Dermatophagoides farinae allergen ATP enzyme sodium / potassium transport beta2 subunit protein and application thereof

Abstract

The invention relates to the field of biomedicine, and discloses dermatophagoides farinae allergen ATPase sodium / potassium transport beta2 subunit protein and application thereof. Wherein the Dermatophagoides farinae allergen ATPase sodium / potassium transport beta2 subunit protein is a newly found Dermatophagoides farinae allergen component, and the amino acid sequence of the Dermatophagoides farinae allergen ATPase sodium / potassium transport beta2 subunit protein is shown as SEQ ID NO.1. According to the invention, co-expression protein of recombinant dermatophagoides farinae allergen ATP enzyme sodium / potassium transport beta2 subunit protein and thioredoxin tag protein is prepared through gene cloning, protein expression and separation and purification; the recombinant dermatophagoides farinae allergen ATPase sodium / potassium transport beta2 subunit protein in the co-expression protein is identified to have certain allergenicity through experimental means such as IgE-enzyme-linked immunosorbent assay and IgE immunoblotting, can be used as a dermatophagoides farinae allergen, and is used for preparing reagents for diagnosing, treating and preventing allergic diseases caused by dermatophagoides farinae. The Dermatophagoides farinae ATP enzyme sodium / potassium transport beta2 subunit protein is especially used for preparing a reagent for diagnosing, treating and preventing allergic diseases caused by Dermatophagoides farinae ATP enzyme sodium / potassium transport beta2 subunit protein.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a dust mite allergen ATPase sodium / potassium transport β2 subunit protein and application thereof. Background Art

[0002] Allergic diseases, also known as allergic diseases, include allergic rhinitis, allergic asthma, atopic dermatitis, etc., which are common clinical diseases. Allergic diseases are caused by a variety of allergens such as inhaled, ingested, contact and drug-related allergens. The World Allergy Organization estimates that the global prevalence of allergic diseases is 10-40%. Among them, there are about 300 million patients with bronchial asthma, 400 million patients with allergic rhinitis, 200 million patients with food allergies and 150 million patients with drug allergies. Allergic asthma is an important phenotype of asthma, accounting for more than 50% of adult asthma patients and more than 80% of children with asthma.

[0003] Dust mites are the most important allergen source species that cause allergic diseases. Their bodies and excrement have been identified as the main indoor inhaled allergens. The results of allergen-specific IgE immunoassays of the serum of patients with allergic diseases showed that more than 70% of the serum samples were positive for dust mite allergens. The protein composition of dust mite bodies and feces is complex. Current studies have found that dust mites have at least 40 allergen components, among which the first and second components of dust mite allergens are the main allergens of dust mites. Although the serum of most patients with allergic diseases is specifically positive for the main allergen components of dust mites, different patients with allergic diseases may only be sensitized to some minor allergens or rare allergen components. Some allergens also have synergistic sensitization effects, and the sensitization mechanisms are complex and diverse. In clinical practice, the crude extract of dust mites has been used as the first choice for effective desensitization treatment. Therefore, the discovery and identification of more dust mite allergen components can provide a theoretical basis for the precise treatment of dust mite allergic diseases. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a dust mite allergen ATPase sodium / potassium transport β2 subunit protein and its application. The newly discovered dust mite allergen is a homolog of the dust mite ATPase sodium / potassium transport β2 subunit protein and can be used to prepare reagents for diagnosing, treating and preventing allergic diseases, especially for preparing reagents for diagnosing, treating and preventing allergic diseases caused by dust mites.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect of the present invention, a dust mite allergen ATPase sodium / potassium transporter β2 subunit protein is provided. The amino acid sequence of the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention provides a co-expressed protein containing the β2 subunit protein of the sodium / potassium transporter ATPase of the dust mite allergen, wherein the amino acid sequence of the co-expressed protein is shown in SEQ ID NO.2.

[0008] The third aspect of the present invention provides a nucleic acid encoding the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein as described in the first aspect.

[0009] The fourth aspect of the present invention provides a recombinant vector, wherein the recombinant vector contains a gene expression cassette encoding the β2 subunit protein of the ATPase sodium / potassium transporter of the dust mite allergen as described in the first aspect.

