Olecranon peach allergen protein Q2 and application thereof

Abstract

The invention belongs to the technical field of fruit allergen detection, and particularly relates to an chickpea allergen protein Q2 and application thereof. According to the invention, the chickpea allergen protein Q2 is screened from chickpeaches by using a protein spectrum method, the chickpea allergen protein Q2 is prepared by using a protein exogenous expression technology, and an immunodetection method of the chickpea allergen protein Q2 is established. According to the detection method, the detection limit of the chickpea allergen protein Q2 is 1.8 mu g / mL, the half inhibitory concentration is 36.3 mu g / mL, and the linear range is 5.9-224.4 mu g / mL. The method has the advantages of simplicity, rapidness, strong specificity and high sensitivity, can be used for rapid detection of the chickpea allergen protein Q2, and provides a simple, reliable and effective method for chickpea allergen detection.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit allergen detection, and particularly relates to a hawksbill peach allergen protein Q2 and an application thereof. Background Art

[0002] As a specialty peach variety in the Lingnan region, the hawk-billed peach has fresh flesh, sweet honey, and is rich in nutrients such as protein, iron, calcium, phosphorus, and vitamin C. It also has the effects of promoting blood circulation and reducing phlegm, relieving asthma and cough, and is rated as one of the "Top Ten Fruits in Lingnan". Peach is one of the main fruits that cause human allergies. With the development of logistics, regional peach varieties with a shorter shelf life are sold all over the country, and allergic reactions caused by peach allergens are also increasing. Fruit allergies usually present mild symptoms, such as oral allergy syndrome, but there are also severe symptoms, such as systemic allergic reactions, urticaria, gastrointestinal symptoms and other allergic reactions, which have a significant impact on the quality of life of allergic patients. Therefore, the study of fruit allergens is becoming increasingly important.

[0003] Patent document CN109837282A discloses a method for expressing and purifying the antigen of citrus allergen Cits1.01, preparing polyclonal antibodies and using the same. The method optimizes the prokaryotic expression sequence of the Cits1.01 gene, performs gene synthesis, constructs a prokaryotic expression system for the allergen Cits1.01, and uses the expressed and purified Cits1.01 protein to prepare rabbit polyclonal antibodies Anti-Cits1.01, detects the content of the allergen Cits1.01 in citrus peel and pulp, and can use the optical density value to measure the strength of Western blot to evaluate the potential allergenicity of the allergen Cits1.01 in the edible part of citrus fruit.

[0004] Patent document CN113504089A discloses an extracting solution and an extraction method for extracting allergens from tomatoes. The extracting solution includes: PBS buffer with a pH value of 7.0 to 8.0, 0.5 to 1.5 mM EDTA, 3 to 8 mM L-cysteine, 1X protease inhibitor, and pectinase accounting for 0.05% to 0.2% of the total mass of the extracting solution. The extracting solution can extract more allergen proteins of different molecular weights from tomatoes, especially small molecular weight allergen proteins. The obtained tomato allergen extract has high transparency and low impurity content other than allergens. It can be directly used in in vitro detection kits, has high biological activity and positive detection rate, and has a clean background.

[0005] Patent document CN118546211A discloses a characteristic peptide combination and its preparation method and application in the detection of peach allergen protein. The characteristic peptide combination can correctly determine whether the sample to be tested contains peach allergen protein. Calculated based on the peach content, the minimum detection concentration in concentrated beverages is 2.4g / 100mL, and the minimum detection concentration in jam is 5.1g / 100g. It has the characteristics of high mass spectrometry response intensity and strong stability.

