CN114835785A - Heat treatment and ultrahigh pressure combined heat processing method for destroying glycinin G5 subunit A3 peptide chain epitope and positioning method - Google Patents

Heat treatment and ultrahigh pressure combined heat processing method for destroying glycinin G5 subunit A3 peptide chain epitope and positioning method Download PDF

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CN114835785A
CN114835785A CN202210374978.9A CN202210374978A CN114835785A CN 114835785 A CN114835785 A CN 114835785A CN 202210374978 A CN202210374978 A CN 202210374978A CN 114835785 A CN114835785 A CN 114835785A
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席俊
王一超
陈慧彬
段宇莹
李英英
付杨
尚阿晨
吴枭
孙富宇
范雨函
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Abstract

The invention relates to a method for destroying the amino acid sequence of an antigenic region of a glycinin G5 subunit A3 peptide chain by combining heat treatment and ultrahigh pressure heat processing, wherein the amino acid sequence of the antigenic region is shown as SEQ ID NO. 19. The invention utilizes a series of bioinformatics software and refers to the three-dimensional crystal structure of the glycinin analyzed by a PDB database, researches and processes the antigen region of the peptide chain protein of the subunit A3 of the glycinin G5 by using a phage display technology, displays the protein of the antigen region on the surface of a phage, and precisely locates the region of the peptide chain protein of the glycinin A3, which is reduced by a heat treatment and ultrahigh pressure combined heat processing method, by three rounds of phage display of the peptide chain A3. The research provides a theoretical basis for screening of soybean-related food processing methods and provides a technical support for evaluation of soybean allergen desensitization effect in the processing process.

Description

Heat treatment and ultrahigh pressure combined heat processing method for destroying glycinin G5 subunit A3 peptide chain epitope and positioning method
Technical Field
The invention relates to the fields of food processing, molecular biology, immunology and bioinformatics, in particular to a method for destroying epitope of a glycinin G5 subunit A3 peptide chain and positioning by heat treatment and ultrahigh pressure combined heat processing.
Background
Food allergy is an adverse reaction of the immune system to food proteins, and nearly 8% of children and 5% of adults in developed countries are reported to be affected by food allergy and also have a tendency to increase year by year. China has no national standard epidemiological investigation on food allergy, but the questionnaire survey mode on 337560 children aged 0-14 in 31 cities in China shows that the total food allergy parent report rate is 5.83%. Allergy not only induces allergic diseases of other systems, but also causes anaphylactic shock and even death in severe cases, and is a general problem affecting the eating habits and physical health of people, and the generated direct or indirect cost has serious influence on the individual and family of allergic patients, the sanitation system and the whole country.
Soybean is one of the eight major food allergens identified by the Food and Agriculture Organization (FAO) of the united nations, and the incidence of soybean allergy in children is reported to approach 0.4%, and as the consumption of soybeans and soybean products increases, the proportion of adults with soybean allergy is found to be increasing. Glycinin is the most abundant allergenic protein in soybean, accounts for about 40% of the total protein in seeds, has a molecular weight of 300-380kDa, and consists of five different subunits, each of which is linked by a disulfide bond between an acidic chain A (molecular weight of 35-43kDa) and a basic chain B (molecular weight of 20kDa), and each of which has allergenicity in G1, G2, G3, G4, G5, wherein the allergenicity of the acidic chain is greater than that of the basic chain. The structure-activity relationship between the molecular structure change and the allergic property of the processed glycinin is analyzed, the molecular mechanism of the reduced allergenicity of the glycinin caused by processing is clarified, and a theoretical basis is provided for the screening of the processing method in the food industry.
The epitope is the basis of protein antigenicity, the allergenicity of soybean can be reduced by adopting a method for positioning and destroying the epitope currently, a commonly used epitope prediction technology and an epitope positioning technology are analyzed by adopting informatics software, wherein a T7 phage display system can express proteins with different molecular weights, the stability is high, the replication cycle is short, Escherichia coli can be infected and phage plaques can be formed, phage expression protein is used for purification, and the epitope of the expression protein is identified by using an antibody purified by ammonium sulfate. The technology is widely applied to the fields of medicines, foods and agriculture, and comprises the application of antibiotic analysis, the application of biotoxins and the application of small molecules of agricultural and veterinary medicines, but researches for accurately positioning the reduction of the allergenicity of a glycinin G5 subunit A3 peptide chain caused by two processing methods are not reported, and particularly, the positioning of allergen epitopes destroyed by heat treatment and ultrahigh pressure combined heat processing treatment is not shown in related documents.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an antigenic region and a positioning method for destroying a glycinin G5 subunit A3 peptide chain by heat treatment and ultrahigh pressure combined heat processing, the method not only can analyze and process the structural immunology basis for reducing the sensitization of the glycinin, provide technical support for the evaluation of the desensitization effect of the soybean allergen in the processing process, but also can further develop an application product for rapidly detecting the desensitization effect of processed foods.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the heat treatment and the ultrahigh pressure combined heat processing destroy the amino acid sequence of the antigenic region of the glycinin G5 subunit A3 peptide chain, wherein the amino acid sequence of the antigenic region is shown as SEQ ID NO. 19. The antigenic regions were displayed with T7 phage.
A method for disrupting the localisation of the antigenic region of a glycinin G5 subunit A3 peptide chain by heat treatment in combination with ultra high pressure thermal processing, comprising the steps of:
(1) carrying out heat treatment and ultrahigh pressure combined heat processing treatment on glycinin, and adding the processed glycinin into a glycinin polyclonal antibody to prepare an epitope specific antibody destroyed by the heat treatment and ultrahigh pressure combined heat processing treatment;
(2) according to the tertiary structure of the protein and the predicted position of B cell conformation epitope, dividing the amino acid sequence of the A3 peptide chain into 3 segments, wherein the segmented amino acid sequence is shown as SEQ ID NO.1-SEQ ID NO.3, and designing the A3 peptide chain overlapped segmented protein;
(3) the antigenic region of the A3 peptide chain protein is destroyed by the combination of heat treatment and ultrahigh pressure heat processing through the phage display technology, and the A3 peptide chain and the protein of the overlapping segment thereof are displayed on the surface of the phage;
(4) an indirect competition ELISA method is utilized to accurately position a region in which the antigenicity of the glycinin G5 subunit A3 peptide chain is reduced due to heat treatment and ultrahigh pressure combined heat processing treatment, and an antigen region in which the sequence of SEQ ID NO.1 is damaged A3 peptide chain is obtained;
(5) Segmenting the sequence of SEQ ID NO.1 again, wherein the segmented amino acid sequence is shown as SEQ ID NO.10-SEQ ID NO.12, repeating the step (3) and the step (4), and further, the sequence of SEQ ID NO.10 is accurate to be an antigen region for damaging the peptide chain of A3;
(6) and (3) segmenting the sequence of SEQ ID NO.10 again, wherein the segmented amino acid sequence is shown as SEQ ID NO.19-SEQ ID NO.21, and repeating the step (3) and the step (4), so that the amino acid sequence of the antigen region of the A3 peptide chain is destroyed and is SEQ ID NO. 19.
