CN112876542B - Novel epitope peptide of coronavirus T cell and application thereof - Google Patents
Novel epitope peptide of coronavirus T cell and application thereof Download PDFInfo
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Abstract
The invention discloses a novel epitope peptide of coronavirus T cells and application thereof, wherein the amino acid sequence of the epitope peptide is shown as SEQ ID NO: 1 to 13. The pMHC compound monomer is prepared by the method, the pMHC compound polymer is further prepared, the pMHC compound polymer can be used for detecting antigen-specific T cells in peripheral blood of a novel coronavirus infection rehabilitator and used for in-vitro T cell activation experiments, and the novel coronavirus T cell epitope peptides can be applied to immunodetection and vaccine research and development related to novel coronaviruses and are worthy of deep research and vigorous popularization.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a novel coronavirus T cell epitope peptide and application thereof.
Background
The novel coronavirus is a pathogen of novel infectious pneumonia occurring in 2019, the source of the virus is not clear at present, and the gene sequencing shows that the coronavirus is highly homologous with bat SARS-like coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). At present, no specific medicine is reported. The immune response mechanism of the human immune system to the novel coronavirus is not clear, including antigen recognition, immune clearance mechanism, immune memory protection mechanism and the like.
Research on novel coronavirus such as SARS with high homology shows that T cell immune response plays an important role in body antiviral defense and body immunopathological injury process after virus infection, especially CD8+T cells, which remained antigen-specific immunoreactivity after 11 years, demonstrated CD8+The important role of T cell immune responses in the immune defense against coronaviruses and their importance in vaccine development. CD8+The first step in the immune response of a T cell is that the T cell, through its surface antigen recognition receptors, specifically recognizes the epitope presented by the virus-infected cell. Therefore, the epitope is an important key molecule for T cells to specifically recognize viruses and play a role in immune protection, and is a key target molecule for immune detection, immune therapy and vaccine development.
The vaccine has the action principle that antigen epitope is utilized to activate the initial lymphocytes of an organism, and antigen substances are removed after activation, proliferation and differentiation. The activated lymphocyte is converted into memory lymphocyte, and when the same antigen substance invades into the body next time, the activated lymphocyte rapidly plays the role of immune response and eliminates the antigen. The current vaccine mainly induces the humoral immune response of the organism and generates antibodies. The new coronavirus vaccine also mainly induces antibody and other humoral immune responses. At present, no research report of a cell immune vaccine aiming at T cells exists.
CD8+T cells recognize pMHC activation through a T Cell Receptor (TCR), kill virus infected cells, and remove viruses, thereby playing a role in resisting virus cellular immunity. Therefore, the identification of T cell epitopes that can effectively activate T cells, and thus further the identification of antigen-specific TCRs, the development of T cell epitope-based targeted therapies, is one of the important approaches to antiviral therapy. At present, no relevant research report exists in the field of new coronavirus.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel epitope peptide of coronavirus T cells and application thereof, wherein the epitope peptide can be assembled into a pMHC compound with HLA-A2 heavy chain and HLA-A2 light chain beta 2m protein or directly loaded to an antigen presenting cell so as to activate the T cells, and can be used for research and development, preparation, drug research and development and clinical treatment of the novel coronavirus vaccine.
The first purpose of the invention is to provide a novel epitope peptide of coronavirus T cells.
The second purpose of the invention is to provide a novel epitope peptide combination of coronavirus T cells.
The third purpose of the invention is to provide the application of the antigen epitope peptide and/or the antigen epitope peptide combination in preparing antigen presenting cells.
It is a fourth object of the present invention to provide a pMHC complex.
A fifth object of the present invention is a method of preparing the pMHC complex.
The sixth object of the present invention is an antigenic peptide-antigen presenting cell complex.
A seventh object of the present invention is a method for preparing an antigenic peptide-antigen presenting cell complex for activating T cells.
The eighth purpose of the invention is the application of one or more of the antigen epitope peptide, the antigen epitope peptide combination, the pMHC complex and the antigen peptide-antigen presenting cell complex in preparing novel coronavirus vaccines and/or medicines.
