Chenopodium glaucum pollen allergen and application thereof

Abstract

The invention discloses a chenopodium glaucum pollen allergen and application thereof. The technical problem to be solved is to provide a novel chenopodium glaucum pollen allergen, and the novel chenopodium glaucum pollen allergen is used for diagnosing and treating chenopodium glaucum pollen allergy. The pollen allergen is a protein with an amino acid sequence as shown in SEQ ID No: 2. The compound can be used for diagnosing pollen allergy or preventing, treating or improving pollen allergy of subjects.

Description

Technical Field

[0001] The present invention particularly relates to Chenopodium album pollen allergen and application thereof. Background Art

[0002] Chenopodium album is an annual herbaceous plant belonging to the genus Chenopodium in the Chenopodium family. It has a wide distribution range and strong adaptability to the environment. It is mainly found in temperate and tropical zones around the world and throughout China. It is commonly found in wilderness, fields, roadsides, and slightly saline-alkali areas. Chenopodium album pollen blooms from June to October each year. Its pollen is abundant and is mainly spread by wind. It is one of the most common inhalation pollen allergens in summer and autumn. Currently, relevant research on pollen allergic diseases has been reported in Europe, North America, and semi-desert countries. Pollen allergies have certain regional and seasonal characteristics, resulting in differences in their allergenicity and prevalence. In northern my country, Chenopodium album pollen ranks first among allergenic pollens in summer and autumn.

[0003] Currently, three C. quinoa allergens have been reported internationally: Che a 1, Che a 2, and Che a 3. Che a 1 is a 143-amino acid glycoprotein with a molecular mass of 17 kDa, belonging to the Ole e 1 family of proteins, with which it shares 27-45% similarity. Che a 2, with a molecular mass of 14.4 kDa, contains 133 amino acids and belongs to the profilin family, with 75% similarity to pollen profilins and 82% similarity to food profilins. Che a 3, with a molecular mass of 9.5 kDa, contains 86 amino acids and belongs to the polcalcin family, with which it shares 65-82% similarity. Subsequently, numerous researchers have cloned, purified, and characterized Che a 1, Che a 2, and Che a 3 by immunochemical methods. However, no research has been reported in China on the identification of C. quinoa pollen allergens, suggesting the possibility of unknown allergens.

[0004] The yeast surface display system belongs to a multivalent eukaryotic expression system. It inserts the foreign gene into the N-terminus of the yeast display vector Aga2 and transfers it into the Saccharomyces cerevisiae cells for induced expression. Under the action of the signal peptide, the foreign protein and Aga2p are transported to the outside of the yeast cell in the form of a fusion protein. It binds to the Aga1p anchored on the surface of the yeast cell through a disulfide bond, thereby displaying the foreign protein on the surface of the Saccharomyces cerevisiae cell. 4 ~10 5copies. Recently, a new method for rapid high-throughput extracellular antigen analysis (REAP) was reported. The study used yeast surface display technology to display 2,688 human extracellular proteins on the surface of yeast cells, screened them using immunoglobulins in patient serum, and identified autoantibodies targeting extracellular proteins through high-throughput sequencing. Several autoantibodies for autoimmune polyglandular syndrome type 1 (APS-1) and systemic lupus erythematosus (SLE) were newly discovered in autoimmune patients. The yeast display system was used to screen individuals infected with SARS-CoV-2, clarifying the mechanism of autoantibodies in COVID-19 infection. In addition, some researchers constructed a yeast surface display platform for synthesizing nanoantibody libraries for screening nanoantibodies against the fibrinogen (Fg) binding domain of Staphylococcus aureus clumping factor A221-550 (ClfA221-550).

[0005] In recent years, the identification of allergens has combined technologies such as proteomics, transcriptomics, immunology, and protein mass spectrometry. This study is the first to apply a yeast surface display system to the identification of plant pollen allergens. Proteomics was used to preliminarily identify allergenic proteins in C. truncatum pollen. Combined with protein mass spectrometry, potential allergenic molecules in C. truncatum pollen were predicted. After cloning potential allergen genes by RT-PCR, a yeast surface display library was constructed. Yeast cells that specifically bound to IgE in the serum of patients allergic to C. truncatum were sorted by flow cytometry, and the C. truncatum pollen allergen was identified by high-throughput sequencing analysis. This study provides technical support for the identification of plant pollen allergens, a scientific basis for desensitization treatment of patients allergic to C. truncatum, and improves local allergy epidemiological data. Therefore, how to obtain a C. truncatum pollen allergen and use it for the diagnosis and treatment of C. truncatum pollen allergy is a technical challenge faced by researchers in this field. Summary of the Invention

[0006] The technical problem solved by the present invention is to provide a new Chenopodium album pollen allergen and use the new allergen for the diagnosis and treatment of Chenopodium album pollen allergy.

[0007] In order to solve the above technical problems, the present invention provides a polypeptide, wherein the polypeptide is any of the following:

[0008] A1) a polypeptide having an amino acid sequence as SEQ ID No: 2;

[0009] A2) a polypeptide having more than 80% identity with the polypeptide of A1) and having the same function as the polypeptide of A1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence of SEQ ID No: 2;

[0010] A3) A fusion polypeptide formed by the polypeptide described in A1) or A2) and a tag for detection or a tag for purification.

[0011] In order to solve the above technical problems, the present invention also provides a use of a polypeptide in preparing a product for diagnosing pollen allergy in a subject, wherein the polypeptide is any one of the following:

[0012] A1) a polypeptide having an amino acid sequence as SEQ ID No: 2;

[0013] A2) a polypeptide having at least 80% identity with the polypeptide of A1) and having the same function as the polypeptide of A1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence of SEQ ID No: 2;

[0014] A3) A fusion polypeptide formed by the polypeptide described in A1) or A2) and a tag for detection or a tag for purification.

[0015] In the above-mentioned use, the polypeptide acts as an allergen in the product;

[0016] In the above-mentioned use, the product is an allergen detection kit;

[0017] In the above use, the product further comprises an additional allergen, wherein the additional allergen is one or more polypeptides selected from the group consisting of Chea 1, Chea 2 and Chea 3, or any combination thereof;

[0018] Further preferably, the amino acid sequence of the Chea 1 polypeptide is shown as SEQ ID No: 14, the amino acid sequence of the Chea 2 polypeptide is shown as SEQ ID No: 8, and the amino acid sequence of the Chea 3 polypeptide is shown as SEQ ID No: 10.

[0019] In the above-mentioned use, the allergen detection kit is used for skin prick test or skin patch test.

[0020] The above-mentioned use is characterized in that, in the product, the polypeptide is in a pharmaceutically acceptable carrier;

[0021] In the above-mentioned use, the carrier is an aqueous solution.

[0022] The above-mentioned use is characterized in that, when the product is used for skin patch test, the polypeptide solution is added to a carrier containing an adsorption material before use to prepare a skin patch.

[0023] The use according to any one of the above items, characterized in that the subject is a mammal;

[0024] In the above-mentioned use, the mammal is a human, a non-human primate, a pet or an experimental animal;

[0025] In the above-mentioned use, the experimental animal is selected from one of the following: rodents, cats, dogs, pigs, and monkeys.

[0026] In the above-mentioned use, the product is an allergen test kit, the subject has a history of pollen allergy, and the product is used in a skin prick test to diagnose whether the subject is allergic to Chenopodium album pollen.

[0027] In the above-mentioned use, the subject has a history of allergy to Chenopodium album, and the product contains the polypeptide and multiple additional Chenopodium album allergens, and the multiple additional Chenopodium album allergens include: Chea 1, Chea 2 and Chea 3 polypeptides, and the product is used to diagnose which one or more Chenopodium album allergens the subject is allergic to among the polypeptide according to claim 1, the Chea 1, the Chea 2 and the Chea 3. Currently, the most common seasonal allergic reaction is type I hypersensitivity, which is an abnormal reaction phenomenon in which the human immune system produces excessive immunity to external allergens. It is an abnormal immune response with the main outcome of causing physiological dysfunction, which will cause certain harm to the human body. The mechanism of type I hypersensitivity is that the secreted IgE has a high affinity with the IgE receptors on the surface of basophils and mast cells, causing the cells to become sensitized to specific allergens. When the allergen enters the body again, it forms a "bridge" with the specific IgE on the surface of mast cells, causing the release of histamine and inflammatory factors. Allergens are high molecular weight (HMW) type I sensitizers, which can cause allergic reactions in the human body. They are macromolecules that produce specific IgE responses.A large number of studies have shown that the binding of pollen proteins to IgE is the core mechanism of allergic reactions. Pollen allergy can be diagnosed by detecting the level of specific IgE antibodies in the serum of allergic patients (Wang Min, Xing Zhimin, Feng Wei, et al. Clinical study of serum total IgE and specific IgE levels in patients with allergic rhinitis allergic to Artemisia pollen. Journal of Clinical Otorhinolaryngology Head and Neck Surgery, 2006(20):3. Xu Yang, Liao Yongmei, Zhong Jianqiao. Detection of serum pollen allergen-specific IgE in patients with papular urticaria. Journal of Southwest Medical University, 2010, 33(3):261-263. Orengo JM, Radin A R, Kamat V, et al. Treating cat allergy with monoclonal IgG antibodies that bind allergen and prevent IgE engagement[J]. Nature Communications, 2018, 9(1):1421. Shade KTC, Conroy ME, Washburn N, et al. Sialylation of immunoglobulin Eis a determinant of allergic pathogenicity. Nature, 2020, 582, 7811. Raúl Porras-Gutiérrez-de-Velasco, Maruthukunnel-Mani B, Vizuet-De-Rueda JC, et al. Patterns of allergen recognition in Ligustrum polysensitized patients: Animmunoproteomics approach. Allergy, 2024,79(7):6.).

[0028] In the above-mentioned use, the product is used to detect whether specific IgE antibodies that specifically bind to the polypeptide exist in a sample from a subject.

[0029] In the above use, the subject has no history of pollen allergy.

