CN116375821A - Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine - Google Patents
Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine Download PDFInfo
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Abstract
The invention discloses a novel coronavirus T cell epitope peptide and application thereof in preparing vaccines, wherein the amino acid sequence of the novel coronavirus T cell epitope peptide is shown as any one of SEQ ID NO. 1-9. The pMHC complex monomer is used for preparing the pMHC complex monomer, and the pMHC complex polymer is further prepared, so that the pMHC complex monomer can be used for detecting antigen-specific T cells in peripheral blood of novel coronavirus vaccinators and infection rehabilitators and is used for in-vitro T cell activation experiments, and the novel coronavirus T cell epitope peptide can be used for preparing general vaccines aiming at various novel coronavirus mutant strains, novel coronavirus-related immunodetection and broad-spectrum therapeutic drug development, and is worthy of intensive research and popularization.
Description
The application is a divisional application of an invention patent application of which the application date is 2022, 2, 24 and the application number is 2022101769472, and the invention is named as a novel coronavirus T cell epitope peptide and application of the novel coronavirus T cell epitope peptide in preparation of vaccines.
Technical Field
The invention relates to the technical field of biology, in particular to a novel coronavirus T cell epitope polypeptide and application thereof in preparing vaccines.
Background
In view of the continuous appearance of the current novel coronal variants and immune escape strains thereof, the vaccine provides a opportunity for developing universal novel coronal vaccines and broad-spectrum therapeutic drugs in the future. The universal vaccine is a broad-spectrum vaccine which can effectively protect the immune crowd even after the virus antigen is mutated. Therefore, development of a universal vaccine and a broad-spectrum therapeutic drug that can still provide effective immune protection for vaccine immunized people after various variants of novel coronaviruses occur is urgent.
According to the research on coronaviruses such as SARS with higher homology, the T cell immune response plays an important role in the antiviral defense of the organism after virus infection and in the pathological damage process of the organism immunity, in particular to CD8 + T cells, which remained after 11 years of antigen-specific immune activity, demonstrated CD8 + T cell immune responses play an important role in the immune defenses against coronaviruses and their importance in vaccine development. CD8 + The first step in the immune response of T cells is the specific recognition of epitope peptides presented by the virus-infected cells by T cells through their surface antigen recognition receptors. Thus, the epitope peptide is a T cell specific recognition virus and exerts immunityImportant key molecules for epidemic protection are key targeting molecules for immunodetection, immunotherapy and vaccine development.
CD8 + T cells recognize pMHC activation through T Cell Receptors (TCRs), kill virus-infected cells, and eliminate viruses, thereby exerting an antiviral cellular immune effect. Therefore, the identification of the peptide capable of effectively activating T cell antigen epitope, which can induce effective T cell immune protection after the virus antigen is mutated, is one of the keys for developing novel coronary general vaccine and broad-spectrum therapeutic drug.
Immune escape is the antagonism, blocking and suppression of the immune response of the body by immunosuppressive pathogens through their structural and non-structural products. The novel coronavirus, because of frequent and sustained mutation, produces a large number of variants, which seriously threatens the immune barrier effect of the vaccine.
Disclosure of Invention
The invention aims to overcome the defect that a T cell epitope peptide for a novel coronavirus general vaccine and application thereof are not developed in the field of novel coronaviruses in the prior art, and provides a novel coronavirus T cell epitope peptide and application thereof in preparing vaccines; it can assemble into pMHC complex with HLA-A2 heavy chain and HLA-A2 light chain beta 2m protein; or directly loaded to antigen presenting cells, can activate T cells, effectively induce T cell immunity, avoid immune escape of mutant strains, and can detect specific CD8+ T cells in novel coronavirus universal vaccine research and development, preparation and drug research and development and clinical treatment of novel coronavirus universal vaccine.
The first object of the invention is to provide a novel coronavirus T cell epitope peptide.
The second object of the invention is to provide a novel coding gene of coronavirus T cell epitope peptide.
A third object of the invention is to provide a novel coronavirus T cell epitope peptide composition.
A fourth object of the present invention is to provide a pMHC complex.
It is a fifth object of the present invention to provide an antigenic peptide-antigen presenting cell complex.
