CN114685628A - Epitope peptide of RBD of SARS-CoV-2 and its application - Google Patents

Epitope peptide of RBD of SARS-CoV-2 and its application Download PDF

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CN114685628A
CN114685628A CN202210296306.0A CN202210296306A CN114685628A CN 114685628 A CN114685628 A CN 114685628A CN 202210296306 A CN202210296306 A CN 202210296306A CN 114685628 A CN114685628 A CN 114685628A
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rbd
sars
epitope peptide
antibody
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CN114685628B (en
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赵�卓
章金勇
邹全明
段连礼
陈致富
苟强
熊青山
敬海明
李思思
陈龙龙
包汶鑫
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Third Military Medical University TMMU
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Abstract

The invention provides an epitope peptide of RBD of SARS-CoV-2, which comprises antigen dominant epitope with amino acid sequence of SEQ ID NO. 11 and/or SEQ ID NO. 17. The invention also provides the application of the SARS-CoV-2 in the preparation of medicines for diagnosing, preventing or treating SARS-CoV-2 infection. The epitope peptide provided by the invention has the effect of inhibiting the combination of RBD of SARS-CoV-2 and human angiotensin converting enzyme 2(ACE2), and can be used for preparing high-efficiency, low-toxicity and high-safety RBD-based medicines for preventing SARS-CoV-2 infection.

Description

Epitope peptide of RBD of SARS-CoV-2 and its application
Technical Field
The invention relates to the technical field of biological medicines, in particular to the technical field of virus antigens, and especially relates to an epitope peptide of RBD of SARS-CoV-2 and application thereof.
Background
The infection of the novel Coronavirus (SARS-CoV-2, New crown for short) is serious, and the epidemic situation is continuously developed globally, thereby bringing a serious challenge to the epidemic situation prevention and control of China.
Because no specific new crown treatment medicine exists at present, the vaccine is an ideal choice for controlling new crown infection. Although several new corona vaccines are currently on the market, the emergence of new corona virus variant strains such as the Delta strain and the Omicron strain continues, making the effectiveness and persistence of existing vaccination challenging. The main initial part of the new coronary infection is respiratory mucosa, most of the existing new coronary vaccines are immunized by intramuscular injection (abbreviated as intramuscular injection), the induced respiratory mucosa immune response is limited, and the intramuscular injection immunity can not prevent the virus transmission of the host mucosa part in time. The key problem to be solved by the new coronary vaccine is to select a proper adjuvant and an appropriate immunization way and induce respiratory mucosa and systemic immune response at the same time. The effective components of SARS-CoV-2 important antigen to induce protective response under different adjuvants and immune approaches and the immune response mechanism thereof are analyzed, which are the basis and precondition for solving the scientific problem.
Spike protein (S protein or Spike protein for short) is an important structural protein of SARS-CoV-2, and is an antigen component of other vaccines except inactivated vaccines in the existing new crown vaccines. The Spike protein contains S1 and S2 subunits, wherein the C terminal of the S1 subunit contains a Receptor binding domain (RBD domain for short), the Spike protein recognizes Receptor angiotensin converting enzyme E2(ACE2) on the surface of a host cell through the RBD domain of the Spike protein and mediates the virus to enter the host cell, and the amino acid variation of the RBD domain can cause the variation of the species specificity and the infection characteristic of the virus. RBD contains the most immunodominant neutralizing epitope in the whole SARS-CoV-2 virus, and 90% of neutralizing antibodies in serum of COVID-19 restorer are induced by RBD. The neutralizing antibody targeting RBD has potential value in preventing and treating SARS-CoV-2 infection. The RBD can evade the NTD domain antibody of the S1 subunit to cause ADE effects. In addition, the RBD has stable property and is easy to be recombined, expressed and purified to prepare, so that the RBD is an ideal antigen of a new corona vaccine.
Studies have demonstrated that antibodies with neutralizing capacity play a protective role in the protection of new coronavirus. The titer of neutralizing antibodies to SARS-CoV-2 correlates with clinical outcome. Because protein antigens exert their functions, specificity is mainly embodied by epitopes therein, among which "immunodominant" epitopes play a major role. Therefore, the identification of protective epitopes for immunodominant responses in RBD antigens is an important prerequisite for the development and optimization of the design of SARS-CoV-2 vaccines based on RBD antigens. Screening the immunodominant epitope of RBD is the premise of stimulating more effective immune response of RBD. Currently known B-cell epitopes of RBDs, either predicted by bioinformatics software, identified by monoclonal antibodies, or identified using human or animal immune models, have not been reported for comprehensive screening of "dominant epitopes" of RBDs involved in immune responses in different immune pathways. Because the initial infection part of the new coronavirus is mainly the respiratory tract mucosa, a method for accurately and effectively screening and identifying the B cell 'dominant epitope' of the RBD participating in immune response under the 'nasal drip route' is urgently needed.