[0010] The fifth aspect of the present invention provides a host cell, wherein the host cell contains the recombinant vector as described in the fourth aspect.

[0011] The sixth aspect of the present invention provides a composition, wherein the composition contains the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein as described in the first aspect.

[0012] Optionally, the composition further comprises a pharmaceutically acceptable carrier.

[0013] The seventh aspect of the present invention provides a method for preparing the dung mite allergen ATPase sodium / potassium transporter β2 subunit protein as described in the first aspect, comprising:

[0014] Total RNA of dust mites was extracted and reverse transcribed to obtain cDNA;

[0015] Design primers and obtain the gene encoding the β2 subunit protein of the ATPase sodium / potassium transporter of the dust mite allergen by PCR amplification. The nucleotide sequence of the gene encoding the ATPase sodium / potassium transporter of the dust mite allergen is shown in SEQ ID NO.3;

[0016] Inserting the gene encoding the ATPase sodium / potassium transport β2 subunit protein of the dust mite allergen into an expression vector to obtain a recombinant plasmid;

[0017] The recombinant plasmid is transferred into a host cell for protein expression and purification to obtain the dust mite allergen ATPase sodium / potassium transport β2 subunit protein.

[0018] Optionally, the primers include an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.5.

[0019] In an eighth aspect, the present invention provides a use of the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein as described in the first aspect, the co-expressed protein as described in the second aspect, the nucleic acid as described in the third aspect, the recombinant vector as described in the fourth aspect, the host cell as described in the fifth aspect, the composition as described in the sixth aspect, or the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein prepared by the preparation method as described in the seventh aspect in the preparation of reagents for diagnosing, treating, and preventing allergic diseases.

[0020] Beneficial effect: The present invention provides a dust mite allergen ATPase sodium / potassium transport β2 subunit protein and its application. The dust mite allergen ATPase sodium / potassium transport β2 subunit protein is a newly discovered dust mite allergen component and is a homologue of the dust mite ATPase sodium / potassium transport β2 subunit protein. The present invention prepares a co-expressed protein of a recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein and a thioredoxin tag protein by gene cloning, protein expression, separation and purification. The recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein in the co-expressed protein is identified to have certain allergenicity by experimental means such as IgE-enzyme-linked immunosorbent reaction and IgE immunoblotting, and can be used as a dust mite allergen for preparing a reagent for the diagnosis, treatment and prevention of allergic diseases caused by dust mites, especially for preparing a reagent for the diagnosis, treatment and prevention of allergic diseases caused by the dust mite ATPase sodium / potassium transport β2 subunit protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is an agarose electrophoresis diagram of the PCR amplification product of the ATPase sodium / potassium transport β2 subunit protein gene of the dust mite allergen in Example 2.

[0022] Figure 2 This is the SDS-PAGE electrophoresis diagram of the expression and purification product of the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein in Example 2.

[0023] Figure 3 This is the IgE-ELISA result of the dust mite allergy patient serum and the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein in Example 3.

[0024] Figure 4 This is the IgE-Western Blot result of the dust mite allergy patient serum and the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein in Example 3.

[0025] Figure 5This is the IgE-Western Blot result of healthy human serum and recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein in Example 3.

[0026] Figure 6 It is the IgE-DotBlot result of the dust mite allergy patient serum and the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein or the recombinant thioredoxin tag protein in Example 3.

[0027] Figure 7 It is the IgE-DotBlot result of healthy human serum and recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein or recombinant thioredoxin tag protein in Example 3. DETAILED DESCRIPTION

[0028] The present invention provides a dust mite allergen ATPase sodium / potassium transport β2 subunit protein and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0029] The embodiment of the present invention provides a dust mite allergen ATPase sodium / potassium transport β2 subunit protein, which is composed of 312 amino acids and has a molecular weight of about 37.33 kDa. The amino acid sequence is shown in SEQ ID NO.1.