[0030] In the above use, if specific IgE to the polypeptide is present in the sample from the subject, it indicates that the subject is allergic to Chenopodium album pollen.

[0031] In the above-mentioned use, the sample is body fluid;

[0032] In the above use, the body fluid is selected from at least one of the following: whole blood, serum and plasma.

[0033] In the above-mentioned use, in the product, the polypeptide is in an aqueous solution or is immobilized on a solid support;

[0034] In the above-mentioned use, the solid phase carrier is a glass slide or magnetic beads.

[0035] Among the above uses, the product is used for radioallergosorbent assay, enzyme-linked immunosorbent assay, fluorescent immunoassay, Western blotting, immunocapture assay, UniCAP assay, microarray chip assay and biochip assay.

[0036] In the above-mentioned use, the enzyme-linked immunosorbent assay is a sandwich ELISA enzyme-linked immunosorbent assay. The terms "allergen" and "allergen" have the same meaning.

[0037] The term "allergen" refers to a substance that can cause an allergic reaction in the body. Allergens include, but are not limited to, microorganisms, mites, parasites, pollen, xenobiotic serum, drugs, and chemicals.

[0038] In the above-mentioned polypeptides, the protein tag refers to a polypeptide or protein that is fused and expressed with the polypeptide of interest using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the protein of interest. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, among others.

[0039] In the above-mentioned polypeptides, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0040] In the above polypeptides, the above 80% identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0041] In the above polypeptide, sequence 2 (SEQ ID No. 2) consists of 133 amino acid residues. It is named HL637 protein or protein HL637 or HL637 recombinant protein or recombinant protein HL637. Its encoding gene is HL637 gene.

[0042] In order to solve the above technical problems, the present invention also provides the use of the above polypeptide in preparing a product for diagnosing pollen allergy and / or in preparing a product for detecting whether a sample to be tested contains antibodies that specifically bind to the polypeptide.

[0043] In the above, the products include but are not limited to reagents, kits and detection devices.

[0044] The term "kit" refers to any delivery system used to deliver substances. In assays, such delivery systems include systems for storing, transporting, or delivering reagents (e.g., oligonucleotides, enzymes, polypeptides, etc. in appropriate containers) and / or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more housings (e.g., boxes) containing the relevant reagents and / or support materials.

[0045] The term "diagnosis" refers to any type of procedure intended to obtain information useful in assessing whether a patient is suffering from or is likely to be suffering from a disease or condition at the time of diagnosis or in the future compared to the average or comparison subjects (the latter preferably having similar symptoms) to discover how the disease progresses or is likely to progress in the future or to assess the responsiveness of the patient to a treatment (e.g., administration of a suitable drug, such as a drug for desensitizing an allergic patient). In other words, the term "diagnosis" includes not only diagnosis, but also prediction and / or monitoring of the course of a disease or condition or the success of a treatment. Preferably, autoantibodies of the IgG or IgE class are detected for monitoring the progression of the disease and / or the success of a treatment.

[0046] The term "diagnosis" preferably does not mean that the diagnostic method or reagent according to the present invention will be conclusive and sufficient to complete the diagnosis based on a single test (let alone a parameter), but may refer to a contribution to what is known as "differential diagnosis" (i.e., a systematic diagnostic procedure that considers the likelihood of a series of possible conditions based on a series of diagnostic parameters). The term "diagnosis" may also refer to a method or reagent for selecting the most promising treatment regimen for a patient. In other words, the method or agent may relate to selecting a treatment regimen for a subject.

[0047] The subject according to the present invention is an animal, including a human, that produces antibodies, preferably allergy-related antibodies of the IgE class or equivalent, more preferably antibodies from a mammal, most preferably a human.

[0048] In preferred embodiments, the term "variant" as used herein may refer to at least a fragment of the referenced full-length sequence, more specifically, an amino acid or nucleic acid sequence that is truncated by one or more amino acids at one or both ends relative to the full-length sequence. Such a fragment comprises or encodes a peptide having at least 10, 15, 25, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive amino acids of the original sequence, or a variant thereof. The total length of the variant may be 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids. A preferred fragment is SEQ ID NO: 2 or a variant thereof.

[0049] Variants may be at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to a reference amino acid sequence or a fragment thereof, wherein amino acids other than those essential for biological activity are deleted or substituted and / or one or more such essential amino acids are replaced in a conservative manner and / or amino acids are added or deleted such that the biological activity of the polypeptide is at least partially retained. The prior art includes various methods that can be used to compare two given nucleic acid or amino acid sequences and calculate the degree of identity, see, for example, Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition. In a preferred embodiment, ClustalW software (Larkin, MA, Blackshields, G., Brown, NP, Chenna, R., McGettigan, PA, McWilliam, H., Valentin, F., Wallace, IM, Wilm, A., Lopez, R., Thompson, JD, Gibson, TJ, Higgins, DG (2007): Clustal W. and Clustal X. version 2.0. Bioinformatics, 23, 2947-2948.) is used with default settings.

[0050] In a preferred embodiment, the variant may further comprise chemical modifications, such as isotope labeling or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, etc. Those skilled in the art are familiar with methods for modifying polypeptides. In addition, variants can also be generated by fusion with other known polypeptides or variants thereof.

[0051] Essentially, the polypeptide variants possess biological activity. In a preferred embodiment, such biological activity is the ability to bind allergy-specific IgE antibodies from a patient, preferably a patient suffering from an allergy, more preferably an allergy to pollen, most preferably an allergy to pollen. In another preferred embodiment, such biological activity is the ability to elicit an allergic reaction in a subject allergic to pollen, preferably C. chenopodium pollen, more preferably C. chenopodium pollen.

[0052] One skilled in the art can design variants by starting with the original T. gondii pollen protein, introducing modified point mutations, truncations, etc., and then confirming that the variant still has biological activity by testing whether the variant binds to antibodies in samples obtained from subjects allergic to T. gondii pollen.

[0053] Variants can be prepared using genetic engineering methods or chemical synthesis methods.

[0054] Binding activity can be determined by Western blotting using serum from patients allergic to T. gondii pollen (preferably as described in the Examples) as a source of antibodies.

[0055] The polypeptide according to the present invention or its variant may be a fusion protein, i.e., a polypeptide fused to another sequence (preferably at the N-terminus or C-terminus), optionally via a linker. The other sequence may be a tag for detection, such as a FLAG tag, or a tag for purification, such as one selected from the group consisting of a GST, His, or MBP tag. An exemplary fusion polypeptide according to the present invention is shown in SEQ ID NO: 2.

[0056] Polypeptide according to the present invention can be provided in any form and with any degree of purification, from tissues, fruits or cells comprising the polypeptide in endogenous form, more preferably cells overexpressing the polypeptide, the crude or enriched lysate of such cells to purified and / or isolated polypeptide (which is substantially pure). In a preferred embodiment, the polypeptide is a native polypeptide, wherein the term "native polypeptide" as used herein refers to a folded polypeptide, more preferably from a tissue or cell, more preferably from a mammalian cell or tissue, optionally from a folded polypeptide purified from non-recombinant tissue or cells. If a native polypeptide is used, it is preferably enriched compared to its native state.

[0057] According to the present invention, the polypeptide can be a recombinant protein, wherein the term "recombinant" as used herein refers to a polypeptide produced using genetic engineering methods at any stage of the production process, such as by fusing a nucleic acid encoding the polypeptide to a strong promoter for overexpression in a cell or tissue or by engineering the sequence of the polypeptide itself and / or using expression in cells modified by genetic engineering. Those skilled in the art are familiar with methods for engineering nucleic acids and encoded polypeptides (e.g., as described in Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning, CSH or Brown T.A. (1986), Gene Cloning—an introduction, Chapman & Hall) and methods for producing and purifying natural or recombinant polypeptides (e.g., Handbooks "Strategies for Protein Purification", "Antibody Purification" published by GE Healthcare Life Sciences, and described in Burgess, RR, Deutscher, MP (2009): Guide to Protein Purification).

[0058] In a preferred embodiment, the term "isolated" means that the polypeptide has been enriched with respect to all C. chenopodium pollen proteins, preferably C. chenopodium pollen allergens, in pollen or a whole pollen extract obtained from whole pollen. In another more preferred embodiment, the polypeptide is an isolated polypeptide, wherein the term "isolated" means that the polypeptide has been enriched with respect to its state after production using biotechnological or synthetic methods and is preferably pure, i.e. at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the polypeptide used consists of said polypeptide as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection.

[0059] The subject according to the present invention is an animal, including a human, that produces antibodies, preferably allergy-related antibodies of the IgE class or equivalent, more preferably antibodies from a mammal, most preferably a human.

[0060] The method according to the present invention contemplates detecting antibodies that bind to a polypeptide according to the present invention in a sample, preferably for diagnosing pollen allergy, more preferably for diagnosing allergy to C. chenopodium pollen. Those skilled in the art are familiar with various methods that can be used to detect antibodies in a sample, for example, selected from the group consisting of immunodiffusion techniques, basophil activation via IgE antibodies, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays such as radiolabeled immunoassays, enzyme immunoassays such as colorimetric assays, chemiluminescent immunoassays, and immunofluorescence techniques.

[0061] In many cases, the absence or presence of the antibody in the test sample (optionally meaning to determine whether the concentration of the antibody exceeds certain threshold values, often shown by detection limit) is sufficient for diagnosis. If the antibody can be detected, this will be useful information for the clinician's diagnosis, and shows that the patient suffers from an increased likelihood of allergy. In a preferred embodiment, the relative concentration of the antibody in the serum can be determined relative to the level that may be found in average healthy subjects. In a preferred embodiment, the term "detection presence or absence" as used herein means being sufficient to check whether a suitable complex detection method can be used to detect the signal that is sufficient to exceed any background level, and the complex detection method indicates that the target antibody exists or there is more target antibody than that present in healthy subjects. In a more preferred embodiment, this can include determining whether the concentration exceeds the concentration in the control or exceeds a threshold value, preferably at least 0.1, preferably 0.2, 0.5, 1, 2, 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000 times higher than the concentration of the target antibody found in average healthy subjects.

