CN115058409A

CN115058409A - Mallotus japonicus pollen allergen triose phosphate isomerase and application thereof

Info

Publication number: CN115058409A
Application number: CN202210651544.9A
Authority: CN
Inventors: 魏继福; 许志强; 焦永欣; 宋乐彬
Original assignee: Jiangsu Cancer Hospital; Jiangsu Province Hospital
Current assignee: Jiangsu Cancer Hospital; Jiangsu Province Hospital
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2022-09-16
Anticipated expiration: 2042-06-09
Also published as: CN115058409B

Abstract

The invention discloses a new allergen triose phosphate isomerase of French phoenix pollen and application thereof as a medicament for preventing, diagnosing and treating allergic diseases. The method comprises the following steps: (a) a protein comprising the amino acid sequence shown in sequence 2; (b) comprises an amino acid sequence formed by replacing, modifying, deleting or adding any amino acid in the amino acid sequence shown in the sequence 2, and a protein with allergen activity and hypoallergenic activity formed by the amino acid sequence; (c) contains an amino acid sequence which has more than 90 percent of identity with the amino acid sequence shown in the sequence 2, and a protein which is composed of the amino acid sequence and has the activity of an allergen, or an allergen which has immunological cross-reactivity with the amino acid sequence shown in the sequence 2. The allergen disclosed by the invention is found in French phoenix pollen for the first time, and can be used for preparing a phoenix pollen allergen detection kit or developing and applying an individualized desensitization vaccine.

Description

Mallotus japonicus pollen allergen triose phosphate isomerase and application thereof

Technical Field

The invention belongs to the fields of molecular biology, immunology and bioinformatics, and particularly relates to a phoenix tree pollen allergen triose phosphate isomerase and application thereof.

Background

Allergic diseases, also known as allergic diseases, are a common chronic inflammatory disease. According to statistics, 30-40% of people worldwide are troubled by allergy problems, and allergic diseases become the sixth disease of the world. Of these, IgE-mediated allergic diseases are most common and include asthma, rhinitis, severe allergic reactions, drug allergies, food allergies, eczema, urticaria, angioedema, and the like. Pollen is one of the most important air allergens, and pollinosis refers to an allergic disease caused by pollen allergens and involving respiratory tract, skin and other systems mediated by IgE, and the incidence rate is increased year by year, particularly in industrial areas.

Plane tree is widely planted in western europe, north america and the like as an urban ornamental tree, and is also widely seen as a street tree in many cities in china such as shanghai, Nanjing, Wuhan and the like. The pollination period is short and dense, in recent years, the pollen concentration is remarkably increased, the sensitization rate varies from 2% to 56% all over the world, and the pollination period is considered to be one of important causes for pollinosis. In the last 10 years, extensive studies have been carried out on the pollen of plane tree, particularly in the western european city, and four allergens have been identified from the extract, namely Pla a 1, Pla a 2, Pla a 3 and Pla a profilin, but new allergens still remain.

At present, the diagnosis and immunotherapy of allergens are still based mainly on crude extracts of allergens, which are all complex mixtures of components. Therefore, the identification and isolation of IgE-mediated allergens is a necessary task to improve the diagnosis and treatment of this growing clinical disease. The discovery of the new sensitization component undoubtedly enriches the component information of the phoenix tree pollen allergen, is beneficial to the diagnosis and the precision and the individuation of the treatment of the phoenix tree pollen allergy, and along with the development of scientific technology and the continuous and deep research on the phoenix tree pollen allergen component, the novel allergen vaccine with clear components and definite action mechanism is expected to become a treatment scheme for replacing a pollen crude extract, thereby efficiently preventing and treating the phoenix tree pollen allergic diseases.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior art, the technical problem to be solved by the invention is to provide a novel allergen protein triose phosphate isomerase of French phoenix pollen.

The technical problems to be solved by the invention include providing the purification preparation of the triose phosphate isomerase of the plane tree pollen, the cloning of a nucleic acid or a gene sequence, the allergen triose phosphate isomerase of the plane tree pollen coded by the gene and the research and application of the allergenicity of the plane tree pollen.

The technical problem to be solved by the invention is to provide an expression cassette, a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium.

The invention also solves the technical problem of applying the allergen of the plane tree pollen, the nucleic acid or the gene, the expression cassette, the recombinant vector, the recombinant protein, the recombinant cell or the recombinant strain to the preparation of a kit for diagnosing the plane tree pollen allergic diseases or a medicament for preventing or treating the plane tree pollen allergic diseases.

