Artemisia sphaerocephala pollen defense protein allergen, kit and application thereof

Abstract

The present invention relates to an Artemisia sphaerocephala pollen-like defense protein allergen, a kit and an application thereof, and relates to the field of biomedical technology. The amino acid sequence of the Artemisia sphaerocephala pollen-like defense protein allergen is shown in SEQ ID NO: 1. The Artemisia sphaerocephala pollen-like defense protein allergen provided by the present invention is a main allergen component in Artemisia sphaerocephala pollen, and has 7 amino acid deletions compared with the subtypes of defense protein allergens from other Artemisia pollens included in the IUIS database, and has an amino acid mutation in the C-terminal conserved region, belonging to a new allergen subtype, which has an impact on the potential allergenicity of allergens; the defense protein allergen can be used to prepare drugs for treating or preventing allergies, or to prepare reagents or kits for allergen detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an Artemisia ordosica pollen defense protein allergen, a kit and applications thereof. Background Art

[0002] Artemisia is a pioneer plant for windbreak and sand fixation in Northwest my country. It is widely distributed in my country's desert areas. The planting area has been expanding over the past 20 years, and the allergy problems caused by it have become more serious. Studies have shown that in the past decade, the increase in the number of people allergic to Artemisia pollen in Northwest my country is significantly related to the expansion of Artemisia planting area. The allergy to Artemisia pollen is caused by certain proteins in it, namely allergens.

[0003] Artemisia pollen allergens have high homology and species specificity. Allergenicity varies between allergen subtypes from different sources. Selecting the most suitable allergen components for clinical diagnosis and immunotherapy can greatly improve the diagnosis and treatment of allergic diseases. However, Artemisia pollen allergen components have not been studied in depth in my country. Defensin-like proteins are the main allergens in Artemisia pollen. The positive rate of patients with Artemisia pollen allergy in my country is as high as 86%. The monomer of Defensin-like protein allergen contains 108 amino acids, with a theoretical isoelectric point of 8.17. It consists of a cysteine-rich defensin domain at the N-terminus and a proline-rich region at the C-terminus. The C-terminus contains hydroxylation and O-glycosylation modifications. The sequences of the 12 Artemisia-like defensin-like allergens included in the database of the Allergen Nomenclature Committee under the International Union of Immunological Societies (IUIS) (http: / / www.allergen.org) are very similar and have similar IgE binding abilities.

[0004] However, there are no reports on defense-like proteins of Artemisia ordosica. Therefore, the present invention conducts in-depth molecular identification of defense-like proteins of Artemisia ordosica, proposes an Artemisia ordosica pollen-like defense protein allergen, a kit and its application, aiming to promote the development of diagnosis of Artemisia ordosica allergen components and recommended treatment. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an Artemisia sphaerocephala pollen-like defense protein allergen, a kit and its application. The purpose is to provide a new subtype of the defense protein-like allergen component in Artemisia sphaerocephala pollen and its application, which is different from other homologous allergen components already included in the IUIS database.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, an Artemisia sphaerocephala pollen defense protein allergen, the amino acid sequence of the Artemisia sphaerocephala pollen defense protein allergen is shown in SEQ ID NO: 1.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] In a second aspect, a nucleic acid is provided, wherein the nucleic acid encodes the Artemisia ordosica pollen defense protein allergen, and the nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 2.

[0010] In a third aspect, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the nucleic acid, and the framework of the recombinant expression vector is pET32a.

[0011] In a third aspect, a transformant, the transformant comprises the recombinant expression vector, and the host of the transformant comprises at least one of bacteria, fungi, insect cells, and animal cells. The host organism may be various protein expression systems such as Escherichia coli for prokaryotic expression, yeast for eukaryotic expression, insect cells, and mammalian cells (such as CHO cells).

[0012] In a fourth aspect, the use of the Artemisia pollen defense protein allergen, or the nucleic acid, or the recombinant expression vector, or the transformant in the preparation of a drug for treating or preventing Artemisia pollen allergy.

[0013] In a fifth aspect, a drug for treating or preventing Artemisia pollen allergy comprises the Artemisia pollen defense protein allergen, or / and the nucleic acid, or / and the recombinant expression vector, or / and the transformant.

[0014] Furthermore, the drug includes a desensitization immune vaccine. The traditional method for effectively treating allergies is specific immunotherapy or desensitization, that is, subcutaneous injection of natural allergen extracts in increasing doses to allergic patients.

