CN108840917B - House dust mite allergen Der p30 and preparation method and application thereof - Google Patents
House dust mite allergen Der p30 and preparation method and application thereof Download PDFInfo
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- CN108840917B CN108840917B CN201810601060.7A CN201810601060A CN108840917B CN 108840917 B CN108840917 B CN 108840917B CN 201810601060 A CN201810601060 A CN 201810601060A CN 108840917 B CN108840917 B CN 108840917B
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- dermatophagoides pteronyssinus
- allergen der
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/35—Allergens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
- G01N2333/43582—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
The invention provides a dermatophagoides pteronyssinus allergen Der p30, the dermatophagoides pteronyssinus allergen Der p30 comprises at least one of an amino acid sequence shown as SEQ ID NO.1 and an amino acid sequence which has at least 98% homology with the amino acid sequence shown as SEQ ID NO.1, and the dermatophagoides pteronyssinus allergen Der p30 has wide application prospects in a dermatophagoides pteronyssinus allergen vaccine and a medicine for preventing, diagnosing and treating allergic diseases caused by dermatophagoides pteronyssinus, particularly the allergic diseases caused by the 30 th component of dermatophagoides pteronyssinus. The recombinant dermatophagoides pteronyssinus allergen Der p30 is obtained by gene cloning, protein expression and purification, has high protein purity and abundant yield, is favorable for subsequent scientific research and application and mass preparation and application, and is particularly applied to dermatophagoides pteronyssinus allergen vaccines and preparation of medicaments for preventing, diagnosing and treating allergic diseases caused by dermatophagoides pteronyssinus.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a dermatophagoides pteronyssinus allergen Der p30 and a preparation method and application thereof.
Background
Allergic diseases (allergic diseases) are regarded as a major health problem in the world by the world health organization, and the total incidence rate of allergic diseases in various countries in the world is as high as 10-30%. The incidence of disease has been increasing in recent years. Among the many inhaled allergens that cause allergic diseases, dust mites are the most important factor causing allergic diseases. Dust mites (Der-phytophagoides farinaceae, Der f), house dust mites (Der p) are now found to be the dominant indoor sensitizing mites widely distributed worldwide.
Since dust mites are medical arthropods, the structure and composition are complex, although several allergen components have been preliminarily identified from hundreds of proteins, studies have shown that dust mites contain many other allergen components; and these allergen components are of great significance for studying the immune pathogenesis of dust mite allergy and developing reagents for diagnosing and treating dust mite allergy.
Disclosure of Invention
In view of the above, the invention provides a dermatophagoides pteronyssinus allergen Der p30, which is obtained through gene cloning, protein expression and purification to obtain a recombinant dermatophagoides pteronyssinus allergen Der p30, wherein the dermatophagoides pteronyssinus allergen Der p30 has wide application prospects in a dermatophagoides pteronyssinus allergen vaccine and a medicine for preventing, diagnosing and treating allergic diseases caused by dermatophagoides pteronyssinus, particularly allergic diseases caused by the 30 th component of dermatophagoides pteronyssinus.
In a first aspect, the invention provides a dermatophagoides pteronyssinus allergen Der p30, wherein the dermatophagoides pteronyssinus allergen Der p30 comprises at least one of an amino acid sequence shown as SEQ ID NO.1 and an amino acid sequence with at least 98% homology with the amino acid sequence shown as SEQ ID NO. 1.
In the present invention, the house dust mite allergen Der p30 is the 30 th component of the house dust mite allergen.
In the present invention, the term "homology" indicates that any two (or more) peptide, polypeptide or protein sequences have amino acid sequences that are "identical" to one another to some extent ("% homology"). Such a degree of homology can be determined using known computer programs. In particular, the BLAST tool, which can be but is not limited to the NCBI database, can be used to determine homology. In the present invention, the homology is at least 98%. Further alternatively, the homology is at least 99%.