The sixth object of the present invention is to provide the application of one or more of the antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide encoding gene, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex, and the antigen peptide-antigen presenting cell complex in preparing novel coronavirus vaccine.
The seventh object of the present invention is to provide the antigen epitope peptide, the encoding gene of the novel coronavirus T cell antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex, and the application of one or more of the antigen peptide-antigen presenting cell complexes in preparing novel coronavirus drugs.
In order to achieve the above object, the present invention is realized by the following means:
T2-A2 is an antigen presenting cell line expressing human MHC class I molecule HLa-A2 by recombinant genetic engineering techniques. Only effective epitope peptides can be presented, so that a stable pMHC complex is formed on the cell surface, and therefore, the antigen peptide can be used as artificial antigen presenting cells for stimulating T cells.
T cell epitope peptides alone cannot work and must be activated in the manner of pMHC complexes or antigen peptide-antigen presenting cell complexes. The invention utilizes MHC monomer and identified novel coronavirus T cell epitope to carry out joint renaturation, thus preparing the pMHC complex. The identified novel coronavirus T cell epitope peptide is loaded on the surface of antigen presenting cells (T2-A2 cells), antigen peptide-antigen presenting cell complexes are prepared, then the prepared pMHC complexes are used for labeling T cells, the antigen epitope peptide can be found to effectively activate T cells in peripheral blood of healthy people, and compared with the antigen epitope peptide of an original virus strain, the variant strain can increase the activation capacity of the T cells and can also effectively kill target cells carrying novel coronavirus antigens. The pMHC complex with the PE fluorescent channel assembled by the T cell epitope peptide can be detected in both novel coronal vaccinators and rehabilitators. The novel coronavirus T cell epitope peptide found newly can effectively induce T cell immunity, avoid immune escape of mutant strain, and can be used as general vaccine or applied to immunotherapy and the like.
Therefore, the invention requires a novel coronavirus T cell epitope peptide, and the amino acid sequence of the novel coronavirus T cell epitope peptide is shown in any one of SEQ ID NO. 1-9.
SEQ ID NO:1:VTWFHAIHV,
SEQ ID NO:2:VTWFHAISG,
SEQ ID NO:3:FKLKDCVMYA,
SEQ ID NO:4:FKLKECVMYA,
SEQ ID NO:5:YHDVRVVLDFI,
SEQ ID NO:6:YHDVRVVLI,
SEQ ID NO:7:ASLPFGWLI,
SEQ ID NO:8:ASLLFGWLI,
SEQ ID NO:9:VTWFHVIHV。
A coding gene of a novel coronavirus T cell epitope peptide, wherein the coding gene codes the novel coronavirus T cell epitope peptide.
A novel coronavirus T cell antigen epitope peptide composition comprises a polypeptide with an amino acid sequence shown in SEQ ID NO: 1-9, and any several of novel coronavirus T cell epitope peptides.
A pMHC complex comprising said novel coronavirus T cell epitope peptide or said novel coronavirus T cell epitope peptide composition.
Preferably, the preparation method of the pMHC complex is that HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the novel coronavirus T cell epitope peptide are renatured.
An antigen peptide-antigen presenting cell complex, and the surface of the antigen presenting cell is provided with the novel coronavirus T cell antigen epitope peptide or the novel coronavirus T cell antigen epitope peptide composition.
Preferably, the antigen presenting cells are T2-A2 cells.
More preferably, the T2-A2 cells are T2 cells over-expressing HLa-A2.
The antigen epitope peptide, the encoding gene of the novel coronavirus T cell antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex and the application of one or more of the antigen peptide-antigen presenting cell complexes in preparing novel coronavirus vaccines.
The antigen epitope peptide, the encoding gene of the novel coronavirus T cell antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex and the application of one or more of the antigen peptide-antigen presenting cell complexes in preparing novel coronavirus medicaments.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers 9 novel coronavirus T cell epitope peptides, which can be used for detecting antigen-specific T cells in peripheral blood of novel coronavirus vaccinators and infection healers and used for in vitro T cell activation experiments after being prepared into pMHC complexes with PE fluorescent channels, and can be used for preparing general vaccines aiming at various novel coronavirus mutant strains, novel coronavirus-related immunodetection and broad-spectrum therapeutic drugs.