[0015] In a sixth aspect, the Artemisia ordosica pollen defense protein allergen, or the nucleic acid, or the recombinant expression vector, or the transformant is used in the preparation of reagents for allergen detection.

[0016] In a seventh aspect, the Artemisia ordosica pollen defense protein allergen, or the nucleic acid, or the recombinant expression vector, or the transformant is used in the preparation of a kit for allergen detection.

[0017] In an eighth aspect, a kit for allergen detection comprises the Artemisia ordosica pollen defense protein allergen, or / and the nucleic acid, or / and the recombinant expression vector, or / and the transformant.

[0018] For patients with allergies, an important means is to detect the allergens that cause allergies. By reducing contact with allergens in a targeted manner, the occurrence of allergies can be effectively prevented. The Artemisia pollen defense protein allergens of the present invention, or / and the nucleic acid, or / and the recombinant expression vector, or / and the transformant, or used together with other allergens, are prepared into a kit for screening allergens in patients with allergies.

[0019] The beneficial effects of the present invention are as follows: the defense protein-like allergen of Artemisia sphaerocephala pollen provided by the present invention is the main allergen component in Artemisia sphaerocephala pollen. Compared with the defense protein-like allergen subtypes from other Artemisia pollens included in the IUIS database, it has 7 amino acid deletions and an amino acid mutation in the conserved region of the C-terminus. It belongs to a new allergen subtype and has an impact on the potential allergenicity of allergens. This type of defense protein allergen can be used to prepare drugs for treating or preventing allergies, or to prepare reagents or kits for allergen detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of immunoblotting of Artemisia sphaerocephala pollen and patient serum; A is the SDS-PAGE electrophoresis detection of Artemisia sphaerocephala pollen extract; B is immunoblotting, P1-P8 represent 8 sera of patients allergic to Artemisia sphaerocephala pollen, and N represents the serum of negative patients;

[0021] Figure 2 This is a comparison of the amino acid sequences of defense protein allergens in pollen from different Artemisia plants;

[0022] Figure 3 The immunoaffinity chromatography method was used to purify the natural defense proteins in Artemisia sphaerocephala pollen (A) and the mass spectrometry detection results (B). The red-marked peptides are the peptides matched by mass spectrometry detection.

[0023] Figure 4 This is a purification diagram of Artemisia ordosica recombinant defense protein allergen Art d 1; wherein, lane 1: prokaryotic expression bacterial solution; lane 2: column flow-through; lanes 3-6: elution with different concentrations of imidazole buffer;

[0024] Figure 5 This is a correlation analysis between the binding values ​​of Artemisia annua pollen Art v 1 and Artemisia sphaerocephala pollen Art d 1 and specific IgE antibodies in patient serum. DETAILED DESCRIPTION

[0025] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0026] Sources of materials and reagents:

[0027] The Artemisia ordosica pollen used in the following examples was collected from Yulin City, Shaanxi Province.

[0028] Example 1

[0029] Cases of Artemisia sphaerocephala pollen allergy were collected from the Ordos region, diagnosed and recorded by clinicians, and serum was collected from patients. All patients signed informed consent forms, and the study was approved by the Ethics Committee (Ethics Approval No. 2021-007).

[0030] Weigh 2g of Artemisia sphaerocephala pollen, add 35ml of PBS buffer, extract overnight at 4℃, centrifuge at 8000rpm for 15min, take the supernatant, and filter with a 0.22μm filter membrane to obtain the Artemisia sphaerocephala pollen protein extract. The extract was tested by SDS-PAGE electrophoresis, and the total protein content was determined by BCA method.

[0031] The crude extract of Artemisia pollen protein was separated by 12% SDS-PAGE gel electrophoresis and transferred to PVDF membrane in situ. After blocking with 5% skim milk powder for 1 hour, 200 μL serum from patients with Artemisia pollen allergy was added and incubated at 4°C overnight. The membrane was washed with TBST 3 times, 10 minutes each time, and then HRP-labeled goat anti-human IgE (Thermo Fisher, catalog number A18793) was added and incubated for 1 hour. The membrane was washed again, ECL was added for color development and the results were recorded in chemiDoc. The Artemisia pollen extract and patient serum had obvious IgE binding bands at 10kDa, 15kDa, 17kDa, 25kDa, 40kDa, 55kDa, and 60kDa ( Figure 1 A and B, where Figure 1 P1-P8 in B represent 8 sera immunoblots of patients allergic to Artemisia pollen). Combined with the identified Artemisia pollen allergens, it can be seen that the one at 25kDa is the defense protein allergen dimer, confirming that this allergic protein component also exists in Artemisia pollen.

