CN101865925A - Method for determining key amino acid of milk allergen epitope - Google Patents
Method for determining key amino acid of milk allergen epitope Download PDFInfo
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Abstract
The invention discloses a method for determining key amino acid of milk allergen epitope, which comprises the following steps of: synthesizing an epitope peptide section according to the primarily determined mimic epitopes area amino acid sequence, and fixing and protecting the epitope peptide section, wherein the C end covalence of the epitope peptide section is combined to an activated fibrous membrane and the N end is acetylized; taking single glycine as a negative control, reacting the synthesized amino acid sequence with polyclonal serum, and determining the main epitope of beta-milk globulin; sequentially substituting each amino acid with alanine or glycine to obtain the amino acid sequence needing to be synthesized; using the obtained amino acid sequence to synthesize the epitope peptide section, and fixing and protecting the epitope peptide section, wherein the C end covalence of the epitope peptide section is combined to the activated fibrous membrane and the N end is acetylized; and taking the single glycine as the negative control, reacting the synthesized amino acid sequence with the polyclonal serum, and determining the key amino acid in milk epitope. The method lays a foundation for the diagnosis and treatment of milk allergy and the detection of an allergen in foods and provides theoretical basis and technical support for the development of hypoallergenic milk.
Description
Technical field
The invention belongs to biological technical field, particularly the accurate location of epi-position.
Background technology
Cow's milk allergy is the reaction of a kind of common food hypersenstivity, the adult of 1-2% and can produce the food hypersenstivity reaction to cow's milk up to 8% children below three years old.Nearly contain in the cow's milk that 20 kinds of protein all have potential sensitization, it is reported that the irritated crowd of cow's milk generally shows as common allergy to a plurality of albumen to the allergy of cow's milk.Beta lactoglobulin, ALA, casein are considered to main anaphylactogen, other albumen such as bovine serum albumin(BSA), immunoglobulin (Ig), lactoferrin also play important effect in allergic reaction, about 30%～50% cow's milk allergy sufferers is arranged to these trace of albumin allergy, and wherein some patient only to these trace of albumin allergy.
Beta lactoglobulin accounts for 10% of milk total protein, 50% of lactalbumin.Beta lactoglobulin is one of main newborn anaphylactogen of generally acknowledging, about 82% cow's milk autopath is to beta lactoglobulin allergy.Do not have beta lactoglobulin in the breast milk, this also is that it becomes one of reason of main anaphylactogen.Simultaneously, beta lactoglobulin belongs to the lipocalin protein family, and lipocalin albumen has identical 8 or 10 beta-barrel structures that antiparallel β-tabs is formed by repeated arrangement.Because its complicated structure, numerous protein has strong sensitization in the lipocalin protein family, and some animal derived anaphylactogens all belong to this albuminoid, as the main anaphylactogen Equ c 1 of horse, main mouse albumen etc.Acidproof and the resistant protease hydrolysis of beta lactoglobulin.Therefore, also exist complete beta lactoglobulin and peptide section thereof in the human body of digestion back, may cause allergic reaction.
The existence form of beta lactoglobulin is relevant with the pH value, in fresh cow milk (pH6.6～6.8), it is to exist with dimeric form, when pH is lower than 3.5, dimer is dissociated into monomer, and pH is between 3.5～5.2 the time, and dimer four dimerizations form eight aggressiveness, pH is higher than at 7.5 o'clock, and dimer dissociates and conformation changes forms the monomer that expands.Beta lactoglobulin generally is a dimer, and its each monomer is 18kDa, contains 162 amino acid, 5 disulfide bond, and wherein 4 is in the chain, 1 is interchain.
" Primary Study of beta lactoglobulin allergen epitope location in the buffalo milk " (" Food Science " 2007, vo1.28, No.10, the Primary Study result of beta lactoglobulin allergen epitope location in the buffalo milk is disclosed P360-363), promptly utilize the specificity rabbit anteserum of immunoaffinity purification that affine elutriation is carried out in phage random seven peptide storehouses, and, determine the epi-position district of beta lactoglobulin anaphylactogen, but can not determine key amino acid wherein by network tool MIMOX analysis.
