A method for identifying a sesame major allergen protein Ses i 3 B cell linear epitope

Abstract

The present application relates to a kind of B cell linear epitope identification method of sesame allergen protein Sesi3, comprising the following steps: first, the B cell linear epitope of sesame allergen Sesi3 is predicted by bioinformatics technology;Then, the B cell linear epitope of sesame allergen Sesi3 is identified by slot-blot immunomicroarray technology combined with immune score matrix.The B cell linear epitope identification method of sesame allergen protein Sesi3 of the present application has the advantages of strong specificity, high sensitivity, high accuracy and high throughput.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically to a method for identifying B-cell linear epitopes of sesame allergen proteins. Background Technology

[0002] Sesame (Sesamum indicum L.) belongs to the Pedaliaceae family and the Sesamum genus, and is an annual herbaceous plant. Sesame is one of the world's oldest oilseed crops, rich in lipids, proteins, vitamins, minerals, and numerous functional components. It possesses anti-inflammatory, antioxidant, anti-cancer, anti-hypertensive, anti-melanin, hearing-protective, and cholesterol-lowering effects. Furthermore, due to its high oil content and sesamin content, sesame's unique aroma has garnered widespread attention in the food industry, and it can be used to produce various foods such as sesame paste and sesame oil. However, as one of the eight newly identified allergens, sesame can also cause severe allergic reactions. Like other food allergies, sesame allergy is an IgE-mediated type I hypersensitivity reaction, caused by ingestion, inhalation, or contact with the allergen. It can cause systemic allergic reactions affecting multiple systems, including the respiratory, gastrointestinal, and skin / mucous membrane systems, and in severe cases, can even lead to shock or death. These allergic symptoms typically persist for the rest of the patient's life.

[0003] Sesame seeds contain seven allergenic proteins, named Ses i 1-Sesi 7 by the International Union of Immunological Societies (IUIS). Ses i 3 is a 7S pea globulin, belonging to the Cupin superfamily, and possesses a β-fold barrel protein supersecondary structure in its three-dimensional spatial structure. Pea globulins consist of three monomers of a single polypeptide chain linked together to form a triangular, flattened trimer, which is resistant to heat denaturation and enzymatic digestion. Ses i 3 is composed of 585 amino acids, lacks disulfide bonds, and has a molecular weight of approximately 45 kDa. Related studies indicate that Ses i 3 can be recognized in the serum of 75% of sesame allergy patients, making it one of the main allergens associated with sesame. Ses i 3 shares over 40% homology with the 7S pea globulin family found in pistachios, hazelnuts, peanuts, and pecans, which suggests the possibility of cross-allergic reactions between Ses i 3 in sesame and other nuts. Furthermore, Ses i 3 reacts differently to different enzymes; it is easily digested by pepsin but is relatively resistant to trypsin and chymotrypsin. Sesi 3 possesses strong resistance to digestion and heat stability, and has a high potential for clinical cross-allergic reactions. This makes Sesi 3 a high-risk sensitizer, potentially causing more severe allergic reactions compared to other allergenic proteins in sesame seeds.