The specific method for treating the destroyed antigen epitope specific antibody by heat treatment and ultrahigh pressure combined heat processing comprises the following steps:
1) heat treatment of glycinin:
putting glycinin with the concentration of 10mg/mL into a pressure-resistant bottle, and treating at 110 ℃ for 50min to obtain heat-treated glycinin;
2) carrying out ultrahigh pressure combined thermal processing on glycinin:
placing the heat-treated glycinin obtained in the step 1) into a sterile homogenizing bag, sealing and vacuumizing, and placing the sealed homogenizing bag into a treatment cavity of an ultrahigh pressure treatment device, wherein the treatment temperature is 23 ℃; starting boosting, wherein the boosting rate is 250MPa/min, keeping the pressure for 15min when the pressure is raised to 400MPa, then releasing the pressure, the pressure releasing rate is 300MPa/min, and then heating at 130 ℃ for 20 min;
3) Preparing the antigen epitope specific antibody destroyed by heat treatment and ultrahigh pressure combined heat processing:
immunizing New Zealand white rabbits with purified natural glycinin to prepare polyclonal antibody of glycinin;
taking the glycinin treated in the step 2) as an antigen, taking 1mL of the antigen and the glycinin polyclonal antibody diluted by 400 times respectively, uniformly mixing, incubating at 37 ℃ for 2h, centrifuging at 6000g for 1min, removing precipitate, and collecting supernatant.
The specific method of the indirect competitive ELISA method comprises the following steps:
diluting the phage protein obtained in step (3) of claim 3 to 50 μ g/mL with carbonate buffer, coating on 96-well plate with 100 μ L per well, sealing, standing overnight at 4 deg.C, spin-drying the liquid in the well the next day, adding 200 μ L PBST buffer, standing for 5min, and repeating for 5 times; adding 200 μ L of 5% skimmed milk powder into each well, sealing at 37 deg.C for 2h, adding 200 μ L PBST buffer solution, standing for 5min, clapping, and repeating for 5 times;
adding 100 μ L of the specific antibody obtained in step (1) of claim 3 diluted 800 times with PBS to each well, incubating at 37 ℃ for 1h, adding 200 μ L of PBST buffer, and standing for 5Clapping the plate after min, and repeating for 5 times; adding 100 mu L of goat anti-rabbit enzyme-labeled secondary antibody diluted by 5000 times with 5% skimmed milk powder into each well, incubating at 37 ℃ for 1h, adding 200 mu L of PBST buffer solution, standing for 5min, and then performing plate-making for 5 times; adding 100 μ L of TMB single-component color developing solution into each well, and developing at 37 deg.C for 10 min; 50 μ L of 2mol/L H per well 2 SO 4 Terminating the reaction; OD was measured at 450 nm.
The purification method of the natural glycinin comprises the following steps: taking 10mL of natural glycinin polyclonal antibody serum, adding an equivalent amount of PBS (phosphate buffer solution) to dilute the polyclonal antibody serum, uniformly mixing under the action of magnetic stirring, slowly adding 20mL of ammonium sulfate solution to ensure that the saturation of the ammonium sulfate is 50%, and standing overnight in a refrigerator at 4 ℃ to ensure that the immunoglobulin in the serum is fully precipitated; centrifuging the serum solution standing overnight at the rotation speed of 10000r/min for 30min at the temperature of 4 ℃, removing the supernatant, and redissolving the protein precipitate by using 20mL of PBS solution; slowly adding 10mL of ammonium sulfate solution under the action of magnetic stirring to make the saturation of the ammonium sulfate solution be 33%, then placing the mixture in a refrigerator at 4 ℃ for standing overnight, centrifuging the mixture for 30min at the rotating speed of 10000r/min at 4 ℃, removing supernatant, and placing the precipitate in 10mL of PBS solution; then putting into a pretreated dialysis bag, sealing the bag mouth of the dialysis bag, placing at 4 ℃, dialyzing with PBS for three days, changing liquid once every 6-8h, and removing ammonium sulfate; after dialysis, the antibody concentration of the purified natural glycinin was determined, dispensed and stored at-20 ℃.
The invention has the beneficial effects that:
the invention utilizes a series of bioinformatics software and refers to the three-dimensional crystal structure of the glycinin analyzed by a PDB database, researches and processes the antigen region of the peptide chain protein of the subunit A3 of the glycinin G5 by using a phage display technology, displays the protein of the antigen region on the surface of a phage, and precisely locates the region of the peptide chain protein of the glycinin A3, which is reduced by a heat treatment and ultrahigh pressure combined heat processing method, by three rounds of phage display of the peptide chain A3. The research provides a theoretical basis for screening of soybean-related food processing methods and provides a technical support for evaluation of soybean allergen desensitization effects in the processing process.
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FIG. 1 is a diagram showing the results of PCR amplification of the glycinin A3 gene;
wherein, A3 is the full length of A3 gene; 2A 3 shows that 2 identical experiments were performed;
FIG. 2 is a diagram of PCR amplification of three overlapping segmented fragments A, B and C of the subunit of glycinin A3;
wherein, M: DNA Marker; 1-3 are A3 gene segment A, B, C;
FIG. 3 is a diagram showing the results of PCR identification of a recombinant plasmid containing glycinin A3 gene;
wherein, M: DNA Marker; a3 is the full length of A3 gene;
FIG. 4 is a diagram of the results of PCR identification of a recombinant plasmid containing three overlapping segmented fragments A, B and C of the glycinin A3 subunit;
wherein, 1 is recombinant plasmid, 2 and 3 are fragments A, 4 and 5 are fragments B, and 6 and 7 are fragments C;
FIG. 5 is a diagram showing the results of enzyme digestion and identification of a recombinant plasmid containing glycinin A3 gene;
wherein, M: DNA Marker; a3 is the full length of A3 gene;
FIG. 6 is a diagram showing the results of enzymatic cleavage of a recombinant plasmid containing three overlapping segmented fragments A, B and C of the subunit A3 of glycinin;
wherein, M: DNA Marker; 1-3 are A3 gene segment A, B, C;
FIG. 7 is a diagram showing the results of PCR identification of the glycinin A3 gene and its overlapping fragments of recombinant phage;
wherein, M: DNA Marker; a3 is the full length of A3 gene; 1-3 are A3 gene segment A, B, C;
FIG. 8 is a blue-white screening;
wherein, the white spots are the successful connection of the vector and the gene, and the blue spots are the blue color of X-Gal changed by Escherichia coli which is not transferred by the plasmid.