Summary of the invention
The objective of the invention is on the basis of determining the allergen epitope district, add to the theoretical prediction of epi-position and by the method that display technique of bacteriophage and pepscan combine beta lactoglobulin in the milk is carried out correct and comprehensive epitope mapping and further definite key amino acid wherein.
The present invention is achieved by the following technical solutions.
1, according to the preliminary mimic epitope region amino acid sequence of determining, synthetic epitope peptide section, and fixing and protection, the C end of section of synthesized peptide is covalently bound on the tunica fibrosa of activation, and the N end is acetylation;
2, with single glycocoll as negative control, with synthetic amino acid sequence and polyclonal serum reaction,, determine the main epi-position of beta lactoglobulin according to the intensity of reacting;
3, each amino acid in the main epi-position of beta lactoglobulin is replaced by alanine or glycocoll successively, needing to obtain synthetic amino acid sequence;
4, the amino acid sequence that obtains by step 3, synthetic again epitope peptide section, and fixing and protection, the C end of section of synthesized peptide is covalently bound on the tunica fibrosa of activation, and the N end is acetylation;
5, with single glycocoll as negative control, with synthetic amino acid sequence and polyclonal serum reaction,, determine the key amino acid in the cow's milk epi-position according to the intensity of reacting.
The present invention is based on the result of bioinformatics prediction B cell epitope, adopting corresponding polyclonal antibody is target molecule, accurately locate the linear epitope of beta lactoglobulin by display technique of bacteriophage and pepscan, can be the material base of illustrating beta lactoglobulin allergy, the detection of carrying out anaphylactogen in breast irritated diagnosis, treatment and the food lays the foundation, for exploitation hypoallergenic cow's milk provides theoretical foundation and technical support, this method also provides new approach for the accurate location of other food allergen epi-position simultaneously.
Description of drawings
Fig. 1 is DANStar software multi-parameter prediction result.
Fig. 2 is water wettability, alpha-helix, the predicting the outcome of β-corner and beta sheet.
Fig. 3 is the interface of MIMOX sequence alignment of the present invention.
Fig. 4 is the beta lactoglobulin site of amino acid correspondence in 112 mimic epitopes of the present invention.
Fig. 5 is the pepscan result.
Fig. 6 discerns the intensity of different peptide sections for the 1# rabbit anteserum.
Fig. 7 discerns the intensity of different peptide sections for the 3# rabbit anteserum.
Fig. 8 discerns the intensity of different peptide sections for the 4# rabbit anteserum.
Fig. 9 determines key amino acid for the amino acid replacement method.
Embodiment
The present invention will be described further by following examples.
Embodiment.
The present embodiment selected materials: purity reaches 90% above buffalo's milk beta lactoglobulin, corresponding polyclonal antibody, and titre reaches 1: 40 more than ten thousand.
Used buffalo's milk beta lactoglobulin be prepared as following steps:
(1.2cm * 30cm) separate uses earlier pH6.8, the initial damping fluid counterion of 0.02mol/L phosphate exchange column with DEAE-Sepharose Fast Flow anion exchange; Get the whey after the suitable dilution ultrafiltration, last sample; PH6.8, the protein that 0.02mol/L phosphate balance buffer solution elution is not adsorbed, about 200mL; Contain the protein that the continuous wash-out of 0～0.5mol/L NaCl phosphate buffer is adsorbed, elution speed is 1.5mL/min; Collect the target eluting peak, (1.2cm * 75cm) further separate: the dress post outgases the post material with vacuum pump with SephadexG-75 gel chromatography column on the ion-exchange target product behind molecular weight 5kDa Amicon Ultra-15 ultra-filtration centrifuge tube ultrafiltration and concentration to the 1/50 volume ultrafiltration and concentration that dams; 0.02mol/L the phosphate buffer balanced gel chromatographic column of pH6.8; After the balance, last sample; 0.02mol/L the phosphate buffer wash-out of pH6.8, elution speed: 8mL/h.Collect target peak, ultrafiltration and concentration to 1/50 volume.The target protein of the purifying that obtains adopts SDS-PAGE, IEF-PAGE and ESI-Q-TOF-MS to identify, its purity is up to more than 90%, and be applicable to the laboratory in a small amount and middle amount prepare.