Disclosure of Invention
The invention provides an identification method of antibody dominant epitope peptide of RBD of SARS-CoV-2 and the application of the epitope peptide in the preparation of medicine for diagnosing, preventing and/or treating SARS-CoV-2 infection.
The invention firstly provides an epitope peptide of RBD of SARS-CoV-2, which comprises the antigen dominant epitope with the amino acid sequence of SEQ ID NO. 11 and/or SEQ ID NO. 17.
In one embodiment according to the present invention, the epitope peptide is coupled with a polypeptide tag at the N-terminus or C-terminus; preferably, the polypeptide label is a biotin label or a fluorescent label.
The invention further provides the application of the epitope peptide in the preparation of a specific antibody diagnostic reagent for SARS-CoV-2 infection.
The invention also provides the application of the epitope peptide in preparing vaccines for preventing or treating SARS-CoV-2 infection.
The invention further provides the application of the epitope peptide as a screening target of SARS-CoV-2 infection resisting drugs.
In one embodiment according to the present invention, there is also provided a pharmaceutical composition for preventing or treating SARS-CoV-2 infection, comprising the above epitope peptide and a pharmaceutically acceptable adjuvant.
In one embodiment according to the invention, an adjuvant is further comprised, preferably AddaVax.
In one embodiment according to the present invention, the pharmaceutical composition is in a dosage form for nasal administration; preferably a spray, nasal drops, powder, gel or microsphere formulation.
The invention further provides a diagnostic reagent for SARS-CoV-2 infection, which comprises the above antigen epitope peptide, wherein the antigen epitope peptide is coated on a detection carrier, and the detection carrier is selected from any one of a polystyrene micro-reaction plate, a colloidal gold reagent strip, a magnetic bead and a micro-fluidic chip.
In one embodiment according to the present invention, further comprising a second antibody specifically recognizing the murine IgG antibody;
preferably, the second antibody is selected from one of a goat anti-mouse monoclonal antibody, a rabbit anti-mouse polyclonal antibody;
preferably, the second antibody is coupled to a coordinating group that activates or quenches the specific fluorophore.
The technical scheme of the invention has the following beneficial effects:
the RBD antibody immunodominant epitope peptide obtained by the method has the effect of inhibiting the combination of RBD and human angiotensin converting enzyme 2(ACE2), and can be used for preparing high-efficiency, low-toxicity and high-safety RBD-based medicines for preventing SARS-CoV-2 infection, such as preventive vaccines.
The antibody dominant epitope peptide of the RBD of SARS-CoV-2 provided by the invention has the effect of inhibiting the combination of the RBD and human angiotensin converting enzyme 2(ACE2), the animal is immunized by the dominant epitope, the immune preparation has no irrelevant component or harmful component, and the specific monoclonal antibody prepared by the antibody dominant epitope peptide can better prevent SARS-CoV-2 infection.
Through sequence comparison and analysis, the antibody dominant epitope peptide of the RBD of SARS-CoV-2 provided by the invention has conserved sequence in various SARS-CoV-2 strains, therefore, the antibody dominant epitope peptide can also be used as a diagnostic reagent of SARS-CoV-2 or used for preventing and treating other SARS-CoV-2 infection.
Drawings
FIG. 1 is a graph showing the results of ELISA detection of overlapping peptides screened according to the present invention using RBD nasal-drop immunized BALB/c mice as a primary antibody;
FIG. 2 is a serum specific IgG antibody titer profile of BALB/c mice immunized nasally with RBD;
FIG. 3 is a serum-specific IgG antibody subtype profile of BALB/c mice immunized nasally with RBD;
FIG. 4 is a graph showing the results of neutralizing antibody titer in the efficacy analysis of the binding of human angiotensin converting enzyme 2(ACE2) to RBD-immunized BALB/c mice antiserum selected according to the present invention;
FIG. 5 is a graph of the results of an analysis of the effect of antiserum of RBD-immunized BALB/c mice selected according to the present invention on the inhibition of RBD binding to human angiotensin-converting enzyme 2(ACE2) in serum dilution inversion graphs;
FIG. 6 shows the selected antibody immunodominant epitope peptide RBD of the present invention61-78、RBD97-114Distributing an analysis result map at the position in the RBD three-dimensional structure;
FIG. 7 shows the immunodominant epitope peptide RBD of the antibody selected by the present invention61-78、RBD97-114The amino acid sequence conservation analysis result map of (1).
