Dermatophagoides farinae allergen sulfur oxygen reduction protein dependent peroxide reduction protein and application thereof

Abstract

The invention relates to the field of biomedicine, and discloses dermatophagoides farinae allergen sulfur oxygen reduction protein dependent peroxide reduction protein and application thereof. Wherein the dermatophagoides farinae allergen sulfur oxygen reduction protein dependent peroxide reduction protein is a newly found dermatophagoides farinae allergen component, and the amino acid sequence of the dermatophagoides farinae allergen sulfur oxygen reduction protein dependent peroxide reduction protein is shown as SEQ ID NO. 1. The recombinant dermatophagoides farinae allergen sulfur oxygen reduction protein-dependent peroxide reduction protein is prepared through gene cloning, protein expression and separation and purification, and the recombinant dermatophagoides farinae allergen sulfur oxygen reduction protein-dependent peroxide reduction protein is identified to have certain allergenicity through experimental means such as IgE-enzyme-linked immunosorbent assay and IgE immunoblotting and can be used as a dermatophagoides farinae allergen; the invention relates to a dermatophagoides farinae dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides dermatophagoides

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a dust mite allergen thioredoxin-dependent peroxide reduction protein and an application thereof. Background Art

[0002] Allergic diseases, also known as allergic diseases, are a major health problem in the world today. The total incidence of allergic diseases in countries around the world is about 15%-30%, which has long troubled patients and their families. The allergens that cause allergic diseases are mainly divided into foodborne allergens and inhaled allergens, and dust mites are common inhaled allergens. The types of dust mites that are most closely related to allergic diseases are house dust mites (Der p) and dust mites (Der f). Studies have shown that dust mites have a variety of different allergen components, and more than 40 species have been identified so far. These allergen components also have cross-reactivity between different dust mites. A more comprehensive understanding and study of the allergen components of dust mites, and the preparation of biological preparations of single or combined allergen components, can accurately detect the sensitization causes of patients with allergic diseases, and provide a basis for the formulation of personalized and accurate desensitization treatment plans. Therefore, a comprehensive and systematic exploration and identification of dust mite allergen components is of great value for the clinical application of allergic diseases caused by dust mites. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a dust mite allergen thioredoxin-dependent peroxide-reducing protein and its application. The newly discovered dust mite allergen is a homologue of the dust mite thioredoxin-dependent peroxide-reducing 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.

[0004] The technical solution of the present invention is as follows:

[0005] In a first aspect of the present invention, a dust mite allergen thioredoxin-dependent peroxide reduction protein is provided. The amino acid sequence of the dust mite allergen thioredoxin-dependent peroxide reduction protein is shown in SEQ ID NO.1.

[0006] The second aspect of the present invention provides a nucleic acid encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein as described in the first aspect.

[0007] The third aspect of the present invention provides a recombinant vector, wherein the recombinant vector contains a gene expression cassette encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein as described in the first aspect.

[0008] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the recombinant vector as described in the third aspect.

[0009] The fifth aspect of the present invention provides a composition, wherein the composition contains the dust mite allergen thioredoxin-dependent peroxide reducing protein as described in the first aspect.

[0010] Optionally, the composition further comprises a pharmaceutically acceptable carrier.

[0011] The sixth aspect of the present invention provides a method for preparing the dust mite allergen thioredoxin-dependent peroxide reduction protein as described in the first aspect, comprising:

[0012] Total RNA from dust mites was extracted and reverse transcribed to obtain cDNA;

[0013] Design primers to obtain the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein by PCR amplification, wherein the nucleotide sequence of the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein is shown in SEQ ID NO.2;

[0014] Inserting the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein into an expression vector to obtain a recombinant plasmid;

[0015] The recombinant plasmid is transferred into a host cell for protein expression and purification to obtain dust mite allergen thioredoxin-dependent peroxide reduction protein.

[0016] Optionally, the primers include an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0017] In a seventh aspect, the present invention provides a use of the dust mite allergen thioredoxin-dependent peroxide-reducing protein as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant vector as described in the third aspect, the host cell as described in the fourth aspect, the composition as described in the fifth aspect, or the dust mite allergen thioredoxin-dependent peroxide-reducing protein prepared by the preparation method as described in the sixth aspect in the preparation of reagents for diagnosing, treating, and preventing allergic diseases.