[0004] IgE-mediated type I hypersensitivity reactions can be divided into three phases: sensitization, challenge, and effector phases. In the sensitization phase, food allergens are processed by antigen-presenting cells (APCs), forming small peptides that bind to major histocompatibility molecules (MHCs), forming peptide-major histocompatibility molecules (pMHCs). These pMHCs, distributed on the cell surface, are recognized by T lymphocytes, inducing their proliferation, promoting their differentiation into Th2 cells, and releasing a series of cytokines that activate B cells to produce specific IgE antibodies. These IgE antibodies are subsequently recognized by FcεRI receptors on the surface of mast cells and basophils, resulting in a sensitized state. The peptides that activate T lymphocytes and promote their differentiation are called T-cell epitopes. Since T-cell epitopes are typically obtained through APC processing, they are usually linear epitopes. During the provocation phase, once the food allergen re-enters the body, it cross-links with IgE antibodies, stimulating mast cell degranulation and releasing various pro-inflammatory factors, including interleukins, histamine, and kininogenase, resulting in allergic symptoms on the skin, in the gastrointestinal tract, in the respiratory tract, and throughout the body. Since B-cell epitopes are polypeptides that directly bind to IgE antibodies, they can be linear or conformational. Research related to epitope identification is mainly divided into T-cell epitope identification and B-cell epitope identification. Currently, there are no detailed reports on the identification of Ses i 3 epitopes using immunological methods. Therefore, clarifying the biological information such as Ses i 3 antigenic epitopes will help to deeply elucidate the sensitization mechanism of Ses i 3 and lay a solid theoretical foundation for the prevention and control of sesame allergens.

[0005] Immunoassay microarray detection employs microsampling to orderly immobilize epitope peptides predicted by bioinformatics software onto a solid-phase support. By scoring the immunoreactivity of each peptide and establishing an immunoscore matrix, it is possible to accurately screen for major B-cell linear epitopes of allergen proteins. This method consumes only microliters of serum and can simultaneously detect thousands of targets, significantly reducing the detection cost per biological sample and facilitating high-throughput epitope screening. Furthermore, the establishment of the immunoscore matrix greatly improves the accuracy of epitope screening, resulting in more reliable data. Summary of the Invention

[0006] To address the shortcomings of existing epitope identification techniques, the present invention aims to synthesize a high-purity recombinant protein, Sesi 3, with certain immunomodulatory activity, and to analyze its structure and homology. The present invention also provides a method for identifying the linear epitope of the sesame allergen protein Sesi 3 on B cells.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] A method for identifying linear epitopes of sesame allergen Ses i 3 on B cells, comprising the following steps:

[0009] (1) Predict the linear epitopes of Ses i 3 on B cells using bioinformatics techniques;

[0010] (2) Synthesize the corresponding antigenic epitope polypeptides by the Fmoc solid-phase method;

[0011] (3) The linear epitopes of Ses i 3, a sesame allergen, were identified by using the slot-blot immune microarray technique combined with the immune score matrix.

[0012] Preferably, the bioinformatics technology in step (1) is selected from one or more of DNAStar software, SOPMA online website and Bcpreds online website.

[0013] Preferably, the bioinformatics technology in step (1) is DNAStar software, SOPMA online website, and Bcpreds online website.

[0014] Preferably, the predicted epitope polypeptide amino acid sequence in step (1) is selected from two or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

[0015] Preferably, the predicted epitope polypeptide amino acid sequence in step (1) is selected from three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

[0016] Preferably, the predicted epitope polypeptide amino acid sequence in step (1) is a combination of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

[0017] Preferably, in step (3), the synthesized predicted epitope polypeptide is immobilized on a nitrocellulose membrane and incubated with the serum of sesame-allergic patients containing IgE antibodies, then incubated with mouse anti-human IgE antibodies labeled with horseradish peroxidase, stained with ECL, photographed, and the gray values ​​are calculated using ImageJ software to establish an immune score matrix based on the scores.

[0018] Preferably, the predicted epitope polypeptide synthesized in step (3) is incubated with the serum of a sesame-allergic patient for 4 hours at a temperature of 37°C.

[0019] Preferably, the predicted epitope polypeptide synthesized in step (3) is incubated with horseradish peroxidase-labeled mouse anti-human IgE antibody for 3 hours at a temperature of 37°C.

[0020] Preferably, the ChemiDoc image scanner and Image Lab™ Touch software are used to take the picture in step (3).

[0021] Preferably, in step (3), peptides that can be recognized by more than half of the serum IgE in the immune score matrix or peptides whose immune scores exceed the sum of the average score and the error are identified as B cell linear epitopes of Ses i 3.