The ninth purpose of the invention is the application of one or more of the antigen epitope peptide, the antigen epitope peptide combination, the pMHC complex and the antigen peptide-antigen presenting cell complex in preparing a novel coronavirus detection reagent.
In order to achieve the purpose, the invention is realized by the following scheme:
T2A2 cell is an antigen presenting cell line expressing human MHC-I molecule HLA-A2 by recombinant genetic engineering techniques. Only effective antigen epitope can be presented by the antigen, so that a stable pMHC complex is formed on the cell surface of the antigen, and the antigen can be used as an artificial antigen presenting cell for stimulating T cells.
T cell epitopes alone are not working and T cell activation must be performed in the form of pMHC complexes or antigenic peptide-antigen presenting cell complexes. The invention utilizes HLA-A2 heavy chain and light chain protein recombined by genetic engineering and identified novel coronavirus T cell epitope to carry out combined renaturation, prepares pMHC compound, directly loads the identified novel coronavirus T cell epitope on the surface of an antigen presenting cell, prepares an antigen peptide-antigen presenting cell compound (T2A2 cell), and finds that the compound can effectively activate T cells in peripheral blood of healthy people. Activated T cells can effectively kill target cells carrying the new coronavirus antigen. The newly found T cell epitope of the novel coronavirus has complete biological activity and can be used as a vaccine and an immunotherapy.
Therefore, the invention claims a novel coronavirus T cell epitope peptide, the amino acid sequence of which is shown as SEQ ID NO: 1 to 13.
SEQ ID NO:1：FVFLVLLPLV；
SEQ ID NO:2：FTISVTTEI；
SEQ ID NO:3：VVFLHVTYV；
SEQ ID NO:4：YIWLGFIAGL；
SEQ ID NO:5：VTWFHAIHV；
SEQ ID NO:6：FLHVTYVPA；
SEQ ID NO:7：SVLLFLAFV；
SEQ ID NO:8：FLLVTLAIL；
SEQ ID NO:9：FLFLTWICLL；
SEQ ID NO:10：LIFLWLLWPV；
SEQ ID NO:11：TLACFVLAAV；
SEQ ID NO:12：FVLAAVYRI；
SEQ ID NO:13：GLMWLSYFI。
The invention also claims a novel coronavirus T cell epitope peptide combination, which comprises an amino acid sequence shown as SEQ ID NO: 1-13.
The application of the epitope peptide and/or the epitope peptide combination in preparing antigen presenting cells also belongs to the protection scope of the invention.
The invention also claims a pMHC complex containing the epitope peptide or the combination of the epitope peptides.
A process for preparing pMHC compound includes such steps as combining HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the epitope peptide or the epitope peptide, renaturing, and purifying.
Preferably, the molar ratio of the heavy chain of HLA-A2, the beta 2m light chain of HLA-A2 and the epitope peptide or the combination of the epitope peptides is 1: 2: 10.
the invention also claims an antigen peptide-antigen presenting cell compound, and an antigen presenting cell with the antigen epitope peptide or the antigen epitope peptide combination on the surface.
Preferably, the antigen presenting cell is a T2a2 cell.
More preferably, the T2A2 cells are T2 cells overexpressing HLA-A2.
The preparation method of the antigen peptide-antigen presenting cell complex for activating T cells is characterized in that the antigen peptide-antigen presenting cell complex containing the novel coronavirus T cell antigen epitope peptide is mixed with antigen presenting cells for incubation, and then the antigen peptide-antigen presenting cell complex is used for T cell activation.
Preferably, the antigen presenting cell is a T2a2 cell.
A fusion protein containing the antigen epitope peptide combination.
The application of one or more of the antigen epitope peptide, the antigen epitope peptide combination, the pMHC compound and/or the antigen peptide-antigen presenting cell compound in preparing a novel coronavirus vaccine and/or medicament.