FIG. 9 shows plaques formed on the surface of recombinant phages;
wherein the dark spots are plaques.
FIG. 10 is a diagram showing the results of PCR amplification of overlapping segments of glycinin fragment A;
wherein, M: DNA Marker; a to c are segments A-1, A-2 and A-3 of the segment A; 2 a, b and c are shown to represent that 2 times of the same experiment are carried out;
FIG. 11 is a diagram showing the results of PCR identification of recombinant plasmids of overlapping fragments of glycinin fragment A;
wherein, M: DNA Marker; a to c are segments A-1, A-2 and A-3 of segment A.
FIG. 12 is a diagram showing the results of the restriction enzyme identification of a recombinant plasmid having an overlapped fragment of glycinin fragment A;
wherein, M: DNA Marker; a to c are segments A-1, A-2 and A-3 of segment A.
FIG. 13 is a diagram showing the results of PCR identification of recombinant phages for overlapping fragments of glycinin fragment A;
wherein, M: DNA Marker; a to c are segments A-1, A-2 and A-3 of segment A.
FIG. 14 is a diagram showing the results of PCR amplification of an overlapping fragment of glycinin fragment A-1;
wherein, M: DNA Marker; a to c are segments A-1-a, A-1-b, A-1-c of segment A-1;
FIG. 15 is a diagram showing the results of PCR identification of a recombinant plasmid having an overlapped fragment of glycinin fragment A-1;
wherein, M: DNA Marker; a to c are segments A-1-a, A-1-b, A-1-c of segment A-1; 2 a, b and c are shown to represent that 2 times of the same experiment are carried out;
FIG. 16 is a diagram showing the results of the restriction enzyme identification of a recombinant plasmid having an overlapped fragment of glycinin fragment A-1;
wherein, M: DNA Marker; a to c are segments A-1-a, A-1-b, A-1-c of segment A-1;
FIG. 17 is a diagram showing the results of PCR identification of recombinant phages for overlapping fragments of glycinin fragment A-1;
Wherein, M: DNA Marker; a to c are segments A-1-a, A-1-b, A-1-c of segment A-1;
FIG. 18 is a graph showing the results of ELISA assay of recombinant phage-expressed proteins of A3 full-length and its overlapping fragments;
wherein a, b, c represent the significance of the difference at a level of p < 0.05; the total length is the full length of the A3 gene; the first fragment, the second fragment and the third fragment are A3 gene fragment A, B, C;
FIG. 19 is a diagram showing the result of ELISA assay of fragment A and its overlapping fragments;
wherein a, b, c represent the significance of the difference at a level of p < 0.05; A-1-A-3 are fragments A-1, A-2, A-3 of fragment A;
FIG. 20 is a diagram showing the result of ELISA assay of fragment A-1 and its overlapping fragments;
wherein a, b, c represent the significance of the difference at a level of p < 0.05; a-1 is the full length of the fragment A-1 gene; A-1-a-A-1-c are fragments A-1-a, A-1-b and A-1-c of the fragment A-1.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1 preparation of epitope-specific antibodies destroyed by Heat treatment and ultra high pressure Combined Heat treatment
1. Heat treatment of glycinin:
placing glycinin with concentration of 10mg/mL in a pressure-resistant bottle, and treating at 110 deg.C for 50min to obtain heat-treated glycinin.
2. Carrying out ultrahigh pressure combined thermal processing on glycinin:
placing the heat-treated glycinin with the mass concentration of 10mg/mL into a sterile homogenizing bag, sealing and vacuumizing, and placing the sealed homogenizing bag into a treatment cavity of an ultrahigh pressure treatment device at the treatment temperature of 23 ℃; starting boosting, wherein the boosting rate is 250MPa/min, when the pressure is increased to 400MPa, keeping the pressure for 15min, and then releasing the pressure, wherein the pressure releasing rate is 300 MPa/min; followed by heating at 130 ℃ for 20 min.
3. Preparing the antigen epitope specific antibody destroyed by heat treatment and ultrahigh pressure combined heat processing:
a polyclonal antibody (rabbit antiserum) against glycinin was prepared by immunizing a New Zealand white rabbit with purified native glycinin.
The glycinin subjected to the heat treatment and ultrahigh pressure combined heat processing is taken as an antigen, 1mL of the antigen and 400-fold diluted glycinin polyclonal antibody (rabbit antiserum) are uniformly mixed and incubated at 37 ℃ for 2h, then 6000g of the mixture is centrifuged for 1min to remove precipitates, and the supernatant is collected, namely the antibody with the epitope specificity of the antigen destroyed by the heat treatment and ultrahigh pressure combined heat processing.
The purification method of the natural glycinin comprises the following steps: adding 10mL of natural glycinin polyclonal antibody serum into an equal amount of PBS solution to dilute the polyclonal antiserum, uniformly mixing under the action of magnetic stirring, slowly adding 20mL of SAS (ammonium sulfate) solution to ensure that the saturation of the SAS is 50%, and then placing the serum in a refrigerator at 4 ℃ for standing overnight to ensure that the immunoglobulin in the serum is fully precipitated. The serum solution left overnight was centrifuged at 10000r/min at 4 ℃ for 30min, the supernatant was discarded, and the protein precipitate was reconstituted with 20mL PBS. Slowly adding 10mL of SAS solution under the action of magnetic stirring to enable the saturation degree of the SAS solution to be 33%, then placing the SAS solution in a refrigerator at 4 ℃ for standing overnight, centrifuging the SAS solution for 30min at the rotating speed of 10000r/min at 4 ℃, removing supernatant, and placing the precipitate in 10mL of PBS solution. Then putting into pretreated dialysis bag, sealing the bag mouth, placing at 4 deg.C, dialyzing with PBS for three days, changing solution every 6-8h, and removing ammonium sulfate. After dialysis, the antibody concentration of the purified natural glycinin was determined, dispensed and stored at-20 ℃.