The novel coronavirus T cell epitope peptide is prepared into a pMHC complex with a PE fluorescent channel, or directly loaded to antigen presenting cells, so that T cells are activated, and the novel coronavirus T cell epitope peptide can be used for research and development of novel coronavirus vaccines, preparation, drug research and development and clinical treatment, and can be applied to:
1) Development and preparation of novel coronal vaccines: after the novel coronavirus is mutated, the plurality of T cell epitopes can induce the organism to generate immune response to generate antigen-specific T cells. Therefore, the T cell epitope is a candidate antigen epitope peptide of the novel coronavirus universal vaccine.
2) Detecting whether it has cellular immune function against infection by the novel coronavirus: the novel coronavirus antigen specific T cells are detected in the body of a person to be detected, which represents that the body has generated T cell immune function, and the T cell immune function of the body and the possibility of infection of the novel coronavirus can be evaluated according to the proportion of the antigen specific CD8T marked by the pMHC complex prepared into the fluorescent channel by the epitope peptide.
3) The effect after vaccination was evaluated: the detection of novel coronavirus antigen-specific T cells in the inoculant, which represent that the organism has developed T cell immune function, can be used to assess the likelihood of the organism re-infecting the novel coronavirus.
4) Monitoring the condition: can be used for monitoring the change of the illness state of the close contact person, the medical observer, the suspected and the diagnosed patient.
5) And (3) prognosis judgment: poor prognosis is predicted if the body fails to mount a T cell immune response, or the proportion of antigen-specific T cells continues to decrease.
Drawings
FIG. 1 is an identification of T2-A2 antigen presentation of 33 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 33 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of the formation of pMHC complex by 33 antigen peptides, ELISA method is used to screen 33 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the above 33 peptides are used to conduct peptide exchange analysis, and enzyme label instrument is used to measure absorbance (OD value) at 450nm wavelength.
FIG. 2 is an identification of T2-A2 antigen presentation of 10 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 10 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; detection of pMHC complex formation by 10 antigen peptides C, screening 10 novel coronavirus T cell epitope peptides by ELISA, peptide exchange analysis with uv-sensitive peptide/MHC complex and the 10 peptides described above, and absorbance (OD value) measurement with a microplate reader at 450nm wavelength.
FIG. 3 is an identification of T2-A2 antigen presentation of 28 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 28 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; detection of pMHC complex formation by 28 antigen peptides C28 novel coronavirus T cell epitope peptides were screened by ELISA, peptide exchange analysis was performed with uv-sensitive peptide/MHC complex and the 28 peptides described above, and absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm.
FIG. 4 is an identification of T2-A2 antigen presentation of 6 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 6 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of formation of pMHC complex by 6 antigen peptides, ELISA method is used for screening 6 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the 6 peptides are used for peptide exchange analysis, and enzyme label instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 5 is an identification of T2-A2 antigen presentation of 30 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: identification experiments of T2-A2 antigen presentation of 30 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of formation of pMHC complex by 30 antigen peptides, ELISA method is used for screening 30 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the above 30 peptides are used for peptide exchange analysis, and enzyme-labeled instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 6 is an identification of T2-A2 antigen presentation of 32 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: identification experiments of T2-A2 antigen presentation of 32 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; detection of pMHC complex formation by 32 antigen peptides C, screening of 32 novel coronavirus T cell epitope peptides by ELISA, peptide exchange analysis with uv-sensitive peptide/MHC complex and the above 32 peptides, and absorbance (OD value) measurement with a microplate reader at 450nm wavelength.
FIG. 7 is an identification of T2-A2 antigen presentation of 22 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: identification experiments of T2-A2 antigen presentation of 22 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is the detection of 22 antigen peptides forming pMHC complex, ELISA method is used for screening 22 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the 22 peptides are used for peptide exchange analysis, and enzyme label instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 8 is an identification of T2-A2 antigen presentation of 6 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 6 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of formation of pMHC complex by 6 antigen peptides, ELISA method is used for screening 6 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the 6 peptides are used for peptide exchange analysis, and enzyme label instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 9 shows the purification results of pMHC complexes after assembly; a: purifying the result of the DEAE cellulose ion exchange column after assembling the pMHC complex; b: purification results of the assembled pMHC complex with molecular sieves (GE Superdex75 pg).