The technical problem to be solved finally by the invention is to provide a medicament or vaccine for preventing or treating phoenix pollen allergy or a kit for allergen diagnosis.

The technical scheme is as follows: in order to solve the above technical problems, the present invention provides an allergen protein triose phosphate isomerase derived from french phoenix pollen, comprising:

(a) as shown in SEQ ID NO: 2, and 2, or a pharmaceutically acceptable salt thereof; or

(b) A protein with one or more amino acids which are substituted, modified, deleted or added in the amino acid sequence in (a) and have the activities of allergen and hypoallergenic activity; or

(c) A protein having an allergen activity, which is composed of an amino acid sequence having 90% or more identity to the amino acid sequence of (a); or

(d) An allergen protein immunologically cross-reactive with the protein of (a).

The present disclosure also includes amino acids that make up the allergenic protein triose phosphate isomerase that comprises:

(2a) as shown in SEQ ID NO: 2;

(2b) at least 90% homology with the amino acid sequence shown in (2 a);

(2c) and (3) a complementary sequence of the amino acid sequence shown in (2 a) and an active allergen protein formed by hybridizing the complementary sequence.

The present disclosure also includes a nucleic acid or gene encoding said allergen protein triosephosphate isomerase or said amino acid, said nucleic acid or gene comprising:

(3a) as shown in SEQ ID NO: 1;

(3b) at least 90% homologous to the nucleotide sequence of (3 a);

(3c) a single-stranded DNA or RNA sequence complementary to the nucleotide sequence of (3 a).

The present disclosure also includes expression cassettes, recombinant vectors, recombinant proteins, recombinant cells or recombinant bacteria comprising said nucleic acids or genes.

The invention also comprises the application of the allergen triose phosphate isomerase of the plane tree pollen, the nucleic acid or the gene, the expression cassette, the recombinant vector, the recombinant protein or the recombinant cell in the preparation of a kit for diagnosing plane tree pollen allergic diseases or a medicine for preventing or treating plane tree pollen allergic diseases.

The invention also comprises the application of the allergen triose phosphate isomerase of the French phoenix tree pollen, the amino acid, the nucleic acid or the gene, the expression cassette, the recombinant vector, the recombinant protein, the recombinant cell or the recombinant bacterium in the preparation of a medicament or reagent for detecting the specific IgE content of the triose phosphate isomerase in the serum of a patient.

The present invention also includes any of the following preparations for preventing, diagnosing and treating allergic diseases in (d) to (f):

(d) a prophylactic or therapeutic agent comprising the allergen protein triose phosphate isomerase, the amino acid or the nucleotide as an active ingredient;

(e) at least one of T-cell reactive polypeptide fragments, B-cell reactive polypeptide fragments, proteins constructed by fusion of polypeptide fragments and carrier proteins, or polynucleotides derived from the allergen protein triose phosphate isomerase and having immunoregulatory properties as an active ingredient;

(f) a specific therapeutic antibody derived from said allergen protein, said antibody comprising one of a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanised modified antibody.

The invention also comprises a medicament for preventing or treating allergic diseases, wherein the medicament is a single or compound preparation and comprises the allergen protein triose phosphate isomerase, the amino acid or the nucleic acid or the gene.

Wherein the medicament is an allergen vaccine. The allergen vaccine comprises a DNA vaccine, a recombinant live bacterium vaccine and a recombinant protein vaccine.

The invention also comprises a kit for allergen diagnosis, which comprises the allergen protein triose phosphate isomerase, the amino acid or the nucleic acid or gene.

Wherein the kit further comprises a solid phase carrier adsorbing or connecting the allergen.

Has the advantages that: the invention relates to a new component with serum specificity IgE binding activity, which is identified from phoenix tree pollen allergen by excavating allergen components in phoenix tree pollen and finally by a method of combining traditional chromatographic separation and purification and tandem mass spectrometry identification technology, and is a gene sequence and an amino acid sequence of allergen triose phosphate isomerase of French phoenix tree pollen. Through research on the allergen triose phosphate isomerase, the allergen triose phosphate isomerase can be used for desensitization treatment and prevention of allergic diseases by preparing an allergen vaccine or related medicaments. Therefore, the invention has important value for clinical diagnosis and treatment of patients with phoenix tree pollen allergy.

Drawings

Fig. 1A is an elution profile of phoenix tree pollen extract on HiTrap Q HP anion exchange column, the parts marked with arrows are combined for further purification, and the black box is the part containing phoenix tree pollen triose phosphate isomerase.

