CN114773452A

CN114773452A - IgE binding epitopes of the major allergen alpha-lactalbumin from bovine whey

Info

Publication number: CN114773452A
Application number: CN202210425077.8A
Authority: CN
Inventors: 谭宏凯; 胡巍; 熊子奕; 胡永芯; 邱毓; 黎晶晶; 李欣; 陈红兵; 武涌; 孟轩夷
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2022-07-22

Abstract

The invention relates to an IgE binding epitope of whey allergen alpha-lactalbumin, and the amino acid sequence of the IgE epitope is as follows:¹¹Glu‑Leu‑Lys‑Asp‑Leu‑Lys‑Gly‑Tyr‑Gly‑Gly‑Val‑Ser‑Leu‑Pro‑Glu‑Trp‑Val²⁷. The invention adopts enzyme linked immunosorbent assay (ELISA) to screen 16 parts of serum, and finally obtains 12 parts of cow milk allergy child patient serum. High-purity cow milk specific IgE purified by a Hitrap Protein G HP affinity chromatographic column is used as a target molecule, and affinity panning is carried out on a bacteriophage dodecapeptide library. And predicting linear epitopes by using bioinformatics software, and finally obtaining the IgE epitope information of the whey allergen alpha-lactalbumin. The invention has great significance for exploring immunological mechanism of cow milk allergy, researching the function of the epitope in food allergy, and guiding the development of hypoallergenic dairy products which directionally break the epitope.

Description

IgE binding epitopes of the major allergen alpha-lactalbumin from bovine whey

Technical Field

The invention relates to the fields of molecular biology, immunology and bioinformatics, in particular to an IgE binding epitope of a main allergen alpha-lactalbumin of cow milk whey.

Background

Cow milk and dairy products are popular among consumers due to high nutritional value, but the problem of food safety caused by cow milk allergy is also widely concerned. Cow's milk and milk products are one of eight major allergic foods recognized by WHO/FAO. Cow's Milk Allergy (CMA) is a common disease in the early life, with a prevalence rate of 2-7%, and shows an increasing trend. It is estimated that 2-3% of infants in european countries develop symptoms of cow's milk allergy. In a recent survey of 6768 infants in china, the prevalence of CMA was found to be 2.69%. CMA is classified into immunoglobulin E (IgE) mediated, non-lgE E mediated, and mixed (IgE and non-IgE) mediated. Approximately 60% of CMAs are IgE-mediated, i.e. associated with aberrant humoral immune responses, although slightly different depending on the study population and age. CMA mediated by non-IgE or a mixture of both is relatively rare. The mechanism of IgE-mediated CMA generation is that cow milk allergen sensitizes immune cells of the body and causes them to produce specific IgE (slgE), which is then bound to effector cells to put them in the sensitisation phase; when the organism contacts with the same kind of allergen again, the effector cells are degranulated and release media such as histamine and the like, and finally clinical symptoms such as eczema, dyspnea, abdominal pain, vomiting and the like appear in a skin system, a respiratory system, a gastrointestinal tract system and the like, and even shock and death of a patient can be caused when cow milk allergy is serious.

More than 30 proteins are contained in the milk, and all the milk has potential sensitization. Casein, beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) are currently considered as major allergens. Adjusting the pH of skim milk to 4.6 at 20 deg.C, two fractions are obtained: the casein in coagulated state accounts for 80%, and the whey protein in solution state accounts for 20%. The major allergens in whey protein are beta-lactoglobulin and alpha-lactalbumin, which account for 50% and 25% of the whey protein component, respectively. Alpha-lactalbumin is a monomeric globular calcium-binding metalloprotein of 123 amino acid residues belonging to the lysozyme family, with a molecular weight of 14.4kDa, with four disulfide bonds and a high affinity binding site for calcium. Researchers have found significant homology between lactalbumin in bovine milk and human milk, where 74% of the residues are identical and the other 6% of the residues show chemical similarity.