Example 2 overlapping fragment cloning of the peptide chain Gene of glycinin A3
1. Construction of A3 and its overlapping segmented gene polyclonal vector
A three-dimensional structural model of the subunit A3 polypeptide chain of glycinin G5 was first constructed prior to prediction. The three-dimensional template is a glycinin three-dimensional structure constructed by predecessors by X-ray crystal diffraction, can be inquired on a protein database PDB to obtain (ID:2D5H), on the basis, a G5 subunit A3 polypeptide chain three-dimensional structure is constructed by SWISS-MODLE, 2D5H.2.D is used as the template, the sequence recognition degree is 100%, the coverage is complete, the GMQE score is 0.68, the similarity of the three-dimensional model and the real glycinin structure is high, and a foundation is laid for predicting B cell conformation epitope.The amino acid sequence of the peptide chain of glycinin A3 (Gene ID:547452) was retrieved through the NCBI GenBank database. According to a tertiary structure model of an A3 peptide chain, a conformational epitope of the polypeptide is predicted in a Discotope 2.0 network server, and the predicted dominant epitope is as follows: 21 HRVE 24 、 90 FEK-QDS 110 、 137 GDE 139 、 155 QLDQNC 159 、 168 NPDI 171 、 174 PET-EGG 202 、 209 SKH 211 、 218 NTNEDT 223 、 230 PDDERK 235 、 240 VEGGLS 245 、 247 ISPKW 251 . The A3 peptide chain overlapping segment was designed as follows (underlined parts are overlapping fragments):
first stage (fragment a): 1-140 AA:
ITSSKFNECQLNNLNALEPDHRVESEGGLIETWNSQHPELQCAGVTVSKRTLNRNGLHLPSYSPYPQMIIVVQGKGAIGFAFPGCPETFEKPQQQSSRRGSRSQQQLQDSHQKIRHFNEGDVLVIPPGVPYWTYNTGDEP(SEQ ID NO.1)
second stage (fragment B): 81-230 AA:
AFPGCPETFEKPQQQSSRRGSRSQQQLQDSHQKIRHFNEGDVLVIPPGVPYWTYNTGDEPVVAISLLDTSNFNNQLDQNPRVFYLAGNPDIEHPETMQQQQQQKSHGGRKQGQHQQQEEEGGSVLSGFSKHFLAQSFNTNEDTA EKLRSP(SEQ ID NO.2)
third stage (fragment C): 171-320 AA:
IEHPETMQQQQQQKSHGGRKQGQHQQQEEEGGSVLSGFSKHFLAQSFNTNEDTAEKLRSPDDERKQIVTVEGGLSVISPKWQEQEDEDEDEDEEYEQTPSYPPRRPSHGKHEDDEDEDEEEDQPRPDHPPQRPSRPEQQEPRGRGCQTRN(SEQ ID NO.3)
according to the predicted epitope, overlapping segmentation is carried out on the A3 subunit under the condition of not breaking the epitope, the number of amino acid residues overlapped by each segment of sequence and the previous segment of sequence is 60, Primer Premier 5 software is used for detecting that a template does not contain EcoRI and HindIII sites, the structure of a Primer is designed to be 5 '-protective base-enzyme cutting site-Primer sequence-3', the restriction enzyme of an upstream Primer is EcoRI, and the restriction enzyme of a downstream Primer is HindIII. The sequence is shown in the following table 1:
TABLE 1 glycinin A3 subunit gene segment primers
Name (R) Primer sequences
A-up 5′-CTGGAATTCATTACCTCCAGCAAGTTCAACG-3′(SEQ ID NO.4)
A-down 5′-TCCAAGCTTTGGTTCATCGCCAGTGTTATAGG-3′(SEQ ID NO.5)
B-up 5′-CTGGAATTCTTTGCATTTCCGGGATGT-3′(SEQ ID NO.6)
B-down 5′-TCCAAGCTTTGGAGACCGAAGTTTCT-3′(SEQ ID NO.7)
C-up 5′-CTGGAATTCGATATAGAGCACCCAGAGACCA-3′(SEQ ID NO.8)
C-down 5′-TCCAAGCTTATTTCTAGTCTGACATCCTCTT-3′(SEQ ID NO.9)
Attached: when the full-length of the A3 peptide chain is used for PCR reaction, the upstream primer is SEQ ID NO.4 sequence, and the downstream primer is SEQ ID NO.9 sequence.
The nucleotide sequence corresponding to the sequence information of the A3 peptide chain was synthesized from Shanghai as a template for PCR reaction and stored in the PUC57 plasmid vector. The PCR reaction system is shown in Table 2 below:
TABLE 2 PCR reaction System
PCR system Volume of
Sterile ultrapure water 22μL
Upstream primer (10. mu. mol/L) 1μL
Downstream primer (10. mu. mol/L) 1μL
Template gene 1μL
DNA polymerase 25μL
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 60s, annealing at 57 ℃ for 60s, and extension at 72 ℃ for 90s, wherein the three conditions of denaturation, annealing and extension are cycled for 34 times; further extension for 10min at 72 ℃; the PCR gene product was stored at 4 ℃ and detected by 2% agarose electrophoresis.
Recovering DNA with reference to the DNA recovery kit, and mixing the recovered DNA with pMD TM 18-T vector ligation. Ligation system (10 μ L): pMD TM -18T vector: 1 μ L, DNA recovery fragment: 4 μ L, Ligation buffer (mixture of ligase and Ligation buffer): 5 μ L. Will be provided withThe system was placed in an incubator at 16 ℃ and connected overnight.
The PCR amplification products were analyzed on 2% agarose gel (FIG. 1, FIG. 2). The result shows that the vector plasmid synthesized by the marine engineering is used as a template, the PCR technology is adopted to obtain the A3 gene and the genes of the three segmented fragments thereof, the observed target bands conform to the expected fragment sizes, the fragment sizes are 960bp, 420bp, 450bp and 450bp respectively, and non-specific amplification does not exist, which indicates that the specificity of the primer is good.
