骨髓、外周血。
通过所述显色反应后的斑点形成细胞数量判断所述T细胞肽库筛选结果；若显色反应后的斑点形成细胞数量为阴性对照中的斑点形成细胞数量的两倍或以上，则结果为阳性。
在一种实施方式中，所述培养扩增所述的外周血单个核细胞包括以下步骤：
(1)取外周血单个核细胞，加入培养基和肽库溶液，孵育；
(2)加入含有白介素7的培养基，孵育；
(3)加入含有白介素2的培养基，孵育；
(4)离心，收集沉淀的细胞，洗涤，静息，即得扩增的外周血单个核细胞；。
在一种实施方式中，所述培养基为含10％胎牛血清的RPMI1640培养基；所述IL-7的浓度含量为15-25ng/mL；所述IL-2的浓度含量为175～225U/mL。
本发明提供了氨基酸序列如SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.30、SEQ ID NO.37、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.53、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID  NO.75、SEQ ID NO.77、SEQ ID NO.87、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92、SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.104、SEQ ID NO.109、SEQ ID NO.110、SEQ ID NO.114、SEQ ID NO.115、SEQ ID NO.116、SEQ ID NO.117、SEQ ID NO.125、SEQ ID NO.126、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.151、SEQ ID NO.152、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.164、SEQ ID NO.169、SEQ ID NO.171、SEQ ID NO.175、SEQ ID NO.185、SEQ ID NO.195、SEQ ID NO.200、SEQ ID NO.201、SEQ ID NO.204、SEQ ID NO.206、SEQ ID NO.214、SEQ ID NO.215、SEQ ID NO.220、SEQ ID NO.222、SEQ ID NO.224、SEQ ID NO.229、SEQ ID NO.234、SEQ ID NO.235、SEQ ID NO.236、SEQ ID NO.238、SEQ ID NO.242、SEQ ID NO.243、SEQ ID NO.244、SEQ ID NO.245、SEQ ID NO.251或SEQ ID NO.255所示的多肽中的任意一条或多条组成的肽库在制备中国人群HLA特异新型冠状病毒疫苗中的应用。
在一种实施方式中，所述肽库覆盖9个HLA-A等位基因和17个HLA-B等位基因。
在一种实施方式中，所述肽库覆盖HLA-A2、HLA-A11、HLA-A24、HLA-A3、HLA-A1、HLA-A31、HLA-A33、HLA-A30、HLA-A2，HLA-B27、HLA-B62、HLA-B46、HLA-B60、HLA-B44、HLA-B61、HLA-B35、HLA-B51、HLA-B67、HLA-B75、HLA-B55、HLA-B58、HLA-B13、HLA-B54、HLA-B5102、HLA-B52、HLA-B39。
在一种实施方式中，所述疫苗包括表位疫苗。
在一种实施方式中，所述疫苗还包括载体，所述载体包括脂质、热激活蛋白、卵清蛋白、牛血清蛋白、钥孔血蓝蛋白。
在一种实施方式中，所述疫苗还包括佐剂，所述佐剂包括弗氏佐剂和氢氧化铝佐剂。
本发明还提供了氨基酸序列如SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.30、SEQ ID NO.37、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.53、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.77、SEQ ID NO.87、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92、SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.104、SEQ ID NO.109、SEQ ID NO.110、SEQ ID NO.114、SEQ ID NO.115、SEQ ID NO.116、SEQ ID NO.117、SEQ ID NO.125、SEQ ID NO.126、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.151、SEQ ID NO.152、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.164、SEQ ID NO.169、SEQ ID NO.171、SEQ ID NO.175、SEQ ID NO.185、SEQ ID NO.195、SEQ ID NO.200、SEQ ID NO.201、SEQ ID NO.204、SEQ ID NO.206、SEQ ID NO.214、SEQ ID NO.215、SEQ ID NO.220、SEQ ID NO.222、SEQ ID NO.224、SEQ ID NO.229、SEQ ID NO.234、SEQ ID NO.235、SEQ ID NO.236、SEQ ID NO.238、SEQ ID NO.242、SEQ ID NO.243、SEQ ID NO.244、SEQ ID NO.245、SEQ ID NO.251或SEQ ID NO.255所示的多肽中的任意一条或多条组成的肽库在制备中国人群HLA特异新型冠状病毒T细胞免疫检测试剂盒中的应用。
