CN103675291A - Method for screening wheat varieties with low celiac sprue toxicity based on T cell epitopes - Google Patents
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Abstract
A method for screening wheat varieties with low celiac sprue toxicity based on T cell epitopes comprises the following steps: separating and extracting wheat gluten protein in two steps, performing SDS-PAGE electrophoresis combined with PageBlue TM staining method to identify the gluten protein, detecting celiac sprue T cell epitopes caused by the gluten protein through immunoblotting and other steps. According to the invention, T cell epitopes-based screening method is adopted to screen out the wheat varieties with low celiac sprue toxicity, so that safe and reliable low gluten food can be supplied for celiac sprue patients from the origin of raw materials, and the safety risk of the celiac sprue patients is reduced.
Description
Technical field
Under the present invention, field is mainly food and biological technical field, relates to a kind of method based on the low chylous diarrhea toxicity of t cell epitope examination wheat breed.
Background technology
Chylous diarrhea is that a kind of inheritance susceptible person does not tolerate wheat, rye and barley gluten protein and the chronic enteritis disease that causes.It reaches 1-2% at the American-European incidence of disease, and the whole world is in rising trend.This disease is at present without effective therapy, and strict GFD is the unique therapeutic modality of patient.But due to few without seitan food and quality is very different on the market, the food that does not simultaneously much contain seitan is also likely sneaked into seitan in process, has had a strong impact on the life of celiac patients.Therefore, the low chylous diarrhea toxicity of examination wheat breed is significant.
Gluten protein mainly comprises gliadin (α/β, γ, the molten albumen of ω-ol) and the polymeric glutenin (high molecular weight glutenin and low molecular weight glutenin) of monomer.The two contains a large amount of proline and glutamine, containing a small amount of arginine, lysine and histidine.Gliadin is mainly dissolved in alcoholic solution, and glutenin is dissolved in the alcoholic solution containing reductive agent.Due to the singularity of gluten protein amino acid kind and content, cause it to be different from common food proteins, conventional the whole bag of tricks is also inapplicable for the research of gluten protein.
What cause chylous diarrhea is some t cell epitope on gluten protein, and these epi-positions do not exist only on gliadin, are present on glutenin yet.The efficient separation and Extraction of gluten protein is most important for the evaluation that causes chylous diarrhea t cell epitope.At present, about the extracting method of gluten protein, mainly contain 60% Ethanol Method and 50% isopropyl alcohol in conjunction with DTT single stage method.60% ethanol extraction method is to use the earliest also more general a kind of extracting method, but the glutenin extraction efficiency that this method causes chylous diarrhea epi-position for same existence is not high, studies have reported that 60% ethanol extraction method can extract gluten protein 0.01g from every gram of wheat, concentration is 0.33mg/mL.And 50% isopropyl alcohol in conjunction with the single stage method of DTT owing to having added reductive agent DTT, thereby greatly improved for the extraction efficiency of glutenin, can from every gram of wheat, extract gluten protein 0.03g, the gluten protein concentration that this method is extracted is 1.01mg/ml.But one-step extraction is not high to the extraction efficiency of the molten albumen of ω-ol and D-type low molecular weight protein (LMWP), the demonstration of the experimental result of existing report, in the gluten protein that this method is extracted, these two kinds of albumen have disappearance significantly.
The gluten protein extracting is by SDS-PAGE(SDS-polyacrylamide gel) electrophoresis identifies, the colouring method of conventional SDS-PAGE glue mainly contains Coomassie brilliant blue (R-250) decoration method and silver and dyes etc.Because gluten protein has atypical amino acid, form, thereby different dyeing conditions has deviation in various degree to the evaluation of gluten protein.Wherein traditional Coomassie brilliant blue (R-250) decoration method is mainly reacted with lysine and arginine residues, and detection sensitivity is 8-16 ng, and this method is simple to operate.But the gluten protein after traditional coomassie brilliant blue staining, in decolorization, albumen can be to some extent by wash-out, the particularly molten albumen of ω-ol and low molecular weight glutenin, some albumen even can lack.Although it is higher that silver dyes spirit sensitivity, is 4-8 ng, running program is complicated, and easy overstain.Research discovery, silver dyes the Color of high molecular weight glutenin also poor, if extend dyeing time, easily causes again the overstain of other albumen.