Optionally, the dermatophagoides pteronyssinus allergen Der p30 further comprises a dermatophagoides pteronyssinus allergen having immunological cross-reactivity with the amino acid sequence shown as SEQ ID NO. 1. As used herein, the term "immunological cross-reactivity" is the conventional understanding in the industry when an antibody can react with different antigens that are not identical in molecular structure but have similar antigenic determinants, and is referred to as immunological cross-reactivity; different antigens have immunological cross-reactivity.
Optionally, the coding gene of the dermatophagoides pteronyssinus allergen Der p30 comprises a nucleotide sequence of an amino acid sequence shown as SEQ ID NO. 1.
Further optionally, the coding gene of the dermatophagoides pteronyssinus allergen Der p30 comprises a nucleotide sequence shown in SEQ ID NO. 2.
Optionally, the coding gene of the dermatophagoides pteronyssinus allergen Der p30 should consider degenerate bases, that is, the coding gene of the amino acid sequence shown in SEQ ID NO.1 includes the nucleotide sequence shown in SEQ ID NO. 2, the protection scope should also protect the nucleotide sequence with base degeneracy with the SEQ ID NO. 2, and the corresponding amino acid sequence of the nucleotide sequences is still SEQ ID NO. 1.
The dermatophagoides pteronyssinus allergen Der p30 provided by the first aspect of the invention has wide application prospects in dermatophagoides pteronyssinus allergen vaccines and medicines for preventing, diagnosing and treating allergic diseases caused by dermatophagoides pteronyssinus.
In a second aspect, the present invention provides a recombinant vector comprising a gene encoding the dermatophagoides pteronyssinus allergen Der p30 according to the first aspect.
Optionally, the recombinant vector comprises at least one of a recombinant expression vector and a recombinant cloning vector. Further optionally, the recombinant vector contains BamHI and XhoI enzyme cutting sites, and specifically, the coding gene of dermatophagoides pteronyssinus allergen Der p30 is inserted between the BamHI and XhoI enzyme cutting sites. Optionally, when the coding gene of the dermatophagoides pteronyssinus allergen Der p30 is inserted into the vector, an initiation codon (such as ATG) can be added to the 5 'end of the coding gene of the dermatophagoides pteronyssinus allergen Der p30, and a stop codon (such as TAA) can be added to the 3' end.
Optionally, the recombinant vector contains a purification tag, which may be specifically, but not limited to, His tag, GST tag, SUMO tag.
Alternatively, the recombinant vector may be, but is not limited to, at least one of pET-32a, pGEX-6P-1, pPIC-9K, pPIC-Z α.
In a third aspect, the present invention provides a host cell comprising a recombinant vector as described in the second aspect.
Optionally, the host cell comprises at least one of a cloning host cell and an expression host cell. Specifically, the host cell can be, but not limited to, escherichia coli, saccharomyces cerevisiae, pichia pastoris, animal cells, plant cells. In particular, but not limited to, Escherichia coli DH5 α, Escherichia coli Top10, Escherichia coli Orgami (DE3), Pichia pastoris GS115, Pichia pastoris SMD 1168.
In a fourth aspect, the invention provides a preparation method of house dust mite allergen Der p30, which comprises the following steps:
(1) preparing and obtaining a coding gene of a dermatophagoides pteronyssinus allergen Der p30, wherein the coding gene of the dermatophagoides pteronyssinus allergen Der p30 comprises a nucleotide sequence shown as SEQ ID NO: 1;
(2) inserting the coding gene of the dermatophagoides pteronyssinus allergen Der p30 into an expression vector to obtain a recombinant plasmid;
(3) and transferring the recombinant plasmid into an expression host cell, and performing protein expression and purification to obtain the dermatophagoides pteronyssinus allergen Der p 30.
Optionally, the gene for preparing and obtaining the dermatophagoides pteronyssinus allergen Der p30 comprises: extracting total protein of house dust mite, separating and identifying to obtain the house dust mite allergen Der p30, and obtaining the coding gene of the house dust mite allergen Der p30 through PCR amplification.
