CN113173982A

CN113173982A - Rap v 2 protein-related epitope peptide and application thereof

Info

Publication number: CN113173982A
Application number: CN202110397450.9A
Authority: CN
Inventors: 李振兴; 于闯; 林洪; 张自业; 许利丽; 黄玉浩
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2021-07-27
Anticipated expiration: 2041-04-14
Also published as: CN113173982B

Abstract

The invention discloses 7 Rap v 2 protein-related epitope peptides, belonging to the technical field of biology. The amino acid sequences of the Rap v 2 protein related epitope peptide are respectively shown in SEQ ID NO. 1-7, and the application of the Rap v 2 protein related epitope peptide includes but is not limited to preparation of paramyosin with low allergenicity, detection of an Rap v 2 protein IgE antibody in serum of an allergic patient, preparation of a kit for detecting the Rap v 2 protein in food, preparation of a therapeutic antibody, a pharmaceutical composition or a vaccine and the like.

Description

Rap v 2 protein-related epitope peptide and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an epitope peptide related to a Rap v 2 protein and application thereof.

Background

Food allergy is an adverse stress immune response generated by some susceptible people after taking some specific food, and is clinically manifested as adverse symptoms of tissues and organs such as skin, oral cavity, digestive tract, respiratory tract, cardiovascular system, nervous system and the like, and severe food allergy can cause shock and even death. The protein in food is the main allergen component for triggering allergy, and the antigenic epitope of allergen is the key group directly participating in food allergy immune response reaction and is the material basis for determining the immunogenicity and specificity of allergen protein. Therefore, the identification of the allergen epitope is helpful to reveal the nature of the binding reaction between the antigen and the antibody, and has important significance for the diagnosis and prognosis judgment of allergic diseases, the research and development of vaccines, immunotherapy and the like.

Mollusks are a large and diverse group of about 10 thousands distributed in salt water, fresh water and land, some of which are important food sources for people and a large group of foods that induce food allergies. The group separates an allergen named as Rap v 2 from whelk, and according to tests such as amino acid composition and molecular weight, the group determines that Rap v 2 is paramyosin. Paramyosin (PM) is an important allergen present in mollusks, as well as a cross-reactive allergen and has good heat resistance and digestive stability. However, no report is found at home and abroad on the specific epitope of the food allergen protein.

Currently, research on mollusk allergens is not deep enough, and paramyosin is an allergenic protein capable of causing an organism to generate allergic reaction and is also an important allergen in soft marine products, and after being ingested, the paramyosin causes serious harm to the physical and mental health of allergic patients. The lack of epitope information leads to great difficulty in diagnosing and treating mollusc allergic patients, so that the research on novel allergens and epitopes in molluscs is very important.

Disclosure of Invention

The invention aims to provide an epitope peptide related to a Rap v 2 protein and application of the epitope peptide, and the technical scheme is as follows:

the invention provides an epitope peptide, wherein an amino acid sequence of the epitope peptide is selected from at least one of SEQ ID NO 1-7.

All the antigen epitope peptides are from Rap v 2 protein, and the sequence of the Rap v 2 protein is shown in SEQ ID NO. 8.

The Rap v 2 protein is paramyosin, a novel allergen protein, derived from whelk (Rapana venosa), and has an amino acid sequence available from the protein database of NCBI under the accession number gb: MN784956, and the primary structure of paramyosin contains 859 amino acids.

On the basis of the scheme, the invention also provides an epitope peptide, the amino acid sequence of which has homology of more than or equal to 80% with any amino acid sequence shown in SEQ ID NO. 1-7 and shows the same immunogenicity and the same antigenicity with any amino acid sequence shown in SEQ ID NO. 1-7. Preferably, the homology is more than or equal to 90%; furthermore, the homology is more than or equal to 95 percent; in a preferred embodiment, the homology is greater than or equal to 97%.

On the basis of the scheme, the invention also provides an epitope peptide, the amino acid sequence of which is formed by substituting, deleting or adding amino acid residues on the basis of any amino acid sequence of SEQ ID NO. 1-7, and the epitope peptide has the same immunogenicity and the same antigenicity as any amino acid sequence shown in SEQ ID NO. 1-7.

On the basis of the scheme, the invention also provides a nucleic acid for encoding the epitope peptide, an expression vector containing the nucleic acid and a host cell containing the expression vector.

On the basis of the scheme, the invention also provides related applications of the epitope peptide, including but not limited to the following cases:

(1) preparing paramyosin with low sensitization;

(2) detecting an IgE antibody of a Rap v 2 protein in serum of an allergic patient;

(3) preparing a kit for detecting Rap v 2 protein in food or other samples;

(4) preparing a therapeutic antibody, a pharmaceutical composition or a vaccine.