[0062] Detection of antibodies may include the following steps:

[0063] Step a) comprises contacting a sample comprising the antibody with a polypeptide according to the invention or a variant thereof under conditions suitable for the formation of a complex. This can be achieved by contacting a liquid sample with the polypeptide in an immobilized form (e.g., fixed to a membrane, such as used in a Western blot or line blot). When the sample and polypeptide are contacted, any antibodies present in the sample that are capable of specifically binding to the polypeptide will undergo such binding to the effect that a complex comprising the polypeptide and the antibody is formed;

[0064] and / or;

[0065] step b) comprising isolating the complex formed in step a), for example by removing the sample and optionally washing the immobilized polypeptide with a washing buffer;

[0066] And / or; step c), which comprises detecting the complex, for example by adding a second antibody that can bind to the antibody from the sample. The second antibody can contain a label, such as an enzyme or a radioisotope. Alternatively, the complex can be directly detected using methods such as chemical cross-linking followed by SDS-PAGE or using mass spectrometry.

[0067] The detection of antibodies against the polypeptide according to the present invention indicates that the subject who provided the sample is allergic to G. chenopodium pollen.

[0068] Within the scope of the present invention are diagnostically useful carriers comprising means for specifically capturing antibodies against the polypeptides of the present invention. In preferred embodiments, the term "specific capture antibody" as used herein refers to the ability to specifically bind to the target antibody to the effect of binding and removing it from the sample while other antibodies are substantially unbound and retained in the sample. According to the present invention, means are provided for the specific detection of captured antibodies, optionally as part of a kit. In preferred embodiments, the term "specific detection of captured antibodies" as used herein means that after capture, the antibody of the means that specifically binds to the specific capture antibody (which binds to the polypeptide according to the present invention) is detected, but not any other antibodies present in the sample. In preferred embodiments, the term "specific binding" as used herein means that the binding is stronger than a binding reaction characterized by the following dissociation constant: 1×10 -5M , more preferably 1×10 -7M , more preferably 1×10 -8M , more preferably 1×10 -9M , more preferably 1×10 -10M , more preferably 1×10 -11M , more preferably 1×10 -12M , as determined by surface isoscillator resonance using a Biacore instrument at 25°C in PBS buffer, pH 7.

[0069] In the above, the antibody is an IgG antibody or an IgE antibody.

[0070] In the above use, the sample to be tested contains the body fluid of the subject's antibodies.

[0071] In the above use, the body fluid containing the subject's antibodies is selected from at least one of the following: whole blood, serum, cerebrospinal fluid and saliva.

[0072] In the above, the body fluid containing the subject's antibodies is selected from whole blood.

[0073] In the above, the antibody is an IgG antibody or an IgE antibody. Preferably, the IgG is IgG4.

[0074] In the above uses, the product also includes a diagnostically useful carrier.

[0075] In the above, the diagnostically useful carrier is selected from at least one of the following: beads, test strips, microtiter plates, and blots. Preferably, the carrier is selected from at least one of the following: Western blots, line blots, and dot blots, glass surfaces, slides, biochips, membranes, microarrays, electrophoresis gels, and microtiter plates.

[0076] Preferably, the diagnostically useful carrier is a solid phase carrier for contacting a means for specifically capturing antibodies (said means being associated with said carrier) with a body fluid sample from a subject, preferably a mammalian subject, more preferably a human subject. The carrier can be directly in contact with the sample and allows the means for specifically capturing antibodies to be exposed to any antibodies in the sample. In a preferred embodiment, the solid phase carrier is a diagnostic device, more preferably selected from the group comprising beads, test strips, microtiter plates, blots, preferably selected from the group comprising Western blots, line blots and dot blots, glass surfaces, slides, biochips, membranes, microarrays, electrophoresis gels and microtiter plates.

[0077] More preferably, diagnostically useful carriers are line blots (Raoult, D., and Dasch, GA (1989), The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. J. Immunol. Methods, 125 (1-2), 57-65; WO2013041540). In a preferred embodiment, the term "line blot" as used herein refers to a test strip coated with one or more tools (preferably each a polypeptide) for capturing antibodies, more preferably based on a membrane. If two or more tools are used, they are preferably spatially separated on the carrier. Preferably, the width of the band is at least 30% of the width of the test strip, more preferably 40%, 50%, 60%, 70% or 80%. The test strip may include one or more control bands for confirming that it has been in contact with the sample for a sufficiently long time under appropriate conditions, particularly in the presence of human serum, antibody conjugates or both. Many line blots are commercially available, for example from EUROIMMUN AG, Lübeck, Germany.

[0078] In the present application, for implementing the sample of the present invention comprises antibody, also referred to as immunoglobulin.Usually, sample is the body fluid of the representative set of whole immunoglobulins comprising experimenter.However, after sample is provided, sample can undergo further processing, and this can comprise whole immunoglobulins or any immunoglobulin class of fractionation, centrifugation, enrichment or separation experimenter, preferably IgE and / or IgG (preferably, IgG4), which can affect the relative distribution of various types of immunoglobulins.Sample can be selected from whole blood, serum, cerebrospinal fluid and saliva, preferably serum.In the most preferred embodiment, sample comprises IgE or IgG class antibody.

[0079] Within the scope of the present invention are diagnostically useful carriers comprising means for specifically capturing antibodies directed against the polypeptides of the present invention. In preferred embodiments, the term "specific capture antibody" as used herein refers to the ability to specifically bind to the target antibody to the effect of binding and removing it from a sample while other antibodies are substantially unbound and remain in the sample.

[0080] In order to solve the above technical problems, the present invention also provides the use of the above polypeptide in preparing a medicine or pharmaceutical composition for preventing, treating or improving pollen allergy in a subject.

[0081] In the above use, the subject is allergic to the above polypeptide.

[0082] In the above uses, the medicine or pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0083] The term "pharmaceutically acceptable excipient" refers to a pharmaceutically or food-acceptable carrier, solvent, suspending agent or excipient for delivering the active ingredient in the medicine of the present invention to an animal or human. Exemplary excipients can be liquid or solid, and include but are not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in combination with the pharmacodynamic compound. Examples of excipients include various lactose, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrin and starch.

[0084] The term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein means approved by a national government or listed in the Chinese Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, particularly humans.

[0085] The drug or pharmaceutical composition may be administered, for example, orally, parenterally, by inhalation spray, topically, by eye drops, rectally, nasally, buccally, vaginally or via an implanted reservoir wherein the term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0086] The medicament or pharmaceutical composition can be provided in a suitable dosage form, such as capsules, tablets, and aqueous suspensions and solutions, preferably in sterile form. It can be used in a method for treating a disease, preferably an allergy, comprising administering to a subject an effective amount of a polypeptide of the invention. Hypoallergenic variants of the polypeptides according to the invention can be used. Those skilled in the art are familiar with methods for producing hypoallergenic variants of known allergens.

[0087] The dosage form of the drug or pharmaceutical composition may include, but is not limited to, an inhalation preparation, an oral preparation, and an injectable preparation. The inhalation preparation may be an inhalation aerosol, an inhalation powder inhalation, an inhalation spray, an inhalation liquid preparation, an inhalation atomizer, a lyophilized powder injection for inhalation, or a nasal spray. The oral preparation may be a tablet, powder, capsule, granule, pill, powder, ointment, solid beverage, or oral liquid. The injectable preparation may be an injectable solution or a powder injection for injection.

[0088] In order to prepare the drug or pharmaceutical composition into an injectable preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents (carriers) commonly used in the art can be used, for example, water, saline, phosphate buffered saline, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose or glycerol can be added to the injectable preparation.

[0089] Treatments, ingredients and compositions of such pharmaceutical compositions, useful adjuvants, carriers, production of hypoallergenic variants, and carrier proteins are disclosed in US2021 / 0128781, Aglas L, Bethanis A, Chrusciel P, Stolz F, Gruen M, Jaakkola UM, Jongejan L, Yatkin E, Van Ree R. In vivo induction of functional inhibitory IgG antibodies by hypoallergenic Bet v 1 variants. Front Immunol. 2020 Sep 3;11:2118, US9856296, US9040054, Orozco-Navarrete B, Kaczmarska Z, Dupeux F, et al. Structural Bases for the Allergenicity of Fra a 1.02in Strawberry Fruits, J Agric Food Chem. 2020;68(39):10951-10961.doi:10.1021 / acs.jafc.9b05714 and Neudecker P, Lehmann K, Nerkamp J, Mutational epitope analysis of Pru av 1 and Apig 1, the majorallergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgEreactivity with three-dimensional structure. Biochem J. 2003; 376 (Pt 1): 97-107. doi: 10.1042 / BJ20031057, Hofer et al., Tackling Bet v 1 and associated food allergies with a single hybrid protein,Journal of Allergy and ClinicalImmunology,140(2)2,2017,525-533.

[0090] In any of the above uses, the above kits, the above kits, any of the above uses or the above medicines, the pollen is Chenopodium album pollen.

[0091] The terms "polypeptide," "peptide," "protein," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear, cyclic, or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers, such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA-mediated amino additions such as arginylation, ubiquitination, or any other manipulation such as conjugation to a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0092] The terms "patient" and "subject" are used interchangeably to refer to human or other mammalian patients and subjects, and include any individual examined or treated using the methods of the present invention. However, it should be understood that "patient" does not mean the presence of symptoms. Suitable mammals falling within the scope of the present invention include, but are not limited to, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., koalas, bears, wild cats, wild dogs, wolves, dingoes, foxes, etc.).

[0093] The terms "polypeptide," "peptide," "protein," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear, cyclic, or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers, such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA-mediated amino additions such as arginylation, ubiquitination, or any other manipulation such as conjugation to a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0094] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogenic infections, and the like. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, and the like.