[0006] However, there are few studies on hawkbill peach allergens. The preparation of hawkbill peach allergen protein is of great significance for in-depth exploration of its sensitization mechanism and immunoassay analysis. Therefore, it is very necessary to provide a preparation method and application of hawkbill peach allergen protein. Summary of the invention

[0007] In order to solve the defects of the prior art, the purpose of the present invention is to provide a chickpea allergen protein Q2 and its application. The present invention uses the prepared chickpea allergen protein Q2 to establish an immunoassay method for chickpea allergen protein Q2, providing a rapid, reliable and easy-to-operate detection method for the detection of chickpea allergens.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is:

[0009] The present invention provides a citrus aurantium allergen protein Q2, and the amino acid sequence of the citrus aurantium allergen protein Q2 is shown in SEQ ID NO: 1.

[0010] Furthermore, the amino acid sequence of the hawkthorn allergen protein Q2 is (SEQ ID NO: 1): MGVFTYESEFTSEIPPPRLFKAFVLDADNLVPKIAPQAIKHSEILEGDGGPGTIKKITFGEGS QYGYVKHKIDSIDKENHSYSYTLIEGDALGDNLEKISYETKLVASPSGGSIIKSTSHYHTK GDVEIKEEHVKAGKEKASNLFKLIETYLKGHPDAYN.

[0011] Furthermore, the antigenic epitopes of the hawksbill peach allergen protein Q2 are 13-15 (EIP), 62-64 (GSQ), 77-79 (KEN), 95-96 (NL), 124-125 (KG), 128-130 (EIK), and 155-157 (HPD).

[0012] Furthermore, the present invention provides a gene encoding an allergen protein Q2 of the genus Cialis, which encodes the above-mentioned genus Cialis allergen protein Q2. The nucleotide sequence of the gene encoding the genus Cialis allergen protein Q2 is shown in SEQ ID NO: 2.

[0013] Furthermore, the nucleotide sequence of the gene encoding the hawkbill peach allergen protein Q2 is (SEQ ID NO: 2):

[0014] ATGGGTGTCTTCACCTACGAGAGCGAATTCACCTCTGAAATTCCGCCACCACGTCTGTTTAAAGCGTTCGTTCTGGACGCGGATAATCTGGTTCCGAAAATTGCACCGCAGGCGATCAAACATAGCGAAATTCTGGAAGGCGACGGCGGTCCGGGTACCATCAAAAAGATTACCTTCGGCGAAGGTAGCCAGTACGGTTACGTCAAACACAAAATCGACAGCATCGACAAAGAGAACCAC AGCTACAGCTATACCCTGATTGAAGGCGACGCACTGGGCGATAACCTGGAAAAAATCAGCTATGAAACCAAACTGGTTGCGAGCCCGTTCTGGCGGTAGCATTATCAAAAGCACCAGCCATTACCACCAAAGGCGACGTCGAAATCAAAGAAGAACATGTCAAAGCGGGCAAAGAAAAAGCGAGCAACCTGTTCAAACTGATCGAAACCTACCTGAAAGGCCATCCGGACGCATATAAC.

[0015] Furthermore, the present invention provides a recombinant vector of citrus aurantium allergen protein Q2, wherein the recombinant vector comprises a gene encoding the citrus aurantium allergen protein Q2.

[0016] Furthermore, the present invention provides an expression system comprising the coding gene of the above-mentioned hawkthorn allergen protein Q2 or the above-mentioned hawkthorn allergen protein Q2 recombinant vector, and the expression system includes but is not limited to an Escherichia coli expression system or a yeast expression system.

[0017] In addition, the present invention also provides the use of the hawksbill peach allergen protein Q2 in the preparation of immunological diagnostic reagents, food allergen diagnostic reagents or diagnostic kits.

[0018] Furthermore, the present invention also provides a kit for detecting the allergen protein Q2 of hummus, wherein the kit comprises the above-mentioned hummus allergen protein Q2.

[0019] Furthermore, the kit also contains an antibody to the allergen protein Q2 of the genus Cicer australis, an enzyme-labeled secondary antibody, a substrate solution, a calibrator and / or a quality control product.

[0020] Furthermore, the garcinia allergen protein Q2 antibody is prepared by immunizing rabbits, mice or camelids with the garcinia allergen protein Q2, and can also be prepared by immunizing rabbits, mice, camelids and other animals with garcinia natural protein extracts containing the allergen protein Q2, including but not limited to monoclonal antibodies, polyclonal antibodies, and genetically engineered antibodies.