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 acquisition of overlapping peptides
Based on the Sequence ID of the RBD protein (Sequence ID:6XDG _ E), another expected overlapping peptide was again obtained by moving downstream a number of amino acids smaller than the length of the overlapping peptide each time relative to the length of the expected overlapping peptide. The length of the expected overlapping peptide can be 15-30 amino acids, the number of amino acids in each step can be 4-8, in this example, starting from amino acid No. 1, 6 amino acids in each step, and 18 amino acid polypeptides (Shanghai Jier Biochemical technology Co., Ltd.) with overlapping steps are synthesized to obtain 35 overlapping peptides. The purities are all more than 95%. The synthetic walking overlapping peptide information is shown in table 1. The synthetic peptide fragment was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 1mg/mL, dispensed and frozen at-70 ℃ until diluted to 1mM with PBS at the time of use.
TABLE 1 walking overlapping peptide information
Figure BDA0003563464060000051
Example 2 collection and preservation of RBD immune sera
Recombinant SEB holoprotein of the mutated SEB three toxin sites was constructed based on the method disclosed in the reference (reference: JintaoZou et al a-Hemolysin-Aided immunization of the Spike Protein RBD Resulted in expressed Immunogenicity and mutagenesis obtained SARS-CoV-2 Variants antigens. front Immunol.2021 Sep 24; 12:757691.), the dose of RBD Protein in immunized mice was adjusted to 20 ug/mouse, the dose of AddaVax adjuvant was 50 μ L, the route of immunization was nasal drop immunization, three immunizations over 0, 14, 21 days. And (3) measuring the antiserum titer, and selecting the RBD titer to be more than 1: 64000 mouse immune sera were used for subsequent testing.
Example 3 screening of B cell immunodominant epitopes for RBD
Adjusting the coating concentration of the overlapping peptide to be 5 mu g/well (taking a holoprotein RBD (SSequilibrium ID:6XDG _ E) as a positive control), coating, washing, sealing, washing again, adding the RBD immune antiserum obtained in example 2, diluting at 1:50, incubating for 1.5h, washing, adding HRP-goat anti-mouse IgG (purchased from Enjingyal organism, product number E1WP319), diluting at 1:5000, washing, adding a TMB substrate developing solution (purchased from Beyotime/Biyun, product number P0209-100ml), reading the OD value at 450nm after terminating the reaction, and taking the positive overlapping peptide as the OD detection value-blank control of the amino acid overlapping peptide according to the formula 18)/(the OD detection value of the negative peptide-blank control) to be not less than 2.1. Through SPSS16.0 data inspection, positive overlapping peptides with significant statistical significance relative to other positive overlapping peptide readings are obtained, and are defined as immunodominant epitope peptides of B cells, namely immunodominant epitope peptides. Unrelated peptide OVA192–201(SEQ ID NO:36EDTQAMPFRV) is a negative control peptide.
As a result: as shown in FIG. 1, there are 2 positive polypeptides RBD61-78(SEQ ID NO:11 CYGVSPTKLNDLCFTNVY)、RBD97-114(SEQ ID NO:17TGKIADYNYKLPDDFTGC) has significant statistical significance relative to other dominant peptide readsDefined as a dominant epitope, OVA in this figure192-201Negative irrelevant peptide; by the method, all B cell epitopes of the RBD are obtained by screening, and B cell dominant epitopes of the RBD are determined.
Example 4 antisera from RBD-immunized mice were analyzed for their ability to inhibit the binding of RBD to human angiotensin-converting enzyme 2(ACE 2);
the proteins of RBD were immunized with AddaVax adjuvant (purchased from InvivoGen) on days 0, 14, 21 with three intramuscular injections for 6-8 weeks of BALB/c mice, and antisera were collected from the tail vein on day 7 after the last immunization.
To evaluate the immunogenicity of RBD, RBD-specific antibody levels were determined by administering AddaVax adjuvant to the RBD protein described in example 2 followed by instillation into the immunized mice, and collecting blood samples from the tail vein of mice after triple immunization. Results of RBD-specific IgG antibody titers in sera of immunized mice are shown in fig. 2, and geometric mean titers of RBD-specific IgG antibodies in sera of immunized groups were increased to about 106. Wherein the log10 logarithm of the RBD-specific IgG antibody titer is 5.95 +/-0.2375 (P < 0.05). The above results show that: the nasal drip immunization of RBD supplemented with AddaVax adjuvant can induce high level of RBD specific antibody.