Cow's milk beta lactoglobulin with purifying is an antigen, the initial immunity Freund's complete adjuvant; The booster immunization incomplete Freund; Immunization route carries out every rabbit 1mg/mL per two all immunity once in subcutaneous multi-point injection mode, and immunity is 4 times altogether.Adopt the square formation titration to determine best bag by concentration, indirect ELISA is followed the tracks of antibody titer and is reached l: more than 400,000; Suppress ELISA and immunoblot experiment by indirect ELISA, competition again, the antigenicity of the cow's milk beta lactoglobulin of proof separation and purification is intact, there are stronger immunological cross-reaction in buffalo's milk beta lactoglobulin and cow's milk beta lactoglobulin, and its crossing-over rate reaches 69.27%.
2, method
2.1 multi-parameter prediction buffalo's milk beta lactoglobulin antigen linear epitope result
Utilize DNAStar software that buffalo's milk beta lactoglobulin amino acid sequence is carried out the theoretical prediction of epi-position correlation parameter, as shown in Figure 1, may constitute the position such as the table 1 of antigenic region.
The epi-position district of table 1DANStar software prediction
By the webserver, we predict to the antigenic region of cow's milk beta lactoglobulin that once more Fig. 2 and table 2 have shown the predictive analysis results of water wettability, alpha-helix, β-corner and the beta sheet of buffalo's milk beta lactoglobulin.
Table 2 water wettability, alpha-helix, the possible site of the existence of β-corner and beta sheet
*Pass through Kyto﹠amp; The result that Dolittle analyzes,
*Result by the Welling analysis.
Comprehensive above forecast analysis, we have obtained the possible epitope district of buffalo's milk beta lactoglobulin: AA30-45, AA67-78, AA97-115, AA124-141, AA150-156.
2.2 phage random peptide library location
2.2.1 phage random seven peptide storehouses screening
The polyclonal antibody IgG of purifying is cushioned liquid (0.1mol/LNaHCO with bag
3, pH 8.6) and be diluted to 10 μ g/mL, every hole 100 μ L bag is by 96 hole ELISA Plate, and 4 ℃ slightly shake overnight incubation.Discard coating buffer, the liquid (0.1mol/LNaHCO that blockades is filled it up with in every hole
3, 5mg/mLBSA, 0.02% NaN
3), the 4C 2h that blockades; (50mmol/L Tris-HCl, 150mmol/L NaCl 0.1%Tween-20) wash plate 6 times fast, and the original peptide of the bacteriophage storehouse that every hole adds 100 μ L (contains 2 * 10 with TBST
11Individual bacteriophage), room temperature jog concussion 60min; Discard not in conjunction with phagocytosis body fluid, wash plate 10 times with TBST, every hole adds glycocoll eluent (0.2mol/L, pH 2.2Glycine-HCl, 1mg/mLBSA) 100 μ L, room temperature jog 10min, the bacteriophage of wash-out specificity combination, collect eluent in microcentrifugal tube, add neutralization buffer (Tris-HCl, pH 9.1); Stay 5 μ L to be used for titer determination, the residue eluate increases, and is used for the next round screening.Same method is carried out the 2nd, 3,4 and is taken turns screening, and it is 0.25%, 0.5%, 0.5% that the concentration of eluent TBST increases progressively successively.
2.2.2 the mensuration of phage titre
With the LB nutrient culture media eluent is carried out doubling dilution, get respectively in the ER2738 bacterium liquid that each dilution bacteriophage joins the exponential phase for preparing, join mixing in the top-layer agar behind the room temperature incubation, evenly be laid on the plate overnight incubation.Locus coeruleus number on the numeration plate.Locus coeruleus number * extension rate in phage titre=plate, the bacteriophage of promptly per 10 μ L forms unit.
2.2.3 select clone at random
Take turns 30 single bacterium colonies of picking from the 3rd and 4 at random and be added to the ER2738 overnight culture on the flat board less than 100 clone's, be inoculated in the LB nutrient culture media by dilution in 1: 100, after 37 ℃ of shaking tables were cultivated 4.5-5hrs, culture changed in the microcentrifugal tube, centrifugal 30 seconds.Supernatant changes in the new pipe, and is centrifugal again.Change 80% supernatant over to fresh centrifuge tube with pipettor, this is amplification bacteriophage storage liquid, gets 500 μ L and extracts single DNA with NaI, and all the other are as amplification and ELISA.