在一种实施方式中，所述肽库覆盖9个HLA-A等位基因和17个HLA-B等位基因。
在一种实施方式中，所述肽库覆盖HLA-A2、HLA-A11、HLA-A24、HLA-A3、HLA-A1、HLA-A31、HLA-A33、HLA-A30、HLA-A2，HLA-B27、HLA-B62、HLA-B46、HLA-B60、HLA-B44、HLA-B61、HLA-B35、HLA-B51、HLA-B67、HLA-B75、HLA-B55、HLA-B58、HLA-B13、HLA-B54、HLA-B5102、HLA-B52、HLA-B39。
在一种实施方式中，所述试剂盒中还含有IFN-γ单克隆抗体。
在一种实施方式中，所述试剂盒中还设置有ELISpot反应板，ELISpot上预包被捕获T细胞分泌的IFN-γ的单克隆抗体。
在一种实施方式中，所述ELISpot板上预包被有捕获IFN-γ的单克隆抗体。
本发明还提供了氨基酸序列如SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.30、SEQ ID NO.37、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.53、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.77、SEQ ID NO.87、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92、SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.104、SEQ ID NO.109、SEQ ID NO.110、SEQ ID NO.114、SEQ ID NO.115、SEQ ID NO.116、SEQ ID NO.117、SEQ ID NO.125、SEQ ID  NO.126、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.151、SEQ ID NO.152、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.164、SEQ ID NO.169、SEQ ID NO.171、SEQ ID NO.175、SEQ ID NO.185、SEQ ID NO.195、SEQ ID NO.200、SEQ ID NO.201、SEQ ID NO.204、SEQ ID NO.206、SEQ ID NO.214、SEQ ID NO.215、SEQ ID NO.220、SEQ ID NO.222、SEQ ID NO.224、SEQ ID NO.229、SEQ ID NO.234、SEQ ID NO.235、SEQ ID NO.236、SEQ ID NO.238、SEQ ID NO.242、SEQ ID NO.243、SEQ ID NO.244、SEQ ID NO.245、SEQ ID NO.251或SEQ ID NO.255所示的多肽中的任意一条或多条组成的肽库在制备细胞疗法或过继疗法试剂盒中的应用。
有益效果：
1、本发明设计了271条由10个氨基酸重叠而成的15-18聚体SARS-CoV-2肽段，271条SARS-CoV-2肽段的氨基酸序列如SEQ ID NO.1～SEQ ID NO.271所示，这些肽段跨越了整个S、M和N蛋白，根据SARS-CoV-2 S蛋白S1区和S2区的自然分裂位点，将S蛋白的多肽分为S1区和S2区，S1蛋白的多肽有92条(氨基酸序列如SEQ ID NO.87～SEQ ID NO.178所示)，S2蛋白的多肽有93条(氨基酸序列如SEQ ID NO.179～SEQ ID NO.271所示)，M蛋白的多肽有29条(氨基酸序列如SEQ ID NO.1～SEQ ID NO.29所示)，N蛋白的多肽有57条(氨基酸序列如SEQ ID NO.30～SEQ ID NO.86所示)。
2、本发明首先鉴定了中国人群来源的PBMC细胞的HLA分型，主要为HLA-A2/A11/A24/A3/A1，HLA-B27/B62/B46/B60/B44/B61/B35等，将271条多肽序列一一进行ELISpot验证，筛选得到85条覆盖中国人群主要HLA分型的新冠病毒阳性表位，其中位于S蛋白53条，位于M蛋白的13条，位于N蛋白的19条。并鉴定出优势T细胞表位35条，包括位于S蛋白的优势表位17条，位于M蛋白的优势表位7条，位于N蛋白的优势表位11条。
3、本发明鉴定得到15条CD8 +T阳性表位(氨基酸序列如SEQ ID NO.272～SEQ ID NO.286所示)，这些序列为今后新型冠状病毒疫苗研发、新型治疗药物研发和诊断检测试剂盒的的开发提供了基础。
4、本发明通过多肽抗原的刺激，并在体外培养细胞9天，使病毒特异性T细胞增殖，有效提高检测的灵敏度。本发明可以依照具体情况选择判定标准进行肽库的选择，并对不同肽库刺激样本得到的ELISpot结果进行选择或组合：如需要提高检测灵敏度，尽量减少漏判，可选择总肽库或S1或M肽库任一阳性即判定为最终阳性；如需要减少误判(排除非阳性病人)，需要提高特异度，可以将S1肽库和M肽库结果相结合(双阳判定为最终阳性)。经过总肽库刺激的新冠肺炎病人康复6个月后的T细胞反应阳性率为：93.42％；康复12个月后的T细胞反应阳性率为：91.78％。而利用S1肽库和M肽库配伍评价的方法则可以得到更高的灵敏度和特异度(金标准为病毒核酸检测)：灵敏度71.43％，特异度96.43％。
附图说明
图1新冠病毒S蛋白阳性表位；
图2新冠病毒M蛋白和N蛋白阳性表位；
图3为：HLA-A*1101与表位多肽superdex200分子筛柱形图；
图4为：HLA-A*1101与表位多肽形成的MHC复合物生物素化后superdex200分子筛柱形图；