The existing method for detection of gluten protein has ELISA method, Western blot and mass spectroscopy.ELISA method need to adopt specific kit, and cost is higher.Commercially available kit is mainly based on R5 monoclonal antibody and Skerritt antibody at present.These two kinds of antibody all just detect alcohol soluble protein, and for existing equally the glutenin of chylous diarrhea toxicity not detect.Western blot principle is consistent with ELISA method, but the selection of this method antagonist is freer, is no longer confined to the antibody for alcohol soluble protein, but can be in conjunction with the antibody for causing chylous diarrhea t cell epitope, and testing result more accurately, intuitively.That mass spectrophotometry has is highly sensitive, amount of samples is few, quality precision is high and the advantage such as reliable results, but compare with immunological detection method, mass spectroscopy relies on expensive equipment, and expense is higher, waste time and energy, be not suitable for food service industry existing fast, detect in a large number.
Summary of the invention
The object of the invention is to set up a kind of method based on the low chylous diarrhea toxicity of t cell epitope examination wheat breed, the SDS-PAGE(SDS-polyacrylamide gel that specifically provides the two-step approach, gluten protein of separation and Extraction gluten protein to identify) electrophoresis is in conjunction with PageBlue
tMdecoration method and gluten protein cause the Western blot that chylous diarrhea t cell epitope is identified.Not only can be applied to the low chylous diarrhea toxicity of examination wheat breed, also can be applicable to barley, rye and the oat varieties of the low chylous diarrhea toxicity of examination.This method testing result is clear, comprehensive, directly perceived.
The present invention is achieved by the following technical solutions.
Concrete steps of the present invention are as follows.
1, the extraction of gluten protein and evaluation.
(1) extraction of gluten protein.Choose each product grow wheat benevolence mortar and grind to form fine powder, pour in centrifuge tube, for extracting gluten protein.Extracting for the first time, with 50%(v/v) isopropyl alcohol extracting, after ultrasonic concussion 10min, vortex mixes 30min, the centrifugal 10min of 10000rpm collects supernatant, retains precipitation.Extracting for the second time, every pipe precipitation adds 0.5ml 50% isopropyl alcohol/1%DTT/50mM Tris pH 7.5, ultrasonic concussion 10min, 60 ℃ of heating 30min, every 5-10 min mixes once, the centrifugal 10min of 10000rpm, collects supernatant, abandons precipitation, just the supernatant of twice merges, the centrifugal 1min of 14000rpm, obtains supernatant, is total gluten protein.
(2) evaluation of gluten protein.Adopt conventional SDS-PAGE electrophoresis, the separation gel with 10% and 4% concentrated glue carry out Protein Separation, use PageBlue
tMgluten protein is dyeed and spent the night, gel imaging.
2, cause the evaluation of chylous diarrhea t cell epitope.
(1) according to a conventional method the gluten protein of electrophoretic separation is transferred to cellulose nitrate adipose membrane (NC film) upper, takes out film, ultrapure water rinses 3 times, MemCode
tMstaining kit dyeing, gel imaging rear decoloring.
(2) two kinds of monoclonal antibodies (anti-Glia-α 9 monoclonal antibodies and anti-Glia-α 20 monoclonal antibodies) of take based on t cell epitope are primary antibodie, according to conventional method, do Western blotting, BCIP/NBT colour developing, Quantity One software analysis.
(3) by gray scale scanning, compare the height of t cell epitope content, a little less than assessment wheat breed chylous diarrhea strong toxicity.