The results of the analysis of the RBD-specific IgG antibody subtype in the sera of the immunized mice are shown in FIG. 3, in which the RBD-specific IgG antibody is mainly IgG1 subtype.
Neutralizing antibody titers were first detected by a competition ELISA method to assess RBD-induced levels of functional antibodies. The competitive inhibition Assay was performed using a Kit (Anti-SARS-CoV-2 Neutralizing Antibody tip diagnostic Assay Kit, available from Acrobiosystems, Beijing, China). Serum samples from immunized mice were diluted 1:10 and mixed with 0.3. mu.g/ml RBD of HRP-SARS-CoV-2 spike protein for repeat analysis. The microplate reader detected the optical density value (OD value) at 450nm and the neutralization inhibition rate was calculated according to the formula (1-average OD value of sample/average OD value of negative control). times.100%. The results are shown in fig. 4, the antibody NT50(Titers of 50% neutrallization, 50% Neutralization titer) induced after 3 times of 20. mu.g RBD reached 1:3412(P <0.05), and the reciprocal plot of serum dilution is shown in fig. 5. The above results illustrate that: RBD was adjuvanted with AddaVax adjuvant for nasal drip immunization, and the high levels of RBD-specific antibodies induced had a neutralizing capacity to compete for inhibition of RBD binding to human angiotensin converting enzyme 2.
Example 5 location of immunodominant epitope peptide in three-dimensional Structure of RBD holoprotein
This example is presented to demonstrate the results of the positional distribution analysis of antibody immunodominant epitope peptides in the three-dimensional structure of the RBD holoprotein.
Downloading a 3D structure chart of reported RBD protein from a public database of PubMed protein, and labeling the sequence position of the immunodominant epitope peptide screened in the experiment by PyMOL 1.1 program software.
The results are shown in FIG. 6, the dominant peptide RBD61-78、RBD97-114The epitope peptide sequences are respectively positioned in different loop regions of the RBD three-dimensional crystal structure, and therefore, the dominant epitope peptide sequences (antibody dominant epitopes) are reliable candidate molecules of the RBD epitope vaccine.
Example 6 amino acid sequence conservation analysis of immunodominant epitope peptides in SARS-CoV-2 strains
This example is for the antibody immunodominant epitope peptide RBD screened by the present invention61-78、RBD97-114The result of amino acid sequence conservation analysis of (1).
The amino acid sequence of RBD protein of 30 SARS-CoV-2 strains was searched in Genbank database, and the amino acid sequence was analyzed by comparison using Basic Local Alignment Search Tool (BLAST) software of NCBI, and 30 SARS-CoV-2 strains were randomly selected for Multiple sequence Alignment (Multiple Alignment) with the website address htps:// BLAST.
The results are shown in FIG. 7, the dominant peptide RBD61-78、RBD97-114The amino acid sequence in each of 30 SARS-CoV-2 strains is conservative, so that it has good application prospect.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
1 5 10 15
Trp Asn
<210> 5
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
1 5 10 15
Ser Asn
<210> 6
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
1 5 10 15
Tyr Ser
<210> 7
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
1 5 10 15
Ser Ala
<210> 8
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
1 5 10 15
Phe Lys
<210> 9
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
1 5 10 15
Ser Pro
<210> 10
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn
1 5 10 15
Asp Leu
<210> 11
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
1 5 10 15
Val Tyr
<210> 12
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
1 5 10 15
Val Ile
<210> 13
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
1 5 10 15
Val Arg
<210> 14
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
1 5 10 15
Gly Gln
<210> 15
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile
1 5 10 15
Ala Asp
<210> 16
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys
1 5 10 15
Leu Pro
<210> 17
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
1 5 10 15
Gly Cys
<210> 18
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp
1 5 10 15
Asn Ser
<210> 19
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
1 5 10 15
Ser Lys
<210> 20
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
1 5 10 15
Tyr Asn
<210> 21
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
1 5 10 15
Leu Phe
<210> 22
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
1 5 10 15
Leu Lys
<210> 23
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
1 5 10 15
Asp Ile
<210> 24
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
1 5 10 15
Tyr Gln
<210> 25
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
1 5 10 15
Pro Cys
<210> 26
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu
1 5 10 15
Gly Phe
<210> 27
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
1 5 10 15
Pro Leu
<210> 28
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
1 5 10 15
Phe Gln
<210> 29
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
1 5 10 15
Val Gly
<210> 30
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
1 5 10 15
Arg Val
<210> 31
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
1 5 10 15
Phe Glu
<210> 32
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
1 5 10 15
Pro Ala
<210> 33
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
1 5 10 15
Pro Lys
<210> 34
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn
1 5 10 15
Leu Val
<210> 35
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
1 5 10 15
Val Asn Phe
<210> 36
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Glu Asp Thr Gln Ala Met Pro Phe Arg Val
1 5 10

Claims (10)

1. An epitope peptide of RBD of SARS-CoV-2, which comprises an epitope having an amino acid sequence of SEQ ID NO. 11 and/or SEQ ID NO. 17.
