JPH07112999A - Recombinant acarid allergen - Google Patents

Abstract

PURPOSE:To provide a recombinant acarid allergen which is useful as a treating agent or diagnostic agent for acarid allergic diseases with high safety and effectiveness because it has no anaphylaxy induction. CONSTITUTION:A recombinant acarid allergen having antigenicity and allergen activity to be obtained by expression of the gene originating from an acarid body having antigenicity and allergen activity. This recombinant acarid allergen is obtained by adjusting mRNA from an acarid body such as American house dust mite (Dermatophagoides farinae), using it as a template to synthesize cDNA including the DNA sequence of Table 1 to obtain the gene originating from an acarid body, linking the gene to a plasmid vector such as pUEX2 so that the gene may be expressed to constitute expression vector, culturing a bacterium, yeast or mammalian cells transformed with the expression vector to produce the recombinant acarid allergen, and purifying and collecting the allergen through the gel filtration chromatography and the antibody-immobilized affinity chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant mite allergen having allergen activity.

[0002]

2. Description of the Related Art Indoor dust mites are important as a major cause of allergic diseases such as atopic bronchial asthma. Conventionally, as a therapeutic agent for allergic diseases, hyposensitization therapy, which is a causative agent of allergy, has been regarded as the most important fundamental treatment, and it is an inhalable allergen such as hay fever, indoor dust, fungal allergy, etc. In the case of antigen-induced diseases, the evaluation is now established. However, since this hyposensitization therapy involves the risk of anaphylaxis due to the sensitizing antigen, it is necessary to administer a safe therapeutic antigen, and research on such a sensitizing antigen is underway.

Regarding the mite allergic disease, it has been reported that two types of mites, Dermatophagoides pteronyssinus and Dermatophagoides farinae , are important as sources of allergens in indoor dust [Allerg. Asthma, 10 , 329]. ~ 334 (1964);
J. Allergy, 42 , 14-28 (1968)]. From these mites, major mite allergens were fractionated, and they were contained in mite excrement and / or mite body and had a molecular weight of 24-28k.
D glycoprotein (pI 4.6-7.2) and / or molecular weight 14.5-20k
It is reported to be a D protein (pI 5 to 7.2) [J. Imm
unol., 125, 587 ~ 592 (1980); J. Allergy Clin. Immun
ol., 76 , 753-761 (1985); Immunol., 46 , 679-687.
(1982); Int. Arch. Allergy Appl. Immunol., 81, 214
~ 223 (1986); J. Allergy Clin. Immunol., 75 , 686 ~
692 (1985), etc.]. However, the fact is that an effective and safe antigen for treating desensitization has not been obtained yet.

As for the gene cloning of the mite allergen, Der p, which is the major allergen of Dermatophagoides farinae
I (Molecular weight: 25,371) and Der p II (Molecular weight: 14,131)
, And also for Der f I (molecular weight: 25,191) and Der f II (molecular weight: 14,021), which are the major allergens of Dermatophagoides farinae, the respective genes have been cloned and the nucleotide sequences have been determined [Int. Arch. Aller
gy Appl. Immunol., 85, 127-129 (1988); J. Exp. Me
d., 167 , 175-182 (1988); J. Exp. Med., 17 0, 145
7 ~ 1462 (1989); Int. Arch. Allergy Appl. Immuno
l., 91 , 118 〜 123 (1990); Int. Arch. Allergy Appl.
Immunol., 91, 124-129 (1990); Jpn. J. Allergol., 3
9, 557 ~ 561 (1990); Clinical and Experimental Alle
rgy, 21, 25 ~ 32 (1991); Clinical and Experimental
Allergy, 21, 33-37 (1991)], studies of mite allergens by genetic recombination techniques have been attempted. However, what has been used as a sensitizing antigen has not been obtained yet.