[0095] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature specified as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0096] The terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0097] When the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecule Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (19 97); and BLASTP, BLASTN, and BLASTX algorithms (see Altsch μL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altsch μL et al., Meth. Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLAST Altsch μL et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0098] The term "effective" refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, and the like.

[0099] As used herein, the terms "comprising, comprises, and comprised of" are synonymous with "including, includes, or "containing, and contain," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising, comprises, and comprised of" also include the term "consisting of."

[0100] Beneficial effects

[0101] The incidence of allergic diseases in China is increasing annually, and the total number of people suffering from allergic diseases has exceeded 300 million. IgE testing is currently the primary method for allergy detection. While there are over 600 specific IgE allergen tests available internationally, only around 60 are detectable in China, far from meeting the diagnostic needs of allergy sufferers. This study utilized yeast surface display technology combined with high-throughput sequencing to identify plant pollen allergens, aiming to establish a platform for the precise identification of plant allergens and their allergenic epitopes.

[0102] Yeast surface display has become a powerful tool for high-throughput screening, particularly due to its advantages as a eukaryotic host organism for protein expression, enabling the direct screening of large protein libraries. Allergic diseases are currently one of the three major diseases that are the focus of research and prevention, and an increasing number of people are suffering from allergic diseases. This study, for the first time, applied a yeast surface display system to the identification of plant pollen allergens and successfully identified the Chenopodium album pollen allergen Che a4, which has a molecular weight of approximately 14.2 kDa, encodes 133 amino acid residues, and belongs to the profilin protein family. The application of this system lays the foundation for the subsequent identification of plant pollen allergens.

[0103] This application uses yeast surface display technology to find a new allergen from Chenopodium album pollen. This application uses Chenopodium album pollen as the research object, extracts its total pollen protein, and preliminarily identifies the distribution of Chenopodium album allergen protein by Western blot; uses protein mass spectrometry technology combined with Chenopodium album transcriptome data to predict its potential allergen genes and construct a yeast display library; uses the yeast surface display system combined with high-throughput sequencing to identify Chenopodium album pollen allergens. The results show that: (1) Chenopodium album allergen proteins are distributed between 15-95kDa; (2) Chenopodium album allergen Che a 4 located at 15kDa was successfully identified; (3) Che a 4 contains 133 amino acid residues, with a protein molecular weight of approximately 14.2kDa. Its open reading frame ORF sequence is 402bp long and belongs to the Profilin family. It has an amino acid sequence similarity of approximately 77% with Che a 2. This study uses the yeast surface display system combined with high-throughput sequencing to screen out the Chenopodium album pollen allergen Che a 4, providing technical support for the identification of plant pollen allergens.

[0104] Chenopodium album has the characteristics of wide distribution range, strong environmental adaptability, large amount of pollen, and strong allergenicity, which leads to a large number of patients with Chenopodium album pollen allergy in northern my country every summer and autumn, seriously affecting the daily life of residents. Three Chenopodium album pollen allergens Che a 1, Che a 2, and Che a 3 have been reported abroad, and their protein molecular weights are 17kDa, 14.4kDa, and 9.5kDa respectively. Studies have found that the main allergens in the three types of Chenopodium album pollen are different in different regions. This study found that the allergenic proteins in Chenopodium album pollen are distributed between 15-95kDa ( Figure 1 b), which is consistent with the results of previous studies, and speculates that in addition to the three reported allergens of Chenopodium album pollen, there may be unknown allergens. At present, there are few studies on the identification of Chenopodium album pollen allergens in China, and pollen allergies are highly regional and seasonal. This study aims to use yeast surface display and high-throughput sequencing technology to identify the main allergen molecules in Chenopodium album pollen in China. In the flow cytometry sorting stage, the IgE that specifically binds to the 15kDa protein in the serum of the patients with Chenopodium album allergy used was dominant, resulting in the successful screening of the main allergen Che a 4 ( Figure 4 Middle b, Figure 5 ), composed of 133 amino acid residues, with a protein molecular weight of approximately 14.2 kDa and an ORF frame sequence length of 402 bp. It belongs to the same Profilin gene family as Che a 2, and has an amino acid sequence similarity of 76% and a base sequence similarity of 72% ( Figure 6), and further identification of other allergen protein molecules located at 18kDa, 42kDa, and between 15-95kDa will be conducted later. Furthermore, the sera of the patients with allergic reactions to G. quinoa in this study did not react positively with three previously reported G. quinoa allergens: Che a 1, Che a 2, and Che a 3. This may be due to regional differences and changes in dietary structure.

[0105] Currently, yeast display has been used to analyze interactions between human antibodies, determine affinity constants of recombinant antibodies, and generate whole-cell biosensors for various disease states. This system is a promising biotechnology platform. As a eukaryotic expression system, its post-transcriptional processing and post-translational modification mechanisms promote the expression and correct folding of many antigens, resulting in functional protein conformations. Compared with protein microarrays and phage display, yeast surface display can be used to identify conformational epitopes of autoantibodies targeting proteins and has high accuracy in detecting autoantibodies against known proteins. This study successfully screened the Chenopodium album allergen Che a4, which specifically binds to IgE in the serum of patients with Chenopodium album allergy, using a yeast surface display system, thereby identifying a functional conformational epitope of the protein. A previous study reported the development of a high-throughput phage display-based sequencing technology (AllerScan), which fragmented 12 peanut allergen proteins into 397 20-amino acid residue peptides for use in constructing phage display libraries. This library is capable of comprehensively detecting linear epitopes recognized by peanut-specific antibodies, providing a new method and approach for identifying linear epitopes of allergens.

[0106] The present invention discloses a Chenopodium album pollen allergen and its application. The technical problem addressed is to provide a novel Chenopodium album pollen allergen for use in the diagnosis and treatment of Chenopodium album pollen allergy. Specifically disclosed is a pollen allergen comprising a protein having an amino acid sequence such as SEQ ID No: 2. This allergen can be used to diagnose pollen allergy or prevent, treat, or alleviate pollen allergy in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Figure 1 shows the protein identification of Chenopodium albumen pollen allergens. (a) Coomassie Brilliant Blue-stained protein gel of Chenopodium albumen pollen protein (HL). M is a 10-190 kDa protein marker. (b) Western blot analysis of serum Chenopodium albumen pollen proteins and samples from 58 patients with Chenopodium albumen allergy. The numbers above the Western blot indicate the serum numbers of the patients with Chenopodium albumen allergy.

[0108] Figure 2Figure 2 shows the amplification of the potential allergen gene of Chenopodium truncatum and the quality identification of the yeast display plasmid library. (a) and (b) are gel images of the amplification of the potential allergen gene of Chenopodium truncatum. M is GL2000. DNA marker, 365 is gene HL365, 451 is gene HL451, 915 is gene HL915, 020 is gene HL020, 183 is gene HL183, 422 is gene HL422, 603 is gene HL603, 637 is gene HL637, 823 is gene HL823, 012 is gene HL012, 338 is gene HL338, 5458 is gene HL5458, 517 is gene HL517, 484 is gene HL484, 413 is gene HL413, 369 is gene HL369, 950 is gene HL950, 458 is gene HL458, 437 is gene HL437, 538 is gene HL538, 537 is gene HL537, 109 is gene HL109; (c) Coverage analysis of yeast display plasmid library.

[0109] Figure 3 This is an immunofluorescence detection of the expression level of potential allergen proteins in Chenopodium truncatum. Saccharomyces cerevisiae EBY100 / pYD1 is the control group, EBY100 / pYD1-HL is the yeast surface display library of potential Chenopodium truncatum allergens, 0h and 48h are samples taken at 0h and 48h of induction, respectively, HA-FITC and V5-FITC are FITC-labeled fluorescence of HA and V5 antibodies, respectively, and HA and V5 are superimposed images of white light and 488nm excitation light channels.

[0110] Figure 4 Protein gel images and immunoblotting results of recombinant proteins, (a) Coomassie brilliant blue-stained protein strips of recombinant proteins r012, r369, r338, r637, r109, and r538, (b) Immunoblotting results of recombinant proteins r012, r369, r338, r637, r109, and r538 with serum from patients allergic to G. quinoa.

[0111] Figure 5 Figures show immunoblot validation of the recombinant protein r637. (a) Immunoblotting validation of a serum pool established from sera from patients with T. gondii allergy with T. gondii pollen protein HL and recombinant protein r637. (b) Immunoblotting validation of serum from a single T. gondii allergic patient with recombinant protein r673. The numbers above the western blot membrane indicate the serum number of the T. gondii allergic patient.

[0112] Figure 6 The following are the sequence similarity comparison diagrams of HL 637 and Che a 2: (a) base sequence similarity comparison of HL 637 and Che a 2; (b) amino acid sequence similarity comparison of HL 637 and Che a 2. DETAILED DESCRIPTION

[0113] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0114] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0115] Example 1 Molecular Identification of Chenopodium quinoa Allergen Protein

[0116] Construction of Transcriptome Library of Chenopodium album

[0117] Total RNA was extracted from the flowers, stems, and leaves of Chenopodium truncatum. mRNA was enriched and fragmented, followed by first-strand cDNA synthesis, second-strand cDNA synthesis, ds cDNA end-repair, and dA-tailing, followed by 3' and 5' end adapter ligation. Transcriptome libraries from different parts of Chenopodium truncatum were obtained through library sorting, PCR enrichment, and purification. Initial quantification was performed using Qubit 2.0, library quality was checked using Qsep 100, and sequencing was performed using a Novaseq 6000.

[0118] Transcriptome assembly and functional annotation

[0119] De novo transcriptome assembly was performed. Fastp was used to filter adapters and low-quality reads to generate clean reads. Unigenes were assembled using Trinity software. Alignment and annotation were performed using the NR, NT, and SwissProt databases; PFAM protein domain prediction was performed; GO and InterPro (https: / / www.ebi.ac.uk / interpro) functional annotation was performed; and KEGG metabolic pathway analysis was performed. The COMPARE (https: / / comparedatabase.org) and Allergome (http: / / www.allergome.org) databases were used to predict allergenic proteins and assess their allergenic potential.