[0018] Beneficial effect: The present invention provides a dust mite allergen thioredoxin-dependent peroxide reduction protein and its application. The dust mite allergen thioredoxin-dependent peroxide reduction protein is a newly discovered dust mite allergen component and is a homologue of the dust mite thioredoxin-dependent peroxide reduction protein. The present invention prepares a recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein by gene cloning, protein expression, separation and purification, and identifies it as having certain allergenicity by experimental means such as IgE-enzyme-linked immunosorbent reaction and IgE immunoblotting. It can be used as a dust mite allergen for preparing reagents for the diagnosis, treatment and prevention of allergic diseases caused by dust mites, especially for preparing reagents for the diagnosis, treatment and prevention of allergic diseases caused by the dust mite thioredoxin-dependent peroxide reduction protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is an agarose electrophoresis diagram of the PCR amplification product of the thioredoxin-dependent peroxide-reducing protein gene of the dust mite allergen in Example 2.

[0020] Figure 2 This is the SDS-PAGE electrophoresis diagram of the expression and purification product of the recombinant dust mite allergen thioredoxin-dependent peroxide reducing protein in Example 2.

[0021] Figure 3 This is the result of IgE-ELISA between the serum of dust mite allergic patients and the recombinant dust mite allergen thioredoxin-dependent peroxide reducing protein in Example 3.

[0022] Figure 4 This is the result of IgE-Western Blot of the serum of the patient allergic to dust mites and the recombinant dust mite allergen thioredoxin-dependent peroxide reducing protein in Example 3.

[0023] Figure 5 This is the result of IgE-Western Blot of healthy human serum and recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein in Example 3.

[0024] Figure 6 This is the IgE-DotBlot result of the serum of the patient with dust mite allergy and the recombinant dust mite allergen thioredoxin-dependent peroxide reducing protein in Example 3.

[0025] Figure 7 This is the IgE-DotBlot result of healthy human serum and recombinant dust mite allergen thioredoxin-dependent peroxide reducing protein in Example 3. DETAILED DESCRIPTION

[0026] The present invention provides a dust mite allergen thioredoxin-dependent peroxide reduction 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.

[0027] The embodiment of the present invention provides a dust mite allergen thioredoxin-dependent peroxide reduction protein, which consists of 221 amino acids and has a molecular weight of about 25 kDa. The amino acid sequence is shown in SEQ ID NO.1.

[0028] SEQ ID NO.1:

[0029] MASLLRQFIRPASISLAKRNVNLLNTRFMSAFVQQPAPDFKATAVVDKQFKDIKLVDFQGKYLVLFFYPLDFTFVCPTEIIAFNDRLQEFRDLQTEVVAVSVDSHFSHLAW CNTPRKQGGLGDMKMPIIADLTKKISGDYGVLLPEAGVALRGLFIIDTKGIVRQITINDLPVGRSVDETLRLIKAFQYTDKHGEVCPANWQPDRPTIDPRNAREYFEKQN.

[0030] An embodiment of the present invention provides a nucleic acid encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein as described above.

[0031] The embodiment of the present invention provides a recombinant vector, wherein the recombinant vector contains a gene expression cassette encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein as described above.

[0032] 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 Xba I restriction sites, and specifically, the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein is inserted between the BamHI and Xba I restriction sites.

[0033] In one embodiment, the recombinant vector is selected from one of T vector and pET-His, but is not limited thereto.

[0034] An embodiment of the present invention provides a host cell, wherein the host cell contains the recombinant vector as described above.

[0035] 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.

[0036] An embodiment of the present invention provides a composition, wherein the composition contains the dust mite allergen thioredoxin-dependent peroxide reducing protein as described above.

[0037] 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.

[0038] 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.

[0039] The embodiment of the present invention provides a method for preparing the dust mite allergen thioredoxin-dependent peroxide reduction protein as described above, comprising the steps of:

[0040] (1) Extract total RNA from dust mites and reverse transcribe to obtain cDNA;

[0041] (2) designing primers and obtaining the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein by PCR amplification, wherein the nucleotide sequence of the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein is shown in SEQ ID NO.2;

[0042] (3) inserting the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein into an expression vector to obtain a recombinant plasmid;

[0043] (4) The recombinant plasmid is transferred into a host cell for protein expression and purification to obtain the dust mite allergen thioredoxin-dependent peroxide reduction protein.

[0044] In one embodiment, the primers include an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0045] In one embodiment, the step (4) specifically comprises: transforming the recombinant plasmid into a host cell, culturing, inducing, disrupting the cells, and chromatographically purifying to obtain the dust mite allergen thioredoxin-dependent peroxide reduction protein.

[0046] The embodiments of the present invention provide a use of the dust mite allergen thioredoxin-dependent peroxide-reducing 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 thioredoxin-dependent peroxide-reducing protein prepared by the preparation method as described above in the preparation of reagents for diagnosing, treating, and preventing allergic diseases.

[0047] In one embodiment, the allergic disease is an allergic disease caused by dust mites.