And the application of one or more of the antigen epitope peptide, the antigen epitope peptide combination, the pMHC complex and/or the antigen peptide-antigen presenting cell complex in preparing a novel coronavirus detection reagent.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers 13 novel coronavirus T cell epitope peptides, prepares a pMHC compound monomer by using the peptides, and further prepares a pMHC compound polymer which can be used for detecting antigen-specific T cells in peripheral blood of a novel coronavirus infection rehabilitee and used for in-vitro T cell activation experiments.
The antigen epitope peptide, HLA-A2 heavy chain and HLA-A2 light chain beta 2m protein are assembled into a pMHC compound, or are directly loaded to an antigen presenting cell, so that T cells are activated, the antigen presenting cell can be used for research and development, preparation, drug research and development and clinical treatment of a new coronavirus vaccine, and can be applied to:
1) development and preparation of new corona vaccines. Antigen-specific T cells are only generated after the body has developed an immune response following infection. Thus, detection of this T cell represents a previous infection with a new coronavirus.
2) And (3) detecting whether the immune function of the cells resisting the infection of the new coronavirus is realized. As above, the detection of the T cell specific to the new coronavirus antigen in the body of the subject represents that the body has already generated the T cell immune function, and the strength of the T cell immune function of the body to the new coronavirus infection can be evaluated according to the proportion of the T cell immune function.
3) The disease condition was monitored. Can be used for monitoring the change of the illness state of patients who are closely contacted with the patient, medical observers and suspected and diagnosed patients.
4) And (5) judging prognosis. If the body fails to produce a T cell immune response, or the proportion of antigen-specific T cells continues to decrease, a poor prognosis is indicated.
Drawings
FIG. 1 is a graph demonstrating whether mitomycin intervention in T2A2 cells affects the binding effect to antigenic peptides and whether it inhibits T2A2 cell proliferation; a: it was verified whether mitomycin intervention in T2A2 cells affected the binding effect with antigenic peptides. Set 6 groups, T2A2 blank, T2A2(MC 10. mu.g/mL, 2h), T2A2(MC 20. mu.g/mL, 20min), T2A2 (10. mu.M positive control peptide (influenza A M1 polypeptide, GILGFVFTL)), T2A2(MC 10. mu.g/mL, 2h + 10. mu.M positive control peptide), T2A2(MC 20. mu.g/mL, 20min + 10. mu.M positive control peptide); b: the results are summarized to show that mitomycin-mediated T2a2 cells did not affect binding to antigenic peptides. C: mitomycin inhibits T2a2 cell proliferation. Flow meter detection of CFSE-labeled T2a2 on day0 and day 7; the results summarized that mitomycin (20. mu.g/mL, 20min) inhibited T2A2 cell proliferation.
FIG. 2 is a T2A2 identification of a total of 39 epitopes of novel coronavirus T cells, a negative control peptide (GLQRLGYVL, derived from Zika virus gene encoding), a positive control peptide (influenza A M1 polypeptide, GILGFVFTL); a: T2A2 identification experiment of 15 new coronavirus antigen polypeptides; b: T2A2 identification experiment of 15 new coronavirus antigen polypeptides; c: T2A2 identification experiment of 9 new coronavirus antigen polypeptides; a1, B1, and C1: summary of triplicate experiments.
FIG. 3 is the detection and application of pMHC complexes formed by 39 antigen peptides; a: screening 39 novel coronavirus T cell epitopes by ELISA, performing peptide exchange analysis with ultraviolet sensitive peptide/MHC complex and the above 39 peptides, performing enzyme-linked immunoassay with human HLA-A2, and measuring absorbance (OD value) at 450nm with enzyme-linked immunosorbent assay. Blank control: phosphate Buffered Saline (PBS); positive control peptide (influenza a M1 polypeptide, GILGFVFTL); ultraviolet ray comparison: ultraviolet-sensitive peptides irradiated with ultraviolet rays; b: purifying a DEAE cellulose ion exchange column assembled by the pMHC compound; c: purifying the assembled molecular sieve (GE Superdex75pg) of the pMHC compound; d: flow cytometry for HLA-A2 detection+COVID-19 recovered patients CD8+ T cells specific for the 39 peptides described above, control: healthy volunteers positive for HLA-A2; e: the results are summarized to show that the prepared 39 tetramers can detect memory CD8+ T cells in the body of a patient recovering from the novel coronavirus infection.