[0021] Furthermore, the enzyme-labeled secondary antibody is an HRP-labeled goat anti-rabbit or mouse or camel IgG secondary antibody.

[0022] Furthermore, the present invention also provides an immunoassay method for the kit for detecting the allergen protein Q2 of the hawksbill peach:

[0023] Step S1, coating the hawksbill peach allergen protein Q2 in the microwells of a microwell plate, incubating, washing, sealing, and drying;

[0024] Step S2, adding gradiently diluted citrus allergen protein Q2 or pre-treated test samples into the microwells of the chemiluminescent plate, adding antibodies against citrus allergen protein Q2 into the microwells, incubating, and washing the plate;

[0025] Step S3, adding enzyme-labeled secondary antibody into the microwells, incubating, and washing the plate;

[0026] Step S4, adding the substrate solution into the microwell, oscillating and mixing, and measuring the signal value at a certain wavelength;

[0027] Step S5, plotting a standard curve of the immunoassay method with the signal value of each standard concentration hole as the ordinate and the log10 value of the drug standard solution concentration as the abscissa, and analyzing the content of the pyracantha allergen protein Q2 in the sample based on the established standard curve.

[0028] The invention uses a protein spectrum method to screen and obtain a chickpea allergen protein Q2 from chickpeas, and uses an exogenous expression technology to prepare the chickpea allergen protein Q2. After in-depth research, the inventors found that the antigenic epitopes of the chickpea allergen protein Q2 are 13-15 (EIP), 62-64 (GSQ), 77-79 (KEN), 95-96 (NL), 124-125 (KG), 128-130 (EIK), and 155-157 (HPD). The inventors applied the chickpea allergen protein Q2 to immunological diagnostic reagents, food allergen diagnostic reagents or diagnostic kits, and established an immunological detection method of the chickpea allergen protein Q2 using the chickpea allergen protein Q2, and the content of the allergen protein Q2 in the sample to be tested can be calculated. The detection limit of the allergen protein Q2 of the chickpea in the immunoassay method was 1.8 μg / mL, the half inhibition concentration was 36.3 μg / mL, the linear range was 5.9-224.4 μg / mL, and the inhibition rate and the logarithmic value of the allergen protein Q2 formed a significant S-shaped curve relationship, with a correlation coefficient R 2 The value is 0.997, which can be used for the rapid detection of hawkbill peach allergen protein Q2.

[0029] In summary, compared with the prior art, the immunoassay method for the allergen protein Q2 of the genus Cibotium wilfordii provided by the present invention is simple, rapid, specific and highly sensitive. It can directly detect the content of the allergen protein Q2 in food or be applied to the detection of genus Cibotium wilfordii allergens, providing a reliable and fast new method for the detection of genus Cibotium wilfordii allergens. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the SDS-PAGE electrophoresis of the crude protein of Prunus armeniaca.

[0031] Figure 2 This is the protein spectrum analysis diagram of the hawkbill peach allergen protein Q2.

[0032] Figure 3 This is a map of the recombinant vector of the hawkbill peach allergen protein Q2.

[0033] Figure 4 This is the SDS-PAGE electrophoresis of the recombinant protein Q2 of the hawkbill peach allergen.

[0034] Figure 5 This is a graph showing the activity analysis results of the natural allergen protein from the hawkbill peach and the recombinant protein Q2.

[0035] Figure 6 This is a graph showing the stability analysis results of the hawkbill peach allergen recombinant protein Q2.