2. Vector cloning of A3 peptide chain and its gene of overlapped segment
And (3) transformation: respectively taking 10 mu L of A3 peptide chain and the ligation product of the gene of the overlapping segment thereof, respectively and uniformly mixing the ligation product with 25 mu L of competent cells, carrying out ice bath for 30min, carrying out water bath at 42 ℃ for 90s, placing on ice for cooling for 5min, then adding 500 mu L of SOC culture medium for uniformly mixing, and carrying out shake culture at 37 ℃ for 1 h. Centrifuging the culture solution at 4000rmp for 4min, sucking 400 μ L of supernatant, discarding, blowing the rest solution, mixing well, and spreading on Amp containing 50 μ g/mL + (ampicillin), 50mg/mL isopropyl thiogalactoside and 20mg/mL X-gal (chromogenic substrate for. beta. -galactosidase) on solid medium, until coagulation was complete, the incubator was inverted at 37 ℃ for 12-16h until a macroscopic white blue spot was formed (FIG. 8). Picking single white spot on solid culture medium, dissolving in 20 μ L TE (Tris-EDTA buffer), heating in water bath at 65 deg.C for 10min, and centrifuging to obtain supernatant. The supernatant was used as a template for PCR amplification and identified with 2% agarose, and the results are shown in FIGS. 3 and 4, where the size of the target band was 960bp, indicating that the gene has been successfully linked to competent cells. Then picking out white spots and inoculating the white spots on Amp containing 25 mu g/mL + The mixture was shake-cultured at 37 ℃ for 10-12 hours in LB liquid medium, and centrifuged at 7000rmp for 10min to obtain a precipitate.
3. Small extraction of plasmids
Plasmids were extracted using a plasmid miniprep kit (Tiangen Biotechnology (Beijing) Ltd.).
Adding 500 μ L of balance liquid BL into adsorption column CA3 (placing adsorption column into collection tube) several hours in advance, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collection tube, and placing adsorption column into the collection tube; respectively taking 5mL of overnight cultured bacteria liquid (the sediment collected by the centrifugation) in a centrifuge tube, centrifuging at 12000rpm for 1min, absorbing the supernatant as much as possible, adding 250 μ L of solution P1 (ribonuclease A (RNase A)) into the bacteria with the sediment, and completely suspending the bacteria sediment by using a vortex oscillator; then 250 mu L of solution P2 is added, the thalli are turned over gently for 6-8 times to be fully cracked, and the obtained bacterial liquid needs to be clear and viscous. If the bacterial quantity is excessive, the P2 solution quantity can be properly increased; adding 350 mu L of solution P3 into a centrifuge tube, immediately and gently turning for 6-8 times, fully and uniformly mixing, then centrifuging for 10min at 12000rpm when white flocculent precipitate appears; placing the obtained supernatant in an adsorption column CP3, centrifuging at 12000rpm for 1min, and discarding the waste liquid in the collection tube; then adding 500 mu L of deproteinized liquid PD into the adsorption column, centrifuging at 12000rpm for 1min, and discarding waste liquid in the collection tube; adding 600 μ L of rinsing solution into adsorption column, centrifuging at 12000rpm for 2min, and removing waste liquid; putting the adsorption column into a collecting pipe, and centrifuging at 12000rpm for 2min to remove residual rinsing liquid in the adsorption column; to ensure that the experiment was affected by ethanol, the adsorption column was uncapped at room temperature, allowed to stand at room temperature for 6min, and the residual rinse solution was thoroughly dried. The adsorption column is placed in a clean centrifuge tube, 30 mu L of TE buffer solution is dripped into the middle position, the mixture is heated in a water bath at 60 ℃ for 6-8min, centrifuged at 12000rpm for 3min, and the plasmid solution is collected into the centrifuge tube after the operation is repeated. Store at-20 ℃.
4. Restriction enzyme identification of recombinant plasmid
The extracted plasmid is cut and identified by EcoRI and Hind III, the primer sequences are shown in Table 1, and the double-enzyme system is shown in Table 3:
TABLE 3 Dual enzyme digestion identification System
System of Volume of
Recombinant plasmid 15μL
EcoR Ⅰ 1μL
Hind Ⅲ 1μL
10×Green buffer 5μL
Sterile water 28μL
General System 50μL
The cleavage products were identified by 2% agarose electrophoresis (FIGS. 5 and 6). As a result, the bands of the A3 fragment and the three segmented genes thereof are consistent with the expectation and are 960bp, 420bp, 450bp and 450bp respectively, which indicates that the A3 gene and the three segmented gene thereof are successfully transferred into Escherichia coli.
Example 3 expression of glycinin A3 peptide chain and its overlapping segmented gene protein by phage display technology
1. Enzyme digestion recovery of A3 and its segmented gene fragment
And (3) carrying out enzyme digestion on the recombinant plasmid by using EcoRI and Hind III, separating the enzyme digestion product by using 2% agarose gel, cutting a target DNA gel block, and recovering a target gene fragment by using a gel recovery kit.
2. Phage packaging
TABLE 4 phage packaging System
Packaging system Volume of
Target fragment gene 0.5μL
T7vector arms (T7 phage) 0.2μL
T4 DNA ligase 0.2μL
10 Xligation buffer solution 0.1μL
Total volume 1μL
And uniformly mixing the system, placing the system at the constant temperature of 16 ℃ for incubation for 12-16h, then adding a T7 packaged extract with the volume of 5 times of that of the connection product, uniformly mixing, placing the system at the constant temperature of 22 ℃ for incubation for 2h, and adding an LB liquid culture medium with the volume of 9 times of that of the connection product to stop the reaction.