FIG. 10 evaluation of 167 antigen peptides versus CD8 + Activation of T cells. Induction of specific CD8 after 7 days of antigenic peptide stimulation + T cell production, NC: positive-ctrl (T2-A2 contains no peptide and CD 8) + T cell co-culture) as a negative control, PC: positive-ctrl (influenza A M1 peptide GILGFVFTL) as Positive control, and flow cytometry for detecting antigen-specific CD8 in peripheral blood of healthy volunteers activated by the above antigen peptide + T cells.
FIG. 11 is a summary of results of evaluating activation of CD8+ T cells by 167 antigen peptides; a: the results summarize show that antigen-specific CD8 can be detected by the pMHC complex with fluorescent channels formed by these mutated epitopes + T, variation compared with T cell epitope peptide of original virus strainSpecific activation of CD8 by corresponding T cell epitope peptide of strain + The proportion of T cells is unchanged or increased; b: the result summarization shows that the antigen-specific CD8 can be detected by the pMHC complex with fluorescent channel formed by the T cell epitope peptide of the novel coronavirus variant virus strain and the T cell epitope peptide of the corresponding original virus strain + T, the corresponding T cell epitope peptide of the variant strain specifically activates CD8 as compared with the T cell epitope peptide of the original strain + The proportion of T cells decreases; c: the result summarization shows that the antigen-specific CD8 can be detected by the pMHC complex with fluorescent channel formed by the T cell epitope peptide of the novel coronavirus variant virus strain and the T cell epitope peptide of the corresponding original virus strain + T, the corresponding T cell epitope peptide of the variant strain specifically activates CD8 as compared with the T cell epitope peptide of the original strain + The proportion of T cells increases or decreases.
FIG. 12 evaluation of 167 antigen peptides versus CD8 + T cell toxic effects; a (upstream): antigenic peptide induced specificity CD8 + T cell mediated killing of cells, CFSE labeled T2-A2 cells as viable target cells; day 0-ctrl: dyeing before stimulation; T2-A2-ctrl (T2-A2 is peptide-free and CD 8) + T cell co-culture); neg-ctrl (epstein barr virus IVTDFSVIK); pos-ctrl (influenza a M1 peptide gilgfveftl) was a positive control; b (left): the results show that the antigen peptide can kill target cells (T2-A2 cells); a (downstream): specific CD8 + T cell mediated apoptosis of target cells, T2-A2-ctrl (T2-A2 peptide-free co-culture with CD8+ T cells); neg-ctrl (EB virus IVTDFSVIK), pos-ctrl (influenza A M1 peptide GILGFVFTL), flow cytometry detects the proportion of apoptosis in T2-A2 cells labeled with CFSE and apoptosis markers Annexin V-APC; b (right): the result summarization shows that the antigen peptide can induce apoptosis of target cells (T2-A2 cells); c (upstream): specific CD8 + T cell IFN-gamma + Is released. T2-A2-ctrl (T2-A2 is peptide-free and CD 8) + T cell co-culture), neg-ctrl (epstein barr virus IVTDFSVIK), pos-ctrl (influenza a M1 peptide gilgfftl). Flow cytometer detection markers with CD8 + IFN-γ + Proportion of the components; d (left side): the result summarization shows that the antigen peptide can induce CD8 + T release IFN-gamma + The method comprises the steps of carrying out a first treatment on the surface of the C (downstream): specific CD8 + T cell GZMB + Is released. T2-A2-ctrl (T2-A2 is peptide-free and CD 8) + T cell co-culture), neg-ctrl (epstein barr virus IVTDFSVIK), pos-ctrl (influenza a M1 peptide gilgfftl); flow cytometer detection markers with CD8 + GZMB + Proportion of the components; d (right): the result summarization shows that the antigen peptide can induce CD8 + T release GZMB + 。