[0120] Chenopodium album pollen protein extraction

[0121] The total protein of Chenopodium album pollen was extracted by acetone precipitation method. 2 mL of Chenopodium album pollen was fully ground in liquid nitrogen and 5 times the volume of extraction buffer (0.605% (w / v) Tris-HCl) was added. pH 6.8; 0.50% (w / v) SDS; 10% (w / v) glycerol; 5% (w / v) β-ME), precipitate at -20°C for 30 minutes, 11000g, centrifuge at 4°C for 30 minutes; take the supernatant, add 3 times the volume of acetone, precipitate at -20°C for 1 hour, 11000g, centrifuge at 4°C for 30 minutes, and discard the supernatant; add 80% acetone (containing 0.07% β-ME) to wash the precipitate twice, and dry it in a low-temperature vacuum dryer; add reconstitution buffer (1% NaCl, 0.072% KH2PO4, 3.530% Na2HPO4·12H2O, 0.6% phenol, adjust the pH to 6.5-7.5) to the protein powder, ultrasonically dissolve it completely for 30 minutes, centrifuge it at 12000g, and take the supernatant, which is the Chenopodium album pollen protein solution.

[0122] Immunoblotting experiments

[0123] The loading amount of the above-prepared pollen total protein solution was about 150 μg, and the loading amount of the purified protein was 5 μg. SDS-PAGE was performed using a 15% denatured protein precast gel; after electrophoresis, the protein was transferred to a nitrocellulose membrane using a semi-dry transfer apparatus at 2.5A-25V, and blocked with 5% skim milk powder at room temperature for 2 hours; the serum of a patient allergic to G. quinoa was diluted 1:10, incubated overnight at 4°C, and the membrane was washed three times with TBST; HRP-labeled mouse anti-human IgE secondary antibody was incubated at room temperature for 2 hours, and the membrane was washed three times with TBST; ECL chemiluminescence solution was used for imaging on a GelDoc Go imaging system, and the images were processed using Image Lab 6.0 software.

[0124] Molecular identification of allergen proteins in Chenopodium album

[0125] The distribution of Chenopodium album pollen proteins on 15% protein gel is mainly concentrated in the range of 10-95 kDa ( Figure 1 The results of SDS-PAGE of Chenopodium album pollen protein by Nouri et al. and immunoblotting of serum from patients with Chenopodium album allergy showed that the potential allergen molecules in Chenopodium album pollen were distributed between 15-95 kDa ( Figure 1 Figure b) is consistent with the results of studies on Chenopodium album pollen allergens. Among 58 patients with Chenopodium album allergy, the allergenic proteins were located at 15 kDa in 43 cases, 18 kDa in 18 cases, 22 kDa in 3 cases, 25 kDa in 13 cases, 32 kDa in 12 cases, 42 kDa in 23 cases, 55 kDa in 10 cases, 70 kDa in 16 cases, and 90 kDa in 12 cases.

[0126] Example 2 Cloning of potential allergen genes from Chenopodium album pollen and construction of yeast display library

[0127] Protein mass spectrometry detection

[0128] An appropriate amount of the above-prepared pollen total protein solution was subjected to SDS-PAGE, stained with rapid Coomassie Brilliant Blue staining solution at room temperature for 15 minutes, decolorized in distilled water for 30 minutes, and protein strips at 15kDa, 18kDa and 42kDa were cut respectively, stored in 200μL distilled water, and transported to Novogene Biotechnology Co., Ltd. at 4°C for protein spectrum identification. The bioinformatics software AlgPred2.0 and DeepAlgPro, the allergen databases Allergome, AllerCatPro and COMPARE, combined with the Chenopodium album transcriptome database (the above-mentioned transcriptome assembly and functional annotation), potential allergen genes were predicted: 8 potential allergen genes were predicted at 15kDa, 9 potential allergen genes were predicted at 18kDa, and 5 allergen genes were predicted at 42kDa.

[0129] Gene cloning and plasmid library construction

[0130] Total RNA from Chenopodium album pollen was extracted using the Huayueyang Rapid Universal Plant RNA Extraction Kit, and single-strand cDNA was synthesized using the Novozymes Reverse Transcription Kit. Specific primers were designed based on the gene sequence and the multiple cloning site of the expression vector pYD1 (Table 1). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using Chenopodium album pollen cDNA as a template, 20 gene fragments containing homology arm sequences were obtained by two rounds of RT-PCR amplification ( Figure 2 (a, b) , HL183 and HL950 were amplified unsuccessfully multiple times. The target genes were ligated into the pTOPO-Blunt vector for next-generation sequencing. The pYD1 vector was linearized using restriction endonucleases Kpn I and Not I. The correctly sequenced gene fragments (with the homology arm sequence pYD1-F added to the 5' end and the HA-tag and homology arm sequence pYD1-R added to the 3' end) were ligated into the linearized pYD1 vector using homologous recombinases. Eighteen pYD1 recombinant plasmids were successfully obtained. Vector construction for two genes, HL413 and HL537, was unsuccessful. Equal amounts of the 18 recombinant plasmids were mixed to create the pYD1-HL recombinant plasmid library.

[0131] Table 1 Primer information of potential allergen genes of Chenopodium quinoa

[0132]

[0133]

[0134] Plasmid library diversity identification

[0135] 12 μg of the pYD1-HL recombinant plasmid library was chemically transformed into yeast EBY100 competent cells. Positive transformants were screened on YNB selection medium (containing leucine but not tryptophan). All transformants were collected using YNB-CAA (2% glucose) medium and labeled EBY100 / pYD1-HL. 1 mL of EBY100 / pYD1-HL was used for plasmid extraction. A high-throughput sequencing library was constructed using a DNA library construction kit to identify the diversity of the transformed library. Sequencing analysis revealed that the coverage of the pYD1-HL yeast display plasmid library was 89%. Two recombinant plasmids were missing from the plasmid library: pYD1-HL365 and pYD1-HL451. This may have been lost during the transformation or plasmid extraction process, such as Figure 2 As shown in Figure c: This indicates that the conversion efficiency and coverage of the yeast display library of the potential allergens of Chenopodium truncatum are high, further demonstrating that the yeast display library was successfully constructed and the plasmid library is of good quality.

[0136] Example 3 Expression and display of potential allergen proteins of Chenopodium quinoa

[0137] Expression and display of fusion proteins

[0138] Take an appropriate amount of EBY100 / pYD1-HL at a dilution ratio of 1:100 and add it to YNB-CAA (2% glucose) and culture at 30℃ until OD600 reaches 2-5. Collect the yeast cells and resuspend them in YNB-CAA (2% galactose) to OD600 of 0.5-1. Induce expression at 25℃ for 48h. Samples were taken at 0h and 48h for 10 7 The cells were stored at 4°C until use.

[0139] Immunofluorescence detection

[0140] Compare 0 to 10 in 48h 7 The cells were collected by centrifugation, resuspended in 1×PBS (pH 7.2-7.4), washed three times, and resuspended in 250μL PBS containing 1mg / mL BSA solution with mouse anti-His and mouse anti-HA primary antibodies, respectively. The cells were incubated at 37°C for 1 hour, washed three times with PBS, and then resuspended in goat anti-mouse IgG antibody (FITC) diluted in PBS containing 1mg / mL BSA. The cells were incubated at 37°C for 1 hour, washed three times with PBS, collected by centrifugation, and resuspended in 40μL PBS. A small amount of yeast cell suspension was dropped on a glass slide and placed under a laser confocal electron microscope to detect the immunofluorescence signal of HA and V5 tag proteins on the yeast surface, which was used to reflect the expression level of HL protein of the potential allergen (HL) protein of gray Chenopodium album. The results are shown in Figure 2. Figure 3As shown, the control EBY100 / pYD1 cells showed no HA-tag-specific green fluorescence at either 0 or 48 hours, but showed V5-tag-specific green fluorescence, with a stronger green fluorescence signal at 48 hours, indicating that the V5-tagged protein was successfully displayed on the yeast cell surface. Its expression level increased after 48 hours of induction with 2% galactose. In EBY100 / pYD1-HL yeast cells induced under the same culture conditions and expression system, both HA- and V5-tag-specific green fluorescence were present on the yeast cell surface at both 0 and 48 hours, with stronger green fluorescence after 48 hours of induction, indicating that the HL protein was successfully displayed on the yeast cell surface.

[0141] Flow cytometry sorting

[0142] The 10 cells induced for 48 hours were collected by centrifugation. 8 Yeast cells were resuspended in PBE flow cytometry buffer (1× PBS pH 7.2-7.4 containing 0.5% BSA and 0.5 mmol / L EDTA) and incubated with either 10-fold diluted serum from a patient allergic to G. tenuissima or 3000-fold diluted mouse anti-V5 antibody overnight at 4°C. The cells were washed three times in PBE. Goat anti-human IgE (FITC) or goat anti-mouse IgG (FITC) was diluted in PBE and incubated at 4°C in the dark for 1 hour. The cells were washed three times in PBE. Yeast cells that specifically bound to IgE were sorted using a flow cytometer (Bio-Rad S3e 488 / 640 nm system). The sorted yeast cells were harvested, and yeast plasmid DNA was extracted. After amplification using pYD1-F / R primers, a high-throughput sequencing library was constructed using a DNA library preparation kit for analysis of the sorted genes. The sequencing results were analyzed and the potential allergenic genes HL (HL637, HL538, HL484, HL369, HL338, HL109 and HL012) of Chenopodium album were obtained based on molecular weight and allergenicity.

[0143] Example 4 Expression and identification of potential allergen proteins in Chenopodium quinoa

[0144] Prokaryotic expression vector construction

[0145] Gene-specific primers for HL637, HL538, HL484, HL369, HL338, HL109, and HL012 were designed (Table 2 ), and a His tag was added to the 5′ end of the gene. The target gene HL was ligated into the pET28a vector (Merck, 69864-3) by homologous recombination.