2.2.4 amplification
Nutrient culture media with 20mL increases to each clone's, step such as the amplification of peptide storehouse.
2.2.5 indirect ELISA is identified positive colony
Be cushioned liquid dilution antibody (100ug/mL) with bag, every hole 100 μ L bag is by 96 hole ELISA Plate, and 4 ℃ are spent the night in the wet box of sealing.Abandon coating buffer, the liquid of blockading is filled it up with in every hole, 4 ℃ of 2h that blockade.0.5%TBST washes plate 3 times, and every hole adds the supernatant of clone's, 37 ℃ of 2h, wash plate three times, add the anti-M13 antibody 100 μ L/ holes of the HRP mark of dilution in 1: 5000, incubated at room 1h, add 100 μ L/ hole OPD substrate colour developing liquid, room temperature effect 10～20min colour developing, 2mol/LH after washing plate
2SO
4Cessation reaction, 490nm place reading.
2.2.6 software MIMOX compares mimic epitope
Buffalo's milk beta lactoglobulin amino acid sequence is divided into three section: B1, B2 and B3.The simulating peptide that serum screening by the different experiments rabbit arrives respectively with buffalo's milk beta lactoglobulin amino acid sequence B1, B2 and B3 comparison, form below adopting, compare in the workspace that is input to Fig. 3, to determine that simulating peptide is corresponding to the site in the buffalo's milk beta lactoglobulin amino acid sequence, i.e. mimic epitope.
>mimoseqB1
IIVTQTMKGLDIQKVAGTWHSLAMAASDISLLDAQSAPLRVYVEELKPTPEGNL
>mimoseqB2
EILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCME
>mimoseqB3
NSAEPEQSLACQCLVRTPEVDNEALEKFDKALKALPMHIRLAFNPTQLEGQCHV
2.3 pepscan epitope mapping
2.3.1 the peptide film is synthetic
The peptide that this experiment will be analyzed is synthesized by Sigma company and is fixed on the tunica fibrosa.The C end of section of synthesized peptide is covalently bound on the tunica fibrosa of activation, and the N end is acetylation, and both can prevent to be degraded, and also can keep natural irritated conformation, the negative contrast of single glycocoll.
2.3.2 the detection of peptide film
1) the methanol rinse film 5min of usefulness a little volume;
2) be total to 10min three times with an amount of TBS wash-out, the volume of wash-out depends on the size of film and pipe, and film need be capped fully;
3) with blockading at least 2 hours under the equal volume blocking agent room temperature;
4) with 0.1-10 μ g/mL (liquid of the blockading dilution) incubation of equal volume 3 hours;
5) wash 10min again with T-TBS;
6) educated 2 hours with two temperature resistances of appropriate H RP mark, with the liquid dilution 1: 10000 of blockading;
7) T-TBS with equal volume washes three common 5min of film;
8) the developer at least 100 μ l/cm of Jian Ceing
2Be added on the film;
9) repeatedly wash film or shake container lentamente with the tip head;
10) film is inverted development;
11) expose 60 seconds, develop photographic fixing;
12) utilize software I magePro software quantitatively to determine the OD value.
2.3.3 the regenerative process of peptide film
1) washes film with water and be total to 10min three times;
2) wash film with regeneration damping fluid I and be total to 30min at least 4 times, temperature is 50 degree;
3) wash 20min altogether at least 3 times with 10 * PBS under the room temperature;
4) wash 20min altogether 3 times with T-TBS under the room temperature;
5) wash 10min altogether at least 3 times with TBS under the room temperature;
6) with the antibody test of mark reproductive success whether, if unsuccessful just with regeneration step 2.
3 results
3.1 the dna sequencing result of phage random peptide library mimic epitope
With 1#, 2#, the IgG of the affinity purification that 3# and 4# rabbit anteserum obtain is a target molecule, has selected 300 clone's, identifies 112 positive colonies, sequencing result is shown in table 3-6.