FIG. 13 evaluates the proportion of specific CD8+ T cells in vivo 14 days after the new coronary convalescence patient and 7 days after the second needle was vaccinated; detection of HLA-A2+COVID-19 by A flow cytometry the above 9 patients (T cell epitope peptide of novel coronavirus variant strain compared with T cell epitope peptide of corresponding original strain upon T cell activation) produced specific CD8 + T cell proportion is increased or unchanged) of the peptide; b: the result summarization shows that 10 prepared pMHC complexes with fluorescent channels can detect memory CD8 in novel coronavirus infection rehabilitation person + T cells; c flow cytometry detection of specific CD8 of the above 9 (T cell activation, T cell epitope peptide of novel coronavirus variant strain, produced specific CD8+ ratio increased or unchanged compared to T cell epitope peptide of corresponding original strain) peptides of HLA-A2+ volunteers 7 days after vaccination of the second needle + T cells; d: the results summarised show that the 9 prepared pMHC complexes with fluorescent channels can detect memory cd8+ T cells in the vaccinations.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 prediction of novel coronavirus HLA-A2 restriction epitope peptides
1. Experimental method
CD8T cell epitope prediction was performed on SARS-CoV-2 original virus strain (NC_ 045512.2) spike (S), membrane (M), nucleocapsid (N), envelope (E) and ORF protein sequences using the "MHC I Binding" tool (http:// tools. Iedb. Org/mhci). The prediction method used was IEDB Recommended 2.22 (NetMHCpan EL), MHC allele selection of HLA-A 02:01.
The T cell epitope peptides of all novel coronavirus variant strains containing the same species as b.1.1.7 (Alpha type mutant) (id=1), b.1.351 (Beta type mutant) (id=2), p.1 (Gamma type mutant) (id=3), p.2 (id=4), P.3 (id=5), b.1.429 (Epsilon type mutant) (id=6), b.1.526.1 (Lota type mutant) (id=7), b.1.526.2 (id=8), b.1.618 (id=9), b.1.617.1 (id=10), b.1.617.2 (Delta type mutant) (id=11), b.1.617.3 (id=12), and b.1.1.529 (omacron type mutant) (id=13) were studied with the T cell epitope peptides of the corresponding original strains. The variant sequence of each epitope (GISAID. Org website) was extracted from the GISAID database for further analysis.
2. Experimental results
167 high-efficiency specific candidate T cell epitope peptides are screened out. The information is shown in Table 1.
Table 1 novel 167 epitope peptides of coronavirus T cells:
EXAMPLE 2 identification of novel coronavirus HLA-A2 restriction epitope peptides
1. Experimental method
Candidate T cell epitope peptides predicted in synthetic example 1 were prepared at a concentration of 20. Mu.M. Taking logarithmic growth state T2-A2 cells, planting into 96-well plates, 10 each 5 The blank wells, negative control peptide (epstein barr virus, IVTDFSVIK), positive control peptide (influenza a M1 polypeptide, gilgfftl) and each synthetic candidate T cell epitope peptide were distributed, 3 wells per group, and a final volume of 200 μl. After incubation for 4 hours at 37℃the cells were washed twice by centrifugation, labeled with FITC anti-human HLA-A2 (. Beta.2m) antibodies, incubated at 4℃for 30 minutes in the absence of light and then detected by flow cytometry. Experiments were performed 3 times in total.
2. Experimental results
The results are shown in figures 1 to 8, a and B, which show that 167 antigen polypeptides can be efficiently presented by antigen presenting cells to T cells, indicating that these peptides are T cell epitope peptides.
Example 3 detection of antigen Polypeptides to form pMHC complexes
1. Experimental method
ELISA method detects 167 novel coronavirus T cell epitope peptides screened in the example 2.