Epitopes are the material basis of allergic reactions and can be divided into linear epitopes consisting of linear arrangements of amino acid residues and conformational epitopes, which are specific three-dimensional structures formed by spatial proximity of amino acid residues and recognized by immunologically active substances. Epitopes can also be divided into T-cell epitopes and B-cell epitopes according to the receptor of the epitope, and a B-cell epitope refers to an epitope recognized by a B-cell receptor or a specific antibody secreted from a B cell, such as an IgE epitope and an IgG epitope. The linear epitopes of B cells are typically 8-15 amino acids in length. Although linear epitopes have been reported to be as short as 5 amino acids, epitopes capable of binding IgE with high activity contain at least 8 amino acids.

In recent years, techniques such as peptide arrays, phage display, X-ray crystallography, etc. have been applied to the localization of epitopes of food allergens. The phage display technology is an in vitro selection system for screening target polypeptides or proteins displayed on the surface of a phage, can realize the unification of antibody genotypes and phenotypes, and is widely used for epitope positioning due to the characteristics of convenience, strong specificity, convenient operation and the like. Although many epitope mapping studies have been conducted on cow milk allergic proteins including casein, beta-lactoglobulin and alpha-lactalbumin, few reports have been made on epitope mapping of cow milk whey allergen specific to Chinese population.

Disclosure of Invention

The invention aims to obtain an IgE-binding linear epitope of the whey allergen alpha-lactalbumin, is a material basis for cow milk allergy generation, and can be used for researching cow milk allergy mechanism and reducing cow milk allergy.

The technical content of the invention is as follows:

an IgE binding epitope of a main allergen alpha-lactalbumin in cow milk whey, which has an amino acid sequence as follows:¹¹Glu-Leu-Lys-Asp-Leu-Lys-Gly-Tyr-Gly-Gly-Val-Ser-Leu-Pro-Glu-Trp-Val²⁷。

the sera of 16 cow's milk allergic children patients were screened by enzyme-linked immunosorbent assay (ELISA).

And (3) performing affinity panning on the phage dodecapeptide library by using a high-purity cow milk specific antibody purified by a Hitrap Protein G HP affinity chromatography column as a target molecule.

And predicting by bioinformatics, and finally obtaining the epitope information of the alpha-lactalbumin.

The beneficial effects of the invention are: the invention of the IgE binding epitope of the alpha-lactalbumin has great significance for the molecular mechanism of cow milk allergy generation, guidance of protease screening for destroying the epitope and development of hypoallergenic whey protein products based on the epitope.

Drawings

FIG. 1 is the level of specific antibodies in bovine milk allergy patients; an X axis: serum from different allergic patients, left Y axis: OD value for ALA-specific IgE binding level, right Y-axis: specific IgE content by ImmunoCAP assay;

FIG. 2A is a Protein G affinity purification electrophoretogram; b is the ELISA result of the purified antibody; m is a protein Marker, a lane 1 is serum pool protein of a cow milk allergy patient, and a lane 2 is nonspecific elution peak collection protein;

FIG. 3 is the result of indirect ELISA detection of phage clones;

figure 4 is the IgE linear epitope for alpha-lactalbumin;

FIG. 5 dot blot validation of IgE linear epitopes;

detailed description of the preferred embodiment

Construction of serum pool for cow milk allergy patients

1. Indirect ELISA screening of cow milk allergy patient serum

Serum of allergic patients has sIgE level of cow milk protein detected by ImmunoCAP, and serum with sIgE level of cow milk protein more than or equal to 0.35kUA/L and age of 3-12 years is selected for ELISA. The method for measuring the alpha-lactalbumin specific immunoglobulin E (alpha-lactalbumin specific immunoglobulin E, ALA-sIgE) in the serum of a cow milk allergy patient by using an indirect ELISA method (the indirect ELISA method refers to a plum-willow-life paper, and the paper shows that the plum-willow-life is used for researching the linear epitope of the ovomucoid IgE based on the ELISA method [ D ]. Tianjin medical university 2020: 11-16.).