图5为：M23-Tetramer染SARS-CoV-2感染康复者T细胞流式分析结果代表图；
图6为：N25-Tetramer染SARS-CoV-2感染康复者T细胞流式分析结果代表图。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市售商品或者可以通过已知方法制备。
鉴定PBMC的HLA分型：
1、样品处理
血样体积是200μL时，向离心管中加入血样后直接进行下一步操作；
血样体积少于200μL时，加入Buffer GR补足至200μL，进行下一步；
血样体积大于200μL时，加入1-2.5倍体积的Buffer RCL，轻轻颠倒混匀，12000rpm离心1min，弃掉上清，如果沉淀中还有红色，可重复上述步骤一次。然后向沉淀中加入200μL Buffer GR，震荡混匀然后进行下一步操作。
2、向上述溶液中加入20μL蛋白酶K，混匀。
3、加入200μLBuffer GL，震荡混匀。
4、56℃水浴10min，期间颠倒混匀数次。
5、加入200μL无水乙醇，颠倒混匀，瞬离，使管壁液体集中到管底。
6、将步骤5中的液体全部转移至已装入收集管的吸附柱中，液体较多可分多次加入。12000rpm离心1min，弃掉收集管中废液，吸附柱重新放回。
7、向吸附柱中加入500μLBuffer GW1(使用前确认已加入相应体积的无水乙醇)，12000rpm离心1min，弃掉收集管中废液，吸附柱重新放回。
8、向吸附柱中加入500μLBuffer GW2(使用前确认已加入相应体积的无水乙醇)，12000rpm离心1min，弃掉收集管中废液，吸附柱重新放回。
9、12000rpm离心2min，弃掉收集管中废液，吸附柱置于室温数分钟，彻底晾干。
10、将吸附柱放置于一个新的离心管中，悬空滴入50μL灭菌水，室温放置5min，12000rpm离心1min，收集DNA溶液。
将提取的研究对象的血液DNA送至北京曼泰里生物技术有限公司，公司采用美国ONE LAMBDA公司的流式SSO分型技术测定HLA分型。每个样本均进行了HLA-A和HLA-B两个基因位点的测序。
实施例1 多肽的制备
通过PeptGen软件设计了271条由10个氨基酸重叠而成的15-18聚体SARS-CoV-2肽段，271条SARS-CoV-2肽段的氨基酸序列如SEQ ID NO.1～SEQ ID NO.271所示，这些肽段覆盖了整个S、M和N蛋白，根据SARS-CoV-2 S蛋白S1和S2的裂解位点，将S蛋白的多肽分为S1和S2，S1蛋白的多肽有92条(氨基酸序列如SEQ ID NO.87～SEQ ID NO.178所示)，S2蛋白的多肽有93条(氨基酸序列如SEQ ID NO.179～SEQ ID NO.271所示)，M蛋白的多肽有29条(氨基酸序列如SEQ ID NO.1～SEQ ID NO.29所示)，N蛋白的多肽有57条(氨基酸序列如SEQ ID NO.30～SEQ ID NO.86所示)。将271条肽段混合成为肽库，分别为S1、S2、M和N四个肽库，并作为抗原刺激物用于复苏后的PBMC的刺激培养。
将271条肽段混合制备成四个肽库，其中，S1肽库包含92条S1蛋白的多肽(氨基酸序列如SEQ ID NO.87～SEQ ID NO.178所示)，同样的，S2肽库包含93条S2蛋白的多肽(氨基酸序列如SEQ ID NO.179～SEQ ID NO.271所示)、M肽库包含29条M蛋白的多肽(氨基酸序列如SEQ ID NO.1～SEQ ID NO.29所示)、N肽库包含57条N蛋白的多肽氨基酸序列如SEQ ID NO.30～SEQ ID NO.86所示)。用DMSO溶解合成的多肽，目标浓度为20μg/μl，充分混合均匀，每条多肽取1μl混合成肽库。
实施例2 肽库在ELISpot检测中的应用
利用实施例1中制备的肽库进行PBMC细胞的刺激，利用ELISpot检测T细胞反应阳性率。
(1)外周血PBMC分离
(a)主要试剂耗材
10ml抗凝管与促凝管(BD)、10ml移液管、淋巴细胞分离管(Cat#DKW-LST-24015SK)、1640培养基、DMSO、15ml离心管、50ml离心管、细胞冻存盒、细胞冻存管(Thermo)、国产细胞冻存管(江苏海门，黄盖/蓝盖)、液氮冻存盒、电动移液器、异丙醇、小牛血清(FBS)、巴斯德消毒吸管，具有升降加速度配有15mL离心管吊桶的水平离心机。
(b)样本处理
采用达科为人外周血淋巴细胞分离管Cat#DKW-LST-24015SK。
(c)步骤
1)预处理：取出淋巴细胞分离管，用离心机800g，20℃，离心1min。将存储有抗凝血的试管400g离心5min(或经过长时间放置后直接吸取)，吸取上层血浆2ml，分2管(蓝盖国产细胞冻存管)冻存。
2)样本离心：向含有抗凝血的试管中补加RPMI 1640培养基至10ml，颠倒混匀后，缓缓倒入淋巴细胞分离管中，800g，离心25min，20℃。
3)吸取PBMC：离心后管底是红细胞，中间层是分离液，最上层是血浆，血浆和分离液中间是致密的白膜，含单个核细胞(包括淋巴细胞和单核粒细胞)。在细胞操作台里，用巴斯德吸管吸取中间的白色细胞层，转移至新的15ml离心管内，补加RPMI 1640培养液至12ml。400g离心10min。
4)PBMC清洗：离心完毕，在细胞操作台里，倒掉上清，晃动离心管，利用残余的约200微升的液 体重悬细胞。补加RPMI 1640培养基10ml，400g，10min清洗细胞。
5)细胞冻存：最后尽量吸弃上清，按照上述操作重悬细胞，先加入1ml的FBS，然后再补加1ml的冻存液(含有20％DMSO的FBS)，分装两管，放入冻存盒内，置-80度冻存过夜。促凝血3000rpm离心10min，血清分装3管(黄盖国产冻存管)，-80℃冻存。
(2)肽库刺激和PBMC培养
(a)主要试剂耗材
15ml离心管、1.5ml ep管、1ml移液枪、10ul移液枪、细胞计数板、细胞计数器、24孔细胞培养板、RPMI 1640培养基、白介素-2(IL-2)、白介素-7(IL-7)、小牛血清(FBS)、青链霉素、台盼蓝染液、水浴箱、配有15mL离心管吊桶的水平离心机、细胞培养箱。