[0022] Preferably, the amino acid sequence of the epitope polypeptide obtained in step (3) is selected from two or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 10.

[0023] Preferably, the amino acid sequences of the epitope polypeptides obtained in step (3) are selected from three or more, four or more, five or more, six or more, or seven or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 10.

[0024] Preferably, the amino acid sequence of the epitope polypeptide obtained in step (3) is a combination of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 10.

[0025] A second aspect of the present invention provides a polypeptide composition for identifying B-cell linear epitopes of the sesame allergen protein Sesi 3, the polypeptide composition being selected from two or more of the amino acid sequences SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 10.

[0026] Preferably, the polypeptide composition is selected from three or more, four or more, five or more, six or more, or seven or more amino acid sequences SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, and SEQ ID NO. 10.

[0027] More preferably, the polypeptide composition is a combination of the amino acid sequences SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 10.

[0028] The beneficial effects of this invention are:

[0029] 1. This invention synthesizes recombinant Sesi3 protein with immunogenicity using prokaryotic expression, and characterizes its molecular weight and immunogenicity by SDS-PAGE and Western blot. The protein structure and homology are analyzed using bioinformatics software.

[0030] 2. This invention can identify the linear epitope of sesame allergen protein Ses i 3 B cells. This method has the characteristics of high specificity, high sensitivity, high accuracy and high throughput. Attached Figure Description

[0031] Figure 1 This is an SDS-PAGE analysis diagram of Ses i 3 protein;

[0032] Figure 2 This is a graph showing the hydrophilicity, surface accessibility, flexibility, and antigenicity of the Ses i 3 protein.

[0033] Figure 3 This is a diagram of the secondary structure analysis of the Ses i 3 protein;

[0034] Figure 4This is a homology modeling diagram of the tertiary structure of the Ses i 3 protein;

[0035] Figure 5 This is a Laplace conformation diagram of the tertiary structure of the Ses i 3 protein;

[0036] Figure 6 These are the results of Ses i 3 protein homology analysis;

[0037] Figure 7 This is the result of Ses i 3 protein slot-blot screening for linear epitopes in B cells. Detailed Implementation

[0038] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0039] Example 1: Prokaryotic expression, identification, and homology analysis of Sesi3 protein

[0040] 1. Prokaryotic expression and identification of sesame allergen protein Sesi 3

[0041] The amino acid sequence of the sesame allergen protein Ses i 3 was obtained from the UniProt server. The Ses i 3 gene was inserted into the pET28a vector via BamH1 and XhoI1 restriction sites and introduced into the Rosetta strain (Rosetta E. coli Strain). Positive replicates were selected, and the cells were cultured at 37°C until the OD600 value reached 0.6-0.8. Then, 1 mmol / L IPTG was added to induce protein expression. The strain was collected by centrifugation, resuspended, and sonicated. The supernatant was purified by Ni column affinity chromatography after high-speed centrifugation to obtain the recombinant protein rSes i 3. Subsequently, the purified rSes i 3 was characterized by SDS-PAGE, confirming its molecular weight to be approximately 60 kDa (e.g., rSes i 3). Figure 1 (as shown in the figure), and its immunomodulatory activity was analyzed using Western blot to ensure that it has IgE binding ability.

[0042] 2. Sesi3 protein structure and homology analysis based on bioinformatics software

[0043] After obtaining the Ses i 3 amino acid sequence from the UniProt protein database, the hydrophobicity, surface accessibility, flexibility, and antigenicity of Ses i 3 were predicted and analyzed using the Protean subroutine in the DNAStar software (e.g., Figure 2 As shown), and predict the secondary structure distribution of Ses i 3 using the online server SOPMA (e.g. Figure 3(As shown). Furthermore, SWISS-MODEL was used for homology modeling and 3D structure prediction of Ses i 3, and the reliability of the 3D modeling was evaluated using Ramachandran conformation diagrams (e.g., Figure 5 (As shown). Finally, the three-dimensional structural model of Ses i 3 was drawn and analyzed using PyMOL software (as shown). Figure 4 (As shown).