On the other hand, as a method for diagnosing a mite allergic disease, conventionally, an inquiry has been mainly made, and indoor dust (house dust) is used.
Most of the skin reaction tests using the extract and / or the mite body extract are rare, and rarely, RAST (radio allergosorbent t
est) method to measure IgE antibody titer (relative value) in serum, inhalation provocation test and nasal mucosa provocation test together, and direct diagnosis of mite allergic disease was quite difficult.

[0006]

Conventionally, an indoor dust (house dust) extract has been used for a desensitizing treatment method for bronchial asthma in which indoor dust mite is a specific antigen. Its composition is extremely unclear, and since it contains many kinds of impurities and may induce anaphylaxis, its dose is extremely limited and the therapeutic effect is extremely low. Therefore, from the viewpoint of efficacy and safety, the emergence of a useful therapeutic antigen for desensitization is desired, and at the same time, a stable supply of such a high-quality therapeutic antigen for desensitization is expected. However, in order to stably supply a sufficient amount of mite allergen for such safe and reliable response, the method of extracting and purifying mite allergen from a mite breeding product is not mass-producible and has a quantitative limit. A stable supply is difficult.

[0007] The object of the present invention lies in this point, and it is to provide a safe and effective recombinant mite allergen containing no anaphylaxis-inducing impurities as a therapeutic agent or diagnostic agent for mite allergic diseases. That is, the first object of the present invention is to provide a recombinant mite allergen having an allergen activity obtained by the expression of a gene derived from a mite body. The second object of the present invention is to provide a gene encoding the mite allergen. A third object of the present invention is to provide a mite allergen fragment as an active fragment having allergen activity. A fourth object of the present invention is to provide a peptide having an epitope contained in mite allergen, or a peptide having an epitope that can be regarded as immunologically identical to the epitope. A fifth object of the present invention is to provide a gene encoding a peptide having the above epitope. A sixth object of the present invention is to provide an expression vector capable of expressing the gene of the present invention. A seventh object of the present invention is to provide a transformant transformed with an expression vector capable of expressing the gene. An eighth object of the present invention is to provide a method for producing the recombinant mite allergen. A ninth object of the present invention is to provide a novel therapeutic agent for mite allergic diseases, which comprises the recombinant mite allergen of the present invention as an active ingredient.
A tenth object of the present invention is to provide a novel diagnostic agent for mite allergic diseases, which comprises the recombinant mite allergen of the present invention as an active ingredient.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors found a mite allergen having a strong allergen activity and a gene encoding the mite allergen, and further The present invention has been completed through repeated research.

That is, the gist of the present invention is (1) a recombinant mite allergen containing any of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 9 obtained by expressing a gene derived from a mite body. (2) A gene derived from a mite body, which encodes a protein having an allergen activity, which comprises the amino acid sequence described in (1) above, (3) in the nucleotide sequence shown in SEQ ID NO: 1, at least about 190 from upstream. bp to about 240 bp, about 290 bp to about 380 b
p or a mite allergen fragment containing an amino acid sequence encoded within a region of about 190 bp to about 380 bp, (4) a peptide having an epitope contained in the mite allergen, or immunologically identical to the epitope A peptide having an epitope that can be considered to be (5)
A gene encoding the peptide according to (4) above,
(6) An expression vector capable of expressing the gene according to (2) above, (7) a bacterium, yeast or mammalian cell transformed with the expression vector according to (6) above, (8) above (7)
A bacterium, yeast, or mammalian cell according to claim 1, which is cultured under conditions capable of expressing the gene to produce a recombinant mite allergen, and then the recombinant mite allergen is recovered, Manufacturing method, (9) A mite allergic disease therapeutic agent containing the recombinant mite allergen according to (1) above as an active ingredient, and (10) Mite allergic disease diagnosis containing the recombinant mite allergen according to (1) above as an active ingredient Regarding medicine.

The recombinant mite allergen of the present invention is obtained by expressing a gene derived from a mite body, and the mite used here is not particularly limited,
Indoor dust mites such as Dermatophagoides farinae and Dermatophagoides farinae are used.