[0030] SEQ ID NO.1:

[0031] MANKEEKYCDKQSGWQKFCKFVWNSETGQFLGRTGGSWFKITVFYIIFYLCLAAFWALMLLIFYQTIDYRSPKYQLAESRIGANPGLGFRPRPKDENIDSTLIWFKNGNNEENWKYWSESLQEFLEPYKKSTIDAGAHVENCGDNQMASEGKFCHF DVNAIPSQCSVENHFGYQRGDPCVLIKLNKIFSWKPEGFTDEELRDTHIPDVVRKSYPTRLNNASQLIYITCEGENAADKENVGEIEYYPQQGIEFKYYPFTNQPGYLSPFVFVHFKKPQPGVLINIECKAWARNIKHNRMDREGSVHFELLVDGF.

[0032] The embodiment of the present invention provides a co-expressed protein containing a dust mite allergen ATPase sodium / potassium transport β2 subunit protein. The co-expressed protein is a recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tag protein, which consists of 477 amino acids and has a molecular weight of approximately 56.78 kDa. Its amino acid sequence is shown in SEQ ID NO.2.

[0033] SEQ ID NO.2:

[0034] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHH HHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSMANKEEKYCDKQSGWQKFCKFVWNSETGQFLGRTGGSWFKITVFYIIFYLCLAAFWALMLLIFYQTIDYRSPKY QLAESRIGANPGLGFRPRPKDENIDSTLIWFKNGNNEENWKYWSESLQEFLEPYKKSTIDAGAHVENCGDNQMASEGKFCHFDVNAIPSQCSVENHFGYQRGDPCVLIKLNKIFSWKPE GFTDEELRDTHIPDVVRKSYPTRLNNASQLIYITCEGENAADKENVGEIEYYPQQGIEFKYYPFTNQPGYLSPFVFVHFKKPQPGVLINIECKAWARNIKHNRMDREGSVHFELLVDGF.

[0035] An embodiment of the present invention provides a nucleic acid encoding the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein as described above.

[0036] The embodiment of the present invention provides a recombinant vector, wherein the recombinant vector contains a gene expression cassette encoding the above-mentioned Dermatophagoides farinae allergen ATPase sodium / potassium transporter β2 subunit protein.

[0037] In one embodiment, the recombinant vector includes at least one of a recombinant expression vector and a recombinant cloning vector. Optionally, the recombinant vector contains BamHI and Hind III restriction sites, and specifically, the gene encoding the dust mite allergen is inserted between the BamHI and Hind III restriction sites.

[0038] In one embodiment, the recombinant vector is selected from one of T vector and pET32a, but is not limited thereto.

[0039] An embodiment of the present invention provides a host cell, wherein the host cell contains the recombinant vector as described above.

[0040] In one embodiment, the host cell includes at least one of a cloning host cell and an expression host cell. Optionally, the host cell is selected from one of Escherichia coli Top10 and Escherichia coli BL21 (DE3) plysS, but is not limited thereto.

[0041] An embodiment of the present invention provides a composition, wherein the composition contains the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein as described above.

[0042] In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. Optionally, the pharmaceutically acceptable carrier comprises at least one of a solvent, a polymer and a liposome, but is not limited thereto.

[0043] In the present invention, the composition may further include other active ingredients, which are selected according to the purpose of the composition and are not limited herein.

[0044] The embodiment of the present invention provides a method for preparing the above-mentioned dust mite allergen ATPase sodium / potassium transporter β2 subunit protein, comprising the steps of:

[0045] (1) Extract total RNA from dust mites and reverse transcribe to obtain cDNA;

[0046] (2) designing primers and obtaining the gene encoding the β2 subunit protein of the ATPase sodium / potassium transporter of the dust mite allergen by PCR amplification. The nucleotide sequence of the gene encoding the ATPase sodium / potassium transporter of the dust mite allergen is shown in SEQ ID NO.3;

[0047] (3) inserting the gene encoding the β2 subunit protein of the dust mite allergen ATPase sodium / potassium transporter into an expression vector to obtain a recombinant plasmid;

[0048] (4) The recombinant plasmid is transferred into a host cell for protein expression and purification to obtain the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein.

[0049] In one embodiment, the primers include an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.5.

[0050] In one embodiment, the step (4) specifically comprises: transforming the recombinant plasmid into a host cell, culturing, inducing, breaking the bacteria, and chromatographic purification to obtain the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein.