The present invention has set up two-step approach and has extracted gluten protein, can effectively and more all sidedly extract fast gluten protein, the first step and second step can extract gluten protein and be respectively 0.05g and 0.01g from every gram of wheat, and the gluten protein concentration of extraction is respectively 1.52mg/mL and 0.33mg/mL.And SDS-PAGE result shows that the gluten protein extracting comprises gliadin and glutenin.Meanwhile, the present invention has creatively selected PageBlue
tM(G-250) dyestuff is identified the gluten protein of SDS-PAGE for dyeing, and this method is simple to operate, and the detection sensitivity of gluten protein, up to 5 ng, and is better than silver to the Color of high molecular weight glutenin and is dyed.The present invention has also set up in the Western blot detection gluten protein based on t cell epitope monoclonal antibody and has caused chylous diarrhea t cell epitope, and then the chylous diarrhea toxicity of assessment wheat.This method can detect simultaneously and cause chylous diarrhea t cell epitope on gliadin and glutenin, and method of operating is simple, and testing result is clear and intuitive.
Accompanying drawing explanation
Fig. 1 Wheat Cultivars gluten protein SDS-PAGE schemes (2 μ g, No. 1-8) M:Maker; Bov:Bovictus, Guard cell, for contrast.
Fig. 2 gluten protein Western blotting result and trace relative intensity; A: anti-Glia-α 9 monoclonal antibodies; B: anti-Glia-α 20 monoclonal antibodies; Bov:Bovictus, hexaploid wheat, for contrast.
Embodiment
The present invention will be described further by following examples.
Embodiment 1: Wheat Cultivars causes the examination of chylous diarrhea t cell epitope.
Whole examination process comprise the extraction of gluten protein, the SDS-PAGE of gluten protein in conjunction with PageBlue
tMdyeing is identified, the Western blotting based on t cell epitope monoclonal antibody.Concrete implementation process comprises the steps.
1, the extraction of gluten protein and evaluation.
Choose 8 wheat breeds, be respectively Yanshi 16, interior township 184, Zhengzhou 941 for No. 1-8, the exhibition 893 of laying down, littlely lay down 54, No. 1, new drought, Zheng Nong 16, Henan agriculture 015 and a check variety Bovictus, after abrasive dust, extract gluten protein.
The extraction of gluten protein adopts 50% isopropyl alcohol/DTT two-step approach.Choose each product grow wheat benevolence mortar and grind to form fine powder, pour in centrifuge tube, for extracting gluten protein.Extracting for the first time, with 50%(v/v) isopropyl alcohol extracting, after ultrasonic concussion 10min, vortex mixes 30min, the centrifugal 10min of 10000rpm collects supernatant (supernatant 1), retains precipitation.Extracting for the second time, every pipe precipitation adds 0.5ml 50% isopropyl alcohol/1%DTT/50mM Tris pH 7.5, ultrasonic concussion 10min, 60 ℃ of heating 30min, every 5-10 min mixes once, the centrifugal 10min of 10000rpm, collects supernatant (supernatant 2), abandons precipitation, supernatant 1, supernatant 2 are merged, the centrifugal 1min of 14000rpm, obtains supernatant, is total gluten protein.
Adopt conventional SDS-PAGE electrophoresis, the separation gel that condition is 10% and 4% concentrated glue, applied sample amount is each kind 2 μ g, 1 plate system of electrophoresis is selected separation gel 10mA, 30min, concentrated glue 20mA, 1h, until bromophenol blue is gone to bottom, takes out the fixing 15min of the acetic acid of 50% ethanol and 10% for glue.After fixing, with ultrapure water rinsing 5min, use PageBlue
tMgluten protein is dyeed and spent the night, and gel imaging, the results are shown in Figure 1.
Fig. 1 swimming lane 1-8 is respectively 8 wheat breeds, and Bovictus is check variety.From SDS-PAGE figure, can find out and adopt 50% isopropyl alcohol can effectively extract gluten protein in conjunction with the method for DTT, the gluten protein extracting comprise high-molecular-weight protein, the molten albumen of ω-ol, low molecular weight protein (LMWP) and α/β-, the molten albumen of γ-ol.And from electrophoretogram, can clearly find out that between each wheat breed, gluten protein there are differences.
2, cause the evaluation of chylous diarrhea t cell epitope.