Alternatively, when the host cell according to the fourth aspect is an expression host cell, the method for preparing the dermatophagoides pteronyssinus allergen Der p30 may further comprise: culturing the host cell of the fifth aspect, and performing protein expression and purification to obtain the dermatophagoides pteronyssinus allergen Der p 30. Specifically, but not limited to, when the host cell is an escherichia coli expression host cell, the escherichia coli is cultured in an LB culture medium, isopropyl thiogalactoside is added in the culture process to perform induced expression of the dermatophagoides pteronyssinus allergen Der p30, and the dermatophagoides pteronyssinus allergen Der p30 is obtained by centrifugation, cracking, bacteriolysis, ultrasonic treatment, and separation and purification through a nickel column.
The preparation method of the house dust mite allergen Der p30 provided by the fourth aspect of the invention is simple to operate, easy to realize, low in cost and applicable to large-scale industrial production; compared with natural dermatophagoides pteronyssinus allergen, the dermatophagoides pteronyssinus allergen Der p30 prepared by the preparation method is high in purity, good in yield, strong in biological activity, not easy to be polluted by exogenous toxic substances, good in stability, and capable of being used for researching an immune pathogenesis of dermatophagoides pteronyssinus allergy, particularly an anaphylactic reaction caused by the 30 th component of dermatophagoides pteronyssinus allergen; has important significance in dust mite allergen vaccines and in the development of drugs for diagnosing and treating dust mite allergy.
In a fifth aspect, the present invention provides a composition comprising the house dust mite allergen Der p30 as described in the first aspect or as prepared by the preparation method described in the fourth aspect.
Optionally, the composition further comprises a pharmaceutically acceptable carrier. Further optionally, the pharmaceutically acceptable carrier includes at least one of a solvent, a polymer, and a liposome, but is not limited thereto. Still further optionally, the solvent includes, but is not limited to, water, physiological saline, and other non-aqueous solvents. Specifically, the polymer may be, but is not limited to, polylysine, polyethyleneimine and modifications thereof, chitosan, polylactic acid, gelatin, and cyclodextrin. In particular, the liposome can be, but is not limited to, cholesterol, soy lecithin, egg yolk lecithin. Still further optionally, the pharmaceutically acceptable carrier further comprises one or more of a diluent and an excipient. Optionally, the diluent comprises one or more of starches, sugars, celluloses and inorganic salts. The excipient comprises at least one of binder, filler, lubricant in tablet, matrix part in semisolid ointment and cream, antiseptic, antioxidant, correctant, aromatic, cosolvent, emulsifier and colorant in liquid preparation.
In the present invention, the composition may further include other active ingredients, which are selected according to the use of the composition, and are not limited herein.
In a sixth aspect, the present invention provides a dermatophagoides pteronyssinus allergen Der p30 according to the first aspect, a recombinant vector according to the second aspect, a host cell according to the third aspect, a dermatophagoides pteronyssinus allergen Der p30 prepared by the preparation method according to the fourth aspect, or a composition according to the fifth aspect, for use in a dermatophagoides pteronyssinus allergen vaccine and a medicament for preventing, diagnosing and treating allergic diseases caused by dermatophagoides pteronyssinus.
Especially in the prevention, diagnosis and treatment of house dust mite component 30 induced allergic diseases. Further optional, the application in the medicine for preventing, diagnosing and treating allergic asthma, rhinitis and chronic urticaria.
Optionally, the drug is at least one of a chemical drug and a biological drug. Further optionally, the biological drug is one or more of a polypeptide drug, a protein drug and a gene drug.
Optionally, the medicament also comprises other pharmaceutically acceptable active ingredients for preventing, diagnosing and treating allergic diseases caused by dust mites.
Optionally, the pharmaceutical form comprises a tablet, capsule, powder, granule, pill, syrup, solution or suspension.
The application can be specifically but not limited to: there is provided a kit comprising a dermatophagoides pteronyssinus allergen Der p30 according to the first aspect, a recombinant vector according to the second aspect, a host cell according to the third aspect, or a composition according to the fourth aspect.