FIG. 4 evaluates the activation and cytotoxic effects of 39 antigenic peptides on CD8+ T cells; a: expression level of the T cell activation marker CD69, the above 39 antigenic peptide mixture induced T cell CD69 expression, Blank (T2a2 alone), T2a2-ctrl (T2a2 without peptide cocultured with CD8+ T cells), Pos-ctrl (influenza a M1 peptide GILGFVFTL) as positive control; b: the results are summarized to show that the antigenic peptide mixture can stimulate T cell activation; c: 39 antigen peptides induce specificitySexual CD8+ T cell mediated killing of cells, CFSE labeled T2a2 cells as viable target cells; day 0-ctrl: dyeing before stimulation; T2A2-ctrl (T2A2 does not contain peptide and CD8+ T cells co-culture); pos-ctrl (influenza a M1 peptide GILGFVFTL) as positive control; d: the results collectively showed that the antigenic peptide kills the target cells (T2A2 cells); e: apoptosis of target cells mediated by 39 specific CD8+ T cells, T2a2-ctrl (T2a2 without peptide co-cultured with CD8+ T cells), Pos-ctrl (influenza a M1 peptide GILGFVFTL) as positive control, flow cytometry to detect the proportion of apoptosis of T2a2 cells labeled with CFSE and the apoptosis marker Annexin V-APC; f: the results showed that the antigenic peptide can induce apoptosis of target cells (T2A2 cells); g: 39 specific CD8+ T cell IFN-gamma+Is released. T2A2-ctrl (T2A2 without peptide cocultured with CD8+ T cells), Pos-ctrl (influenza A M1 peptide GILGFVFTL) was a positive control. The flow cytometry detects the CD8+ IFN-gamma+A ratio; h: the result shows that the antigenic peptide can induce CD8+ T to release IFN-gamma+. I: inducing specific CD8+ T cells to generate after 7 days of stimulation by the antigenic peptide, taking Neg-ctrl (T2A2 does not contain peptide and is co-cultured with CD8+ T cells) as a blank control, taking Pos-ctrl (influenza A M1 peptide GILGFVFTL) as a positive control, and detecting the antigenic specificity CD8+ T cells in the peripheral blood of healthy volunteers activated by the antigenic peptide by a flow cytometer; j: a summary of the results shows that antigen-specific CD8+ T can be detected by pMHC tetramers based on n-Sp1, n-Sp7, n-Sp11, n-Sp14, n-Sp16, n-Sp28, n-Ep2, n-Mp2, n-Mp3, n-Mp4, n-Mp5 and n-Mp 6.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 prediction of the epitope restricted by HLA-A2 of the novel coronavirus
First, experiment method
T-cell epitope prediction was performed on spike (S), membrane (M) and nucleoscaped (N) protein sequences of SARS-CoV-2 Wuhan strain (NC-045512.2), SARS-CoV-GD01 strain (AY278489.2), MERS-CoV strain (KF600612.1) and human coronavirus OC43 strain (KF530099.1) using MHC-I binding tools (http:// tools. iedb. org/mhci). The prediction method used was IEDB recommended 2.22(NetMHCpan-EL), and the MHC alleles were selected as HLA-A02: 01 and HLA-A02: 06 (the most common HLA class I genotype in Chinese population). Variant sequences for each epitope (GISAID. org website) were extracted from GISAID database for further analysis.
Second, experimental results
Screening a large number of efficient and specific T cell epitope candidates. The information is shown in table 1.