FIG. 1B is a graph showing the elution on a Superdex G75 incrase column after combination, where the 5-6 parts (marked in black boxes) are the parts containing phoenix pollen triose phosphate isomerase.

FIG. 1C is a map of the combined fractions purified on a 1ml HiTrap SP HP column, the black box marked fraction being the purified phoenix pollen triose phosphate isomerase protein.

FIG. 2 shows 12 peptide fragments of phoenix pollen triose phosphate isomerase protein analyzed by Nano-LC MS/MS.

FIG. 3 is an electrophoretogram of a sequence amplification product encoding triose phosphate isomerase from Sterculia procumbens pollen, in which the leftmost M lane is DNA 2000 marker; lanes 1-10 are PCR amplification products.

FIG. 4 shows the cDNA sequence and the corresponding amino acid sequence of Phosphotriose isomerase derived from Sterculia procumbens, and the peptide identified by LC-MS/MS is underlined and bolded.

FIG. 5 shows that the binding activity of phoenix pollen triose phosphate isomerase to specific IgE in serum of phoenix pollen allergic patients was confirmed by ELISA, and 24 of the sera of phoenix pollen allergic patients reacted positively.

Detailed Description

Pollen allergen protein

The pollen allergen protein provided by the invention is selected from the proteins (a) - (c) below.

(a) A protein comprising an amino acid sequence shown in sequence 2;

(b) a protein having an allergenic activity or a hypoallergenic activity, which comprises an amino acid sequence in which any amino acid in the amino acid sequence shown in sequence No.2 is substituted, modified, deleted or added;

(c) comprises a protein having an allergen activity and comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID No.2, or an allergen protein having immunological cross-reactivity to a protein comprising the amino acid sequence shown in SEQ ID No. 2.

Second, amino acids constituting pollen allergen protein

The amino acid of the present invention is an amino acid sequence constituting the above pollen allergen protein, and includes the amino acids of the allergens described in the following (a) to (c).

(a) Comprises an amino acid sequence shown in a sequence 2;

(b) at least 90% homology with the amino acid sequence 2;

(c) a complementary sequence containing an amino acid sequence shown in a sequence 2 and an active allergen protein formed by hybridizing the complementary sequence;

nucleotide sequence for coding pollen allergen protein

The nucleotide of the invention is a nucleotide sequence for coding the pollen allergen protein, and comprises the nucleotide sequences described in the following (a) - (c).

(a) Comprises a nucleotide sequence shown in a sequence 1;

(b) has at least 90 percent of homology with the nucleotide sequence 1;

(c) a single-stranded DNA or RNA sequence complementary to the nucleotide sequence 1

Fourthly, obtaining of polynucleotide for coding pollen allergen protein

The polynucleotide encoding a pollen protein of the present invention can be obtained by cloning, and the cloning method is not limited.

Preparation of pollen allergen protein

The allergen protein of the invention is obtained by separating and purifying the phoenix tree pollen extract. The method of separation and purification is not particularly limited.

Alternatively, a recombinant plasmid prepared by inserting the polynucleotide of the present invention into a vector may be introduced into a host cell to express the pollen allergen protein intracellularly or extracellularly and collected. The vector and host of the present invention are not particularly limited.

The vector into which the polynucleotide of the present invention is inserted is not particularly limited as long as it can replicate in the above-mentioned host, and may be appropriately determined depending on the type of host to be introduced, the method of introduction, and the like.

Sixth, preventive, diagnostic or therapeutic agent for allergic diseases

The pollen allergen protein of the present invention can be used for the production of a prophylactic or therapeutic agent for allergic diseases including all allergic diseases caused by external specific antigens;

the preventive or therapeutic agent for allergic diseases according to the present invention includes:

(a) the pollen allergen protein is directly used or dried into powder;

(b) the pharmaceutical preparation can be prepared by adding various adjuvants such as stabilizer, excipient, dissolution assistant, opacifier, buffer, painless agent, preservative, and colorant, if necessary.

Antibody of seven, pollen allergen protein

The antibody against the pollen allergen protein of the present invention is an antibody capable of specifically binding to the pollen allergen protein of the present invention. The antibody refers to an immunoglobulin (IgA, IgD, IgE, IgG, IgM, and Fab fragments thereof, F (ab')2 fragments, Fc fragments), and examples thereof include a polyclonal antibody, a monoclonal antibody, a single chain antibody, an anti-idiotype antibody (anti-idiotype antibody), and a humanized antibody, but are not limited thereto.