The invention has the beneficial effects that:
(1) the invention discovers and obtains the dermatophagoides pteronyssinus allergen Der p30 for the first time, enriches the components of the dermatophagoides pteronyssinus allergen, and has wide application prospect in a dermatophagoides pteronyssinus allergen vaccine and a medicament for preventing, diagnosing and treating allergic diseases caused by dermatophagoides pteronyssinus, particularly the allergic diseases caused by the 30 th component of dermatophagoides pteronyssinus;
(2) the recombinant dermatophagoides pteronyssinus allergen Der p30 is obtained by gene cloning, protein expression and purification, has high protein purity, abundant yield and high stability, and is favorable for subsequent mass preparation and research and use.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a double-enzymatic cleavage map of a recombinant plasmid pET-32a-Der p30 provided in example 1 of the present invention;
fig. 2 is a graph of Der p30 protein gel provided in example 1 of the present invention, wherein (a) in fig. 2 is a graph of Der p30 protein gel without purification, and (b) in fig. 2 is a graph of Der p30 protein gel after purification.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the examples of the present invention, the "dust mite allergen Der p 30", the "house dust mite allergen Der 30" and the "Der p 30" may be interchanged.
EXAMPLE A preparation of house dust mite allergen Der p30
1. Recombinant vector construction
Extracting total protein of house dust mite, separating and identifying to obtain house dust mite allergen Der p30, obtaining a nucleotide sequence of the house dust mite allergen Der p30 through PCR, wherein the nucleotide sequence is shown as SEQ ID NO.1 and consists of 324 basic groups (containing stop codon), 108 amino acid sequences (containing stop codon) are coded by the nucleotide sequence, and the relative molecular mass of the protein is 28kD through informatics analysis and is inferred to be 5.24 of isoelectric point.
Adding a BamH I restriction site and a protective base thereof at the N end of a nucleotide sequence of a dermatophagoides pteronyssinus allergen Der p30 by a gene synthesis technology, adding an Xho I restriction site and a protective base thereof at the C end, carrying out BamH I and Xho I double restriction on the nucleotide sequence, carrying out BamH I and Xho I double restriction on pET-32a, carrying out agarose gel electrophoresis verification on restriction products of the two restriction enzymes, recovering after the sequences are correct in size, mixing the restriction products of the two restriction enzymes together, adding T4DNA Ligase (Ligase), carrying out ligation reaction for 16h at 16 ℃ to obtain a recombinant plasmid pET-32a-Der p30, then transforming the recombinant plasmid into escherichia coli Top10, coating a bacterial solution on an LB plate containing aminobenzyl, and culturing at 37 ℃ overnight. Picking a single colony, extracting plasmids, and then carrying out double enzyme digestion and sequencing verification, wherein the double enzyme digestion result is shown in figure 1, wherein M represents a protein molecular weight standard (marker), a lane 1 represents a double enzyme digestion diagram, a target matrix band is about 324bp and accords with the theoretical molecular weight of Der p30, and meanwhile, the sequencing result shows that the target gene is correctly inserted.
2. Expression and purification of Der p30
pET-32a-Der p30 with correct sequencing is transformed into calcium chloride-competent Orgami expression bacteria, screened by an ampicillin plate, and positive colonies are picked for overnight culture. After secondary bacteria shaking, IPTG with the final concentration of 1mmol/L is added for induction, the bacteria are collected after induction at 37 ℃ and 200 r/min for 4h, and the supernatant and the precipitate are respectively taken by ultrasonic crushing and are analyzed by 12 percent SDS-PAGE to determine the solubility of the protein. Ni by FPLC2+And affinity chromatography purification, which comprises fully washing the column with an equilibrium solution, eluting with an imidazole equilibrium buffer solution containing 40mmol/L and 300mmol/L respectively, collecting eluates, and performing SDS-PAGE analysis to obtain Der p 30. The results are shown in figure 2 of the drawings,in FIG. 2, M in (a) indicates a protein molecular weight standard (marker), lane 1 indicates a protein distribution in the medium before induction, lane 2 indicates a protein distribution in the medium after induction, lane 3 indicates a protein distribution in the supernatant, lane 4 indicates a protein distribution in the precipitate, and it can be seen that Der p30 was successfully induced. Then passing through Ni2+After purification by affinity chromatography, as shown in FIG. 2 (b), wherein M represents a protein molecular weight standard (marker), lane 1 represents a map of the purified Der p30 protein, and the Der p30 protein has a size of 28kD, agrees with the predicted result, and is a soluble protein.