2. The epitope peptide according to claim 1, wherein a polypeptide tag is coupled to the N-terminus or C-terminus of the epitope peptide; preferably, the polypeptide label is a biotin label or a fluorescent label.
3. Use of the epitope peptide according to claim 1 or 2 for the preparation of a specific antibody diagnostic reagent for SARS-CoV-2 infection.
4. Use of the epitope peptide according to claim 1 or 2 for the preparation of a vaccine for the prevention or treatment of SARS-CoV-2 infection.
5. The use of the epitope peptide of claim 1 or 2 as a screening target for drugs against SARS-CoV-2 infection.
6. A pharmaceutical composition for preventing or treating SARS-CoV-2 infection, comprising the epitope peptide according to claim 1 or 2 and a pharmaceutically acceptable adjuvant.
7. The pharmaceutical composition according to claim 6, further comprising an adjuvant, preferably AddaVax.
8. The pharmaceutical composition of claim 6 or 7, wherein the pharmaceutical composition is in a form for nasal administration; preferably a spray, nasal drops, powder, gel or microsphere formulation.
9. A diagnostic reagent for SARS-CoV-2 infection comprising the epitope peptide according to claim 1 or 2, wherein the epitope peptide is coated on a detection carrier selected from any one of a polystyrene microplate, a colloidal gold reagent strip, a magnetic bead, and a microfluidic chip.
10. The diagnostic reagent of claim 7, further comprising a second antibody that specifically recognizes a murine IgG antibody;
preferably, the second antibody is selected from one of a goat anti-mouse monoclonal antibody, a rabbit anti-mouse polyclonal antibody;
preferably, the second antibody is coupled to a coordinating group that activates or quenches the specific fluorophore.
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CN111830258A (en) * 2020-07-03 2020-10-27 浙江睦科生物科技有限公司 Novel coronavirus specific antibody detection kit
CN112646006A (en) * 2021-01-20 2021-04-13 中国人民解放军陆军军医大学 Marker epitope polypeptide for diagnosing COVID-19 mild and severe symptoms and application thereof
CN112851770A (en) * 2021-02-05 2021-05-28 中国人民解放军陆军军医大学 Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof
CN112940087A (en) * 2021-03-17 2021-06-11 郑州大学 Common epitope peptide of SARS-CoV and SARS-CoV-2 and its application
CN112961223A (en) * 2021-02-24 2021-06-15 东南大学 SARS-CoV-2 lymphocyte antigen epitope peptide and its application
KR20210123155A (en) * 2020-04-02 2021-10-13 조선대학교산학협력단 Use of RBD as diagnostic, treatment or vaccine of COVID-19
WO2021209035A1 (en) * 2020-04-17 2021-10-21 暨南大学 Novel coronavirus t cell epitope peptide, and pmhc and preparation and application thereof
CN114058593A (en) * 2020-08-06 2022-02-18 中国人民解放军陆军军医大学 Monoclonal antibody of N antigen of SARS-CoV-2, detection method and use thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210123155A (en) * 2020-04-02 2021-10-13 조선대학교산학협력단 Use of RBD as diagnostic, treatment or vaccine of COVID-19
WO2021209035A1 (en) * 2020-04-17 2021-10-21 暨南大学 Novel coronavirus t cell epitope peptide, and pmhc and preparation and application thereof
CN111830258A (en) * 2020-07-03 2020-10-27 浙江睦科生物科技有限公司 Novel coronavirus specific antibody detection kit
CN114058593A (en) * 2020-08-06 2022-02-18 中国人民解放军陆军军医大学 Monoclonal antibody of N antigen of SARS-CoV-2, detection method and use thereof
CN112646006A (en) * 2021-01-20 2021-04-13 中国人民解放军陆军军医大学 Marker epitope polypeptide for diagnosing COVID-19 mild and severe symptoms and application thereof
CN112851770A (en) * 2021-02-05 2021-05-28 中国人民解放军陆军军医大学 Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof
CN112961223A (en) * 2021-02-24 2021-06-15 东南大学 SARS-CoV-2 lymphocyte antigen epitope peptide and its application
CN112940087A (en) * 2021-03-17 2021-06-11 郑州大学 Common epitope peptide of SARS-CoV and SARS-CoV-2 and its application

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