(b)步骤
1、PBMCs细胞复苏：复苏细胞混匀在10ml完全培养基(10％FBS，RPMI 1640培养基)里，混匀1500r离心10min，弃上清，加入x ml的完全培养基重悬。
2、细胞计数：ep管内加10μl台盼蓝，10μl混匀后的细胞，读两个九宫格的细胞数y。
细胞浓度为z＝(y个÷2格*2倍*10 4)/ml
细胞数为w＝z*x＝(y个÷2格*2倍*10 4)/ml*xml
根据具体细胞浓度要求计算所需毫升数并定容(24孔板：2ml/孔，细胞3×10 6/孔)。
3、PBMC刺激：将24孔板放入培养箱静置沉淀3-5h后，用实施例1中的肽库刺激45min(肽库：条数×0.2μl+100μl培养基/孔)，随后加入900μl含有IL-7(20ug/ml)的完全培养基。
4、细胞培养：细胞每天观察，培养9天。每隔两天进行半量换液(吸出上清1mL弃掉，贴壁转圈慢慢补加新鲜培养基)。更换的培养基为含有200U/ml的IL-2的完全培养基。
(3)ELISpot检测
(a)主要试剂耗材
PVDF膜为底的96孔板、1.5mlep管、1ml移液枪、10ul移液枪、细胞计数板、细胞计数器、分液槽、包被抗体(purified anti-IFN-γ)、1XPBS、RPMI 1640培养基、小牛血清(FBS)、DMSO、佛波酯(PMA)、去离子水、吐温、检测抗体(Biotinylated anti-IFN-γ)、亲和素-HRP结合物(streptavidin-HRP)、AEC底物溶液(AEC substrate)、AEC染料(AEC chromogen)、细胞培养箱、冰箱、ELISpot读板仪ELISpot Reader System(CTL-Immunospot S5Versa)等。
(b)步骤
1.包被抗体：准备PVDF膜为底的96孔板(100μl/孔)，包被抗体(purified anti-IFN-γ)与1×PBS混合，比例1:200。
2.孵育：盖盖平放，4℃过夜。
3.封闭：轻倒出溶液，1640培养基洗2次(180μl/孔)后，加入每孔180μl完全培养基(RPMI 1640培养基+10％小牛血清+1-2％青链霉素)封闭。
4.孵育：室温孵育2h。
5.多肽和细胞：
5.1多肽：肽库刺激：单肽终浓度2μg/ml，每孔0.4μg。
5.2细胞：轻吸出1ml上清，使用剩余液体吹起细胞混匀并加入10ml完全培养基，1500r离心10min，弃上清并重悬，加入完全培养基计数。
6.加入多肽并接种：轻倒出封闭液，实验组设2个复孔，并设无刺激物的阴性对照组(mock)及PHA阳性对照组(PMA)(a、实验组：肽库+细胞；b、阴性对照组(mock)：培养基+细胞；c、阳性对照组：细胞+PMA)。
依次加入实施例1中的肽库(0.02*条数μl+100μl完全培养基/孔)、mock(100μl完全培养基/孔)、细胞(1×10 5/孔)、PMA(10μl PMA+90μl完全培养基/孔，粉末PMA每瓶用500μl RPMI 1640培养基或PBS溶解)。
7.孵育：37℃，5％CO2，平放孵育18h。
8.洗板：去除细胞，常温去离子水洗3次，每次5分钟，PBST洗3次，每次2分钟，每次清洗后用力 甩掉清洗液后在吸水纸上用力扣干。
9.孵抗体：检测抗体(Biotinylated anti-IFN-γ)1:250稀释到含10％FBS的PBS中充分混匀。每孔100μl，室温孵育2h。去除原液体，常温去离子水洗3次，每次5分钟，PBST清洗3次，每次间隔2分钟，吸水纸上用力扣干。亲和素-HRP结合物(streptavidin-HRP)1:100稀释到含10％血清的PBS中充分混匀。每孔100μl，室温孵育1h。去除原液体，PBST清洗3次，PBS清洗2次，每次间隔2min，吸水纸上用力扣干。
10.显色：体系：100μl/孔AEC底物溶液(AEC substrate)+1滴/mlAEC染料(AEC chromogen)避光混匀，每孔加100μl。
11.洗板：避光孵育7.5min分钟后看到清晰的斑点，用水冲洗膜的双面以中止反应，室温下晾干(开盖并解开底膜反扣过夜)、使用ELISpot Reader System(CTL-Immunospot S5 Versa)计数斑点。
根据结果显示，经过总肽库刺激的新冠肺炎病人康复6个月后的T细胞反应阳性率为：93.42％；康复12个月后的T细胞反应阳性率为：91.78％。而利用S1肽库和M肽库配伍评价的方法则可以得到更高的灵敏度和特异度(金标准为病毒核酸检测)：灵敏度71.43％，特异度96.43％。
在实际应用中，可以依照具体情况选择判定标准，如需要提高检测灵敏度，尽量减少漏判，那么可选择总肽库或S1或M肽库任一阳性即判定为最终阳性，如果实际情况是希望减少误判，需要提高特异度，那么将S1肽库和M肽库结果相结合(双阳判定为最终阳性)的方法无疑是最好的选择。
表1 不同肽库在ELISpot检测中的应用结果
Figure PCTCN2022122150-appb-000001
实施例3 新冠病毒阳性表位的筛选与鉴定
分别利用实施例1中的氨基酸序列如SEQ ID NO.1～SEQ ID NO.271所示的单条多肽进行PBMC细胞的刺激，利用ELISpot检测T细胞反应阳性率，筛选T细胞阳性表位。
(1)外周血PBMCs分离
(a)主要试剂耗材
10mL抗凝管与促凝管(BD)、10mL移液管、淋巴细胞分离管(Cat#DKW-LST-24015SK)、1640培养基、DMSO、15mL离心管、50mL离心管、细胞冻存盒、细胞冻存管(Thermo)、国产细胞冻存管(江苏海门，黄盖/蓝盖)、液氮冻存盒、电动移液器、异丙醇、胎牛血清(FBS)、巴斯德吸管，具有升降加速度配有15mL离心管吊桶的水平离心机。
(b)样本处理
采用达科为人外周血淋巴细胞分离管Cat#DKW-LST-24015SK。
(c)步骤
1)预处理：取出淋巴细胞分离管，用离心机800g，20℃，离心1min。将存储有抗凝血的试管400g离心5min，吸取上层血浆2mL，分2管(蓝盖国产细胞冻存管)冻存，或经过长时间放置后直接吸取。