3. Recombinant phage titer assay
Coli BLT5615 was added to 8mL of a solution containing Carb + (Carbenicillin) and isopropyl thiogalactoside in M9LB liquid medium, shaking culture at 37 deg.C to OD 600 0.6-0.8. Recombinant phage display with LB medium 10 3 、10 4 、10 5 Fold gradient dilution, different dilutions of recombinant phage 100. mu.L were mixed with 5mL of preheated Top agarose and 250. mu.L of BLT5615 and spread evenly on Carb-containing plates + The solid medium of (2) was allowed to stand for 30min, and plaques were observed after 4 hours of inverted culture in an incubator at 37 ℃ (see fig. 9), and the plaque titer was calculated as follows:
Figure BDA0003589959910000081
(X is the number of plaques and Y is the corresponding dilution factor of the phage)
4. PCR identification of recombinant phages
One plaque was selected and dissolved in 30. mu.L of TE, heated at 65 ℃ for 10min, centrifuged at 12000rmp for 1min to take the supernatant for PCR identification. The reaction system is shown in Table 5:
TABLE 5 plaque PCR identification System
Components of the System Volume of
Lytic plasmids 2μL
T7 Select up primer (T7 phage upstream primer) 0.5μL
T7 Select down primer (T7 phage downstream primer) 0.5μL
Ex-Taq DNA premix 12μL
Sterile ultrapure water 10μL
Total volume 25μL
Recombinant phage PCR System: pre-denaturation at 80 deg.C for 5 min; denaturation at 94 ℃ for 50s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles of denaturation, annealing and extension; extending for 6 min; storing at 4 ℃. The gene fragment was detected by 2% agarose gel electrophoresis. As shown in FIG. 7, the lengths of the A3 gene and its fragment (A, B, C) are 960bp, 420bp, 450bp and 450bp, respectively, indicating that the recombinant phage was successfully ligated. The recombinant phage titer of the A3 gene and the fragment thereof (A, B, C) is calculated as follows: 6X 10 6 pfu、1×10 6 pfu、5×10 5 pfu、1×10 5 pfu。
5. Amplification culture and preservation of recombinant phage
Inoculation of BLT5615 into 8mL of 8mL Carb-containing pellets + The liquid LB medium was incubated at 37 ℃ overnight, and 1mL of the overnight-incubated broth was added to 46mL of the medium containing carb + And isopropyl thiogalactoside in M9LB induction culture to OD 600 0.6-0.8, 10 μ L of recombinant phage was inoculated into the broth, incubated at 37 ℃ until the broth was clear for about 4h, then phage lysates were centrifuged at 4 ℃ at 8000g for 10min, and 1mL of supernatant was taken to remove phage: chloroform 15: 1(v/v) 67. mu.L of chloroform was added and stored at-80 ℃.
6. Purification of recombinant phages
Adding solid PEG8000 to final concentration of 10% and NaCl to final concentration of 1M (namely, the solution of bacteriophage lysate contains PEG8000 with concentration of 10% and NaCl with mass concentration of 1M), slowly stirring until completely dissolving, ice-bathing for more than 1h or overnight at 4 ℃ for precipitating bacteriophage particles, slowly placing the ice-bathed solution in a centrifuge, centrifuging at 4 ℃, 10000rmp for 10min, and collecting the bacteriophage particles. And (3) taking 500 mu L of sterile SM buffer solution to resuspend the phage particles, slowly transferring the suspended phage particles to a centrifuge tube, incubating the suspended phage particles at 22 ℃ for 1h, adding 625 mu L of chloroform to extract cell fragments and PEG8000 remained in the suspension, slightly shaking the suspension for 30s, and centrifuging the suspension for 10min at 4 ℃ and 5000-6000rmp to separate a water phase, wherein the water phase is the purified and enriched phage.
Example 4 screening of destroyed allergen epitopes
With carbonate buffer (NaHCO) 3 2.93g, anhydrous NaCO 3 1.59g, deionized water to 1L, pH 9.6) were diluted to 50 μ g/mL and coated in 96-well plates, 100 μ L per well, sealed and overnight at 4 ℃. Spin-drying the liquid in the wells the next day, adding 200 μ L PBST buffer (phosphate buffer containing Tween), standing for 5min, and repeating for 5 times; adding 200 μ L of 5% skimmed milk powder into each well, sealing at 37 deg.C for 2h, adding 200 μ L PBST buffer solution, standing for 5min, clapping, and repeating for 5 times;
adding 100 μ L of specific antibody (heat-absorbing antibody by ultrahigh pressure combined heat treatment in example 1) diluted 800 times with PBS into each well, incubating at 37 deg.C for 1h, adding 200 μ L of PBST buffer, standing for 5min, and repeating for 5 times; adding 100 mu L of goat anti-rabbit enzyme-labeled secondary antibody diluted by 5000 times with 5% skimmed milk powder into each well, incubating at 37 ℃ for 1h, adding 200 mu L of PBST buffer solution, standing for 5min, and then performing plate-making for 5 times; adding 100 μ L of TMB single-component color developing solution into each well, and developing at 37 deg.C for 10 min; 50 μ L of 2mol/L H per well 2 SO 4 Terminating the reaction; OD was measured at 450 nm. A rabbit antiserum treatment group and a heat-absorbing antibody treatment group for heat treatment are arranged at the same time.
As shown in fig. 18: the rabbit antiserum-treated group (ammonium sulfate-purified rabbit polyclonal antibody of example 1), the heat-treated heat-absorbing antibody-treated group (antibody obtained by heat treatment alone), and the combined heat-absorbing antibody-treated group (antibody obtained by heat treatment and ultrahigh-pressure combined heat treatment of example 1) were compared with each other, while rabbit sera that were not subjected to immunization treatment were set as negative controls.
The indirect competition ELISA identification result shows (FIG. 18) that the antigenicity of the fragment A (fragment one in FIG. 18) is strongest, and the destroying effect of the ultrahigh pressure combined heat treatment method on the fragment A is most obvious, and the next step mainly researches on the segmented expression of the fragment A and the location of the destroyed antigen region.
Example 5 accurate localization of the antigenic region of the A3 peptide chain that was disrupted by processing
And precisely positioning the antigen region of the A3 peptide chain destroyed by the ultrahigh pressure combined heat processing, further performing overlapping segmentation on all the screened positive phage clone fragments, and repeating the steps to precisely position the antigen dominant region. Second round segmentation amino acid sequence:
section A-1: 1 to 65AA
ITSSKFNECQLNNLNALEPDHRVESEGGLIETWNSQHPELQCAGVTVSKRTLNRNGLHLPSYSPY(SEQ ID NO.10)
Section A-2: 51-110 AA
TLNRNGLHLPSYSPYPQMIIVVQGKGAIGFAFPGCPETFEKPQQQSSRRGSRSQQQLQDS(SEQ ID NO.11)
Section A-3: 81-140 AA
AFPGCPETFEKPQQQSSRRGSRSQQQLQDSHQKIRHFNEGDVLVIPPGVPYWTYNTGDEP(SEQ ID NO.12)
The restriction enzymes upstream and downstream of the segmented primers were the same as those in the first round, and the primers were designed as shown in Table 6:
TABLE 6 glycinin A3 peptide chain second round segmentation primer design
Name (R) Primer sequences SEQ
A1-up 5′-CTGGAATTCATTACCTCCAGCAAGTTCAACGAG-3′ SEQ ID NO.13
A1-down 5′-TCCAAGCTTTATAAGGTGAGTAAGATGGCAAGTG-3′ SEQ ID NO.14
A2-up 5′-CTGGAATTCACCCTCAACCGCAACGG-3′ SEQ ID NO.15
A2-down 5′-TCCAAGCTTACTGTCTTGTAGTTGCTGCTG-3′ SEQ ID NO.16
A3-up 5′-CTGGAATTCGCATTTCCGGGATGTCCTGAG-3′ SEQ ID NO.17
A3-down 5′-TCCAAGCTTTGGTTCATCGCCAGTGTTATA-3′ SEQ ID NO.18
The fragment A is expressed in a segmented manner, and the PCR amplification results of the fragment A overlapping the segments A-1, A-2 and A-3 are shown in FIG. 10; the PCR identification result of the recombinant plasmid is shown in FIG. 11; the restriction enzyme identification result of the recombinant plasmid is shown in FIG. 12; the PCR identification result of the recombinant phage is shown in FIG. 13 by connecting the phage vector arm with the gene, and the sizes of the three-segment overlapping segmented primers of the fragment A shown in the results of FIGS. 10-13 are 195bp, 180bp and 180bp, which are consistent with the expected band size. Indirect competitive ELISA identification was performed by phage expression of the protein fragment of interest.