The sequencing result of table 31# rabbit positive colony
The sequencing result of table 42# rabbit positive colony
The sequencing result of table 53# rabbit positive colony
The sequencing result of table 64# rabbit positive colony
3.2 the location of mimic epitope
Adopt first function among the MIMOX that amino acid sequence and buffalo's milk beta lactoglobulin amino acid sequence that order-checking obtains are compared, the simulating peptide of identification more than three is as the mimic epitope of buffalo's milk beta lactoglobulin.Fig. 4 has added up the amino acid sites of the buffalo's milk beta lactoglobulin of simulating peptide identification.The site of the identification of resulting peptide section and the frequency of appearance are analyzed, and the epi-position district that orients the buffalo's milk beta lactoglobulin is: AA14-20, AA26-32, AA36-42, AA70-80, AA82-86 and AA149-156.The simulating peptide that different rabbits is obtained is carried out epitope mapping, result such as table 7 respectively simultaneously.
The mimic epitope peptide section district of the different immune rabbit identifications of table 7
3.3 synthetic peptide positioning result
3.3.1 the testing conditions of peptide film
The peptide film with 37 ℃ of incubation 1h of 3% gelatin after, film is immersed among the 4ug/mL one anti-solution 15mL, room temperature shaking table gentleness is shaken 2h; T-TBS washes four times, and 10min/ time, add two anti-(1: 10000) room temperature incubation 1h, wash four times with T-TBS, 15min/ time, ECL soaks into film, drips repeatedly to make film fully contact about 2min with substrate, dark exposure 10-30 second is placed in the developer 30 seconds then, soaks 20 seconds with fixer again.
3.3.2 pepscan positioning result
The original membrane of synthetic peptide film is shown in Fig. 5 A, and negative serum compares (Fig. 5 B).The figure as a result of 1#-4# rabbit anteserum identification polypeptide section is 5C-F.By ImagePro software with 1#, intensity level such as Fig. 6-8 of the reaction of 3# and 4# rabbit anteserum identification polypeptide section.Wherein the specific recognition of rabbit 2 is very strong, only discerns a peptide section, this point is not quantized here and the comparison of mapping.
According to the immunoreactive result of above four specific serums and synthetic peptide, the peptide section that experimental rabbit serum is discerned respectively is as shown in table 8, is 50% when above according to discrimination, is defined as main epi-position.The result shows that seven main identification epi-position: A6 have been determined in this experiment, A7, and A8, B5, C, F4, F8, its amino acid sequence is as shown in table 9.A7 wherein, A8, F4, F8 can be thought topmost epi-position by three rabbits identifications.
The peptide section of the different immune rabbit identifications of table 8
The peptide section of the different immune rabbit identifications of table 9
3.3.2 the pepscan locator key is by result of calculation (seeing accompanying drawing 9)
To epi-position A6, A7, A8, B5, C, F4, F8 carry out the single amino acids of alanine (glycocoll) and replace, and have determined the key amino acid in the epi-position, see Table 10.
Key amino acid * in the table 10 buffalo's milk beta lactoglobulin epi-position
* runic and underscore mark is key amino acid
Claims (1)
1. the method for a definite key amino acid of milk allergen epitope, utilize the specificity rabbit anteserum of immunoaffinity purification that affine elutriation is carried out in phage random seven peptide storehouses, and, determine the epi-position district of beta lactoglobulin anaphylactogen by network tool MIMOX analysis, it is characterized in that:
(1) according to the preliminary mimic epitope region amino acid sequence of determining, synthetic epitope peptide section, and fixing and protection, the C end of section of synthesized peptide is covalently bound on the tunica fibrosa of activation, and the N end is acetylation;
(2) with single glycocoll as negative control, with synthetic amino acid sequence and polyclonal serum reaction,, determine the main epi-position of beta lactoglobulin according to the intensity of reacting;
(3) each amino acid in the main epi-position of beta lactoglobulin is replaced by alanine or glycocoll successively, needing to obtain synthetic amino acid sequence;
(4) amino acid sequence that obtains by step 3, synthetic again epitope peptide section, and fixing and protection, the C end of section of synthesized peptide is covalently bound on the tunica fibrosa of activation, and the N end is acetylation;
(5) with single glycocoll as negative control, with synthetic amino acid sequence and polyclonal serum reaction,, determine the key amino acid in the cow's milk epi-position according to the intensity of reacting.