Table 1 novel coronavirus T cell epitope peptides:
example 2 whether mitomycin intervention in T2A2 cells affects binding to antigenic peptides
First, experiment method
Taking T2A2 cells in logarithmic growth state (174x CEM. T2: (
CRL-1992TM) Planted in 96-well plates, 10 per well5Mitomycin (MC) was chosen at different concentrations (20. mu.g/mL and 10. mu.g/mL) and for different action times (20min and 2h) to intervene in T2A2, setting 6 groups, group 1: t2a2 blank, group 2: T2A2+ MC 10. mu.g/mL 2h, group 3: T2A2+ MC 20. mu.g/mL 20min, group 4: t2a2+10 μ M positive control peptide (influenza a M1 polypeptide GILGFVFTL), group 5: t2a2+ MC10 μ g/mL 2h +10 μ M positive control peptide (influenza a M1 polypeptide GILGFVFTL), group 6: T2A2+ MC 20. mu.g/mL 20min + 10. mu.M positive control peptide (influenza A M1 polypeptide GILGFVFTL). Each group 3Multiple wells, final volume 200. mu.L. After incubation at 37 ℃ for 4 hours, the cells were washed twice by centrifugation, labeled with FITC anti-human HLA-A2 (. beta.2m) antibody, incubated at 4 ℃ for 30 minutes in the dark, and then detected by flow cytometry. The experiment was performed 3 times in total.
Second, experimental results
Mitomycin did not affect binding to antigenic peptides following T2a2 intervention (fig. 1A and 1B). FIG. 1A shows that T2A2 was treated with 10. mu.g/mL for 2 hours (group 2), 20. mu.g/mL for 20 minutes (group 3), and T2A2 plus positive epitope peptide was treated with 10. mu.g/mL for 2 hours (group 5), 20. mu.g/mL for 20 minutes (group 6), respectively, and binding of the antigenic peptide was not affected compared to the drug-untreated groups (groups 1 and 4) (no difference compared to groups 4, 5 and 6). FIG. 1B is a statistical representation of FIG. 1A.
Example 3 mitomycin inhibition of T2A2 cell proliferation
First, experiment method
Taking T2A2 cells in logarithmic growth state, planting the cells in a 96-well plate, wherein each well is 105Mitomycin (MC 20. mu.g/mL) was taken and the duration of action (20min) intervened in T2A2, 3 groups were set, a T2A2 blank was assigned, T2A2 (day 0) was labeled with CFSE (5. mu.M), T2A2 (day 7) was labeled with CFSE (5. mu.M), 3 duplicate wells of each group, and a final volume of 200. mu.L was used. Placing at 37 ℃ and 5% CO2And (4) culturing in a cell culture box, centrifuging and washing twice after corresponding time, and detecting by using a flow cytometer. The experiment was performed 3 times in total.
Second, experimental results
Mitomycin MC inhibited proliferation of this cell following intervention with T2A2 (FIGS. 1C and D). FIG. 1C shows the success of CFSE labeling (middle group day0 fluorescence peak right-shifted). CFSE still showed a single peak after day 7, indicating that T2a2 cells did not proliferate and could be used as artificial antigen presenting cells without interfering with T cell growth in mixed culture. FIG. 1D is a statistical representation of FIG. 1C.
Example 4 identification of epitope-restricted peptides of the novel coronavirus HLA-A2
First, experiment method
The 39 polypeptides predicted in example 3 were artificially synthesized and prepared to have a concentration of 10. mu.M. Taking T2A2 cells in logarithmic growth state, planting the cells in a 96-well plate, wherein each well is 105Blank wells, negative control peptide (Zika virus gene encoding, GLQRLGYVL), positive control peptide (influenza A M1 polypeptide, GILGFVFTL) and each synthetic antigen polypeptide were distributed, 3 replicate wells per group, with a final volume of 200. mu.L. After incubation at 37 ℃ for 4 hours, the cells were washed twice by centrifugation, labeled with FITC anti-human HLA-A2 (. beta.2m) antibody, incubated at 4 ℃ for 30 minutes in the dark, and then detected by flow cytometry. The experiment was performed 3 times in total.
Second, experimental results
The results are shown in fig. 2A, 2B and 2C, and show that all 39 antigen polypeptides can be efficiently presented by antigen presenting cells.