2)样本离心：向含有抗凝血的试管中补加1640培养基至10mL，颠倒混匀后，缓缓倒入淋巴细胞分离管中，800g，离心25min，20℃。
3)吸取PBMCs：离心后管底是红细胞，中间层是分离液，最上层是血浆，血浆和分离液中间是致密的白膜，含单个核细胞(包括淋巴细胞和单核粒细胞)。在生物安全柜中，用巴斯德吸管吸取中间的白色细胞层，转移至新的15mL离心管内，补加1640培养液至12mL。400g离心10min。
4)PBMCs清洗：离心完毕，在生物安全柜中，倒掉上清，晃动离心管，利用残余的约200微升的液体重悬细胞。补加1640培养基10mL，400g，10min清洗细胞。
5)细胞冻存：最后尽量吸弃上清，按照上述操作重悬细胞，先加入1mL的FBS，然后再补加1mL 20％DMSO的血清冻存液，分装两管，放入冻存盒内，置-80度冻存过夜。促凝血3000rpm离心10min，血清分装3管(黄盖国产冻存管)，-80℃冻存。
(2)单条多肽刺激和PBMCs培养
(a)主要试剂耗材
15mL离心管、1.5mL离心管、1mL移液枪、10μL移液枪、细胞计数板、细胞计数器、24孔细胞培养板、1640培养基、白介素-2、白介素-7、胎牛血清(FBS)、青链霉素、台盼蓝染液、水浴箱、配有15mL离心管吊桶的水平离心机、细胞培养箱。
生长培养基：1640培养基，含10％FBS和1％青链霉素。
(b)步骤
1、PBMCs复苏：复苏细胞混匀在10mL 1640培养基里，1500r离心10min，弃掉液体加x mL的生长培养基重悬。
2、细胞计数：离心管内加10μL台盼蓝，10μL混匀后的细胞，读两个九宫格的细胞数y。
细胞浓度为z＝(y个÷2格*2倍*10 4)/mL
细胞数为w＝z*x＝(y个÷2格*2倍*10 4)/mL*x mL
根据具体细胞浓度要求计算所需毫升数并定容(24孔板：2mL/孔，细胞3×10 6/孔)。
3、PBMCs刺激：将24孔板放入培养箱静置沉淀3-5h后，先吸去1mL上清，再加入实施例1中的肽库(肽库：条数×0.2μL+100μL培养基/孔)，刺激45min，随后加入900μL含有白介素7(IL-7，20μg/mL)的生长培养基。
4、细胞培养：细胞每天观察，培养9天。每隔两天进行半量换液(吸出上清1mL弃掉，贴壁转圈慢慢补加新鲜培养基)。换液配比为：1mL/孔的1640培养基(含10％FBS)，含终浓度为200U/mL白介素2(IL-2)，1％青链霉素。
(3)ELISpot检测
(a)主要试剂耗材
PVDF膜为底的96孔板、1.5mL离心管、1mL移液枪、10μL移液枪、细胞计数板、细胞计数器、分液槽、包被抗体(purified anti-IFN-γ)、1×PBS、1640培养基、胎牛血清(FBS)、DMSO、PMA、去离子水、吐温20、检测抗体(Biotinylated anti-IFN-γ)、亲和素-HRP结合物(streptavidin-HRP)、AEC底物溶液(AEC substrate)、AEC染料(AEC chromogen)、细胞培养箱、冰箱、ELISpot读板仪ELISpot Reader System(CTL-Immunospot S5 Versa)等。
(b)步骤
1.包被抗体：准备包被抗体(purified anti-IFN-γ)与1×PBS混合，比例1:200，加入PVDF膜为底的96孔板(100μL/孔)。
2.孵育：盖盖平放，4℃过夜。
3.封闭：轻倒出溶液，1640培养基洗2次(180μL/孔)后，加入每孔180μL生长培养基(1640培养基+10％胎牛血清+1％青链霉素)封闭。
4.孵育：室温孵育2h。
5.多肽和细胞：
5.1多肽：化学合成实施例1中设计的271条肽段，将125μL DMSO加入2.5mg的合成多肽中溶解(20μg/μL)；单条肽刺激：每孔添加的单条多肽的终浓度10μg/mL，每孔2μg；
5.2细胞：轻吸出1mL上清，使用剩余液体吹起细胞混匀并加入10mL生长培养基，1500r离心10min，弃上清并重悬，加入生长培养基计数，每孔1×10 5个细胞。
6.加入多肽并接种：轻倒出封闭液，实验组设2个复孔，并设无刺激物的阴性对照组(mock)及PMA阳性对照组(PMA)(a、实验组：单肽+细胞；b、阴性对照组(mock)：生长培养基+细胞；c、阳性对照组：细胞+PMA)。
依次加入多肽(0.1μL+100μL生长培养基/孔)、mock(100μL培养基/孔)、细胞(1×10 5/孔)、PMA(10 μLPMA+90μL培养基/孔，粉末PMA每瓶用500μLPBS溶解)。
7.孵育：37℃，5％CO 2，平放孵育18h。
8.洗板：去除细胞，常温去离子水洗3次，每次5分钟，PBST洗3次，每次2分钟，每次清洗后用力甩掉清洗液后在吸水纸上用力扣干。
9.孵抗体：检测抗体(Biotinylated anti-IFN-γ)1:250稀释到含10％FBS的PBS中充分混匀。每孔100μL，室温孵育2h。去除原液体，常温去离子水洗3次，每次5分钟，PBST清洗3次，每次间隔2分钟，吸水纸上用力扣干。亲和素-HRP结合物(streptavidin-HRP)1:100稀释到含10％FBS的PBS中充分混匀。每孔100μL，室温孵育1h。去除原液体，PBST清洗3次，PBS清洗2次，每次间隔2min，吸水纸上用力扣干。
10.显色：体系：100μL/孔AEC底物溶液(AEC substrate)+1滴/mLAEC染料(AEC chromogen)避光混匀，每孔加100μL。
11.洗板：避光孵育7.5min后看到清晰的斑点，用水冲洗膜的双面以中止反应，室温下晾干(开盖并解开底膜反扣过夜)、使用ELISpot Reader System(CTL-Immunospot S5 Versa)计数斑点。
为了量化抗原特异性反应，从实验组中减去阴性对照组中的斑点数量，结果以每10 5个PBMCs表达斑点形成细胞(SFC)来计数。评价标准如下:阴性对照SFC<5个/10 5个，阳性反应定义为SFC＞10个/10 5个；否则，阳性反应被定义为结果至少是阴性对照组的两倍。