[0044] Example 2: Linear epitope screening of Ses i3 B cells based on immune microarray technology

[0045] Three bioinformatics tools, DNAStar, SOPMA, and BCPreds, were used to predict B-cell linear antigenic epitopes of Ses i 3. The amino acid sequence of the sesame allergen Ses i 3 in FASTA format was downloaded from the UNIPROT online website, opened with DNAStar software, and the protean module was selected to obtain the hydrophilicity, antigenic index, surface accessibility, and flexibility of the allergen. The intersection of these parameters was taken as the prediction result for DNAStar. In addition, the amino acid sequence of Ses i 3 was input into the SOPMA and BCPreds websites to obtain the secondary structure and antigenic epitopes. In SOPMA, β-turns and random coils were selected as predicted antigenic epitopes, while BCPreds directly provided the predicted epitope sequences. The prediction results of the three tools are shown in Table 1. Finally, the intersection of the prediction results from the three methods was taken, yielding a total of 10 peptides as the final prediction result (see Table 2).

[0046] Table 1. Prediction results of three bioinformatics tools

[0047]

[0048] Table 2. Prediction results of Ses i 3 B cell linear epitopes

[0049]

[0050] The corresponding antigenic epitope peptides were synthesized using the Fmoc solid-phase method. The IgE binding capacity of the synthesized peptides was identified using slot-blot immunoassay microarray technology, ultimately identifying the Ses i 3 B cell linear epitope. The main steps included: immersing a nitrocellulose membrane (0.22 μm) in ultrapure water to ensure thorough wetting; dissolving the predicted epitope peptide in CBS solution (CBS, 0.015 mol / L Na2CO3, 0.035 mol / L NaHCO3, pH 9.6) to a concentration of 1 mg / mL; then spotting 10 μL onto the air-dried membrane and filtering using a slot-blot vacuum filter. The membrane was then washed three times with PBST buffer (5 minutes each time) to remove unbound sample and blocked with BSA blocking solution at room temperature for 2 hours. After washing again with PBST, serum from 10 patients with sesame allergy was collected (see Table 3 below).

[0051] Table 3. Serum details of patients with sesame allergy

[0052]

[0053] Antibody dilutions (BSA) were prepared at a 1:10 ratio and dropped onto the membrane. The membrane was incubated at 37°C for 4 hours to ensure complete binding of each peptide. After washing, the membrane was further incubated at 37°C for 2 hours with diluted HRP-mouse anti-human IgE antibody (1:1000) as a secondary antibody. Finally, the membrane was washed, stained with ECL staining solution for 1 minute, and analyzed using a ChemiDoc image scanner and Image Lab™ Touch software. The grayscale values ​​of the spots were analyzed using ImageJ software, with the maximum grayscale response value considered as 100%.

[0054] The IgE binding capacity of peptides was divided into four categories and scored according to their gray values: negative strength (score: 0, gray value: 0-10%), weak strength (score: 1, gray value: 10%-40%), moderate strength (score: 2, gray value: 40%-70%), and strong strength (score: 3, gray value: 70%-100%).

[0055] An immune score matrix was constructed based on the scores. Peptides that were identified by the serum of half or more of the allergic patients, or whose scores were higher than the sum of the average score and the scoring error, were identified as major B-cell linear epitopes of Ses i 3 (see Table 4 below).

[0056] Table 4. Ses i 3 B cell linear epitope identification immune score matrix

[0057]

[0058] Although specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. Therefore, the invention is intended to cover all such changes and modifications falling within the scope of the appended claims and their equivalents.

Claims

1. Use of a polypeptide composition for identifying B-cell linear epitopes of the sesame allergen protein Ses i 3, characterized in that, The polypeptide composition is the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO.

10. The polypeptide composition is the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 10.

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