The coating solution is 0.05mol/L carbonate buffer solution with pH of 9.6, and the washing solution, the blocking solution, the serum, the secondary antibody and the like are diluted by 0.01M PBS buffer solution, wherein the washing solution contains 0.05% (v/v) Tween-20, and the blocking solution contains 3% gelatin. The coating concentration of alpha-lactalbumin (Sigma-Aldrich) is 1 mug/mL, serum with the dilution multiple of 200 times is added after blocking, an enzyme label plate is taken out and is dried, washing is carried out for three times, and biotin labeled sheep anti-human IgE (Sigma-Aldrich) with the dilution of 1:5000 is added. Taking out the enzyme label plate, washing for three times after buckling and drying, adding HRP-labeled avidin diluted by 1:60, and preserving heat and moisture for 1h at 37 ℃. The enzyme-labeled plate is taken out, is dried and washed for three times, and is added with TMB substrate solution (Xinbo Sheng Biotechnology Co., Ltd.) with the concentration of 100 mu L/hole, and is kept warm and moisturized for 15min at 37 ℃. 2mol/L H was added₂SO₄The reaction was stopped, 50. mu.L/well. Absorbance at 450nm was measured. The serum is judged to be positive when P/N is more than 2 and P is more than 0.2, wherein P and N are respectively OD of the sample and the negative serum_450nmThe value is obtained.

2. Construction of serum pool

According to the indirect ELISA result, the serum of the patient with positive ALA-sIgE level is screened out, and the equal volume of serum is taken and mixed to obtain a serum pool.

3. Results

3.1 specific IgE level determination of alpha-lactalbumin in cow's milk allergy sufferers

In this study, 25% (4) of patients showed positive IgE reaction to α -lactalbumin, and from fig. 1, it was found that the cow milk protein sIgE results of ImmunoCAP are different from the ELISA results because the former was detected by crude protein and the latter only detected a certain component protein.

3.2 construction of serum pool for cow milk allergy patient

IgE is a target of cow milk allergy generation, ALA-sIgE levels of the sera No. 6, 10, 11 and 16 are low, the sera do not participate in the construction of a serum pool, and the rest 12 parts are mixed to be used as the serum pool.

Secondly, purification of cow milk specific antibody

1. Protein G HP immunochromatographic column one-step affinity purification antibody

The affinity column (GE, USA) was washed with 200. mu.L of activation solution (1M Tris-HCl, pH 9.0) followed by 10 column volumes of equilibration buffer (20mM PBS, pH 7.4) at a flow rate of 1 mL/min. The serum was diluted 1:1(v/v) with the equilibration buffer and passed through a 0.45 μm aqueous filter. After the filtered serum was added to the affinity column, the caps on the top and bottom of the affinity column were tightened and bound by shaking at 37 ℃ for 1 h. And (3) respectively carrying out nonspecific and specific elution on the affinity column by using an equilibrium buffer solution and an eluent (0.1M glycine-HCl, pH 2.7) at the flow rate of 1mL/min, and collecting an effluent liquid at 200 mu L/tube. During the collection of the specific elution peak, a neutralizing solution (1M Tris-HCl, pH 9.0) was added to the collection tube, as a standard, 60 to 200. mu.L of the neutralizing solution was added per ml of the specific elution solution until the pH reached about 7. After completion of the specific elution, the affinity column was washed with 10 column volumes of equilibration buffer and 20% ethanol, respectively. The collected nonspecific eluate and specific eluate were concentrated in an ultrafiltration centrifuge tube and the buffer was changed to 0.01M PBS.

2. Purity and concentration determination of purified antibodies

The purified antibody was identified by reduced polyacrylamide gel electrophoresis (SDS-PAGE) and the concentration was identified by BCA kit (Shanghai Biyuntian Biotechnology Co., Ltd.). The obtained antibody was stored in a refrigerator at-20 ℃.