100. Mu.L of 0.5. Mu.g mL was used at room temperature -1 Streptavidin was incubated on 96U-plates for 16-18 hours, washed 3 times with wash buffer and blocked with dilution buffer (0.5M Tris pH 8.0,1M NaCl,1%BSA,0.2% Tween 20) for 30 min at room temperature. The photoactive peptide (KILGFVFJV) and MHC ((BioLegend, cat#280003, U)S)) was used as an HLA blank, influenza a M1 peptide (M58-66 gilgfftl) was used as a positive control, and epstein barr virus peptide (IVTDFSVIK) was used as a negative control. Then, 20. Mu.L of the dilutions (400. Mu.M) of 167 novel coronavirus T cell epitope peptides of example 1 and 20. Mu.L of Flex-T were each added, which were replaced with a 365nm three-way ultraviolet analyzer (Qian Bell, cat# 1903274) TM Dilutions of the monomer (200. Mu.g/mL) were made in 100. Mu.L to 96-well plates and incubated for 1 hour in a cell incubator at 37 ℃. After three washes with wash buffer, 100 μl of diluted HRP-binding antibody (BioLegend, cat#280303, us) was added and incubation was continued for 1 hour at 37 ℃ before washing. 100 μl of substrate solution (10.34 mL deionized water, 1.2mL 0.1m citric acid monohydrate/trisodium citrate dihydrate, pH 4.0, 240 μl 40mm abts,120 μl hydrogen peroxide solution) was added and incubated at room temperature in the dark for 8min. The reaction was quenched with 50. Mu.L of a termination solution (2% w/v oxalic acid dihydrate). Absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader over 30 minutes.
2. Experimental results
The results are shown in fig. 1 to 8C, which show that 167 antigen polypeptides can form pMHC complexes.
EXAMPLE 4 preparation of epitope peptide pMHC Complex monomer
1. Experimental method
HLA-A2 heavy chain, HLA-A2 light chain β2m and 167 epitope peptides in table 1 of example 1 were each followed by 1:2: the molar ratio of 10 was gradually added dropwise to a renaturation solution (5M urea, 0.4M arginine, 100mM Tris, 3.7mM cystamine, 6.3mM cysteamine and 2mM EDTA) for renaturation to obtain the pMHC complex monomer of the epitope peptide.
The pMHC complex monomer was further purified by passing through DEAE ion exchange column, eluting with 0.5M NaCl, and collecting proteins according to the OD280nm uv absorbance peak. The proteins purified by DEAE ion exchange column were then purified by Superdex75pg molecular sieve, eluted with PBS, and the different molecular weight proteins were collected according to the OD280nm UV absorbance peak.
2. Experimental results
After purification, pMHC complex monomers of the epitope peptide were obtained (fig. 9).
EXAMPLE 5 novel coronavirus HLA-A2 restricted epitope peptide-activated T cells
1. Experimental method
T2 cells activate T cells by expressing HLa-A2 molecules (T2-A2). Mononuclear lymphocytes (PBMCs) in peripheral venous blood of healthy volunteers were isolated and cd8+ T cells were further isolated. T2-A2 cells were labeled with CFSE and incubated with 167 different antigen peptides from example 1 after 20 min treatment with 20. Mu.g/mL mitomycin.
The specific method comprises the following steps:
will be 0.5X10 6 CD8+ T cells and different epitope peptides were loaded 0.5X10 6 T2-A2 cells were co-cultured and co-stimulated with 1. Mu.g/mL anti-human CD28 antibody and 50IU/mL IL-2. 50IU/mL IL-2 and 20 mu M epitope peptide were supplemented every two days.
The pMHC complex monomer of 30 μl of the epitope peptide obtained in example 4 was mixed with 3.3 μl of PE streptavidin (BioLegend cat#405203, US) in a 96-well plate, and after incubation at 4 ℃ for 30 minutes in the dark, 2.4 μl of blocking solution (1.6 μl of 50mM biotin (Thermo Fisher, cat#b20656, US)) and 198.4 μl of PBS were added to stop the reaction, and incubated overnight at 4-8 ℃ to obtain pMHC complex with PE fluorescence channel.
After 7 days of culture, the percentage of survival of T2-A2, the proportion of specific CD8+ T cells labeled with pMHC complex with PE fluorescent channels and the percentage of apoptosis marker Annexin V-APC on T2-A2 cells were calculated and the release of IFN-gamma and GZMB from specific CD8+ T cells was examined.
2. Experimental results
As shown in FIGS. 10 and 11, 167 novel coronavirus T cell epitope peptides selected in example 2 were all able to activate T cells.
Wherein, for the T cell epitope peptide VTWFHAISG (SEQ ID NO: 2) of the B.1.1.7 mutant strain, it corresponds to the T cell epitope peptide VTWFHAIHV (SEQ ID NO: 1) of the original virus strain, CD8 is activated + The cytocapacity of T is relatively stable before and after mutation.