Effect example 1 allergen detection (ELISA)
In 96-well plates, each well was coated with Der p 30100. mu.L (1. mu.g/mL), 3% BSA was blocked overnight at 4 ℃. The serum (diluted at a ratio of 1: 50) of house dust mite allergic patients is used as a primary antibody, the primary antibody in a control group is normal human serum (4 parts), and the primary antibody is incubated at 37 ℃ for 2 h. Then 100. mu.L of biotin-labeled anti-human IgE (1:2000 dilution) was added per well and incubated at 37 ℃ for 2 h. Wash 5 times, add 100. mu.L of HRP-labeled streptavidin per well (1:5000 dilution) and incubate for 1h at 37 ℃. After development, incubation was carried out at 37 ℃ for 10min, and then the reaction was terminated with 2mol/L concentrated sulfuric acid. Detecting the absorbance of the antigen at 450nm by using an automatic microplate reader, analyzing data, determining that the absorbance value detected by serum IgE of a patient is 2.5 times higher than that detected by serum IgE of a normal person as positive, and using the normal person as a contrast, and finding that Der p30 has anaphylactic source.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110> Liu Zhi just
<120> house dust mite allergen Der p30, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Lys Phe Gln Asn Gln Arg Gly Gly Arg Ile Leu Leu His Asp Ile
1 5 10 15
Pro Lys Pro Val Lys Gln Asp Trp Thr Ser Gly Leu Glu Ala Met Glu
20 25 30
Ala Ala Leu Glu Leu Glu Lys Thr Val Asn Gln Ser Leu Leu Asp Leu
35 40 45
His Gly Val Ala Thr Lys Asn Ser Asp Val Gln Phe Ala Asp Phe Leu
50 55 60
Glu Thr His Tyr Leu Thr Glu Gln Val Glu Ala Ile Lys Lys Leu Ala
65 70 75 80
Asp Tyr Val Thr Gln Leu Arg Arg Cys Gly Pro Gly Leu Gly Glu Tyr
85 90 95
Leu Phe Asp Lys His Thr Leu Gln Gly Gln Ser
100 105
<210> 2
<211> 324
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgaaattcc agaaccagcg tggcggccgt atcctgctgc acgatatccc gaaaccggtt 60
aaacaggatt ggaccagcgg cctggaagcg atggaagcgg cgctggaact ggaaaaaacc 120
gttaaccaga gcctgctgga tctgcacggc gttgcgacca aaaacagcga tgttcagttc 180
gcggatttcc tggaaaccca ctacctgacc gaacaggttg aagcgatcaa aaaactggcg 240
gattacgtta cccagctgcg tcgttgcggc ccgggcctgg gtgaatacct gttcgataaa 300
cacaccctgc agggccagag ctaa 324
Claims (10)
1. A dermatophagoides pteronyssinus allergen Der p30 is characterized in that the amino acid sequence of the dermatophagoides pteronyssinus allergen Der p30 is the amino acid sequence shown in SEQ ID NO. 1.
2. The dermatophagoides pteronyssinus allergen Der p30 according to claim 1, wherein the coding gene of the dermatophagoides pteronyssinus allergen Der p30 is a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO. 1.
3. The dermatophagoides pteronyssinus allergen Der p30 according to claim 2, wherein the coding gene of the dermatophagoides pteronyssinus allergen Der p30 is a nucleotide sequence shown in SEQ ID NO. 2.
4. A recombinant vector comprising a gene encoding the dermatophagoides pteronyssinus allergen Der p30 according to any one of claims 1 to 3.