The recombinant mite allergen of the present invention has the following amino acid sequences: ma10, ma3, ma15, ma
29, ma44, ma50, ma113, ma114,
A protein containing any of nine kinds of sequences named as ma115 (SEQ ID NO: 1 to SEQ ID NO: 9),
It has allergen activity.

The recombinant mite allergen of the present invention is
Substitution of one or more amino acids in each amino acid sequence,
Even if variously mutated by deletion, addition or rearrangement, those having allergen activity are included in the recombinant mite allergen of the present invention. Such variants may be obtained by natural allelic variants or by recombinant DNA technology, for example DN
It is obtained by inducing a mutation at a specific site of A.

The recombinant mite allergen of the present invention also includes those expressed as fusion proteins with other proteins.
In the present specification, a recombinant mite allergen expressed by fusing with another protein may be particularly referred to as a fused recombinant mite allergen. Other proteins to be fused are not particularly limited, but examples thereof include β-galactosidase, glutathione-
Examples include S-transferase, protein A and the like.

The recombinant mite allergen of the present invention may be a protein fragment only in the region essential for the allergen activity, or may be composed of a domain essential for the allergen activity. For example, in the present specification, an active fragment having an allergen activity is referred to as a mite allergen fragment, and specifically, in the nucleotide sequence shown in SEQ ID NO: 1, at least about 190 bp to about 2 bp from upstream.
40 bp, about 290 bp to about 380 bp or about 190
An example is a fragment containing the amino acid sequence encoded within the region of bp to about 380 bp.

In addition to those obtained by expressing only the mite allergen protein, those obtained by desorbing other proteins from those expressed as a fusion protein may be used.

[0016] In summary, the recombinant mite allergen of the present invention is a protein having allergen activity, which substantially contains the above-mentioned amino acid sequence obtained by the expression of a gene derived from a mite body.

An example of the recombinant mite allergen of the present invention is a recombinant mite allergen fused with β-galactosidase expressed in Escherichia coli as follows. That is, the fused recombinant mite allergen expressed in E. coli was Ultrogel
Gel filtration chromatography using AcA 44 (manufactured by IBF), anti-β-galactosidase antibody-immobilized affinity chromatography, anti-mite body antibody-immobilized affinity chromatography, anti-Derf I antibody-immobilized affinity chromatography and anti-Derf II antibody. It is purified by immobilized affinity chromatography and has the following properties as a recombinant mite allergen. (1) Recombinant mite allergen ma10 Color and properties: white Water-soluble: readily soluble Molecular weight: about 19,000. Estimate by SDS-PAGE {(molecular weight of fusion protein)-(molecular weight of β-galactosidase)}. In addition, the following recombinant mite allergens are similarly estimated. The amino acid sequence (SEQ ID NO: 1) includes the following. Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu Leu Asp Gln Thr Phe Ala Gly Thr Tyr Trp Val Ile Asn Asn Asn Ser Phe Ile Leu Arg Arg Gln Asn Phe Arg Leu Phe Leu Thr Thr Pro Pro Pro Pro Ala Thr Ala Thr Thr Lys Asn His Having antigenicity. ELIS with specific IgG in mite allergic patient pool serum, rabbit anti-mite body serum, rabbit anti-β-galactosidase
A Reactivity and SDS-PAGE of expressed protein, blot on nitrocellulose membrane and judge by reactivity with the above antiserum.
In addition, the following recombinant mite allergens are also determined in the same manner. Has allergen activity. Judgment is carried out by a leukocyte histamine release test of mite allergy patients using HPLC (High performance liquid chromatography). In addition, the following recombinant mite allergens are also determined in the same manner. Does not induce an anaphylactic reaction. Guinea pigs are immunized with the fusion protein or mite allergen protein by a conventional method, and the anaphylactic reaction is observed during the booster immunization. The following recombinant mite allergens are also observed.