And is opposite toT cell epitope peptide FKLKECVMYA (SEQ ID NO: 4) of the P.3 mutant, which corresponds to T cell epitope peptide FKLKDCVMYA (SEQ ID NO: 3) of the original strain; t cell epitope peptide YHDVRVVLI (SEQ ID NO: 6) for the B.1.526.1 mutant strain, which corresponds to T cell epitope peptide YHDVRVVLDFI (SEQ ID NO: 5) for the original strain; t cell epitope peptide ASLLFGWLI (SEQ ID NO: 8) for the B.1.526.2 mutant strain, which corresponds to T cell epitope peptide ASLPFGWLI (SEQ ID NO: 7) for the original strain; for the T cell epitope peptide VTWFHVIHV (SEQ ID NO: 9) of the B.1.1.529 mutant strain, which corresponds to the T cell epitope peptide VTWFHAIHV (SEQ ID NO: 1) of the original strain, the T cell epitope peptide of the above original strain activates CD8 as compared with the T cell epitope peptide of the mutant strain + T's cellular capacity is enhanced.
Specific CD8 activated by the above peptides + T kills target cells and is specific for CD8 + T cells release IFN-gamma and GZMB (as shown in FIG. 12).
Example 6 novel coronavirus HLA-A2 restricted epitope peptide activated T cells
1. Experimental method
Isolation of PBMCs in peripheral venous blood of new coronavirus original strain (NC 045512.2) infected convalescence persons for 14 days and 7 days after the second needle of beijing ke xing inactivated vaccine and identification of HLA subtype thereof, HLA-a2 positive PBMCs samples were further stained with pMHC complex with PE fluorescent channel and CD8-PerCP antibody of example 5 and then subjected to on-stream detection.
2. Experimental results
The results are shown in FIG. 13, which shows that the pMHC complex with fluorescent channels of the 9 epitope peptides with the amino acid sequences shown in SEQ ID NO. 1-9 can recognize antigen-specific CD8 generated in novel coronavirus infection convalents and vaccinators + T cells.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. The novel coronavirus T cell epitope peptide is characterized in that the amino acid sequence is shown as any one of SEQ ID NO. 8.
2. A novel coronavirus T cell epitope peptide encoding gene, wherein said encoding gene encodes the novel coronavirus T cell epitope peptide of claim 1.
3. A novel coronavirus T cell epitope peptide composition, which is characterized by comprising an amino acid sequence shown in SEQ ID NO:8, and any several of novel coronavirus T cell epitope peptides.
4. A pMHC complex comprising the novel coronavirus T cell epitope peptide of claim 1 or the novel coronavirus T cell epitope peptide composition of claim 3.
5. The pMHC complex according to claim 4, wherein the preparation method of the pMHC complex is that HLA-A2 heavy chain, HLA-A2 light chain beta 2m and novel coronavirus T cell epitope peptide renaturing is obtained.
6. An antigen peptide-antigen presenting cell complex, characterized in that the antigen presenting cell has the novel coronavirus T cell epitope peptide of claim 1 or the novel coronavirus T cell epitope peptide composition of claim 3 on the surface.
7. The antigen peptide-antigen presenting cell complex of claim 6, wherein the antigen presenting cell is a T2-A2 cell.
8. The antigenic peptide-antigen presenting cell complex of claim 7 wherein the T2-A2 cells are T2 cells over-expressing HLa-A2.
9. Use of one or more of the epitope peptide of claim 1, the gene encoding the novel coronavirus T cell epitope peptide of claim 2, the novel coronavirus T cell epitope peptide composition of claim 3, the pMHC complex of claim 4 or 5, and the antigen peptide-antigen presenting cell complex of claim 6 in the preparation of a novel coronavirus vaccine.
10. Use of one or more of the epitope peptide of claim 1, the gene encoding the novel coronavirus T cell epitope peptide of claim 2, the novel coronavirus T cell epitope peptide composition of claim 3, the pMHC complex of claim 4 or 5, and the antigen peptide-antigen presenting cell complex of claim 6 in the preparation of novel coronavirus drugs.
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