结果如图1、图2和表2所示，最终筛选出新冠病毒阳性表位85条，其中位于S蛋白53条，位于M蛋白的13条，位于N蛋白的19条，覆盖了HLA-A2/A11/A24/A3/A1/A31/A33/A30/A2，HLA-B27/B62/B46/B60/B44/B61/B35/B51/B67/B75/B55/B58/B13/B54/B5102/B52/B39。综合针对ELISpot检测结果中IFN-γ的分泌强弱以及针对不同表位的存在阳性反应的人数，鉴定出优势T细胞表位35条，包括位于S蛋白的优势表位17条，位于M蛋白的优势表位7条，位于N蛋白的优势表位11条。
表2 新冠病毒阳性表位
Figure PCTCN2022122150-appb-000002
Figure PCTCN2022122150-appb-000003
Figure PCTCN2022122150-appb-000004
*标记为免疫优势表位
实施例4 预测CD8 +T阳性表位
对于实施例3中表2筛选出的85条阳性多肽，根据阳性康复个体的HLA分型，进行CD8 +T表位的预测。在预测中，通常将％Rank和Affinity作为NetMHC网站预测结果判定的指标，当Affinity<50和％Rank<0.5定义为高亲和力，Affinity<500和％Rank<2定义为弱亲和力。
本次鉴定选用的标准：Affinity<500和％Rank<2，最终鉴定出15条阳性CD8 +T阳性表位，如表3所示。
表3 CD8 +T阳性表位
Figure PCTCN2022122150-appb-000005
Figure PCTCN2022122150-appb-000006
实施例5 多肽-MHC四聚体的制备
(1)多肽/MHC复合物的制备：
1)在HLA-A*1101(Genbank登陆号MT462157.1)基因C端添加能够连接生物素的氨基酸序列GGGLNDIFEAQKIEWHE，构建重组质粒pET28a-HLA-A*1101-Bio，将B2m基因(Genbank登录号为AAA39668.1)与载体连接，构建重组载体pET28a-B2m，并将质粒分别转化到大肠杆菌BL21中，获得重组菌E.coli/pET28a-B2m和E.coli/pET28a-HLA-A*1101-Bio；
2)将步骤1)中携带质粒的大肠杆菌BL21在37℃条件下培养并加入1mmol/L IPTG诱导蛋白表达，收集菌体，将菌体超声破碎，高速离心(12000rpm，10min)后将沉淀溶解在溶解buffer(6mol/L盐酸胍,10％甘油，50mmol/L Tris pH8.0，100mmol/L NaCl，10mmol/L EDTA)中，获得重链HLA-A*1101和轻链B2m；
3)将序列如SEQ ID NO.279所示的多肽和重链、轻链用稀释复性的方法在复性Buffer(100mmol/L Tris pH 8.0，400mmol/L精氨酸，2mmol/L EDTA)中同时复性，使其形成MHC复合物；采用上述相同方法分别用SEQ ID NO.278所示的多肽和重链、轻链制备MHC复合物；
4)使用超滤杯将步骤3)中获得的复性后样品通过10kDa滤膜浓缩样品，并通过浓缩置换溶液为Exchange Buffer(20mmol/L Tris-HCl，50mmol/L NaCl,pH8.0)；将样品取出后4℃离心12000rpm 10min，将上清转移到超滤管中浓缩到约0.5-1ml，将样品过superdex200分子筛进行多肽/MHC复合物纯化，结果如图3所示。
(2)多肽/MHC分子的生物素化
1)将步骤(1)中获得的经过分子筛纯化后的多肽/MHC复合物蛋白样品收集于超滤浓缩管中，浓缩至约300μL，在BirA酶的催化下与D-biotin反应，4℃孵育过夜，获得生物素化后的蛋白样品。
2)将生物素化后的蛋白样品离心后过superdex200分子筛进行生物素化后的复合物纯化，以去除多余的生物素(图4)。
3)将纯化的多肽/MHC复合物用超滤浓缩管浓缩到约500μL，取样进行Gel shift试验验证生物素化效果。
样品制备：
A.2μL链亲和素Streptavidin+8μL分子筛Buffer。
B.8μL生物素化后多肽/MHC样品+2μL 20mg/mL链亲和素Streptavidin；
C.8μL生物素化后多肽/MHC样品+2μL分子筛buffer；
将上述三支样品置冰上孵育30min-2h后进行SDS-PAGE鉴定。
生物素化后的MHC能够与Streptavidin结合成为大分子，从而使得其在SDS-PAGE中的条带滞后，通过比较(C-B)/C的MHC含量的比值可以判断生物素化的效果，既有多少比例的MHC得到较好的生物素化，本发明的技术方案生物素化效应约为70％。
(3)生物素化的MHC分子四聚化：
将生物素化后的MHC分子浓缩，按照链亲和素与多肽/MHC复合物的摩尔比1:5将生物素化后的MHC分子四聚化，链亲和素为带荧光标记的链亲和素，置4℃孵育过夜，制备获得M23四聚体。
按上述相同步骤制备N25四聚体。
实施例6 多肽/MHC四聚体在T细胞分析中的应用
利用SARS-CoV-2特异性多肽/MHC四聚体的高亲和力和高特异性的特点对SARS-CoV-2感染和疫苗免疫后人群T细胞进行检测，评估SARS-CoV-2感染者、康复者和接种疫苗后人群的细胞免疫效果、T细胞的分离与克隆等，采用下述步骤评价SARS-CoV-2感染康复者的T细胞免疫水平：
1)选取HLA-A*1101分型的SARS-CoV-2感染康复者的PBMC，以合成的肽库为刺激物刺激培养PBMCs，培养9天；
2)收获培养后的细胞，用FACS buffer/staining buffer(PBS+0.5％BSA)洗2遍；
3)细胞表面分子染色。加抗体(如FITC-CD8，APC-CD4，PerCp-CD3，PE-Tetramer)。4℃冰上孵育30min；