[0036] Figure 7 This is the standard curve of ELISA immunoassay for chickpea allergen Q2. DETAILED DESCRIPTION

[0037] The present invention is further described below by describing specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0038] Example 1: Identification of allergenic proteins of Prunus armeniaca

[0039] 1. Screening of allergen proteins of hawkbill peach:

[0040] The 8-9 ripe fresh peach fruits collected from the local orchard in Lianping, Heyuan were washed and cut into 0.5 cm slices, vacuum freeze-dried, and crushed using an ultrafine grinder. 1.0 g of peach ultrafine powder was dissolved in 5 mL of PBS buffer and extracted at 120 r / min and 37 ° C for 2 h. The extract was ultrafiltered using an ultrafiltration tube with a molecular cutoff of 3 kDa to prepare a peach crude protein extract.

[0041] SDS-PAGE electrophoresis was used to identify the crude protein extract of the hawkbill peach. The electrophoresis target band was cut into a centrifuge tube, and the gel slice was washed with ammonium bicarbonate solution and decolorized at 37°C. After removing the liquid, 100% acetonitrile (ACN) was added and shaken until the gel particles turned white. After air drying for 10 minutes, the gel pieces were treated with dithiothreitol, reacted at 56°C for 1 hour, and the liquid was removed. Acetyl iodamine was added and treated in the dark for 45 minutes, the liquid was removed and washed repeatedly with ammonium bicarbonate solution. Acetonitrile was added until the gel particles turned white and dried until completely dehydrated. The gel slices were transferred to an ammonium bicarbonate solution containing 0.1 mg / mL trypsin and placed on ice for rehydration for 30 minutes; the remaining liquid was aspirated, and the ammonium bicarbonate solution was added until the gel particles were submerged, and incubated at 37°C for 12 to 18 hours; ultrasonic for 15 minutes, the supernatant was aspirated and transferred to a new centrifuge tube, vacuum dried, dissolved with 0.1% formic acid, and analyzed by liquid chromatography. The peptide mass spectra were compared with the protein database to identify the allergen proteins of the hawkbill peach.

[0042] 2. Identification of allergen proteins in hawkbill peach:

[0043] 2.1. SDS-PAGE protein electrophoresis was used to identify the crude protein extract of hawkbill peach. The identification results of SDS-PAGE protein electrophoresis on the crude protein extract of hawkbill peach are as follows: Figure 1 As shown. Figure 1It can be seen that the crude protein composition of the hawkbill peach is relatively complex and the content is low. The protein molecular weight is 10-80kDa, among which the protein with a higher content is distributed in the molecular weight range of 10-15kDa.

[0044] 2.2. Liquid chromatography was used to analyze the crude protein extract of hawkbill peach. The analysis results of the crude protein extract of hawkbill peach by liquid chromatography are as follows: Figure 2 The UniProt database was used to perform sequence comparison analysis on the proteins identified by mass spectrometry, and the allergen protein Q2 of the hawkbill peach was obtained. Figure 2 It can be seen that the allergen protein Q2 of the hawkbill peach is composed of 160 amino acids and has a molecular weight of 17.6kDa.

[0045] Example 2: Exogenous expression of pyracantha allergen protein

[0046] 1. Exogenous expression of hawkbill peach allergen protein:

[0047] The gene sequence of the hawksbill allergen protein Q2 obtained in Example 1 was inserted between the SfiI and SfiI restriction sites of the pComb3xsss vector, and the ligation product was transformed into Escherichia coli BL21 (DE3) by chemical transformation. The plate was inverted and cultured at 37°C for 12 h, and shaken overnight; 7.5 mL of the overnight cultured bacterial solution was inoculated into 750 mL of LB liquid culture medium (the final concentration of the ampicillin solution was 100 μg / mL), and the culture was placed at 37°C and shaken at 250 rpm for 2 to 3 h. When the OD600 is 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 1 mmol / L, and continue shaking culture at 37°C overnight; after overnight culture, centrifuge at 8000rpm for 20min, collect the bacteria and resuspend them with 5mL of TES solution (formula: 85.575g sucrose, 12.1g trishydroxymethylaminomethane and 0.093g ethylenediaminetetraacetic acid, add 450mL deionized water to dissolve, adjust the solution to pH 8.0 with concentrated hydrochloric acid, add deionized water to 500mL to prepare TES solution), freeze at -80°C for 6h; thaw on ice, add 15mL of 5-fold diluted TES solution, mix at 4°C for 4h, and centrifuge at 12000rpm for 30min to obtain the periplasmic secretion expression solution containing the hawkbill peach allergen protein Q2.