The antibody can be produced by various known methods, and the production method is not particularly limited.

The antibody can be used for identification of an organism, a tissue or a cell thereof expressing the pollen allergen protein of the present invention.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 purification of Sterculia Sprensis pollen allergen triose phosphate isomerase

Weighing 8.0 g of phoenix tree pollen to extract total protein: pollen was first mixed with 60 mL of 0.01M Phosphate Buffered Saline (PBS), added to a final concentration of 1 mM protease inhibitor (PMSF), incubated overnight at 4 ℃ with inversion, centrifuged at 12000 Xg, the supernatant collected, and filtered through a 0.22 μ M membrane for further use.

Purification of triose phosphate isomerase from phoenix tree pollen: sterculia pollen extract was isolated on Ä KTA-go system (GE Healthcare, Uppsala, Sweden) by chromatographic steps of anion exchange, gel filtration and cation exchange. Specifically, crude pollen extract was desalted using 0.02M Tris-HCl, HiTrap desalting column equilibrated at pH 8.4, the collected fractions were adsorbed on HiTrap Q HP column pre-equilibrated with the same buffer as above (Uppsala, Sweden), and the adsorbed proteins were eluted with 0.02M Tris-HCl solution containing 1M sodium chloride in a gradient from 0% to 100%. The eluted fractions were subjected to SDS-PAGE and staining with Coomassie Brilliant blue R250, fractions having similar protein distribution were pooled and separated in Superdex G75, the column was equilibrated with 50 mM phosphate buffer (pH 7.2) containing 150 mM sodium chloride, eluted at a flow rate of 0.5 mL/min, and the fractions were analyzed by SDS-PAGE and pooled for the next separation. The combined fractions were further adsorbed onto a 1mL HiTrap S HP (Uppsala, Sweden) column, eluted with 50 mM 2-morpholinoethanesulfonic acid, pH 6.0, at a flow rate of 1mL/min, analyzed by SDS-PAGE electrophoresis, and finally purified as a single band migrating to approximately 27kDa on SDS-PAGE.

Example 2 internal peptide fragment identification

Identification of IgE binding proteins by LC-MS/MS: the 27kDa protein purified in example 1 was first separated on SDS-PAGE and stained with Coomassie Brilliant blue R250. Protein bands were excised from the gel, digested with trypsin and mass-analyzed, and the raw data was analyzed using pFind software to obtain 12 internal peptide fragments with 89.3% coverage of the full length of the amino acids.

Example 3 cloning of Phoenix Tree pollen triose phosphate isomerase full Length cDNA

We analyzed the mass spectrometry results by pFind software and obtained internal peptide fragments. And matching the peptide segment with the sequencing result of the high-throughput pollen transcriptome to obtain a theoretical cDNA sequence of the coding phoenix pollen triose phosphate isomerase, and further confirming the sequence through T-A cloning and Sanger sequencing. Specifically, the total RNA of the phoenix tree pollen is extracted and subjected to reverse transcription to obtain cDNAs. Designing a primer for PCR amplification according to the matched cDNA sequence of the theoretical phoenix tree pollen triose phosphate isomerase, wherein an upstream primer is 5'-CTCTAGCCTTACTGTGTT-3', a downstream primer is 5'-CCCATACCTGATTCCAAG-3', and carrying out PCR amplification of an actual sequence by a 25 mu L system: ExTaq enzyme (0.125. mu.l), 10 XExTaq buffer (2.5. mu.l), dNTP mix (2. mu.l), phoenix pollen cDNA product (1. mu.l), forward and reverse primer mix (1. mu.l), sterile distilled water to 25. mu.l. PCR amplification conditions were 98 ℃ per 5s (one cycle) for pre-denaturation, 98 ℃ per 10s for denaturation, 55 ℃ per 30s for annealing and 72 ℃ per 1min for extension (30 cycles), and 72 ℃ per 10min for re-extension (one cycle). The PCR amplification product was recovered, purified and ligated into pCE2 TA/Blunt-zero plasmid vector and transformed into JM109 competent cells. Positive clonal bacteria are screened on a Luria-Bertani (LB) plate containing 100 mug/mL kanamycin, and the actual sequence of the natural protein of the phoenix pollen triose phosphate isomerase is obtained through DNA sequencing confirmation, wherein the gene has the full length of 762 bp, codes 253 amino acids and has the theoretical molecular weight of 27 kDa. The amino acid sequence is shown as SEQ ID NO. 2.