4)洗涤。加200μL FACS buffer离心，洗2遍；
用细胞流式仪进行流式分析，结果如图5-6所示，使用多肽-MHC四聚体M23和N25检测SARS-CoV-2感染康复者的T细胞，阳性率分别为3.63％、2.37％，明显高于阴性对照组0.061％、0.096％。
实施例7 多肽在制备疫苗中的应用
将实施例3或实施例4中提供的新冠病毒阳性表位多肽对应的序列，以合成多肽，或核酸序列合成RNA或DNA片段在体内或体外多肽的表达，用于提供新冠的免疫原，将多肽与一定的疫苗载体(脂质、热激活蛋白、卵清蛋白、牛血清蛋白或钥孔血蓝蛋白)连接获得疫苗，多肽还可以通过连接肽与载体蛋白偶联。将疫苗制备为注射液，疫苗中还包括佐剂，佐剂选自弗氏不完全佐剂、弗氏完全佐剂、百日咳杆菌佐剂、脂多糖、MF59(含角鲨烯的水包油乳剂)、AS03(含角鲨烯、维生素E和Tween80)、含单磷酰脂质A(monophos-phoryl lipid A,MPL)AS01和AS02佐剂、胞嘧啶鸟嘌呤寡聚脱氧核苷酸(CpG-ODN)、氢氧化铝、明矾、MONTANIDE ISA 51 VG或MONTANIDE ISA 720 VG。进一步的，为了便于储存，疫苗在与佐剂混合对机体免疫之前，可以以固态粉末的形式保存。可以在免疫时，再将固态粉末配制成液体，并加入等体积的佐剂形成注射液进行免疫。
本发明选择6-8周龄健康HLA转基因小鼠进行肌肉注射/腹腔注射/皮下注射疫苗，末次免疫后1-2周后分离小鼠脾细胞以ELISpot检测IFN-γ以测定记忆性T细胞反应，结果显示，疫苗免疫后，小鼠体内能够检测到较强的记忆性T细胞反应。
实施例8 多肽在细胞疗法中的应用
根据实施例3中的方法，分离单个患者的PBMCs，将细胞与实施例3和实施例4中提供的新冠病毒阳性表位多肽在培养基中进行体外刺激，诱导完成后检测T细胞针对新冠病毒的杀伤能力，通过分选获得特异性T细胞，用于患者的新冠治疗。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一种SARS-CoV-2特异性多肽，其特征在于，包括氨基酸序列如SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.30、SEQ ID NO.37、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.53、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.77、SEQ ID NO.87、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92、SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.104、SEQ ID NO.109、SEQ ID NO.110、SEQ ID NO.114、SEQ ID NO.115、SEQ ID NO.116、SEQ ID NO.117、SEQ ID NO.125、SEQ ID NO.126、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.151、SEQ ID NO.152、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.164、SEQ ID NO.169、SEQ ID NO.171、SEQ ID NO.175、SEQ ID NO.185、SEQ ID NO.195、SEQ ID NO.200、SEQ ID NO.201、SEQ ID NO.204、SEQ ID NO.206、SEQ ID NO.214、SEQ ID NO.215、SEQ ID NO.220、SEQ ID NO.222、SEQ ID NO.224、SEQ ID NO.229、SEQ ID NO.234、SEQ ID NO.235、SEQ ID NO.236、SEQ ID NO.238、SEQ ID NO.242、SEQ ID NO.243、SEQ ID NO.244、SEQ ID NO.245、SEQ ID NO.251或SEQ ID NO.255任一所示的多肽或两种多肽以上的组合。
  2. 编码权利要求1所述SARS-CoV-2特异性多肽的核酸。
  3. 携带权利要求2所述核酸的重组载体。
  4. 权利要求1所述SARS-CoV-2特异性多肽的衍生物，其特征在于，在权利要求1所述SARS-CoV-2特异性多肽的氨基酸序列上取代、或缺失，或添加一个或几个氨基酸，且具有与权利要求1所述SARS-CoV-2特异性多肽相同抗原性的多肽衍生物。
  5. 一种新型冠状病毒CD8 +T细胞表位肽，其特征在于，包括氨基酸序列如SEQ ID NO.272～SEQ ID NO.286任一所示的表位肽或两条表位肽以上的组合。
  6. 编码权利要求5所述新型冠状病毒CD8 +T细胞表位肽的核酸。
  7. 携带权利要求6所述核酸的重组载体。
  8. 一种疫苗，其特征在于，活性成分含有权利要求1所述的SARS-CoV-2特异性多肽、权利要求4所述的SARS-CoV-2特异性多肽衍生物和/或权利要求5所述新型冠状病毒CD8 +T细胞表位肽。
  9. SARS-CoV-2特异性细胞免疫检测试剂盒，其特征在于，含有权利要求1所述的多肽和/或权利要求4所述的多肽衍生物。
  10. 权利要求1所述SARS-CoV-2特异性多肽或权利要求2所述核酸或权利要求3所述重组载体或权利要求4所述SARS-CoV-2特异性多肽的衍生物或权利要求5所述新型冠状病毒CD8 +T细胞表位肽或权利要求6所述核酸或权利要求7所述重组载体在制备新冠病毒的疫苗、作为新冠病毒的检测标志物、制备中国人群HLA特异新型冠状病毒T细胞免疫检测试剂盒或制备细胞疗法或过继疗法试剂盒中的应用。
  11. 一种多肽组合物，其特征在于，所述多肽组合物由S1多肽库、S2多肽库、M多肽库、N多肽库中的任意一个以上的多肽库组成；