[0048] 1 mL of Ni-NTA filler was added to the periplasmic secretion expression solution containing the allergen protein Q2 of the chickpea, mixed at 4°C for 12 hours, and then added to the gravity purification column, rinsed with 20 mL of PBS buffer, and then eluted with gradient concentrations of imidazole solution (PBS solution containing 10, 20, 50, 100, 200 mM (mmol / L) imidazole), and the allergen protein Q2 of the chickpea was identified by SDS-PAGE. The eluate containing the allergen protein Q2 of the chickpea was collected, dialyzed with PBS solution at 4°C for 3 days, and its concentration was determined. After aliquoting, it was stored at -20°C for later use.

[0049] 2. Results of exogenous expression of hawkbill peach allergen protein:

[0050] The present invention constructs a recombinant vector of the allergen protein Q2 of the chickpea. The recombinant vector of the allergen protein Q2 of the chickpea is as follows: Figure 3 The results of SDS-PAGE identification of the allergen protein Q2 of the hawkbill peach are shown in Figure 4 As shown. Figure 4 It can be seen that the allergen protein Q2 of the hawksbill peach can be correctly expressed in Escherichia coli, with a high purity after purification. The protein molecular weight is about 22kDa, which is consistent with the theoretical molecular weight of its amino acids.

[0051] Example 3: Activity analysis of hawkbill peach allergen protein

[0052] 1. Experimental methods:

[0053] The activity of the natural allergen protein and recombinant allergen protein Q2 of hawkbill peach was detected by enzyme-linked immunosorbent assay (ELISA). The natural allergen extract in Example 1 and the recombinant allergen protein Q2 prepared in Example 2 were used to coat 4 μg / mL of the natural allergen and the recombinant allergen protein Q2 in the microwells of the ELISA plate. After coating at 4°C overnight, 120 μL of 5% (mass volume ratio) skimmed milk powder solution was added to each well and blocked at 37°C for 3 hours. After drying the liquid in the wells, bake at 37°C for 1h for use; add 100 μL of 1000-fold diluted allergen antibody (the hawksbill peach allergen protein Q2 antibody is prepared by immunizing rabbits with the recombinant hawksbill peach allergen protein Q2 obtained in Example 2) to the microwells, incubate at 37°C for 30-50min, pour out the liquid in the wells, wash 5 times with detergent, and pat dry on absorbent paper; add 100 μL of 5000-fold diluted HRP-labeled goat anti-rabbit secondary antibody solution to the microwells, incubate at 37°C for 30-50min, wash 5 times with detergent, and pat dry; add 100 μL of substrate solution (Beijing Solebow Technology Co., Ltd., product number PR1210) to the microwells, shake and mix, incubate at 37°C for 30-50min, and measure the absorbance of each well at a wavelength of 450nm.

[0054] 2. Experimental results:

[0055] The results of the activity analysis of the allergen protein of the hawkbill peach are as follows Figure 5 As shown. Figure 5 It can be seen that there is no significant difference in the activity of the natural allergen protein of hawkthorn and the recombinant allergen protein Q2, indicating that the recombinant protein Q2 of hawkthorn obtained by the Escherichia coli expression system has good biological activity.