On the basis of the scheme, the invention also provides paramyosin with hyposensitization prepared from the epitope peptide, a kit for detecting the Rap v 2 protein, a therapeutic antibody, a pharmaceutical composition and a vaccine.

The kit provided by the invention comprises the epitope peptide.

The antibody provided by the invention can be specifically combined with the epitope peptide.

On the basis of the scheme, the invention provides a method for detecting a Rap v 2 protein, which comprises the following steps: the epitope peptide is coupled with inert protein to immunize animals, so that the animals generate antibodies combined with the epitope peptide, the antibodies are extracted and purified, and then the antibodies are used for detecting the Rap v 2 protein.

The invention has the beneficial effects that:

1. the epitope peptide fragment provided by the invention can be used for detecting whether an antibody of the paramyosin antigen exists in a sample or detecting whether an allergic patient is allergic to the paramyosin antigen.

2. The invention provides a brand-new specific epitope of food allergen paramyosin Rap v 2, can be used for diagnosing a patient with soft marine product allergy, and has important significance for the application of vaccine design, the research and development of monoclonal antibodies and antibody detection kits and the like.

Drawings

FIG. 1 is a graph showing the result of predicting potential epitopes of Rap v 2 protein by using a bioinformatics method;

FIG. 2 is a graph showing the result of IgE reaction between epitope peptide and serum of a patient with conch allergy;

FIG. 3 is a graph showing the effect of epitope peptides on KU812 basophil degranulation (A, β -hexosidase release rate; B, tryptase content; C, histamine content; D, IL4 content; E, IL13 content).

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

The invention provides 7 antigen epitope peptides with amino acid sequences shown as SEQ ID NO. 1-7; specifically, as shown in table 1:

TABLE 1

The 7 antigen epitope peptides are all from Rap v 2 protein. The Rap v 2 protein is paramyosin, a novel allergen protein, derived from whelk (Rapana venosa), and has an amino acid sequence available from the protein database of NCBI under the accession number gb: MN784956, and the primary structure of paramyosin contains 859 amino acids.

The sequence of the Rap v 2 protein is shown as SEQ ID NO: 8:

MDYGSDINVVRKVSRTYNVYRGSSPSTQNRLESRIRELEDALDSERDGRVRAEKMVAELNFRVDQLQDSLDEQGTSTSAQMEISKKRESEIMKLRKDMELASAQFEATESSMRKRHQESVNDLSDQVDYLNKNKNRVEKEKQTLVVELDGLTGQLESVSKAKATLESRVDAAERSASGLKSQVDDLTRQLSELTSLKSRLTQENFDLQHQVQELDSNNAALGKARSQLQASNDDLKRQLDDETRQRQNLQVNLSALQADYDNLNVRLEEEAETSTNLRNQLSKVNADYSALKSRFDKELALKMEEMEDLKRRMQVRITELEDTCEQLRVKNSSLEKAKTKLANEIKEITIELENTQIIVQDLTKRNRQLENENAGLQRRIEELTGENNTLRNEKALLEQDVQKLRVSNAELTERNDNLQRENKHLSDQLREANLALKDANRELADLRQLRSQLEAERDSLAAALKDTEEALRDAEAKLASAQAALNQLKIDMENRLREKDEEIENVRRSSARAIEELQRTLVEVETRYKTEISRMKKKFESDIRELEAALDNANRANSEYLKQIKSLQQRVKELETLLEEERRVSDDLRNQLGVAERKRVALAQELEDARALLEGAERARKNAETELADTSTRLSEITLQVTSLTNDKRRLEADLSAMQSDLDDAINNQRGAEERAERLAAENGRLADELRQEQENYKNAESLRKQLEVEIREITIRLEEAEAFATKEGRRLVAKLQVRVRDLEAELENEARRVRESVANQRKAERLHKELVAQTEEERRQLAELASLNDQLQLRIKTYKRQLEEAEDVASLTMNKYRKAQALIDEADARADAAEKNLQTVRRARSMSVTREITRVIKV

screening and identifying process of epitope peptide shown in SEQ ID NO. 1-7

(1) Method for predicting paramyosin epitope by bioinformatics method

According to the analysis of the properties of the DNAStar and the Ante Prot software on the hydrophilicity and hydrophobicity, the surface accessibility, the plasticity, the antigen index and the like of the Rap v 2 protein, an epitope prediction result is comprehensively obtained, and the prediction result is shown in figure 1. Then, the final screening of the predicted epitope is carried out by combining the prediction results of 3 bioinformatics online servers (Bepided, ABCPred and Immunodicine Group Tools Server). The five bioinformatics methods described above predict the resulting epitope sites, respectively, as shown in table 2.