    所述S1多肽库由氨基酸序列如SEQ ID NO.87～SEQ ID NO.178所示的多肽组成，S2多肽库由氨基酸序列如SEQ ID NO.179～SEQ ID NO.271所示的多肽组成，M多肽库由氨基酸序列如SEQ ID NO.1～SEQ ID NO.29所示的多肽组成，N多肽库由氨基酸序列如SEQ ID NO.30～SEQ ID NO.86所示的多肽组成。
  12. 权利要求11所述的多肽组合物在制备用于评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的产品中的应用。
  13. 一种评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的试剂盒，其特征在于，含有权利要求11所述多肽组合物。
  14. 一种评估新冠患者细胞、新冠康复者或新冠疫苗接种者免疫水平的检测方法，其特征在于，使用权利要求11所述多肽组合物刺激新冠患者的PBMC细胞，细胞在体外扩增之后，然后使用ELISpot检测多肽刺激后是否出现斑点，即IFN-γ的释放。
  15. 一种多肽-MHC四聚体，其特征在于，由生物素化的MHC-I与权利要求1所述的SARS-CoV-2特异性多肽结合，或由生物素化的MHC-I与权利要求2所述的SARS-CoV-2特异性多肽衍生物结合。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112194711A (zh) * 2020-10-15 2021-01-08 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 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
CN113801208A (zh) * 2021-09-29 2021-12-17 中国疾病预防控制中心病毒病预防控制所 一种新型冠状病毒的t细胞免疫检测方法
CN113845577A (zh) * 2021-09-29 2021-12-28 中国疾病预防控制中心病毒病预防控制所 SARS-CoV-2特异性多肽及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112194711A (zh) * 2020-10-15 2021-01-08 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 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
CN113801208A (zh) * 2021-09-29 2021-12-17 中国疾病预防控制中心病毒病预防控制所 一种新型冠状病毒的t细胞免疫检测方法
CN113845577A (zh) * 2021-09-29 2021-12-28 中国疾病预防控制中心病毒病预防控制所 SARS-CoV-2特异性多肽及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAN HÜSEYIN, KÖSEOĞLU AHMET EFE, ERKUNT ALAK SEDEF, GÜVENDI MERVENUR, DÖŞKAYA MERT, KARAKAVUK MUHAMMET, GÜRÜZ ADNAN YÜKSEL, ÜN CEM: "In silico discovery of antigenic proteins and epitopes of SARS-CoV-2 for the development of a vaccine or a diagnostic approach for COVID-19", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, ENGLAND, 28 December 2020 (2020-12-28), England, pages 22387 - 22387, XP055944098, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769971/pdf/41598_2020_Article_79645.pdf> [retrieved on 20220719], DOI: 10.1038/s41598-020-79645-9 *
GHOSH, N. ET AL.: "Genome-wide analysis of Indian SARS-CoV-2 genomes to identify T-cell and B-cell epitopes from conserved regions based on immunogenicity and antigenicity", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 91, 16 December 2020 (2020-12-16), XP086467399, DOI: 10.1016/j.intimp.2020.107276 *
STEPHEN N. CROOKE, OVSYANNIKOVA INNA G., KENNEDY RICHARD B., POLAND GREGORY A.: "Immunoinformatic identification of B cell and T cell epitopes in the SARS-CoV-2 proteome", SCIENTIFIC REPORTS, vol. 10, no. 1, 1 December 2020 (2020-12-01), XP055770118, DOI: 10.1038/s41598-020-70864-8 *

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