[0032] Example 2

[0033] The polysaccharide and polyphenol plant RNA extraction kit (Huayueyang, Cat. No. 0416-50) from Beijing Huayueyang Biological Company was used to extract Artemisia pollen mRNA according to the instructions, and transcriptome sequencing was performed using the DNBSeq platform. The sequencing data was filtered by SOAPnuke and trimmomatic, and then the sequences were assembled and annotated using Trinity, and a total of 25,769 CDS were detected. The Q-Exactive HF platform was used to perform a full spectrum of protein determination on Artemisia sphaerocephala extracts, and the protein was compared and matched with the transcriptome database, and a total of 8,251 proteins were identified. Defense protein-like allergens were detected in both the proteome and transcriptome databases.

[0034] Example 3

[0035] The mRNA extracted in Example 2 was reverse transcribed into cDNA using the One-Step gDNA Removal and cDNA Synthesis SuperMix (Quanshijin, Cat. No. AE311-02). According to the transcriptome sequencing results, specific primers were designed (upstream F—gccatggctgatatcggatccATGGCAAAGTGTTCATATGTCTTC (SEQ ID NO: 3), downstream R1—ctcgagtgcggccgca agcttTTAGTGAGCGACCGGA (SEQ ID NO: 4). NO: 4), I5 high-fidelity enzyme (MCLAB, catalog number PDP-100) was used to clone the defense-like protein in Artemisia ordosica pollen. The cloned products with the correct fragment size were recovered by gel ligation to the linearized pET32a plasmid (Thermo Fisher), and then transferred into Escherichia coli DH5α strain (Full Gold, CD201-01), spread on LB medium containing ampicillin (Shanghai Biotechnology, catalog number A507003-0250), cultured at 37°C overnight, picked single colonies for sequencing, and translated the correctly sequenced nucleotide sequence into amino acids. The results showed that there was a special defense-like protein subtype in Artemisia ordosica. Compared with other homologous allergen subtypes that have been included in the IUIS allergen database, the defense-like protein of Artemisia ordosica lacked 7 amino acids, and the mature peptide contained only 101 amino acids, and the 99th position of the C-terminus was mutated from serine (S) to valine (V). Figure 2 These specific sequence variations can be used to develop new diagnostic and immunotherapeutic agents.

[0036] Example 4

[0037] The specific mouse monoclonal antibody A7G4E6 (Hangzhou Aileji Biotechnology Co., Ltd., product number MA-A7G4E6) for Artemisia pollen defense protein was coupled to CNBr-activated-sepherose 4B gel to prepare an antibody affinity purification column. The crude extract of Artemisia pollen protein obtained in Example 1 was freeze-dried to powder and dissolved in 0.1M Tris-HCl buffer. The pollen extract after solvent was added to the antibody affinity purification column. After incubation at room temperature for 1 hour, 5mL 0.1MTris-HCl buffer was added to wash twice, and then 5mL 0.1M glycine was added to elute. The filtrate was collected, freeze-dried, and a small amount of PBS buffer was added to dissolve. The purified product was detected by SDS-PAGE electrophoresis. After purification, the target band appeared at 25kDa, and the size was consistent with that of the defense protein, such as Figure 3 As shown in A and B, the band was cut from the gel and subjected to LC-MS / MS mass spectrometry detection. After detection, the purified protein was determined to be a defense-like protein and named Art d 1 (the sequence is shown in SEQ ID NO: 1).

[0038] Example 5

[0039] The recombinant vector sequenced correctly in Example 3 was transformed into Escherichia coli competent cells Rosetta (DE3) (Full Gold, Catalog No. CD801-02). After plating, a single colony was picked and transferred to 200 ml of LB medium containing ampicillin antibiotics. The culture was cultured at 37°C and 200 rpm until OD600 reached 0.6. IPTG inducer was added to a final concentration of 0.5 mM. The cells were induced overnight at 16°C and 160 rpm. The precipitate was centrifuged at 10,000 rpm and resuspended in 20 ml In PBS, crush in ultrasonic crusher for 20 minutes, power 40%, ultrasonic for 3 seconds, interval 2 seconds, centrifuge at 10000rpm for 10 minutes after crushing, take the supernatant and filter with 0.22μm filter membrane, add 200μl Ni-NTA, incubate at 4℃ for 1h, transfer the sample to protein purification column, let the sample not bound to nickel gel flow out naturally, then wash with 20mM imidazole solution 6 times, 5ml each time, wash away non-specifically bound proteins, then elute the target protein with gradient concentration (50mM, 100mM, 200mM, 250mM) imidazole solution, wash 4 times for each concentration gradient, 500μl each time. Collect the eluate and detect with 12% SDS-PAGE gel, as shown in Figure 4 As shown, the prokaryotic expressed protein contains a 25 kDa tag, so the final expressed target protein is about 37 kDa in size. The sample eluate containing the target protein is collected and dialyzed into PBS buffer (dialysis at 4°C for 48 hours, with the buffer changed every 12 hours), and the sample concentration is determined by the BCA method.