Example 539 detection of antigenic Polypeptides by formation of pMHC complexes
First, experiment method
ELISA method detects 39 new coronavirus T cell epitopes predicted in example 3, using ultraviolet sensitive peptide (KILGFVVFJV)/MHC complex and the 39 peptides predicted in example 3 to perform peptide exchange analysis, then using purified human HLA-A2 capture antibody to coat a micropore plate to prepare solid phase antibody, adding the 39 peptides exchanged with the ultraviolet sensitive peptide into the coated micropore in sequence, then combining with HRP labeled detection antibody to form antibody-antigen-enzyme labeled antibody complex, and adding substrate TMB for developing after thorough washing. TMB is converted to blue by the catalysis of HRP enzyme and to the final yellow by the action of acid. The shade of the color was positively correlated with pMHC in the sample, and the absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader.
Second, experimental results
The results are shown in FIG. 3A, and show that 39 antigenic polypeptides can form pMHC complexes.
EXAMPLE 6 preparation of pMHC Complex monomers of epitope peptides
First, experiment method
The heavy chain of HLA-A2, the beta 2m light chain of HLA-A2 and each epitope polypeptide in example 3 were expressed according to the following ratio of 1: 2: 10 molar ratio was gradually added dropwise to a renaturation solution (5M urea, 0.4M arginine, 100mM Tris,3.7mM cystamine, 6.3mM cysteamine, 2mM EDTA) for renaturation to obtain a pMHC complex monomer of the epitope peptide. The pMHC complex monomer was further purified by passing through a DEAE ion exchange column, eluting with 0.5M NaCl, and collecting the protein according to the OD280nm UV absorption peak. The protein purified by DEAE ion exchange column was then purified by Superdex75pg molecular sieve according to molecular weight, eluted with PBS and different molecular weight proteins were collected according to OD280nm UV absorption peak.
Second, experimental results
After purification, the pMHC complex monomer of the epitope peptide was obtained (fig. 3B, C).
Example 7 use of epitopes in New Corona Virus infected convalescent persons
First, experiment method
Since the expressed HLA-A2 carries a biotin binding site, the fluorescence-labeled avidin (PE-streptavidin), biotin and pMHC complex monomers were expressed as follows: 4: 4, and incubating for 16-18 hours at 4 ℃ in a dark place to prepare the fluorescence-labeled T cell epitope tetramer (i.e. tetramer-PE) of the new coronavirus. Purified by Superdex75pg molecular sieves, eluted with PBS and the different molecular weight proteins were collected according to OD280nm UV absorbance peak.
PBMC in peripheral venous blood of a novel coronavirus infection rehabilitation patient is separated and identified as HLA subtype, and an HLA-A2 positive PBMC sample is further stained by the prepared tetramer-PE and CD8-APC antibodies and then subjected to flow type on-machine detection.
Second, experimental results
The results showed that the 39 epitope peptides tetramer obtained in example 3 identified above could recognize antigen-specific CD8 produced in the convalescent patients with coronavirus infection+T cells (fig. 3D), fig. 3E is a statistical representation of fig. 3D.
Example 8 activation of T cells by the New coronavirus HLA-A2 restricted epitope peptide
First, experiment method
T2 cells activated T cells by expressing the HLA-A2 molecule (T2A 2). Isolation of mononuclear lymphocytes (PBMC) in peripheral venous blood of healthy volunteers and further isolation of CD8+T cells. Using T2A2 cellsCFSE labeling was carried out by treating with 20. mu.g/mL mitomycin for 20 minutes, and then incubating with the 39 different epitope peptides obtained in example 3. Will CD8+T cells (0.5X 10)6) T2A2 cell (0.5X 10) loaded with different epitope peptides from the 39 antigen epitopes6) Co-culture and co-stimulation with 1. mu.g/mL anti-human CD28 antibody and 50IU/mL IL-2. Every two days, 50IU/mL IL-2 and 20. mu.M epitope peptide were supplemented. Evaluation of the activation marker CD69 on T cells after 16 hours and 7 days, respectively, calculation of the percentage of survival number of T2A2, labeling of specific CD8 with tetramer+T cell ratio and apoptosis marker Annexin V-APC on T2a2 cells.