Further recombination reactions were carried out. Firstly, the plasmid of the above-mentioned phoenix pollen triose phosphate isomerase clone bacteria and the plasmid of pET28a E.coli are extracted by using Clon express II One Step Cloning Kit (refer to the instructions provided by the Kit in the operation steps), then the nucleotide sequence of the coding region of the clone bacteria is connected between NcoI and XhoI sites of pET28a + plasmid through recombination reaction to obtain recombinant plasmid, the recombinant plasmid is transformed into BL21 (DE 3) host bacteria (Novozae Biotech Co., Ltd., Nanjing, China) through heat shock to carry out plate Cloning, the monoclonal bacteria are picked up and cultured in liquid medium at 37 ℃, and sequencing is carried out again to confirm the successful construction of the plasmid.

Example 4 ELISA experiments demonstrated the allergenicity of Phosphotriose isomerase from Sterculia pollen

The phoenix pollen triose phosphate isomerase prepared in example 1 was coated overnight in a 96-well plate. Blocking was then performed with 1% Bovine Serum Albumin (BSA) in PBST (PBS with 0.1% Tween 20) for 1.5h at 37 ℃. After washing with PBST, 58 cases of serum (serum from Jiangsu province hospital) from a phoenix pollen allergic patient were added and incubated at 37 ℃ for 1.5 h. Non-allergic patient sera were negative controls. Incubate with 100. mu.L of anti-human IgE-horseradish peroxidase conjugate (1: 2500 dilution) for 1 h at room temperature. After washing, the solution was added with a developing solution for 13 minutes, and then the solution was stopped, and then the solution was detected by an enzyme-linked immunosorbent OD 450. The results showed that in serum of 58 patients with phoenix tree allergy, 24 patients had a positive reaction to phoenix tree pollen triose phosphate isomerase, and the sensitization rate was 41% in the patients with phoenix tree allergy.

Sequence listing

<110> Jiangsu province tumor hospital; jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

<120> Sterculia orientalis pollen allergen triose phosphate isomerase and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 828

<212> DNA

<213> Phoenix pollen allergen triose phosphate isomerase (Pla a TIM)

<400> 1

ctctagcctt actgtgtttc ttcgcctcct gctgttaaaa atgggtagaa atttcttcgt 60

aggcggtaac tggaaatgca atggaaccaa cgaagaagtg aagaagatcg tttccacttt 120

gaatgaagcc gaggttccct ctcaagatgt tgtggaggtt gtggtaagtc ctccgtttgt 180

gtttcttccc gtggtaaaaa gttcattgag gtctgatttc catgtcgcgg cacaaaactg 240

ttgggtgaag aaaggaggtg cttttactgg tgaggttagt gcggagatgc ttgtcaatct 300

gggcattcct tgggtcattc ttggtcattc tgaaagaagg ctcttgttaa atgaatcaca 360

tgagtttgtt ggagataaag ttggatatgc actttctcaa ggcttgaaag tgattgcttg 420

tgttggagag actcttgagc agcgagaatc aggatttacc atggatgttg tttctgctca 480

aacaaaagca attgcagatc gcgtatctaa ttgggctaat gtcgttttgg cgtacgagcc 540

agtatgggcc attggaactg gaaaggttgc aacaccggct caggctcagg aagtgcacta 600

cggattaagg aaatggctgc aagagaatgc tggtgctgaa gttgctgcca caactaggat 660

tatctatgga ggttccgtaa atggtgcaaa ctgcaaagaa ttggcagcaa aacctgatgt 720

tgatggattc ctggtcggtg gagcttctct aaagccggag ttcatcgaca ttatcaagtc 780

tgccacggtg aagaatgctt aagcttgggg cttggaatca ggtatggg 828

<210> 2

<211> 253

<212> PRT

<213> Phoenix pollen allergen triose phosphate isomerase (Pla a TIM)