As shown in fig. 19, the reaction results with the processing-disrupted epitope-specific antibody, as determined by indirect competitive ELISA, indicate that the significantly disrupted regions of the thermal processing and the ultrahigh pressure combined thermal processing are both a-1 fragments, and that the antigen sites of the fragments are reduced after the combined treatment, thereby disrupting the dominant sites of the antigen. The main antigenic region that disrupts the peptide chain of A3 was identified as fragment A-1, and the combined treatment disrupted more epitopes. Next, the segmented expression and the localization of the disrupted antigen region for fragment A-1 were mainly studied.
Third round of segmented amino acid sequence:
paragraph A-1-a: 1-28 AA: ITSSKFNECQLNNLNALEPDHRVESEGG(SEQ ID NO.19)
Paragraphs A-1-b: 23-48 AA:VESEGGLIETWNSQHPELQCAGVTVS(SEQ ID NO.20)
sections A-1-c: 40-65 AA:LQCAGVTVSKRTLNRNGLHLPSYSPY(SEQ ID NO.21)
the restriction enzymes upstream and downstream of the segmented primers were identical to those in the first round, and the primers were designed as shown in Table 7:
TABLE 7 third round of primer design for the peptide chain fragmentation of glycinin A3
Name (R) Primer sequences SEQ
1a-up 5′-CTGGAATTCATTACCTCCAGCAAGTTCAAC-3′ SEQ ID NO.22
1a-down 5′-TCCAAGCTTACCACCTTCGGACTCAAC-3′ SEQ ID NO.23
1b-up 5′-CTGGAATTCGTTGAGTCCGAAGGTGGT-3′ SEQ ID NO.24
1b-down 5′-TCCAAGCTTGGAAACAGTGACACCGGC-3′ SEQ ID NO.25
1c-up 5′-CTGGAATTCCTGCAATGCGCCGGTGT-3′ SEQ ID NO.26
1c-down 5′-TCCAAGCTTTATAAGGTGAGTAAGATGGCAAGTG-3′ SEQ ID NO.27
The fragment A-1 is expressed in a segmented manner, and the PCR amplification results of the fragments A-1 overlapping the segments A-1-a, A-1-b and A-1-c are shown in FIG. 14; the PCR identification result of the recombinant plasmid is shown in FIG. 15; the restriction enzyme identification result of the recombinant plasmid is shown in FIG. 16; the PCR identification result of the recombinant phage is shown in FIG. 17 by connecting the phage vector arm with the gene. The results in FIGS. 14-17 show that the sizes of the three overlapping segmented primers for fragment A are 84bp, 78bp and 78bp, which are consistent with the expected band sizes. Indirect competitive ELISA identification was performed by phage expression of the protein fragment of interest.
As shown in fig. 20, the regions significantly damaged by the thermal processing and the ultrahigh pressure combined thermal processing are both fragments a-1-a, and it is known that the antigen sites of the fragments are reduced and the dominant sites of the antigens are damaged, as determined by indirect competitive ELISA. The main antigenic region of the A3 peptide chain was identified as fragment A-1-a, and the combined treatment destroyed more epitopes.
And finally, obtaining the A-1-a fragment which is the most remarkable fragment damaged by heat treatment and ultrahigh pressure combined heat processing treatment, wherein the amino acid sequence of the fragment is as follows: 1 ITSSKFNECQLNNLNALEPDHRVESEGG 28 。
sequence listing
<110> industrial university of Henan
<120> heat treatment and ultrahigh pressure combined heat processing method for destroying glycinin G5 subunit A3 peptide chain epitope and positioning method
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Claims (6)

1. The heat treatment and the ultrahigh pressure combined heat processing destroy the amino acid sequence of an antigenic region of the glycinin G5 subunit A3 peptide chain, and the amino acid sequence of the antigenic region is shown as SEQ ID NO. 19.
2. The heat treatment and ultra high pressure combined heat processing according to claim 1 to disrupt the amino acid sequence of an antigenic region of the glycinin G5 subunit A3 peptide chain, wherein the antigenic region is phage displayed with T7.
3. A method for disrupting the localisation of the antigenic region of a glycinin G5 subunit A3 peptide chain by heat treatment in combination with ultra high pressure thermal processing, comprising the steps of:
(1) carrying out heat treatment and ultrahigh pressure combined heat processing treatment on glycinin, and adding the processed glycinin into a glycinin polyclonal antibody to prepare an epitope specific antibody destroyed by the heat treatment and ultrahigh pressure combined heat processing treatment;
(2) according to the tertiary structure of the protein and the predicted position of B cell conformation epitope, dividing the amino acid sequence of the A3 peptide chain into 3 segments, wherein the segmented amino acid sequence is shown as SEQ ID NO.1-SEQ ID NO.3, and designing the A3 peptide chain overlapped segmented protein;
(3) the antigenic region of the A3 peptide chain protein is destroyed by the combination of heat treatment and ultrahigh pressure heat processing through the phage display technology, and the A3 peptide chain and the protein of the overlapping segment thereof are displayed on the surface of the phage;
(4) an indirect competition ELISA method is utilized to accurately position a region in which the antigenicity of the glycinin G5 subunit A3 peptide chain is reduced due to heat treatment and ultrahigh pressure combined heat processing treatment, and an antigen region in which the sequence of SEQ ID NO.1 is damaged A3 peptide chain is obtained;
(5) Segmenting the sequence of SEQ ID NO.1 again, wherein the segmented amino acid sequence is shown as SEQ ID NO.10-SEQ ID NO.12, repeating the step (3) and the step (4), and further, the sequence of SEQ ID NO.10 is accurate to be an antigen region for damaging the peptide chain of A3;
(6) and (3) segmenting the sequence of SEQ ID NO.10 again, wherein the segmented amino acid sequence is shown as SEQ ID NO.19-SEQ ID NO.21, and repeating the step (3) and the step (4), so that the amino acid sequence of the antigen region of the A3 peptide chain is destroyed and is SEQ ID NO. 19.