[0146] The pET28a-HL637 recombinant plasmid was prepared by replacing the sequence between fragment 1 (5'-ACTTTAAGAAGGAGATATACC-3') and fragment 2 (5'-GTCGACGGAGCTCGAATTCGGATCC-3') of the pET28a vector with SEQ ID NO: 1, while keeping the other nucleotides of the pET28a vector unchanged. The resulting recombinant plasmid was named pET28a-HL637 recombinant plasmid. The amino acid sequence encoded by SEQ ID NO: 1 is SEQ ID NO: 2.

[0147] The pET28a-HL538 recombinant plasmid was prepared by replacing the sequence between fragment 1 (5'-ACTTTAAGAAGGAGATATACC-3') and fragment 2 (5'-GTCGACGGAGCTCGAATTCGGATCC-3') of the pET28a vector with SEQ ID NO: 3, while keeping the other nucleotides of the pET28a vector unchanged. The resulting recombinant plasmid was named pET28a-HL538 recombinant plasmid. The amino acid sequence encoded by SEQ ID NO: 3 is SEQ ID NO: 4.

[0148] The pET28a-HL484 recombinant plasmid was constructed by replacing the sequence between fragment 1 (5'-ACTTTAAGAAGGAGATATACC-3') and fragment 2 (5'-GTCGACGGAGCTCGAATTCGGATCC-3') of the pET28a vector with SEQ ID NO: 5, while keeping the other nucleotides of the pET28a vector unchanged. The resulting recombinant plasmid was named pET28a-HL484 recombinant plasmid. The amino acid sequence encoded by SEQ ID NO: 5 is SEQ ID NO: 6.

[0149] The pET28a-HL369 recombinant plasmid was constructed by replacing the sequence between fragment 1 (5'-ACTTTAAGAAGGAGATATACC-3') and fragment 2 (5'-GTCGACGGAGCTCGAATTCGGATCC-3') of the pET28a vector with SEQ ID NO: 7, while keeping the other nucleotides of the pET28a vector unchanged. The resulting recombinant plasmid was named pET28a-HL369 recombinant plasmid. The amino acid sequence encoded by SEQ ID NO: 7 is SEQ ID NO: 8.

[0150] The pET28a-HL338 recombinant plasmid was prepared by replacing the sequence between fragment 1 (5'-ACTTTAAGAAGGAGATATACC-3') and fragment 2 (5'-GTCGACGGAGCTCGAATTCGGATCC-3') of the pET28a vector with SEQ ID NO: 9, while keeping the other nucleotides of the pET28a vector unchanged. The resulting recombinant plasmid was named pET28a-HL338 recombinant plasmid. The amino acid sequence encoded by SEQ ID NO: 9 is SEQ ID NO: 10.

[0151] The pET28a-HL109 recombinant plasmid was constructed by replacing the sequence between fragment 1 (5'-ACTTTAAGAAGGAGATATACC-3') and fragment 2 (5'-GTCGACGGAGCTCGAATTCGGATCC-3') of the pET28a vector with SEQ ID NO: 11, while keeping the other nucleotides of the pET28a vector unchanged. The resulting recombinant plasmid was named pET28a-HL109 recombinant plasmid. The amino acid sequence encoded by SEQ ID NO: 11 is SEQ ID NO: 12.

[0152] The pET28a-HL012 recombinant plasmid was constructed by replacing the sequence between fragment 1 (5'-ACTTTAAGAAGGAGATATACC-3') and fragment 2 (5'-GTCGACGGAGCTCGAATTCGGATCC-3') of the pET28a vector with SEQ ID NO: 13, while keeping the other nucleotides of the pET28a vector unchanged. The resulting recombinant plasmid was named pET28a-HL012 recombinant plasmid. The amino acid sequence encoded by SEQ ID NO: 13 is SEQ ID NO: 14.

[0153] SEQ ID NO: 1 is as follows:

[0154] ATGTCGTGGCAAACTTACGTTGATGATCACTTGATGTGTGAGATTGAAGGGACTGGGCATTATCTTACTGCTGCTGCAATTCTGGGTATTGATGGAAGTGTTTGGGCTCAGAGTTCTACCTTTCCTCAGTTTAAGCCAGATGAAATAGCTGCGGTTGTGAAGGATTTCGAGGAGCCCGGTACCCTTGCACCGACCGGGTTGCACCTTGGTGGAGCTAAGTACATGGTTATACAAGGTGAACCTGGGGCTGTTATTCGTGGAAAGAAGGGCCCTGGTGGCATTTGTGTTAAGAAGACCGATCAaGCTTTGGTcGTcGGTATCTATGATGAaCCTGTTACCCCTGGTCAGTGCAACATAATTGTTGAAAGGCTGGGTGATTATCTCATTGAACAGGGCCTCTAA。

[0155] SEQ ID NO: 2 is as follows:

[0156] MSWQTYVDDHLMCEIEGTGHYLTAAAILGIDGSVWAQSSTFPQFKPDEIAAVVKDFEEPGTLAPTGLHLGGAKYMVIQGEPGAVIRGKKGPGGICVKKTDQALVVGIYDEPVTPGQCNIIVERLGDYLIEQGL。

[0157] SEQ ID NO: 3 is as follows:

[0158] ATGTCGAGCACTCAGGAAACCCAGGCCAAGGCTGAGGCTAAGACCGAGGAGTTGTCCCAATGCGCTAAGGAATCGGCAAACGAAGCTAAGGAGAGGACCGCTGATGCTGCTCATTCAGCGGCCGACTCGGTCCAAGAAGGAAAGGAACAAAGCGCTGGATTTCTCCAAGAGACTGGAGAACAAATGAGGAGCATGACTCAGGGTGCTGTGGAGGCTGTGAAGAACACCCTCGGAGTCAATGACAAGAAGTGA

[0159] SEQ ID NO: 4 is as follows:

[0160] MSSTQETQAKAEAKTEELSQCAKESANEAKERTADAAHSAADSVQEGKEQSAGFLQETGEQMRSMTQGAVEAVKNTLGVNDKK。

[0161] SEQ ID NO: 5 is as follows:

[0162] ATGGCGAAGTGTCAAGCTGTTTTTCTCTTGGTTGGCGCTCTCTGCGTCCTGTCCTTGGCCGGTGTCACCAACGCCGCTGAGAACCATTTCAAAGTCCAAGGCATGGTGTACTGTGACACTTGCCGTATCCAATTTATGACCCGCGTTAGCACAATACTGGAAGGGGCAACTGTGAAATTGGAATGCAGGAACATTACTGCAGGAACTCAGACCTTCAAAGCTGAAGCTGTAACAAACAAGGTAGGACAGTACAGCATCCCTGTTGATGGTGATTTCGAGGACGATATCTGTGAAATCGAGTTGGTTAAGAGCCCGAACTAA。

[0163] SEQ ID NO: 6 is as follows:

[0164] MAKCQAVFLLVGALCVLSLAGVTNAAENHFKVQGMVYCDTCRIQFMTRVSTILEGATVKLECRNITAGTQTFKAEAVTNKVGQYSIPVDGDFEDDICEIELVKSPN。

[0165] SEQ ID NO: 7 is as follows:

[0166] ATGTCGTGGCAAACTTACGTCGATGATCACTTGATGTGTCCCATCGAAGAAACCGAAAACCACCTCACCGCCGCCGCTATCGTCGGCTTGGACGGTAGCGTTTGGGCTCAAAGCTCCACCTTCCCCCAGTTGAAGCAAGAAGAAGTGAAGGCAATCTGCAATGAATTCGACGTACCAAATACGTTGGCACCAACAGGTCTTTTCCTTGGAGGAGAAAAGTACATGGTTATTCAGGGAGAGCCTGGAGCTGTTGTTCGTGGAAAGAAGGGACCTGGTGGTGTGTGTATTAAGAAGACTAACCAAGCCCTAGTGTTCGGAATCTACAACGAGCCAGTGACTCCAGGACAATGTAACATGGTTGTCGAGAAGTTGGGTGACTACCTTATCGAACAGGACATCTAG。

[0167] SEQ ID NO: 8 is as follows:

[0168] MSWQTYVDDHLMCPIEETENHLTAAAIVGLDGSVWAQSSTFPQLKQEEVKAICNEFDVPNTLAPTGLFLGGEKYMVIQGEPGAVVRGKKGPGGVCIKKTNQALVFGIYNEPVTPGQCNMVVEKLGDYLIEQDI。

[0169] SEQ ID NO: 9 is as follows:

[0170] ATGGCTGCTGAGGATACACCTCAAGACATTGCTGATAGGGAGAGAATCTTCAAGCGTTTCGACACCAATGGTGATGGAAAAATATCATCCTCTGAGCTTGGTGACGCCCTCAAAACCCTCGGCTCAGTCACCCCTGATGAAGTTAGAAGGATGATGGCAGAGATCGATACTGATGGTGATGGTTTCATTTCTTTTGATGAGTTCACTGACTTTGCTCGGGCTAACCGTGGATTGGTTAAAGATGTTTCCAAGATCTTCTAA。

[0171] SEQ ID NO: 10 is as follows:

[0172] MAAEDTPQDIADRERIFKRFDTNGDGKISSSELGDALKTLGSVTPDEVRRMMAEIDTDGDGFISFDEFTDFARANRGLVKDVSKIF。

[0173] SEQ ID NO: 11 is as follows:

[0174] ATGGCTAACTCAACTGTTGTCAAGCTAGCTTGTGCACTTGTAATGTGCATTGTAGTGGCCGCACCACTAGCCGAGGCAGCCGTTACATGCGGCTTGGTTTCTAGCAAAGTTGCCCCGTGCATTCCCTACCTAAAGGGAGGCGCTGCCCCAACGTCGGGTTGTTGCGGTGGGATTAAGGCTCTCAACGCAGCAGCTGCTTCTGCTGCTGACAAGAAAGTTGCATGTGGTTGCCTGAAAAATGCTGCTGCTGCCATTTCCGGTATCGACTATTCTAAGGCCGCTGGTCTCCCTGGCAAATGTGGTGTTAGCATTCCTTACGCCATTAGCCCCAGCACCAACTGCAACGCGTATGCTTCAATTCTCTCTCTATTATAG。

[0175] SEQ ID NO: 12 is as follows:

[0176] MANSTVVKLACALVMCIVVAAPLAEAAVTCGLVSSKVAPCIPYLKGGAAPTSGCCGGIKALNAAAASAADKKVACGCLKNAAAAISGIDYSKAAGLPGKCGVSIPYAISPSTNCNAYASILSLL。 [[ID=I5]]

[0177] SEQ ID NO: 13 is as follows:

[0178] ATGGCGAAGTGTCAAGCTGTTTTTCTTTTGGTTGGCGCTCTCTGCGTCCTGTCCTTGGCCGGTGTAGCCAATGCCGCCGAGAACCATTTCAAAGTCCAGGGCATGGTGTACTGTGACACTTGCCGTATCCAATTTATGACCCGCATTAGTACAATAATGGAAGGGGCAACTGTGAAATTGGAATGCAGAAACATTACTGCAGGAACTCAGACCTTCAAAGCTGAAGCTGTAACTGATAAGGTAGGACAGTACAGCATCCCTGTTAATGGTGATTTCGAAGACGATATCTGTGAAATCGAGTTGGTTAAGAGCCCCAACAGCGAATGCTCTGAGGTTTCACATGATGTTTATGCCAAGCAATCTGCTAAGGTTAGCCTAACATCCAACAATGGTGAAGCTTCAGACATTCGCAGCGCCAATGCTCTCGGATTCATGAGGAAGGAGCCCCTTAAAGAGTGCCCTGAGGTTCTCAAGGAGTTGGATCTTTATGATGTTAAAGCTAATTAA。

[0179] SEQ ID NO: 14 is as follows:

[0180] MAKCQAVFLLVGALCVLSLAGVANAAENHFKVQGMVYCDTCRIQFMTRISTIMEGATVKLECRNITAGTQTFKAEAVTDKVGQYSIPVNGDFEDDICEIELVKSPNSECSEVSHDVYAKQSAKVSLTSNNGEASDIRSANALGFMRKEPLKECPEVLKELDLYDVKAN。

[0181] (这里原内容没有实际意义,推测是格式相关,保留原样) Transformation and expression of prokaryotic expression system

[0182] Sequencing analysis was performed, and the correctly sequenced pET28a-HL recombinant plasmids (pET28a-HL637 recombinant plasmid, pET28a-HL538 recombinant plasmid, pET28a-HL484 recombinant plasmid, pET28a-HL369 recombinant plasmid, pET28a-HL338 recombinant plasmid, pET28a-HL109 recombinant plasmid and pET28a-HL012 recombinant plasmid) were chemically transformed into Rosetta (DE3) competent cells to obtain Rosetta (DE3) / pET2 8a-HL637 recombinant plasmid, Rosetta (DE3) / pET28a-HL538 recombinant plasmid, Rosetta (DE3) / pET28a-HL484 recombinant plasmid, Rosetta (DE3) / pET28a-HL369 recombinant plasmid, Rosetta (DE3) / pET28a-HL338 recombinant plasmid, Rosetta (DE3) / pET28a-HL109 recombinant plasmid and Rosetta (DE3) / pET28a-HL012 recombinant plasmid. + Positive single clones were obtained by resistance screening, and positive transformants identified by PCR were picked and inoculated into LB (Kan + ) medium at 37 ° C overnight culture, the next day were inoculated into 10 mL of new LB (Kan +) medium at 1:100 and cultured at 37 ° C to OD600 of 0.6-1 (the bacterial solution at this time was recorded as the bacterial solution before induction), IPTG inducer with a final concentration of 0.001 mol / L was added, and expression was induced at 16 ° C overnight to obtain Rosetta (DE3) / pET28a-HL637 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL538 recombinant plasmid fermentation broth, and Ro Rosetta (DE3) / pET28a-HL484 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL369 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL338 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL109 recombinant plasmid fermentation broth and Rosetta (DE3) / pET28a-HL012 recombinant plasmid fermentation broth (the bacterial broth at this time is respectively recorded as induced bacterial broth) and stored at 4°C for future use.

[0183] Table 2 Information on primers for prokaryotic expression of potential allergens of Chenopodium quinoa

[0184] Primer name Sequence information HL637-F1 ACTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCACTCGTGGCAAACTTACGTTG HL637-R1 GTCGACGGAGCTCGAATTCGGATCCTTAGAGGCCCTGTTCAATGAGATAATC HL538-F1 ACTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCACTCGAGCACTCAGGAAAC HL538-R1 GTCGACGGAGCTCGAATTCGGATCCCATCTTCTTGTCATTGACTCCGA HL484-F1 ACTTAAGAAGGAGATATACCATGGGCCATCATCATCATCACGCTCGCTCTTTCTCCAG HL484-R1 GTCGACGGAGCTCGAATTCGGATCCTTAGGTGACCGGGTCTGGAATCC HL369-F1 ACTTAAGAAGGAGATATACCATGGGCCATCATCATCATCACATGTCGTGGCAAACTTACGT HL369-R1 GTCGACGGAGCTCGAATTCGGATCCTTAGATGTCCTGTTCGATAAGGTAG HL338-F1 ACTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCACATGGCTGCTGAGGATACACCTCAA HL338-R1 GTCGACGGAGCTCGAATTCGGATCCTTAGAAGATCTTGGAAACATCTTTAACC HL109-F1 ACTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCACGCTAACTCAACTGTTGTC HL109-R1 GTCGACGGAGCTCGAATTCGGATCCCTATAATAGAGAGAGAATTGAAGCATACGCG HL012-F1 ACTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCACATGGCGAAGTGTCAAGCTG HL012-R1 GTCGACGGAGCTCGAATTCGGATCCTTAATTAGCTTTAACATCATAAAGATCC

[0185] The above fermentation broths (Rosetta (DE3) / pET28a-HL637 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL538 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL484 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL369 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL338 recombinant plasmid fermentation broth, Rosetta (DE3) / pET28a-HL109 recombinant plasmid fermentation broth and Rosetta (DE3) / pET28a-HL012 recombinant plasmid fermentation broth) were centrifuged respectively, and the induced bacteria (R Rosetta (DE3) / pET28a-HL637 recombinant plasmid bacteria, Rosetta (DE3) / pET28a-HL538 recombinant plasmid bacteria, Rosetta (DE3) / pET28a-HL484 recombinant plasmid bacteria, Rosetta (DE3) / pET28a-HL369 recombinant plasmid bacteria, Rosetta (DE3) / pET28a-HL338 recombinant plasmid bacteria, Rosetta (DE3) / pET28a-HL109 recombinant plasmid bacteria and Rosetta (DE3) / pET28a-HL012 recombinant plasmid bacteria), and add 5 mL of lysis buffer NPI-10 (0.05 mol / L NaH2PO4, 0.3 mol / L NaCl, 0.01 mol / L Imidazole pH 8.0), ultrasonically disrupt for 3 s, rest for 6 s, for a total of 15 min, centrifuge at 15000g at 4°C for 20 min, transfer the supernatant to a new centrifuge tube, and retain the precipitate and a small amount of supernatant for later use.

[0186] SDS-PAGE detection of protein expression status:

[0187] A small amount of pre-induction bacterial solution, post-induction bacterial solution, supernatant of post-induction bacterial cell disruption, and precipitate of Rosetta (DE3) / pET28a-HL637, Rosetta (DE3) / pET28a-HL538, Rosetta (DE3) / pET28a-HL484, Rosetta (DE3) / pET28a-HL369, Rosetta (DE3) / pET28a-HL338, Rosetta (DE3) / pET28a-HL109, and Rosetta (DE3) / pET28a-HL012 were taken, and added to 6× Loading Buffer was denatured at 95°C for 10 min, and protein expression was detected by electrophoresis using 15% denaturing protein precast gel. The results showed that Rosetta(DE3) / pET28a-HL637, Rosetta(DE3) / pET28a-HL538, Rosetta(DE3) / pET28a-HL369, Rosetta(DE3) / pET28a-HL338, Rosetta(DE3) / pET28a-HL109, and Rosetta(DE3) / pET28a-HL012 expressed the target protein bands in the induced bacterial liquid and the supernatant of induced bacterial cell disruption, while Rosetta(DE3) / pET28a-HL484 did not express the target protein bands.

[0188] Purification of soluble proteins

[0189] The supernatant of the disrupted cells after induction with Rosetta (DE3) / pET28a-HL637, Rosetta (DE3) / pET28a-HL538, Rosetta (DE3) / pET28a-HL369, Rosetta (DE3) / pET28a-HL338, Rosetta (DE3) / pET28a-HL109 and Rosetta (DE3) / pET28a-HL012 was filtered through a 0.22 μm filter membrane and loaded onto a Ni-NTA chromatography column (HisTrap HP 5 mL, GE healthcare). The column was washed with 5 column volumes of NPI 10 and the column was purified by NPI-250 (0.05 mol / L NaH2PO4, 0.3 mol / L NaCl, 0.25 mol / L Imidazole pH 7.0). 8.0) were subjected to 10%, 60%, and 100% gradient elution to obtain recombinant proteins r637, r538, r369, r338, r109, and r102 ( Figure 4In (a), recombinant protein R637 was obtained by fermentation of recombinant plasmid pET28a-HL637, recombinant protein R538 was obtained by fermentation of recombinant plasmid pET28a-HL538, recombinant protein R369 was obtained by fermentation of recombinant plasmid pET28a-HL369, recombinant protein R338 was obtained by fermentation of recombinant plasmid pET28a-HL338, and recombinant protein R109 was obtained by fermentation of recombinant plasmid pET28a-HL109. The flow-through solution, wash solution, and eluted protein solution of recombinant proteins R637, R538, R484, R369, R338, and R109 were set aside for later use.