[0056] Example 4: Stability analysis of hawkbill peach allergen protein

[0057] 1. Experimental methods:

[0058] The stability of the recombinant allergen protein Q2 from Fructus chinensis obtained in Example 2 was detected by enzyme-linked immunosorbent assay (ELISA), and the biological activity of the recombinant allergen protein Q2 in PBS solutions with different pH values ​​(4.0, 5.0, 6.0, 7.0, 7.4, 8.0, 9.0 and 10.0), PBS solutions with different salt ion concentrations (0.005, 0.01, 0.02 and 0.04 mol / L), PBS solutions with different methanol concentrations (1%, 2%, 4%, 6%, 8% and 10%), PBS solutions with different ethanol concentrations (1%, 2%, 4%, 6%, 8% and 10%), and PBS solutions with different acetonitrile concentrations (1%, 2%, 4%, 6%, 8% and 10%) were analyzed. 4 μg / mL of the recombinant allergen protein Q2 of hawksbill peach was coated in the microwells of the ELISA plate. After coating at 4°C overnight, 120 μL of 5% (mass volume ratio) skimmed milk powder solution was added to each well and blocked at 37°C for 3 hours. After drying the liquid in the wells, bake at 37°C for 1h for use; add 100 μL of 1000-fold diluted allergen antibody (the hawksbill peach allergen protein Q2 antibody is prepared by immunizing rabbits with the recombinant hawksbill peach allergen protein Q2 obtained in Example 2) to the microwells, and use PBS solutions under different conditions for dilution, incubate at 37°C for 30-50min, pour out the liquid in the wells, wash 5 times with detergent, and pat dry on absorbent paper; add 100 μL of 5000-fold diluted HRP-labeled goat anti-rabbit secondary antibody solution to the microwells, incubate at 37°C for 30-50min, wash 5 times with detergent, and pat dry; add 100 μL of substrate solution (Beijing Solebow Technology Co., Ltd., product number PR1210) to the microwells, shake and mix, incubate at 37°C for 30-50min, and measure the absorbance of each well at a wavelength of 450nm.

[0059] 2. Experimental results:

[0060] The stability analysis results of the allergen protein of hawkbill peach are as follows Figure 6 As shown. Figure 6It can be seen that the recombinant allergen protein Q2 of hawkbill peach has good biological activity in the range of pH 7.0-9.0. The activity is reduced to a certain extent under the addition of acid or alkali, but it can still maintain more than 80% biological activity. Under the conditions of 0.01mol / L and 0.02mol / L PBS, the recombinant allergen protein Q2 of hawkbill peach can maintain good biological activity; low ion concentration and high ion concentration have a certain inhibition on its activity. PBS solution containing 1%-8% methanol can promote its biological activity to a certain extent, and 10% methanol has a slight inhibitory effect on its activity; PBS solution containing 1%-4% ethanol can promote its biological activity to a certain extent, and 6%-10% ethanol has different degrees of inhibition on its activity.

[0061] Example 5: Immunoassay analysis of hawkbill peach allergen protein

[0062] 1. Experimental methods:

[0063] An immunoassay analysis method for allergen protein of hawkthorn was established by enzyme-linked immunosorbent assay (ELISA). 4 μg / mL of the recombinant hawkthorn allergen protein Q2 prepared in Example 2 was coated in the microwells of the ELISA plate. After coating at 4°C overnight, 120 μL of 5% (mass-to-volume ratio) skimmed milk powder solution was added to each well and sealed at 37°C for 3 hours. After drying the liquid in the wells, the wells were dried at 37°C for 1 hour for use; 50 μL of a series of concentrations (1000, 333.33, 111.11, 37.03, 12.34, 4.11, 1.37, 0.45, 0.15, 0 μg / mL) of recombinant allergen protein Q2 solution or the sample solution to be tested was added to the microwells of the ELISA plate, and 50 μL of 1000-fold diluted allergen antibody (the hawkthorn allergen protein Q2 antibody was prepared by immunizing rabbits with the recombinant hawkthorn allergen protein Q2 prepared in Example 2) was added to the microwells. Prepared by microwells), incubate at 37℃ for 30-50min, pour out the liquid in the wells, wash 5 times with washing solution, and pat dry on absorbent paper; add 100μL of 5000-fold diluted HRP-labeled goat anti-rabbit secondary antibody solution to the microwells, incubate at 37℃ for 30-50min, wash 5 times with washing solution, and pat dry; add 100μL of substrate solution (Beijing Solebow Technology Co., Ltd., item number PR1210) to the microwells, shake and mix, incubate at 37℃ for 30-50min, and measure the absorbance of each well at a wavelength of 450nm. Draw a standard curve with the absorbance value of each standard concentration well as the ordinate and the log10 value of the drug standard solution concentration as the abscissa, and perform curve fitting y=(AD) / [1+(x / C)B]+D. Among them, A and D represent the absorbance values ​​when the concentration of recombinant allergen protein Q2 is minimum and maximum, respectively, and C is the midpoint concentration; when the concentration of recombinant allergen protein Q2 is equal to C, the absorbance value is (A+D) / 2, which is at the inflection point of the curve, and the half inhibition concentration is IC 50, B represents the steepness of the curve, called the slope factor; IC 10 The detection limit is IC 20 ~IC 80 The content of allergen protein Q2 in the sample to be tested can be calculated based on the absorbance value of the sample to be tested.