[0051] The embodiments of the present invention provide a use of the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein as described above, the nucleic acid as described above, the recombinant vector as described above, the host cell as described above, the composition as described above, or the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein prepared by the preparation method as described above in the preparation of reagents for diagnosing, treating, and preventing allergic diseases.

[0052] In one embodiment, the allergic disease is an allergic disease caused by dust mites.

[0053] In one embodiment, the allergic disease is an allergic disease caused by the dermatophagoides farinae ATPase sodium / potassium transporter β2 subunit protein.

[0054] The present invention will be further described below by means of specific examples.

[0055] Example 1: Discovery of the β2 subunit protein of the sodium / potassium transporter ATPase of dust mite allergen

[0056] By analyzing the dung beetle genome sequence, the gene sequence of ATPase sodium / potassium transporter β2 subunit protein of dung beetle allergen was obtained. The gene sequence consists of 939 nucleotides. The nucleotide sequence is shown in SEQ ID NO.3 (5' to 3' end).

[0057] SEQ ID NO.3:

[0058] 。

[0059] Example 2: Molecular cloning of the β2 subunit protein of the sodium / potassium transporter ATPase of the dust mite allergen I. Extraction of total RNA from dust mite

[0060] The living mites of Dermatophagoides farinae were freshly isolated and total RNA was extracted using the Trizol method.

[0061] 2. Full-length cloning and identification of target genes

[0062] The total RNA extracted from dust mites was used as a reverse transcription template, and the EvoM-MLV reverse transcription kit of Hunan Aikerui Biotechnology Co., Ltd. was used to reverse transcribe and synthesize cDNA, and then the cDNA was used as a template for PCR amplification reaction. According to the analysis of the genome sequence of dust mites in Example 1, the forward primer DF-41-F and the reverse primer DF-41-R of the ATPase sodium / potassium transport β2 subunit protein of the dust mite allergen were designed and synthesized, the nucleotide sequence of DF-41-F is shown in SEQ ID NO.4 (5' to 3' end), and the nucleotide sequence of DF-41-R is shown in SEQ ID NO.5 (5' to 3' end).

[0063] SEQ ID NO. 4: GGATCCATGGCAAACAAAGAAGAAAAAT.

[0064] SEQ ID NO. 5: AAGCTTAGAATCCATCGACTAATAATTCA.

[0065] The reaction system for PCR amplification was as follows (50 μl): 1 μl of template cDNA, 1 μl of forward primer, 1 μl of reverse primer, 0.5 μl of ExTaq DNA polymerase (TAKARA), 5 μl of 10×Ex Taq Buffer, 4 μl of dNTP Mixture, and 37.5 μl of sterile deionized water.

[0066] The reaction conditions of PCR amplification were as follows: denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 40 sec, and 30 cycles. After amplification, the PCR products were electrophoresed on 1.5% agarose and photographed.

[0067] The PCR results of the gene of the ATPase sodium / potassium transporter β2 subunit protein of the dust mite allergen are as follows Figure 1 shown. Figure 1 In the figure, lane M is a DNA standard molecular weight marker, and lane 1 is the PCR product of the ATPase sodium / potassium transporter β2 subunit protein gene of the dust mite allergen.

[0068] The above PCR recovery product was connected with the T vector (TAKARA), transformed into competent Escherichia coli Top 10, screened and selected single clone colonies for culture, extracted plasmids for double restriction digestion identification, and positive single clones were sequenced by Sangon Biotechnology (Shanghai) Co., Ltd. The cloned plasmids with correct sequencing and the pET32a expression vector were double-digested with BamH I and HindIII, respectively, and subjected to agarose gel electrophoresis. After gel cutting and recovery, they were connected with T4 DNA ligase at 16°C overnight, transformed into competent Escherichia coli Top 10, screened and selected single colonies, extracted plasmids and double-digested for identification, and the recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein expression plasmid was stored for future use.

[0069] Among them, the thioredoxin tag protein in the expression vector pET32a assists in the co-expression of the dust mite allergen ATPase sodium / potassium transport β2 subunit protein. The thioredoxin tag protein is composed of 165 amino acids, with a molecular weight of about 19.45 kDa, and its amino acid sequence is shown in SEQ ID NO.6.

[0070] SEQ ID NO.6:

[0071] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGS.