[0190] SDS-PAGE to check protein purification status

[0191] The flow-through solution, wash solution and elution protein solution of recombinant proteins r637, r538, r484, r369, r338 and r109 were taken respectively, added with 6× Loading Buffer, denatured at 95°C for 10 min, and the protein purification status was detected by electrophoresis using 15% denatured protein precast gel. The results showed that the recombinant proteins r637, r538, r484, r369, r338 and r109 were all in the elution protein solution.

[0192] Example 5 Western-blot verification of recombinant protein

[0193] The following criteria for patients with ash allergy

[0194] Skin prick tests using a quinoa pollen extract should produce a result of 2+ or higher, and a UniCAP test for serum-specific IgE greater than 0.35. The patient should also have two or more of the typical symptoms of runny nose, sneezing, and nasal congestion and itching, with symptoms persisting for at least one year. Serum from patients with quinoa allergy was provided by the Department of Allergy at Beijing Shijitan Hospital (all patients signed a voluntary agreement).

[0195] In order to further determine whether the above-mentioned recombinant proteins r637, r538, r369, r338, r109 and r012 are allergen proteins of Chenopodium album pollen, this study constructed a serum pool (named as Chenopodium album allergic patient mixed serum pool) by mixing equal volumes of sera from Chenopodium album allergic patients 7797, 7560, 7463, 7672 and 7386, and performed Western-blot verification with the recombinant proteins r637, r538, r369, r338, r109 and r012, respectively.

[0196] Western-blot verification of Chenopodium album pollen allergens:

[0197] About 5 μg of purified recombinant proteins r637, r538, r369, r338, r109, and r012 (using 1× PBS solution (Solebol, P1020) to adjust the protein concentration to about 0.15 μg / μL) were electrophoresed using a 15% denatured protein precast gel (Solebol, PG01510-S-1pk); after the electrophoresis, a portion was stained with a rapid Coomassie brilliant blue staining solution (Solebol, G4540) ( Figure 4 In (a), a portion of the protein was transferred to a nitrocellulose membrane (Solyb, YA1800-1PK) using a semi-dry transfer apparatus at 2.5A-25V, and blocked with blocking solution (TBST containing 5% skim milk powder) at room temperature for 2 h; a serum pool (a mixed serum pool of patients allergic to G. quinoa 7797, 7560, 7463, 7672, and 7386) was diluted 10-fold with 2-3 mL of blocking solution, incubated overnight at 4°C, and washed three times with 1× TBST (Solyb, T1081, diluted 10-fold), each for 10 min; an HRP-labeled mouse anti-human IgE secondary antibody (abcam, Ab99806) was diluted 3000-fold with 2-3 mL of blocking solution, incubated at room temperature for 2 h, and washed three times with 1× TBST, each for 10 min; ECL chemiluminescence solution (Novozyme, E411-05) was used for imaging on a GelDoc Go imaging system, and the images were obtained using Image Lab 6.0 software to process the image. The results are as follows Figure 4 (b) Figure 4 In (b), M is a protein marker, and r637, r538, r369, r338, r109 and r012 are the detection results of recombinant proteins r637, r538, r369, r338 and r109 respectively) as shown: the recombinant protein r637 has a specific immune band at 15kDa, while r012, r369, r338, r109 and r538 have no specific immune reaction. It is speculated that r637 is a Chenopodium album pollen allergen.

[0198] To further confirm that r637 is a T. quinoa pollen allergen protein, 7 serum pools from multiple T. quinoa allergic patients and 30 serum samples from single T. quinoa allergic patients were set up for Western-blot verification with the recombinant protein r637.

[0199] 30 single-case sera of patients with quinoa allergy: 7219, 7338, 7465, 7577, 7582, 7620, 7648, 7650, 7652, 7684, 7761, 7763, 7797, 7463, 7878, 7938, 8233, 7474, 8195, 8202, 7386, 7408, 7560, 7036, 7128, 5311, 5309, 5167, 5083, 5180. 7 groups of serum pools are obtained by grouping and mixing the sera of patients with quinoa allergy, namely, group 1 serum pool, group 2 serum pool, group 3 serum pool, group 4 serum pool, group 5 serum pool, group 6 serum pool, group 7 serum pool, and group 8 serum pool:

[0200] Serum pool group 1 was composed of equal volumes of sera from 8 patients, namely, 7219, 7338, 7561, 7577, 7128, 7235, 7271, and 7319; serum pool group 2 was composed of equal volumes of sera from 8 patients, namely, 7582, 7620, 7648, 7650, 7386, 7408, 7463, and 7465; serum pool group 3 was composed of equal volumes of sera from 4 patients, namely, 7652, 7684, 7560, and 7672; serum pool group 4 was composed of equal volumes of sera from 2 patients, namely, 7761 and 7683; serum pool group 5 was composed of equal volumes of sera from 2 patients, namely, 7878 and 7745; serum pool group 6 was composed of equal volumes of sera from 2 patients, namely, 7938 and 7797; and serum pool group 7 was composed of equal volumes of sera from 2 patients, namely, 8195 and 8202.

[0201] The detection method is the same as the above-mentioned "Western-blot verification of Chenopodium album pollen allergen". Figure 5 ( Figure 5 (a) shows the test results of different serum pool groups and recombinant protein r637: the immunoblotting of recombinant protein r637 and 7 groups of serum pools were all positive, indicating that HL 637 protein (Rosetta (DE3) / pET28-HL637 expressed protein) is an allergen protein molecule at 15kDa of gray Chenopodium album and is one of the main allergens of gray Chenopodium album.

[0202] Furthermore, the 30 serum samples from patients with single-case allergy to Chenopodium album were separately tested with recombinant protein r637 by Western-blot, and the detection method was the same as the above-mentioned "Western-blot verification of Chenopodium album pollen allergen". Figure 5 ( Figure 5 (b) shows the test results of the above 30 single-case sera of patients with gray quinoa allergy and the recombinant protein r637.

[0203] Example 6 Sequence Analysis of Chenopodium quinoa Allergen Che a 4

[0204] The open reading frame (ORF) sequence of HL637 is 402 bp long, encoding 133 amino acid residues. The protein molecular weight is estimated to be approximately 14.2 kDa using DNAMAN software. It belongs to the same Profilin family as the reported Chenopodiaceae allergen Che a 2, and its base sequence similarity with Che a 2 is 72% ( Figure 6 (a)), the amino acid sequence similarity is 76% ( Figure 6 (b)), named Che a4.

[0205] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a polypeptide in the preparation of a product for diagnosing pollen allergy in a subject, wherein the polypeptide is any one of the following: A1) a polypeptide having an amino acid sequence as SEQ ID No: 2; A2) a polypeptide having at least 80% identity with the polypeptide of A1) and having the same function as the polypeptide of A1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence of SEQ ID No: 2; A3) A fusion polypeptide formed by the polypeptide described in A1) or A2) and a tag for detection or a tag for purification.

2. The use according to claim 1, characterized in that The polypeptide acts as an allergen in the product; Preferably, the product is an allergen detection kit; More preferably, the product further comprises an additional allergen, said additional allergen being one or more polypeptides selected from the group consisting of: Chea 1, Chea 2 and Chea 3, or any combination thereof; Further preferably, the amino acid sequence of the Chea 1 polypeptide is shown as SEQ ID No: 14, the amino acid sequence of the Chea 2 polypeptide is shown as SEQ ID No: 8, and the amino acid sequence of the Chea 3 polypeptide is shown as SEQ ID No:

10.

3. The use according to claim 1 or 2, characterized in that The allergen detection kit is used for skin prick test or skin patch test.

4. The use according to claim 3, characterized in that In the product, the polypeptide is in a pharmaceutically acceptable carrier; Preferably, the carrier is an aqueous solution.

5. The use according to claim 1 or 2, characterized in that When the product is used for skin patch test, the polypeptide solution is added to a carrier containing an adsorption material before use to prepare a skin patch.

6. The use according to any one of claims 1 to 5, characterized in that The subject is a mammal; Preferably, the mammal is a human, a non-human primate, a pet or an experimental animal; More preferably, the experimental animal is selected from one of the following: rodents, cats, dogs, pigs, and monkeys.

7. The use according to any one of claims 2 to 6, characterized in that The product is an allergen test kit. The subject has a history of pollen allergy. The product is used in a skin prick test to diagnose whether the subject is allergic to Chenopodium album pollen.

8. The use according to claim 7, characterized in that The subject has a history of allergy to Chenopodium album, and the product contains the polypeptide and multiple additional Chenopodium album allergens, wherein the multiple additional Chenopodium album allergens include: Chea 1, Chea 2 and Chea 3 polypeptides, and the product is used to diagnose which one or more Chenopodium album allergens the subject is allergic to among the polypeptide of claim 1, the Chea 1, the Chea 2 and the Chea 3.

9. The use according to claim 2, characterized in that The product is used to detect whether specific IgE antibodies that specifically bind to the polypeptide exist in a sample from a subject.

10. The use according to claim 9, characterized in that The subject had no history of pollen allergy.

11. The use according to claim 10, characterized in that If specific IgE to the polypeptide is present in the sample from the subject, it indicates that the subject is allergic to Chenopodium album pollen.

12. The use according to claim 11, characterized in that The sample is whole blood, serum or plasma.

13. The use according to any one of claims 8 to 12, characterized in that In the product, the polypeptide is in an aqueous solution or immobilized on a solid support; Preferably, the solid phase carrier is a glass slide or magnetic beads.

14. The use according to any one of claims 8 to 12, characterized in that The products are used in radioallergosorbent assays, enzyme-linked immunosorbent assays, fluorescent immunoassays, Western blotting, immunocapture assays, UniCAP assays, microarray chip assays, and biochip assays.

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