[0064] 2. Experimental results:

[0065] The results of immunoassay analysis of the allergen protein Q2 of the hawkbill peach are as follows Figure 7 As shown. Figure 7 It can be seen that the detection limit of the established ELISA analysis method for the allergen protein Q2 of the hawkbill peach is 1.8μg / mL, the half inhibition concentration is 36.3μg / mL, the linear range is 5.9-224.4μg / mL, and the inhibition rate has a significant S-shaped curve relationship with the logarithmic value of the allergen protein Q2. The correlation coefficient R 2 The value was 0.997. This method can directly detect the content of allergen protein Q2 in food, and has the advantages of high sensitivity and high specificity.

[0066] The above are only further embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which belong to the protection scope of the present invention.

Claims

1. A chickpea allergen protein Q2, characterized in that: The amino acid sequence of the hawksbill peach allergen protein Q2 is shown in SEQ ID NO:

1.

2. The allergen protein Q2 of hawkthorn fruit according to claim 1, characterized in that: The antigenic epitopes of the chickpea allergen protein Q2 are 13-15 (EIP), 62-64 (GSQ), 77-79 (KEN), 95-96 (NL), 124-125 (KG), 128-130 (EIK), and 155-157 (HPD).

3. A gene encoding a hawkbill peach allergen protein Q2, characterized in that: The nucleotide sequence of the gene encoding the citrus aurantium allergen protein Q2 is shown in SEQ ID NO:

2.

4. A recombinant vector of hawkbill peach allergen protein Q2, characterized in that: The recombinant vector comprises the gene encoding the hawkthorn allergen protein Q2 according to claim 3.

5. An expression system comprising the gene encoding the allergen protein Q2 of the hawksbill peach according to claim 3 or the recombinant vector of the allergen protein Q2 of the hawksbill peach according to claim 4, characterized in that: The expression system includes but is not limited to an E. coli expression system or a yeast expression system.

6. Use of the hawksbill peach allergen protein Q2 as claimed in claim 1 in the preparation of immunological diagnostic reagents, food allergen diagnostic reagents or diagnostic kits.

7. A kit for detecting the allergen protein Q2 of hawkbill peach, characterized in that: The kit comprises the hawkthorn allergen protein Q2 according to claim 1.

8. The kit for detecting the allergen protein Q2 of the hawksbill peach according to claim 7, characterized in that: The kit also contains an antibody against the allergen protein Q2 of the hawksbill peach, an enzyme-labeled secondary antibody, a substrate solution, a calibrator and / or a quality control product.

9. The kit for detecting the allergen protein Q2 of the hawksbill peach according to claim 8, characterized in that: The citrus allergen protein Q2 antibody is prepared by immunizing rabbits, mice or camelids with the citrus allergen protein Q2 according to claim 1.

10. The kit for detecting the allergen protein Q2 of the hawksbill peach according to claim 8, characterized in that: The enzyme-labeled secondary antibody is an HRP-labeled goat anti-rabbit or mouse or camel IgG secondary antibody.

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