[0048] In one embodiment, the allergic disease is an allergic disease induced by dust mite thioredoxin-dependent peroxiredoxin.

[0049] The present invention will be further described below by means of specific examples.

[0050] Example 1: Discovery of thioredoxin-dependent peroxide-reducing protein of dust mite allergen

[0051] By analyzing the dung beetle genome sequence, the gene sequence of the thioredoxin-dependent peroxide-reducing protein of the dung beetle allergen was obtained. The gene sequence consists of 666 nucleotides, and the nucleotide sequence is specifically shown in SEQ ID NO.2 (5' to 3' end).

[0052] SEQ ID NO.2:

[0053] .

[0054] Example 2: Molecular cloning of thioredoxin-dependent peroxide-reducing protein of dust mite allergen

[0055] 1. Extraction of total RNA from Dermatophagoides farinae

[0056] The dust mite bodies were enriched, ground with liquid nitrogen, and the total RNA of dust mites was extracted using the column extraction method TaKaRa MiniBEST Universal RNA Extraction Kit (Code No.9767).

[0057] 2. Reverse transcription and cDNA synthesis

[0058] The extracted total RNA of dust mites was reverse transcribed using Thermo SuperScript IV reverse transcriptase (Cat.18090010) at 37°C for 10 min. The reverse transcription system was as follows:

[0059] Reaction content Usage Dermatophagoides farinae total RNA 1 μg SuperScript IV Reverse Transcriptase 1μl Reverse transcription buffer 1μl 6mers Random Primer (20μM) 1μl Oligo dT Primer (2.5 μM) 1μl dNTP Mixture (10mM each) 1μl <![CDATA[RNA free H2O]]> Make up to 10 μl

[0060] 3. PCR amplification of target gene

[0061] According to the analysis of the dust mite genome sequence in Example 1, the forward primer DF-43-F and the reverse primer DF-43-R of the dust mite allergen thioredoxin-dependent peroxide reduction protein gene were designed and synthesized. The nucleotide sequence of DF-43-F is shown in SEQ ID NO.3 (5' to 3' end), and the nucleotide sequence of DF-43-R is shown in SEQ ID NO.4 (5' to 3' end).

[0062] SEQ ID NO. 3: GGATCCATGGCATCTTTACTTCGACAA.

[0063] SEQ ID NO. 4:TCTAGACTAATTTTGTTTCTCAAAATA.

[0064] The reaction system for PCR amplification is as follows (50 μl):

[0065] Reaction content Usage Dermatophagoides farinae cDNA 500ng TAKARA ExTaq DNA Polymerase 1μl PCR Buffer 1μl Forward Primer 1μl Reverse primer 1μl dNTP Mixture (10mM each) 4μl <![CDATA[RNAfree H2O]]> Make up to 50 μl

[0066] The reaction conditions of PCR amplification are as follows: denaturation at 95℃ for 1 min, annealing at 53℃ for 30 sec, extension at 72℃ for 40 sec, and reaction for 30 cycles. After the amplification, the PCR product was electrophoresed on 1.5% agarose gel and photographed. 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 thioredoxin-dependent peroxide-reducing protein gene of the dust mite allergen.

[0067] 4. Linkage cloning of target genes

[0068] The above PCR recovered products were connected with T vector (TAKARA), transformed into competent Escherichia coli Top 10, single clone colonies were screened and selected for culture, plasmids were extracted 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 pET-His expression vector were double-digested with BamH I and Xba I, 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, single colonies were screened and selected, plasmids were extracted and double-digested for identification, and the recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein expression plasmid was stored for future use.

[0069] 5. Induced expression and purification of target protein

[0070] The above-identified recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein expression plasmid was transformed into competent Escherichia coli BL21 (DE3) plysS and inoculated into LB medium containing Amp for expansion of the recombinant bacteria. When the recombinant bacteria were activated to the logarithmic growth phase, the inducer IPTG was added at a final concentration of 1 mol / L to induce the expression of the recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein.

[0071] The recombinant bacteria were collected by centrifugation, resuspended with PBS, and lysozyme was added to lyse in an ice bath for 10 minutes. The bacteria were then ultrasonically disrupted in an ice bath, and the supernatant was separated by centrifugation. The supernatant containing the recombinant dust mite allergen thioredoxin-dependent peroxide-reducing protein was filtered and added to a Ni-NTA column that had been equilibrated in advance. The Ni-NTA column equilibration solution was used to continue equilibration of the chromatography column, and the protein was eluted with an elution buffer containing 250 mmol / L imidazole. The protein eluate was collected according to the peak segmentation and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0072] The various buffers required for purification are prepared as follows:

[0073] (1) Resuspension buffer: 50 mM PB, 150 mM NaCl, pH 7.2;

[0074] (2) Column equilibration buffer: 50 mM PB, 300 mM NaCl, 5 mM Imidazole, pH 7.5;

[0075] (3) Column elution buffer: 50 mM PB, 300 mM NaCl, 250 mM Imidazole, pH 7.5;

[0076] (4) Dialysis buffer: 50 mM PB, 150 mM NaCl, pH 7.5.