3. Specificity evaluation of purified antibodies

IgE level determination of purified antibodies was performed using indirect ELISA.

4. Results

4.1 purity and concentration of purified antibody

As can be seen from FIG. 2A, the untreated serum contained more hetero-proteins (lane 1). Lane 2 is a non-specific eluate with a band around 72kDa and no band at 50kDa, indicating that IgG has been separated. The purity of lane 2 was 90% as calculated by Image J software. The BCA assay showed that the concentration of non-specific eluent was 3000. mu.g/mL.

4.2 specificity results of purified antibodies

As can be seen from FIG. 2B, the non-specific eluate, ALA-sIgE, was collected for epitope screening.

Screening of IgE epitopes of tri-and alpha-lactalbumin

1. Screening of mimotopes by phage display technology

Panning of IgE mimotopes was performed using phage dodecapeptide library kit (New England Biolabs). The concentrations of the coated IgE are 600 mug/mL, 450 mug/mL, 300 mug/mL and 300 mug/mL from the first round to the fourth round, the concentrations of the washing liquid are 0.1%, 0.25%, 0.5% and 0.5% respectively, the incubation and binding time of the phage is 60min, 45min, 30min and 30min respectively, and the elution time of the glycine is 6min, 8min, 10min and 10min respectively. Progressively more stringent panning conditions help to select for more specific phage sequences. From the fourth round of titer plate, 10 phage were picked and sent to Jinzhi corporation for sequencing, with-96 III as the sequencing primer.

2. Indirect ELISA for identifying positive phage clones

Binding of the selected polypeptide to the antibody was detected by phage ELISA. The coating protein is 2 mu g/mL of anti-milk protein IgE, 100 mu L/well, coated on a 96-well microplate, incubated overnight at 4 ℃, and simultaneously coated with 3% BSA-PBS as a negative control. The wells were decanted and washed three times with PBST containing 0.05% Tween-20. Blocked with 3% gelatin, 300. mu.L/well, incubated for 1h at 37 ℃. The plate was washed three times, amplified and purified phage was added, 100. mu.L/well, and incubated at 37 ℃ for 1 h. The number of phage added was in four sets of gradients, 10 each¹²、10¹⁰、10⁸、10⁶The amount of phage added in the negative control is10⁶And adding PBS buffer to the blank control. The plate was washed six times, and then a secondary HRP-labeled anti-M13 antibody (Beijing Yi Qiao Shenzhou science Co., Ltd.) was added thereto at 100. mu.L/well, followed by incubation at 37 ℃ for 1 hour. Washing the plate for six times, adding TMB color development solution, 100 μ L/hole, and developing in dark at 37 deg.C for 15 min. 2M H is added₂SO₄The reaction was stopped, 50. mu.L/well and OD was determined_450nmThe value is obtained. When P/N is more than 2 and P is more than 0.2, the bacteriophage is judged to be positive, wherein P and N are respectively OD of the sample and the negative control_450nmThe value is obtained.

3. Bioinformatics localization of IgE epitopes

An alpha-lactalbumin amino acid sequence is obtained from an NCBI database (www.ncbi.nlm.nih.gov) and consists of 123 amino acids, the inserted phage exogenous gene sequence is translated by using an Editseq software module of DNAStar, and linear epitope positioning is carried out by using DNAman 7.0 software.

4. Dot blot method for validating IgE epitopes

IgE epitope sequences are synthesized and bonded to a PVDF membrane, and the binding capacity of the synthesized peptide fragments and antibody IgE is detected by dot blot. The polypeptide was spotted at a concentration of 1mg/mL on a nitrocellulose membrane fixed in a spotter at 5. mu.L/well with 3% BSA-PBS as a negative control, and dried in an incubator at 37 ℃ for 30 min. After the sample application hole is dried, 3 percent gelatin is dripped, and the sample application hole is sealed in an oven at 37 ℃ for 1 hour. The blocking solution was decanted and the membranes were washed three times for 5min each with PBST containing 0.05% Tween-20. Cow's milk allergy patient serum (10 pooled serum pool) diluted 20-fold with PBS was added at 50. mu.L/well and incubated at 4 ℃ for 12 h. The next day, serum was recovered and membranes were washed three times, biotin-labeled goat anti-human IgE secondary antibody was added at 50. mu.L/well, and incubated at 37 ℃ for 1 h. The membrane was washed 5 times for 3min each, and HRP-labeled avidin was added at 50. mu.L/well and incubated at 37 ℃ for 1 h. The membrane was washed 5 times for 3min each time.