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| CN102331497A (en) * | 2011-06-15 | 2012-01-25 | 天津医科大学 | Kit for detecting milk allergen and preparation method thereof |
| CN103675284A (en) * | 2013-12-03 | 2014-03-26 | 南昌大学 | Method for assessing food allergenicity by using monoclonal antibodies to allergen epitopes |
| CN106290893A (en) * | 2016-07-18 | 2017-01-04 | 南昌大学 | A kind of based on Platinum Nanoparticles probe in detecting Lac Bovis seu Bubali beta lactoglobulin and the method for sensitization residue thereof |
| CN111089910A (en) * | 2018-10-24 | 2020-05-01 | 中国农业大学 | Method for assisting in identifying linear epitope of plant food allergen |
| CN112986578A (en) * | 2021-02-04 | 2021-06-18 | 南昌大学 | Test strip for screening serum specific Ig E and preparation method thereof |
| CN113257355A (en) * | 2021-05-17 | 2021-08-13 | 北京工商大学 | Method for determining cross-allergen-acting surface sites related to eggs |
| CN113278058A (en) * | 2021-05-24 | 2021-08-20 | 北京工商大学 | Cow milk beta-lactoglobulin T cell immune tolerance hydrolysate and preparation method and application thereof |
| CN114751974A (en) * | 2022-04-21 | 2022-07-15 | 胡巍 | IgG binding epitopes of the major allergen alpha-lactalbumin from bovine milk whey |
| CN114874310A (en) * | 2022-06-15 | 2022-08-09 | 澳优乳业（中国）有限公司 | IgG epitope peptide of whey allergen beta-lactoglobulin |
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Non-Patent Citations (2)
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| 李欣: "水牛乳中β-乳球蛋白的IgG结合表位的定位研究", 《中国博士学位论文全文数据库》 * |
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| CN102331497A (en) * | 2011-06-15 | 2012-01-25 | 天津医科大学 | Kit for detecting milk allergen and preparation method thereof |
| CN103675284A (en) * | 2013-12-03 | 2014-03-26 | 南昌大学 | Method for assessing food allergenicity by using monoclonal antibodies to allergen epitopes |
| CN103675284B (en) * | 2013-12-03 | 2015-07-01 | 南昌大学 | Method for assessing food allergenicity by using monoclonal antibodies to allergen epitopes |
| CN106290893A (en) * | 2016-07-18 | 2017-01-04 | 南昌大学 | A kind of based on Platinum Nanoparticles probe in detecting Lac Bovis seu Bubali beta lactoglobulin and the method for sensitization residue thereof |
| CN111089910A (en) * | 2018-10-24 | 2020-05-01 | 中国农业大学 | Method for assisting in identifying linear epitope of plant food allergen |
| CN112986578B (en) * | 2021-02-04 | 2023-03-10 | 南昌大学 | Test strip for screening serum specific Ig E and preparation method thereof |
| CN112986578A (en) * | 2021-02-04 | 2021-06-18 | 南昌大学 | Test strip for screening serum specific Ig E and preparation method thereof |
| CN113257355A (en) * | 2021-05-17 | 2021-08-13 | 北京工商大学 | Method for determining cross-allergen-acting surface sites related to eggs |
| CN113257355B (en) * | 2021-05-17 | 2023-08-11 | 北京工商大学 | Method for determining cross allergen acting epitope related to eggs |
| CN113278058A (en) * | 2021-05-24 | 2021-08-20 | 北京工商大学 | Cow milk beta-lactoglobulin T cell immune tolerance hydrolysate and preparation method and application thereof |
| CN114751974A (en) * | 2022-04-21 | 2022-07-15 | 胡巍 | IgG binding epitopes of the major allergen alpha-lactalbumin from bovine milk whey |
| CN114874310A (en) * | 2022-06-15 | 2022-08-09 | 澳优乳业（中国）有限公司 | IgG epitope peptide of whey allergen beta-lactoglobulin |
| CN114874310B (en) * | 2022-06-15 | 2024-03-26 | 澳优乳业（中国）有限公司 | IgG epitope peptide of whey allergen beta-lactoglobulin |
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