Second, experimental results
The 39 types of epitope peptides (n-Sp1, n-Sp7, n-Sp11, n-Sp14, n-Sp16, n-Sp28, n-Ep1, n-Ep2, n-Mp2, n-Mp3, n-Mp4, n-Mp5 and n-Mp6, wherein the amino acid sequences are shown as SEQ ID NO: 1-13) all share 13 different epitope peptides and can activate T cells to express CD69 (FIG. 4A-B). T cells activated by these 13 epitope peptides could effectively kill T2A2 cells, as shown by a reduced proportion of surviving T2A2 (FIGS. 4C-D). At the same time, the proportion of residual T2A2 apoptosis was increased (FIG. 4E-F, CFSE and Annexin V double positive). Killing of T cells was achieved by secretion of IFN- γ (fig. 4G-H). Finally, after 7 days of culture, these 13 epitopes induced antigen-specific memory T cells (fig. 4I-J). By combining all data, the 13 epitopes are effective epitopes, and can be presented by effective antigens, activate T cells, kill target cells and induce the generation of memory T cells.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> river-south university
<120> novel epitope peptide of coronavirus T cell and application thereof
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1 5 10
<210> 2
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Phe Thr Ile Ser Val Thr Thr Glu Ile
1 5
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Val Val Phe Leu His Val Thr Tyr Val
1 5
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu
1 5 10
<210> 5
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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1 5
<210> 6
<211> 9
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<213> Artificial Sequence (Artificial Sequence)
<400> 6
Phe Leu His Val Thr Tyr Val Pro Ala
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<210> 7
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Ser Val Leu Leu Phe Leu Ala Phe Val
1 5
<210> 8
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Phe Leu Leu Val Thr Leu Ala Ile Leu
1 5
<210> 9
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<213> Artificial Sequence (Artificial Sequence)
<400> 9
Phe Leu Phe Leu Thr Trp Ile Cys Leu Leu
1 5 10
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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<210> 11
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Thr Leu Ala Cys Phe Val Leu Ala Ala Val
1 5 10
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Phe Val Leu Ala Ala Val Tyr Arg Ile
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<210> 13
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gly Leu Met Trp Leu Ser Tyr Phe Ile
1 5
Claims (9)
1. A novel epitope peptide of coronavirus T cells, which is characterized in that the amino acid sequence of the epitope peptide is shown as SEQ ID NO: 1 is shown.
2. A novel epitope peptide composition of coronavirus T cells, which is characterized by comprising an amino acid sequence shown as SEQ ID NO: 1.
3. Use of the epitope peptide according to claim 1 and/or the epitope peptide combination according to claim 2 for the preparation of antigen presenting cells.
4. A pMHC complex containing the epitope peptide according to claim 1 or the combination of epitope peptides according to claim 2.
5. A method for preparing a pMHC complex, which comprises subjecting an HLA-A2 heavy chain, HLA-A2 light chain β 2m, and the epitope peptide according to claim 1 or the epitope peptide according to claim 2 to combined renaturation and purification.
6. An antigenic peptide-antigen presenting cell complex, which is characterized in that the antigenic epitope peptide of claim 1 or the antigenic epitope peptide composition of claim 2 is on the surface of an antigen presenting cell.
7. A method for preparing an antigen peptide-antigen presenting cell complex for activating T cells, comprising mixing and incubating the antigen presenting cells with the antigen peptide-antigen presenting cell complex containing the novel coronavirus T cell of claim 1, followed by T cell activation.
8. Use of one or more of the epitope peptide of claim 1, the epitope peptide composition of claim 2, the pMHC complex of claim 4, and the antigenic peptide-antigen presenting cell complex of claim 6 for the preparation of a novel coronavirus vaccine and/or therapeutic agent.
9. Use of one or more of the epitope peptide of claim 1, the epitope peptide composition of claim 2, the pMHC complex of claim 4, and the antigenic peptide-antigen presenting cell complex of claim 6 for the preparation of novel reagents for the detection of coronaviruses.
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