5. As a result, the

5.1 sequencing of phage by affinity panning

Table 1 shows the enrichment effect of phages during panning, and it can be found that the yield and recovery rate of phages are increased with each panning. In the fourth round, of bacteriophagesThe yield reaches 1.2 multiplied by 10 respectively⁵It was shown that specifically bound phage were effectively enriched in this process. In the fourth round of plates, 10 plaques were picked from the IgE group, designated IgE1 to IgE10, sent to jingzhi corporation for sequencing and 10 12 peptide sequences were obtained. Table 2 shows the phage exogenous sequences.

TABLE 1 enrichment of phages in four rounds of IgE affinity panning in children

Note: recovery rate (recovery amount/input amount of library)

TABLE 2 mimotopes screened by phage display technology

5.2 phage ELISA results

The binding capacity of the selected polypeptide to the antibody was tested by indirect ELISA. It was found that as the number of phage added decreased, the absorbance also exhibited a tendency to decrease. FIG. 3 shows that the phage was added at 10¹²OD of (1)_450nmThe value is obtained. The OD of the remaining clones, except IgE5, was found_450nmAll values were 2-fold higher than the negative control group (Con group), so 9 clones were judged as positive clones.

5.3 IgE epitope mapping of alpha-lactalbumin

The foreign sequence of the phage and the amino acid sequence of the alpha-lactalbumin are compared by using the multi-sequence comparison function of software DNAman 7.0, default parameters are unchanged, the recognition site and the occurrence frequency of the peptide segment are analyzed, and the peptide sequence containing more than three amino acids which are continuously overlapped and more than five amino acids which are not continuously overlapped are defined as a linear epitope. Fig. 4 shows the linear epitope of a-lactalbumin. We mapped the IgE linear epitope of alpha-lactalbumin to AA 11-27.

5.4IgE Linear epitope AA11-27 results validation

The dot blot results are shown in FIG. 5. Wherein, the A picture is the result of dot blot, P is the result of measuring the binding capacity of IgE and the peptide fragment AA11-27 in the serum of an allergic patient, N is the result of measuring the binding capacity of negative serum IgE and the peptide fragment AA11-27, and the picture shows that the binding capacity of the peptide fragment and the serum of the allergic patient is obvious and is not bound with the negative serum; panel B is a quantitative comparison by Image J (V1.38) analysis, plotted using GraphPad Prism 8.2.1: in the abscissa, P is serum of allergic patients, N is negative serum, and in the ordinate, the binding capacity of the peptide fragment AA11-27 to IgE is shown. The blotting result proves that the peptide fragment AA11-27 has stronger binding capacity with the IgE in the serum of the allergic patient, which indicates that the peptide fragment is the linear epitope of the alpha-lactalbumin.

Sequence listing

<110> Tanzhongkai

<120> IgE binding epitopes of the main allergen alpha-lactalbumin from cow's milk whey

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> cattle (Bos gaurus)

<400> 1

Glu Leu Lys Asp Leu Lys Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp

1 5 10 15

Val

Claims

1. An IgE binding epitope of a main allergen alpha-lactalbumin of milk whey, which is characterized in that: the amino acid sequence is SEQ ID No: 1:¹¹Glu-Leu-Lys-Asp-Leu-Lys-Gly-Tyr-Gly-Gly-Val-Ser-Leu-Pro-Glu-Trp-Val²⁷。