5. A host cell comprising the recombinant vector of claim 4.
6. A preparation method of dermatophagoides pteronyssinus allergen Der p30 is characterized by comprising the following steps:
(1) preparing and obtaining an encoding gene of a dermatophagoides pteronyssinus allergen Der p30, wherein the encoding gene of the dermatophagoides pteronyssinus allergen Der p30 is a gene which is encoded as SEQ ID NO: 1;
(2) inserting the coding gene of the dermatophagoides pteronyssinus allergen Der p30 into an expression vector to obtain a recombinant plasmid;
(3) and transferring the recombinant plasmid into an expression host cell, and performing protein expression and purification to obtain the dermatophagoides pteronyssinus allergen Der p 30.
7. The method for preparing a house dust mite allergen Der p30 according to claim 6, wherein the gene encoding the house dust mite allergen Der p30 is prepared and obtained by: extracting house dust mite total protein, separating and identifying to obtain house dust mite allergen Der p30, and obtaining the coding gene of the house dust mite allergen Der p30 through PCR amplification.
8. A composition comprising a house dust mite allergen Der p30 according to any of claims 1 to 3 or a house dust mite allergen Der p30 obtainable by the process according to any of claims 6 to 7.
9. The composition of claim 8, wherein the composition further comprises a pharmaceutically acceptable carrier.
10. Use of the dermatophagoides pteronyssinus allergen Der p30 according to any one of claims 1 to 3, the recombinant vector according to claim 4, the host cell according to claim 5, the dermatophagoides pteronyssinus allergen Der p30 prepared by the preparation method according to any one of claims 6 to 7, or the composition according to any one of claims 8 to 9 for the preparation of a dermatophagoides pteronyssinus allergen vaccine and for the preparation of a medicament for the prevention, diagnosis and treatment of allergic diseases caused by dermatophagoides pteronyssinus.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5972352A (en) * | 1987-06-17 | 1999-10-26 | Thomas; Wayne R. | Cloning of mite allergens |
| CN101432299A (en) * | 2006-04-28 | 2009-05-13 | 碧欧美公司 | House dust mite allergen |
| CN103073636A (en) * | 2013-01-29 | 2013-05-01 | 中国科学院昆明动物研究所 | Dermtophagoides farinae allergens Der f 29 and Der f 30, and genes and applications thereof |
| CN104894089A (en) * | 2015-03-27 | 2015-09-09 | 深圳大学 | Dust mite allergen and application thereof |
| CN106146640A (en) * | 2016-05-31 | 2016-11-23 | 深圳大学 | Dust mite allergen and application thereof |
-
2018
- 2018-06-12 CN CN201810601060.7A patent/CN108840917B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5972352A (en) * | 1987-06-17 | 1999-10-26 | Thomas; Wayne R. | Cloning of mite allergens |
| CN101432299A (en) * | 2006-04-28 | 2009-05-13 | 碧欧美公司 | House dust mite allergen |
| CN103073636A (en) * | 2013-01-29 | 2013-05-01 | 中国科学院昆明动物研究所 | Dermtophagoides farinae allergens Der f 29 and Der f 30, and genes and applications thereof |
| CN104894089A (en) * | 2015-03-27 | 2015-09-09 | 深圳大学 | Dust mite allergen and application thereof |
| CN106146640A (en) * | 2016-05-31 | 2016-11-23 | 深圳大学 | Dust mite allergen and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Group 30 mite allergen-like protein (Ferritin) [Euroglyphus maynei];Arlian,L.G.等;《GenBank: OTF74206.1》;20170512;序列表 * |
| High-quality assembly of Dermatophagoides pteronyssinus genome and transcriptome reveals a wide range of novel allergens;Xiao-Yu Liu等;《J Allergy Clin Immunol》;20180102;第2269页左栏，表1，第2271页 * |
| The draft genome assembly of Dermatophagoides pteronyssinus supports identification of novel allergen isoforms in Dermatophagoides species;Thomas A. Randall等;《Int Arch Allergy Immunol》;20180111;136-146 * |
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| CN108840917A (en) | 2018-11-20 |
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