[0040] Example 6

[0041] The recombinant Art d 1 obtained in Example 5 was diluted to 10 μg / ml, 50 μl was added to ImmunoCAP labeled with streptavidin (No. o212), incubated at 37°C for 30 min, and then taken out and stored at 4°C for use. Serum from patients allergic to Artemisia sphaerocephala pollen was collected from the Ordos area, and the specific IgE antibodies of the patient serum and the defense protein-like component Art v 1 (w231) in Artemisia pollen (No. w6) and Artemisia vulgaris pollen were tested on the ImmunoCAP system. At the same time, the IgE binding of the patient serum to Art d 1 was measured. The results are shown in Table 1. All 9 patients tested were positive for Art v 1 and also positive for the recombinant Art d 1 produced in the present invention, with a compliance rate of 100%, and the specific IgE values ​​of Art d 1 and Art v 1 were significantly correlated ( Figure 5 ), but the test value was slightly lower (Table 1), indicating that the Artemisia sphaerocephala pollen defense protein Art d 1 identified and produced in the present invention can be used for in vitro diagnosis of patients allergic to Artemisia sphaerocephala pollen.

[0042] Table 1 ImmunoCAP test results

[0043]

[0044]

[0045] In summary, the present invention provides a defense protein allergen-like allergen of Artemisia deserticola pollen, which is the main allergen component in Artemisia deserticola pollen. Compared with the defense protein allergen-like subtypes from other Artemisia pollens included in the IUIS database, it has 7 amino acid deletions and an amino acid mutation in the conserved region of the C-terminus. It belongs to a new allergen subtype and has an impact on the potential allergenicity of allergens. This defense protein allergen can be used to prepare drugs for treating or preventing allergies, or for preparing reagents or kits for allergen detection.

[0046] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An Artemisia sphaerocephala pollen defense protein allergen, characterized in that: The amino acid sequence of the Artemisia ordosica pollen defense protein allergen is shown in SEQ ID NO:

1.

2. A nucleic acid, characterized in that The nucleic acid encodes the Artemisia ordosica pollen defense protein allergen according to claim 1, and the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 2.

4. A transformant, characterized in that The transformant comprises the recombinant expression vector according to claim 3, and the host of the transformant comprises at least one of bacteria, fungi, insect cells, and animal cells.

5. Use of the Artemisia sphaerocephala pollen defense protein allergen according to claim 1, or the nucleic acid according to claim 2, or the recombinant expression vector according to claim 3, or the transformant according to claim 4 in the preparation of a drug for treating or preventing Artemisia sphaerocephala pollen allergy.

6. A drug for treating or preventing Artemisia sphaerocephala pollen allergy, characterized in that: It comprises the Artemisia ordosica pollen defense protein allergen according to claim 1, or / and the nucleic acid according to claim 2, or / and the recombinant expression vector according to claim 3, or / and the transformant according to claim 4.

7. A drug for treating or preventing Artemisia sphaerocephala pollen allergy according to claim 6, characterized in that: The medicine includes a desensitization immunization vaccine.

8. Use of the Artemisia ordosica pollen defense protein allergen according to claim 1, or the nucleic acid according to claim 2, or the recombinant expression vector according to claim 3, or the transformant according to claim 4 in the preparation of a reagent for allergen detection.

9. Use of the Artemisia ordosica pollen defense protein allergen according to claim 1, or the nucleic acid according to claim 2, or the recombinant expression vector according to claim 3, or the transformant according to claim 4 in the preparation of a kit for allergen detection.

10. A kit for allergen detection, characterized in that: It comprises the Artemisia ordosica pollen defense protein allergen according to claim 1, or / and the nucleic acid according to claim 2, or / and the recombinant expression vector according to claim 3, or / and the transformant according to claim 4.

Images