TABLE 2

The potential antigen epitopes obtained by the five bioinformatics prediction methods are comprehensively analyzed, and the total number of the potential antigen epitopes is 12, and the amino acid length of the predicted epitopes is between 11 and 18. The predicted 12 epitopes were subjected to solid phase synthesis and designated as P1 to P12, respectively, in amino acid starting order. The Rap v 2 protein epitope sites finally predicted by the bioinformatics method are shown in table 3.

TABLE 3

Note: in table 3 PM refers to Rap v 2 protein.

(2) Method for screening Rap v 2 protein epitope by using immune dot blot array technology

The amino acid sequences of P1-P12 are synthesized by adopting Fmoc (fluoroxylmethod) solid-phase method, and the purity (> 98%) and the molecular weight of the synthesized polypeptide are verified by adopting High Performance Liquid Chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) method, so as to meet the experimental requirements.

Serum samples of 6 whelk allergy patients (from the university of Qingdao subsidiary hospital, all 6 subjects showed clinical history of at least two or more symptoms including acute urticaria, allergic rhinitis, anaphylactic shock and asthma, etc. after eating whelk) were taken as test groups with numbers from S1 to S6; serum samples from 2 normal volunteers without any shellfish allergy history (from allergy department of affiliated hospital university of Qingdao) were taken as negative control group; each serum sample was stored in aliquots at-80 ℃ until use.

Each test group serum, each control group serum and each predicted epitope (numbered from P1 to P12) were subjected to immunoblot array detection.

The immunoblot assay procedure was as follows:

1) treating a nitrocellulose membrane: a nitrocellulose membrane (NC membrane) was immersed in ultrapure water to sufficiently wet the NC membrane.

2) Sample application: the Rap v 2 protein was dissolved to 1.5mg/mL with CBS solution to prepare a spot solution. And taking the NC membrane out of the ultrapure water, airing the redundant water on the NC membrane, and then carrying out spotting. Add 2. mu.L of allergen-like solution to each array spot and dry naturally at room temperature.

3) Washing the membrane: the NC membrane was washed three times with PBST buffer for 5min each, and allergen proteins not bound to the NC membrane in the spotting fluid were removed.

4) And (3) membrane sealing: after the NC membrane was blocked in the membrane reaction blocking solution at room temperature for 2h, the membrane was washed three times with PBST buffer.

5) Primary antibody incubation: the serum of a patient with the conch allergy is diluted by 1:5(v/v) by using 1% skim milk, then the synthesized predicted epitope peptide is mixed with the diluted serum, and after incubation for 1h, the mixture is spotted on an NC membrane. After incubation for 4h at room temperature, the membranes were washed three times with PBST.

6) And (3) secondary antibody incubation: diluted with 1% skim milk at a ratio of 1:10000(v/v) according to the specification of HRP-conjugated coat Anti-Human IgE subcordary Antibody, dropped onto NC membrane, incubated at room temperature for 1h, and washed three times with PBST buffer.

7) ECL development: and mixing the solution A and the solution B in the ECL kit in equal volume, immersing the NC membrane in developing solution for reaction for 1min, taking out the NC membrane, performing exposure treatment by using a gel imager, and collecting images.

IgE immunoblot reaction results were scored according to four grades of reaction intensity, according to the code classification of Ayuso et al: strong IgE reaction intensity (black array, 3 min, corresponding to white Dot-blot reaction), medium IgE reaction intensity (black gray array, 2 min, corresponding to light gray Dot-blot reaction), weak IgE reaction intensity (light gray array, 1min, corresponding to black gray Dot-blot reaction), no IgE reaction (white array, 0 min, corresponding to black Dot-blot reaction). The stronger the competitive position of the antigen epitope peptide and the antigen protein for serum IgE, the weaker reaction signal of the Dot-blot immune array is presented, and the stronger the IgE reaction intensity of the array is. In addition, polypeptides that are recognized by half or more of the allergy sera or that have an intensity score of the microarray higher than the sum of the average score and the error of the score can be identified as epitope polypeptides, as indicated by "+" according to the criteria of Ayuso et al.

The test results are shown in FIG. 2, and 12 predicted epitopes have no IgE reaction with the healthy control serum, but have IgE reaction with the serum of the conch allergy patient to different degrees. From the frequency and intensity of IgE reactivity, seven major predicted IgE binding epitopes were identified: p1: rap v 2(10-27), P2: rap v 2(32-47), P3: rap v 2(64-81), P4: rap v 2 (106-: rap v 2 (273-: rap v 2 (361-: rap v 2 (491-. Therefore, the 7 predicted epitopes can be screened as the epitopes of the Rap v 2 protein.