Gluten protein, after SDS-PAGE electrophoresis, by wet method transferring film, is transferred to gluten protein on NC film, takes out film, and ultrapure water rinses 3 times, MemCode
tMstaining kit dyeing, gel imaging rear decoloring.According to conventional method, do Western blotting subsequently, add primary antibodie (being respectively anti-Glia-α 9 monoclonal antibodies and anti-Glia-α 20 monoclonal antibodies) to spend the night, the film after spending the night adds two anti-and substrate colour developings.Trace result is carried out gray scale scanning by Quantity One software, calculate the relative intensity value of its t cell epitope content, by relatively drawing, cause the minimum kind of chylous diarrhea t cell epitope intensity, the trace result of last comprehensive two kinds of monoclonal antibodies is determined low chylous diarrhea toxicity wheat breed.It the results are shown in Figure 2.
Fig. 2 A is the relative intensity figure that anti-Glia-α 9 monoclonal antibody trace results and gluten protein cause chylous diarrhea t cell epitope, and B is the relative intensity figure that anti-Glia-α 20 monoclonal antibody trace results and gluten protein cause chylous diarrhea t cell epitope.From A figure in conjunction with the relative intensity figure of A can find out No. 4, kind and contain for No. 6 to cause chylous diarrhea t cell epitope Glia-α 9 lower than other kinds.From B figure, in conjunction with the relative intensity figure of B, can find out kind and contain for No. 6, No. 4 and No. 7 to cause chylous diarrhea t cell epitope Glia-α 20 lower than other kinds.Although No. 7 kind contains less Glia-α 20 epi-positions, contain more Glia-α 9 epi-positions due to No. 7, and this epi-position is the main chylous diarrhea t cell epitope that causes, thereby gets rid of No. 7.Because No. 4 and No. 6 kinds are in two traces reactions, quite, thereby whose chylous diarrhea toxicity is lower, requires further study in the two performance.Consider, show that the chylous diarrhea toxicity of No. 4 and No. 6 wheat breeds is minimum.
Claims (1)
1. the method based on the low chylous diarrhea toxicity of t cell epitope examination wheat breed, is characterized in that as follows:
(1) extraction of gluten protein and evaluation
A) extraction of gluten protein: choose each product grow wheat benevolence mortar and grind to form fine powder, pour in centrifuge tube, for extracting gluten protein; Extracting for the first time, with 50%(v/v) isopropyl alcohol extracting, after ultrasonic concussion 10min, vortex mixes 30min, the centrifugal 10min of 10000rpm collects supernatant, retains precipitation; Extracting for the second time, every pipe precipitation adds 0.5ml 50% isopropyl alcohol/1%DTT/50mM Tris pH 7.5, ultrasonic concussion 10min, 60 ℃ of heating 30min, every 5-10 min mixes once, the centrifugal 10min of 10000rpm, collects supernatant, abandons precipitation, the supernatant of twice is merged, the centrifugal 1min of 14000rpm, obtains supernatant, is total gluten protein;
B) evaluation of gluten protein: adopt conventional SDS-PAGE electrophoresis, the separation gel with 10% and 4% concentrated glue carry out Protein Separation, use PageBlue
tMgluten protein is dyeed and spent the night, gel imaging;
(2) cause the evaluation of chylous diarrhea t cell epitope
A) according to a conventional method the gluten protein of electrophoretic separation is transferred on cellulose nitrate adipose membrane, takes out film, ultrapure water rinses 3 times, MemCode
tMstaining kit dyeing, gel imaging rear decoloring;
Glia-α 9 monoclonal antibodies and Glia-α 20 monoclonal antibodies of b) take based on t cell epitope are primary antibodie, according to conventional method, do Western blotting, BCIP/NBT colour developing, Quantity One software analysis;
C) by gray scale scanning, compare the height of t cell epitope content, a little less than assessment wheat breed chylous diarrhea strong toxicity.