<400> 2

Met Gly Arg Asn Phe Phe Val Gly Gly Asn Trp Lys Cys Asn Gly Thr

1 5 10 15

Asn Glu Glu Val Lys Lys Ile Val Ser Thr Leu Asn Glu Ala Glu Val

20 25 30

Pro Ser Gln Asp Val Val Glu Val Val Val Ser Pro Pro Phe Val Phe

35 40 45

Leu Pro Val Val Lys Ser Ser Leu Arg Ser Asp Phe His Val Ala Ala

50 55 60

Gln Asn Cys Trp Val Lys Lys Gly Gly Ala Phe Thr Gly Glu Val Ser

65 70 75 80

Ala Glu Met Leu Val Asn Leu Gly Ile Pro Trp Val Ile Leu Gly His

85 90 95

Ser Glu Arg Arg Leu Leu Leu Asn Glu Ser His Glu Phe Val Gly Asp

100 105 110

Lys Val Gly Tyr Ala Leu Ser Gln Gly Leu Lys Val Ile Ala Cys Val

115 120 125

Gly Glu Thr Leu Glu Gln Arg Glu Ser Gly Phe Thr Met Asp Val Val

130 135 140

Ser Ala Gln Thr Lys Ala Ile Ala Asp Arg Val Ser Asn Trp Ala Asn

145 150 155 160

Val Val Leu Ala Tyr Glu Pro Val Trp Ala Ile Gly Thr Gly Lys Val

165 170 175

Ala Thr Pro Ala Gln Ala Gln Glu Val His Tyr Gly Leu Arg Lys Trp

180 185 190

Leu Gln Glu Asn Ala Gly Ala Glu Val Ala Ala Thr Thr Arg Ile Ile

195 200 205

Tyr Gly Gly Ser Val Asn Gly Ala Asn Cys Lys Glu Leu Ala Ala Lys

210 215 220

Pro Asp Val Asp Gly Phe Leu Val Gly Gly Ala Ser Leu Lys Pro Glu

225 230 235 240

Phe Ile Asp Ile Ile Lys Ser Ala Thr Val Lys Asn Ala

245 250

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctctagcctt actgtgtt 18

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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cccatacctg attccaag 18

Claims

1. An allergen protein triose phosphate isomerase derived from phoenix tree pollen, characterized in that: the allergen triosephosphate isomerase comprises:

(b) A protein having an allergenic activity or a hypoallergenic activity, wherein one or more amino acids are substituted, modified, deleted, or added to the amino acid sequence in (a); or

(d) An allergen protein immunologically cross-reactive with the protein of (a).

2. Amino acids constituting the allergenic protein triose phosphate isomerase according to claim 1 characterized in that: said amino acid comprises

(2a) As shown in SEQ ID NO: 2;

(2b) at least 90% homology with the amino acid sequence shown in (2 a);

3. A nucleic acid or gene encoding the allergenic protein triose phosphate isomerase of claim 1 or the amino acid of claim 2, characterized in that the nucleic acid or gene comprises:

(3a) as shown in SEQ ID NO: 1;

(3b) at least 90% homologous to the nucleotide sequence of (3 a);

4. An expression cassette, recombinant vector, recombinant protein, recombinant cell or recombinant bacterium comprising the nucleic acid or gene of claim 3.

5. Use of the allergen triose phosphate isomerase of plane tree pollen as defined in claim 1, the amino acid as defined in claim 2, the nucleic acid or gene as defined in claim 3, the expression cassette, recombinant vector, recombinant protein, recombinant cell or recombinant bacterium as defined in claim 4 for the preparation of a kit for diagnosing plane tree pollen allergic diseases or a medicament for preventing or treating plane tree pollen allergic diseases or for the preparation of a medicament or reagent for detecting the content of triose phosphate isomerase-specific IgE in the serum of a patient.

6. Any of the following preparations for preventing, diagnosing and treating allergic diseases (d) to (f):

(d) a prophylactic or therapeutic agent comprising the allergen protein triose phosphate isomerase according to claim 1, the amino acid according to claim 2 or the nucleotide according to claim 3 as an active ingredient;

(e) at least one of a T-cell reactive polypeptide fragment, a B-cell reactive polypeptide fragment, a protein constructed by fusing a polypeptide fragment and a carrier protein, which are derived from the allergen protein triose phosphate isomerase of claim 1 and have immunoregulatory activity, and a polynucleotide as an active ingredient;

(f) a specific therapeutic antibody derived from the allergen protein triosephosphate isomerase of claim 1, wherein said antibody comprises one of a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanized modified antibody.

7. A medicament for preventing or treating an allergic disease, which is a single or compound preparation comprising the allergen protein triose phosphate isomerase according to claim 1, the amino acid according to claim 2, or the nucleic acid or gene according to claim 3.

8. The medicament of claim 7, wherein the medicament is an allergen vaccine.

9. A kit for allergen diagnosis, comprising the allergen protein triose phosphate isomerase according to claim 1, the amino acid according to claim 2, or the nucleic acid or gene according to claim 3.

10. The kit according to claim 9, further comprising a solid support to which the allergen protein triose phosphate isomerase of claim 1 is adsorbed or linked.