(3) Antigen epitope peptide sensitized KU812 cell degranulation experiment

1) Cell culture

KU812 cells were cultured in complete RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin and placed at 37 ℃ in 5% CO₂A wet incubator.

2) Cell stimulation

Continuously culturing for 1 week in 1640 culture medium (0.5 mu g/mL) containing human myeloma IgE to stimulate the expression of Fc epsilon RI receptor on the surface of KU812 cells, and replacing the cell culture solution every two days for 3-4 times during the culture.

3) Cell sensitization and stimulation

After the last replacement of the fresh medium 1640 containing human myeloma IgE (0.5. mu.g/mL), the cells were first digested the next day, 5mL of the cell suspension were centrifuged at 400 Xg for 5 minutes and washed 1 time with Hank's (HBSS) balanced salt solution. The cell pellet was suspended in 5mL 1640 complete medium and pipetted into the desired volume of 96-well plates at 50 μ L/well. Diluting human serum with 1640 basic culture medium at a ratio of 1:5, adding 50 μ L of diluted serum into each well, and placing in CO₂Incubate overnight in the incubator. Cells were then stimulated with purified paramyosin and the predicted epitope for 1 hour at room temperature.

4) Detection of content of five biomarkers of KU812 cells

The IgE immunized KU812 cells were centrifuged with the collected supernatant using a commercial ELISA kit, the release of tryptase, histamine, IL-4 and IL-13 cytokines in the supernatant was determined, and cell degranulation was observed by measuring the amount of β -hexosidase in the supernatant.

The results are shown in fig. 3, and the ability of all potential epitopes to induce a response in KU812 cells was analyzed. A predicted epitope with a significant difference (p <0.01) compared to the negative control was considered as a potential allergen epitope. The overall release profiles of β -hexosidase release rate (fig. 3A), histamine content (fig. 3B), and tryptase content (fig. 3C) were relatively consistent. The P6, P9 and P11 epitope peptides were not significantly different from the negative control group (P >0.05), whereas the Rap v 2 protein and the predicted epitope (including P1, P2, P3, P4, P7, P8 and P10) had significant degranulation on basophils (P < 0.01). Furthermore, the results for IL-4 (fig. 3D) and IL-13 (fig. 3E) cytokine levels were similar, with predicted epitopes P1, P2, P4, P7, P8 and P10 showing significant degranulation effects (P < 0.01). Combining the results of five biomarkers, the P1, P2, P3, P4, P7, P8 and P10 of the Rap v 2 protein have obvious degranulation effect, which indicates that the protein is an allergenic epitope region. The test results are also consistent with the dot blot inhibition test.

Based on the above two approaches, 7 peptides of the invention (P1, P2, P3, P4, P7, P8 and P10) were further demonstrated to be the specific IgE binding sequences for primarily Rap v 2.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

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Claims

1. An epitope peptide, characterized in that its amino acid sequence is selected from the group consisting of:

(1) at least one of amino acid sequences shown in SEQ ID NO 1-7;

(2) on the basis of the amino acid sequences shown in SEQ ID NO. 1-7, the amino acid sequences which have the same immunogenicity and the same antigenicity as any amino acid sequence shown in SEQ ID NO. 1-7 are formed by replacing, deleting or adding one or more amino acids;

(3) has an amino acid sequence with homology of more than or equal to 80 percent with any amino acid sequence shown in SEQ ID NO. 1-7, and has the same immunogenicity and the same antigenicity with the amino acid sequences shown in SEQ ID NO. 1-7.

2. A nucleic acid encoding the epitope peptide according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. Use of the epitope peptide according to claim 1 for preparing paramyosin having hypoallergenic activity.

6. The use of the epitope peptide of claim 1 in the preparation of a kit for detecting Rap v 2 protein in food.

7. A detection kit comprising the epitope peptide according to claim 1.

8. Use of the epitope peptide of claim 1 for the preparation of a therapeutic antibody, a pharmaceutical composition or a vaccine.

9. An antibody capable of specifically binding to the epitope peptide of claim 1.

10. A method for detecting a Rap v 2 protein, which comprises the following steps: immunizing an animal with the epitope peptide of claim 1 by coupling to an inert protein, allowing the animal to produce an antibody which binds to the epitope peptide, extracting the purified antibody, and then detecting the Rap v 2 protein using the antibody.