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Cited By (9)
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| CN107176974A (en) * | 2017-06-06 | 2017-09-19 | 中国医学科学院北京协和医院 | The special CD4+T cell epitopes of the alcohol soluble proteins of ω 5 and its application |
| JP2017532544A (en) * | 2014-09-10 | 2017-11-02 | ヴィブラント ホールディングス リミテッド ライアビリティ カンパニー | Peptide microarray and novel biomarkers for celiac disease |
| CN108471717A (en) * | 2016-06-17 | 2018-08-31 | 大韩民国（农村振兴厅长） | Anaphylactoid wheat with wheat dependence-exercise induced is not tolerated for mitigating and preventing seitan |
| CN108676863A (en) * | 2017-05-18 | 2018-10-19 | 四川出入境检验检疫局检验检疫技术中心 | The method that high throughput sequencing technologies identify chylous diarrhea sensibiligen in wheat flour |
| US10746732B2 (en) | 2012-09-28 | 2020-08-18 | Vibrant Holdings, Llc | Methods, systems, and arrays for biomolecular analysis |
| US10799845B2 (en) | 2012-11-14 | 2020-10-13 | Vibrant Holdings, Llc | Substrates, systems, and methods for array synthesis and biomolecular analysis |
| US11168365B2 (en) | 2017-05-26 | 2021-11-09 | Vibrant Holdings, Llc | Photoactive compounds and methods for biomolecule detection and sequencing |
| CN114438044A (en) * | 2022-01-14 | 2022-05-06 | 南昌大学 | Multienzyme complex for efficiently degrading gluten protein and application thereof |
| US11565231B2 (en) | 2012-02-07 | 2023-01-31 | Vibrant Holdings, Llc | Substrates, peptide arrays, and methods |
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| US11815512B2 (en) | 2012-09-28 | 2023-11-14 | Vibrant Holdings, Llc | Methods, systems, and arrays for biomolecular analysis |
| US11674956B2 (en) | 2012-09-28 | 2023-06-13 | Vibrant Holdings, Llc | Methods, systems, and arrays for biomolecular analysis |
| US10746732B2 (en) | 2012-09-28 | 2020-08-18 | Vibrant Holdings, Llc | Methods, systems, and arrays for biomolecular analysis |
| US10799845B2 (en) | 2012-11-14 | 2020-10-13 | Vibrant Holdings, Llc | Substrates, systems, and methods for array synthesis and biomolecular analysis |
| JP7256229B2 (en) | 2014-09-10 | 2023-04-11 | ヴィブラント ホールディングス リミテッド ライアビリティ カンパニー | Peptide microarray and novel biomarkers for celiac disease |
| US10900964B2 (en) | 2014-09-10 | 2021-01-26 | Vibrant Holdings, Llc | Peptide microarrays and novel biomarkers for celiac disease |
| JP2021144040A (en) * | 2014-09-10 | 2021-09-24 | ヴィブラント ホールディングス リミテッド ライアビリティ カンパニー | Peptide microarrays and novel biomarkers for celiac disease |
| JP2017532544A (en) * | 2014-09-10 | 2017-11-02 | ヴィブラント ホールディングス リミテッド ライアビリティ カンパニー | Peptide microarray and novel biomarkers for celiac disease |
| CN108471717A (en) * | 2016-06-17 | 2018-08-31 | 大韩民国（农村振兴厅长） | Anaphylactoid wheat with wheat dependence-exercise induced is not tolerated for mitigating and preventing seitan |
| CN108676863B (en) * | 2017-05-18 | 2022-05-03 | 四川出入境检验检疫局检验检疫技术中心 | Method for identifying celiac allergen in wheat flour by high-throughput sequencing technology |
| CN108676863A (en) * | 2017-05-18 | 2018-10-19 | 四川出入境检验检疫局检验检疫技术中心 | The method that high throughput sequencing technologies identify chylous diarrhea sensibiligen in wheat flour |
| US11168365B2 (en) | 2017-05-26 | 2021-11-09 | Vibrant Holdings, Llc | Photoactive compounds and methods for biomolecule detection and sequencing |
| CN107176974A (en) * | 2017-06-06 | 2017-09-19 | 中国医学科学院北京协和医院 | The special CD4+T cell epitopes of the alcohol soluble proteins of ω 5 and its application |
| CN114438044A (en) * | 2022-01-14 | 2022-05-06 | 南昌大学 | Multienzyme complex for efficiently degrading gluten protein and application thereof |
| CN114438044B (en) * | 2022-01-14 | 2024-03-29 | 南昌大学 | Multienzyme complex for efficiently degrading gluten protein and application thereof |
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