4. The method for locating according to claim 3, wherein the heat treatment and the ultrahigh pressure combined heat treatment are used for treating the destroyed epitope specific antibody by the following specific methods:
1) heat treatment of glycinin:
putting glycinin with the concentration of 10mg/mL into a pressure-resistant bottle, and treating at 110 ℃ for 50min to obtain heat-treated glycinin;
2) carrying out ultrahigh pressure combined thermal processing on glycinin:
placing the heat-treated glycinin obtained in the step 1) into a sterile homogenizing bag, sealing and vacuumizing, and placing the sealed homogenizing bag into a treatment cavity of an ultrahigh pressure treatment device, wherein the treatment temperature is 23 ℃; starting boosting, wherein the boosting rate is 250MPa/min, keeping the pressure for 15min when the pressure is raised to 400MPa, then releasing the pressure, the pressure releasing rate is 300MPa/min, and then heating at 130 ℃ for 20 min;
3) Preparing the antigen epitope specific antibody destroyed by heat treatment and ultrahigh pressure combined heat processing:
immunizing a New Zealand white rabbit with purified natural glycinin to prepare a glycinin polyclonal antibody;
taking the glycinin treated in the step 2) as an antigen, taking 1mL of the antigen and the glycinin polyclonal antibody diluted by 400 times respectively, uniformly mixing, incubating at 37 ℃ for 2h, centrifuging at 6000g for 1min, removing precipitate, and collecting supernatant.
5. The localization method according to claim 3, wherein the indirect competitive ELISA method comprises the following specific steps:
diluting the phage protein obtained in step (3) of claim 3 to 50 μ g/mL with carbonate buffer, coating on 96-well plate with 100 μ L per well, sealing, standing overnight at 4 deg.C, spin-drying the liquid in the well the next day, adding 200 μ L PBST buffer, standing for 5min, and repeating for 5 times; adding 200 μ L of 5% skimmed milk powder into each well, sealing at 37 deg.C for 2 hr, adding 200 μ L of LPBST buffer solution, standing for 5min, clapping, and repeating for 5 times;
adding 100 μ L of the specific antibody obtained in step (1) of claim 3 diluted 800 times with PBS to each well, incubating at 37 ℃ for 1h, adding 200 μ L of PBST buffer, standing for 5min, and performing plate-making for 5 times; adding 100 mu L of goat anti-rabbit enzyme-labeled secondary antibody which is diluted by 5000 times with 5% skimmed milk powder into each hole, incubating for 1h at 37 ℃, adding 200 mu L of PBST buffer solution, standing for 5min, and then performing plate tapping for 5 times; adding 100 μ L of TMB single-component color developing solution into each well, and developing at 37 deg.C for 10 min; 50 μ L of 2mol/L H per well 2 SO 4 Terminating the reaction; OD was measured at 450 nm.
6. The method of claim 4, wherein the native glycinin is purified by the method of: taking 10mL of natural glycinin polyclonal antibody serum, adding an equivalent amount of PBS (phosphate buffer solution) to dilute the polyclonal antibody serum, uniformly mixing under the action of magnetic stirring, slowly adding 20mL of ammonium sulfate solution to ensure that the saturation of the ammonium sulfate is 50%, and standing overnight in a refrigerator at 4 ℃ to ensure that the immunoglobulin in the serum is fully precipitated; centrifuging the serum solution standing overnight at the rotation speed of 10000r/min for 30min at the temperature of 4 ℃, removing the supernatant, and redissolving the protein precipitate by using 20mL of PBS solution; slowly adding 10mL of ammonium sulfate solution under the action of magnetic stirring to make the saturation of the ammonium sulfate solution be 33%, then placing the mixture in a refrigerator at 4 ℃ for standing overnight, centrifuging the mixture for 30min at the rotating speed of 10000r/min at 4 ℃, removing supernatant, and placing the precipitate in 10mL of PBS solution; then putting into a pretreated dialysis bag, sealing the bag mouth of the dialysis bag, placing at 4 ℃, dialyzing with PBS for three days, changing liquid once every 6-8h, and removing ammonium sulfate; after dialysis, the antibody concentration of the purified natural glycinin was determined, dispensed and stored at-20 ℃.
CN202210374978.9A 2022-04-11 2022-04-11 Heat treatment and ultrahigh pressure combined heat processing method for destroying glycinin G5 subunit A3 peptide chain epitope and positioning method Pending CN114835785A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108329384A (en) * 2018-02-28 2018-07-27 河南工业大学 It is a kind of that the GlymBd60K proteantigens region destroyed and screening technique are processed based on display technique of bacteriophage positioning
CN108715608A (en) * 2018-05-31 2018-10-30 河南工业大学 It is a kind of that the beta-conglycinin β subunit antigens region destroyed and screening technique are processed based on display technique of bacteriophage positioning
CN108727481A (en) * 2018-05-31 2018-11-02 河南工业大学 It is a kind of that beta-conglycinin α ' the subunit antigens region destroyed and screening technique are processed based on display technique of bacteriophage positioning

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108329384A (en) * 2018-02-28 2018-07-27 河南工业大学 It is a kind of that the GlymBd60K proteantigens region destroyed and screening technique are processed based on display technique of bacteriophage positioning
CN108715608A (en) * 2018-05-31 2018-10-30 河南工业大学 It is a kind of that the beta-conglycinin β subunit antigens region destroyed and screening technique are processed based on display technique of bacteriophage positioning
CN108727481A (en) * 2018-05-31 2018-11-02 河南工业大学 It is a kind of that beta-conglycinin α ' the subunit antigens region destroyed and screening technique are processed based on display technique of bacteriophage positioning

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