[0077] The SDS-PAGE electrophoresis results of the recombinant dust mite allergen thioredoxin-dependent peroxide-reducing 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 thioredoxin-dependent peroxide reducing protein purified by Ni-NTA chromatography.

[0078] Example 3: Allergenicity Detection of Thioredoxin-Dependent Peroxide Reducing Protein of Dust Mite Allergen

[0079] 1. IgE-ELISA experiment

[0080] The recombinant dust mite allergen sulforedoxin-dependent peroxide reduction protein prepared in Example 2 was coated on the ELISA plate, 2 μg per well, overnight at 4°C, and the ELISA plate was blocked with 5% BSA the next day. 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 sulforedoxin-dependent peroxide reduction protein are shown in Figure 3 shown.

[0081] 2. IgE-Western Blot Experiment

[0082] The recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein prepared in Example 2 was subjected to SDS-PAGE electrophoresis. 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. 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 all had binding reactions with the recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein, and the negative samples all had no binding reactions with the recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein. The IgE-Western Blot results of the serum of dust mite allergic patients and the recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein are shown as follows: Figure 4 The results of IgE-Western Blot of healthy human serum and recombinant dust mite allergen thioredoxin-dependent peroxiredoxin are shown in Figure 5 shown.

[0083] 3. IgE-DotBlot Experiment

[0084] 2 μg of the recombinant dust mite allergen sulforedoxin-dependent peroxide reduction protein prepared in Example 2 was added to the PVDF transfer membrane. 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 serum of dust mite allergic patients and the recombinant dust mite allergen sulforedoxin-dependent peroxide reduction protein are shown in Figure 1. Figure 6 The results of IgE-DotBlot of healthy human serum and recombinant dust mite allergen thioredoxin-dependent peroxiredoxin are shown in Figure 7 shown.

[0085] In summary, the present invention provides a dust mite allergen thioredoxin-dependent peroxide reduction protein and its application. Wherein, the dust mite allergen thioredoxin-dependent peroxide reduction 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 recombinant dust mite allergen thioredoxin-dependent peroxide reduction protein by gene cloning, protein expression, separation and purification, and identifies it as having certain allergenicity through experimental means such as IgE-enzyme-linked immunosorbent reaction and IgE immunoblotting. It can be used as a dust mite allergen for preparing reagents for the diagnosis, treatment and prevention of allergic diseases caused by dust mites, especially for preparing reagents for the diagnosis, treatment and prevention of allergic diseases caused by dust mite thioredoxin-dependent peroxide reduction protein.

[0086] 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 thioredoxin-dependent peroxide-reducing protein, characterized in that: The amino acid sequence of the dust mite allergen thioredoxin-dependent peroxide-reducing protein is shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that The nucleic acid encodes the dust mite allergen thioredoxin-dependent peroxide reduction protein according to claim 1.

3. A recombinant vector, characterized in that: The recombinant vector contains a gene expression cassette encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein according to claim 1.

4. A host cell, characterized in that The host cell contains the recombinant vector according to claim 3.

5. A composition, characterized in that The composition contains the dust mite allergen thioredoxin-dependent peroxide reducing protein according to claim 1.

6. The composition according to claim 5, characterized in that The composition also includes a pharmaceutically acceptable carrier.

7. A method for preparing the dust mite allergen thioredoxin-dependent peroxide reduction protein according to claim 1, characterized in that: include: Total RNA of dust mites was extracted and reverse transcribed to obtain cDNA; Design primers to obtain the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein by PCR amplification, wherein the nucleotide sequence of the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein is shown in SEQ ID NO.2; Inserting the gene encoding the dust mite allergen thioredoxin-dependent peroxide reduction protein 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 dust mite allergen thioredoxin-dependent peroxide reduction protein.

8. The preparation method according to claim 7, characterized in that: The primers include an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.

4.

9. Use of the dust mite allergen thioredoxin-dependent peroxide-reducing protein according to claim 1, the nucleic acid according to claim 2, the recombinant vector according to claim 3, the host cell according to claim 4, the composition according to any one of claims 5 to 6, or the dust mite allergen thioredoxin-dependent peroxide-reducing protein prepared by the preparation method according to any one of claims 7 to 8 in the preparation of reagents for diagnosing, treating and preventing allergic diseases.

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