[0072] The coding gene of the thioredoxin tag protein consists of 495 nucleotides, and its nucleotide sequence is shown in SEQ ID NO.7 (5' to 3' end).

[0073] SEQ ID NO.7:

[0074] ATGAGCGATAAAATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAA AAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAGGCCATGGCTGATATCGGATCC.

[0075] The co-expressed complete gene composed of the thioredoxin tag protein gene and the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein gene consists of 1434 nucleotides, and its nucleotide sequence is shown in SEQ ID NO.8 (5' to 3' end).

[0076] SEQ ID NO.8:

[0077]

[0078] 3. Induced expression and purification of target protein

[0079] The above identified recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein expression plasmid was transformed into competent Escherichia coli BL21 (DE3) plysS, inoculated for resuscitation, activated to the logarithmic growth phase, and then the inducer IPTG with a final concentration of 1 mol / L was added to induce the expression of the recombinant protein.

[0080] The recombinant bacteria were collected by centrifugation, lysozyme was added, and the bacteria were broken by ultrasonication in an ice bath. The supernatant was collected by centrifugation, filtered, and added to the equilibrated Ni-NTA column. The chromatography column was equilibrated with Ni-NTA column equilibration solution, and the protein was eluted with 250 mmol / L imidazole elution buffer. The protein eluate was collected according to the elution peak segmentation and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0081] The final purified protein is the co-expressed protein, i.e., the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein. The SDS-PAGE electrophoresis results of the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein are as follows: Figure 2 shown. Figure 2 In the figure, lane M is a protein standard molecular weight marker, and lane 1 is a recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tag protein purified by Ni-NTA chromatography. The recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tag protein comprises a recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein (about 37.33 kDa) and a thioredoxin tag protein (19.45 kDa), with a total molecular weight of about 56.78 kDa, and its amino acid sequence is shown in SEQ ID NO.2.

[0082] Example 3: Allergenicity Detection of ATPase Sodium / Potassium Transporter β2 Subunit Protein of Dermatophagoides Farinae Allergens 1. IgE-ELISA Experiment

[0083] The recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tag protein prepared in Example 2 was coated on the ELISA plate, 2 μg per well, and incubated at 4°C overnight. The next day, the ELISA plate was blocked with 5% BSA, and the serum of the positive patient to be tested was diluted 1:5 and added for incubation for 4 hours. After washing the plate, the secondary antibody against human IgE was added to detect the absorption of OD 450nm wavelength. The IgE-ELISA results of the serum of dust mite allergic patients or healthy people and the recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tag protein are shown as follows Figure 3 shown.

[0084] 2. IgE-Western Blot Experiment

[0085] The recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tagged protein prepared in Example 2 was subjected to SDS-PAGE electrophoresis, and the target protein was transferred from the polyacrylamide gel to the PVDF transfer membrane by using a slot transfer system. The PVDF was cut into membrane strips according to the width of the electrophoresis lane, and the membrane was blocked with 5% BSA. The membrane was incubated at 4°C overnight, and the serum of the positive patient to be tested was diluted 1:5 and added to the membrane strips for incubation for 1 hour. After the membrane strips were washed, the anti-human IgE secondary antibody was added and incubated for 2 hours. After washing, DAB color development solution was added for 10 minutes. The positive samples showed brown color at the position of the target protein band, and the negative samples had no reaction. The IgE-Western Blot results of the dust mite allergy patient serum and the recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tagged protein are shown as follows Figure 4 The results of IgE-Western Blot of healthy human serum and recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein are shown in Figure 5 shown.

[0086] 3. IgE-DotBlot Experiment

[0087] 2 μg of the recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tagged protein prepared in Example 2 was added to the PVDF transfer membrane as the experimental group. After adsorption, it was dried and the membrane was blocked with 5% BSA. The membrane was incubated at 4°C overnight. The serum of the positive patient to be tested was diluted 1:5 and added to the membrane for incubation for 2 hours. After the membrane was washed, the anti-human IgE secondary antibody was added and incubated for 1 hour. After washing, DAB color development solution was added for 10 minutes. The positive sample showed brown at the target protein band position, and the negative sample had no reaction. The IgE-DotBlot results of the dust mite allergy patient serum and the recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein-thioredoxin tagged protein are shown as follows: Figure 6 As shown in A, the IgE-DotBlot results of healthy human serum and recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein are as follows Figure 7 As shown in A.

[0088] Recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin-tagged protein was replaced with recombinant thioredoxin-tagged protein as a negative control, and the rest of the operations were the same as the experimental group. Neither the positive nor the negative samples had any binding reaction with the recombinant thioredoxin-tagged protein. The IgE-DotBlot results of the sera of dust mite allergic patients and the recombinant thioredoxin-tagged protein are shown in Figure 2. Figure 6As shown in B, the IgE-DotBlot results of healthy human serum and recombinant thioredoxin-tagged protein are as follows Figure 7 As shown in B.

[0089] Depend on Figure 6 and 7 It can be seen that in the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein-thioredoxin tag protein, the dust mite allergen component is the recombinant dust mite allergen ATPase sodium / potassium transporter β2 subunit protein.

[0090] In summary, the present invention provides a dust mite allergen ATPase sodium / potassium transport β2 subunit protein and its application. Wherein, the dust mite allergen ATPase sodium / potassium transport β2 subunit protein is a newly discovered dust mite allergen component, and its amino acid sequence is shown in SEQ ID NO.1. The present invention prepares a co-expressed protein of a recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein and a thioredoxin tag protein by gene cloning, protein expression, separation and purification, and identifies the recombinant dust mite allergen ATPase sodium / potassium transport β2 subunit protein in the co-expressed protein by experimental means such as IgE-enzyme-linked immunosorbent reaction and IgE immunoblotting to have certain allergenicity, and can be used as a dust mite allergen for preparing a reagent for the diagnosis, treatment and prevention of allergic diseases caused by dust mites, especially for preparing a reagent for the diagnosis, treatment and prevention of allergic diseases caused by the dust mite ATPase sodium / potassium transport β2 subunit protein.

[0091] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A dust mite allergen ATPase sodium / potassium transporter β2 subunit protein, characterized in that: The amino acid sequence of the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein is shown in SEQ ID NO.

1.

2. A co-expressed protein containing the β2 subunit protein of the ATPase sodium / potassium transporter of dust mite allergen, characterized in that: The amino acid sequence of the co-expressed protein is shown in SEQ ID NO.

2.

3. A nucleic acid, characterized in that The nucleic acid encodes the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein according to claim 1.

4. A recombinant vector, characterized in that: The recombinant vector contains a gene expression cassette encoding the dust mite allergen ATPase sodium / potassium transport β2 subunit protein according to claim 1.

5. A host cell, characterized in that The host cell contains the recombinant vector according to claim 4.

6. A composition, characterized in that The composition contains the dust mite allergen ATPase sodium / potassium transport β2 subunit protein according to claim 1.

7. The composition according to claim 6, characterized in that The composition also includes a pharmaceutically acceptable carrier.

8. A method for preparing the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein according to claim 1, characterized in that: include: Total RNA of dust mites was extracted and reverse transcribed to obtain cDNA; Design primers, and obtain the gene encoding the β2 subunit protein of the ATPase sodium / potassium transporter of the dust mite allergen by PCR amplification. The nucleotide sequence of the gene encoding the ATPase sodium / potassium transporter of the dust mite allergen is shown in SEQ ID NO.3; Inserting the gene encoding the ATPase sodium / potassium transport β2 subunit protein of the dust mite allergen into an expression vector to obtain a recombinant plasmid; The recombinant plasmid is transferred into a host cell for protein expression and purification to obtain the dust mite allergen ATPase sodium / potassium transport β2 subunit protein.

9. The preparation method according to claim 8, characterized in that: The primers include an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.

5.

10. Use of the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein according to claim 1, the co-expressed protein according to claim 2, the nucleic acid according to claim 3, the recombinant vector according to claim 4, the host cell according to claim 5, the composition according to any one of claims 6 to 7, or the dust mite allergen ATPase sodium / potassium transporter β2 subunit protein prepared by the preparation method according to any one of claims 8 to 9 in the preparation of reagents for diagnosing, treating and preventing allergic diseases.

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