(2) Recombinant mite allergen ma3 Color and properties: white Water-soluble: readily soluble Molecular weight: about 34,000. The amino acid sequence (SEQ ID NO: 2) includes the following. Val Thr Ala Leu Glu Leu Leu Leu Lys Gly Glu Thr Glu Asp Lys Thr Arg Arg Tyr Val Ala Glu Leu Thr Ala Val Gly Ser Pro Ser Asn Lys Gln Ala Lys Ala Gln Ile Glu Val Thr Lys Gly Glu Glu Tyr Lys Ile Thr Leu Lys Ser Pro Glu His Glu Phe Asn Thr Glu Phe Thr Ile His Ala Asp Lys Asn Asn Leu Lys Met His Met Asp Phe Pro Asn Val Phe Gln Ala Asp Leu Thr Gly Thr Phe Gln His Asp Lys Glu Asn Asn Val Arg Lys Asn Gln Leu Asn Leu Gln Tyr Lys Phe Ala Gly Asp Glu Lys Pro His Thr Val Asp Tyr Glu Asn Glu Phe Ser Phe Asn Leu Lys Arg Ser Ser Lys Asp Lys Asn Ser Gly Val Asp Tyr Arg Ala Lys Tyr Met Ser Ser His Phe Pro Ile Leu Asn His Lys Val Asn Ile Gln Phe Lys Tyr Arg Pro Phe Lys Val Asn Glu Leu Asn Leu Glu Gly Glu Phe Gly Arg Glu Leu Gln His Lys Phe Gln Leu Met Arg Asn Ser Gln Ile Glu Val Glu Glu Val Arg Pro Phe Lys Met His Gly Asn Ser Asp Ile Lys Leu Met Ala Asn Asp Leu Asp Ile Asp Tyr Asp Leu Lys Ser Glu Phe Lys Tyr Glu Ser Asn Lys Gly Thr Pro Ile Glu Leu Gln Tyr Lys Ile Ser Gly Lys Asp Arg Ser Lys Arg Ala Ala Asp Leu Gly Ala Glu Asp Val Glu Gly Val Ile Asp Tyr Lys Asn Asn Gly Ser Pro Ile Asp Ser Lys Met His Ala His Leu Lys Met Lys Gly Asn Asn Tyr Gly Tyr Asp Ser Glu Leu Lys Gln Thr Gln Pro Gln Gln Tyr Glu Gly Lys Ile Thr Leu Ser Lys Asn Asp Lys Lys Ile Phe Ile Asn His Lys Ser Glu Met Thr Lys Pro Thr Asn Thr Phe His Leu Lys Thr Asp Ala Asp Val Ser Tyr Ser Asp Ser Asp Met Lys Lys His Tyr Gln Met Glu Antigen Have sex. Has allergen activity. Does not induce an anaphylactic reaction.

(3) Recombinant mite allergen ma15 Color and properties: white Water-soluble: readily soluble Molecular weight: about 22,000. The amino acid sequence (SEQ ID NO: 3) includes the following. Glu Trp Tyr His Leu Phe Lys Arg Trp Leu Arg Lys Arg Phe Thr Lys Met Ile Asp Glu Leu Lys Pro Ser Leu Leu His Val Lys Gly Lys Lys Arg Pro Ile Val Tyr Glu Cys Ala Glu Ile Ser Trp Lys Val Met Asn Asn Gly Asp Val Phe Ile Leu Leu Val Pro Asn Phe Val Phe Val Trp Thr Gly Lys His Ser Asn Arg Met Glu Arg Thr Thr Ala Ile Arg Val Ala Asn Asp Leu Lys Ser Glu Leu Asn Arg Phe Lys Leu Ser Ser Val Ile Leu Glu Asp Gly Lys Glu Val Glu Gln Thr Ser Gly Ala Glu Tyr Asp Ala Phe Asn Lys Ala Leu Ser Leu Asp Lys Lys Asp Ile Asp Leu Lys Gln Met Pro Lys Gly Tyr Asp Tyr Ala Ala Ser Asp Lys Ser Phe Glu Ser His Glu Arg Ser Phe Val Thr Leu Tyr Lys Cys Phe Glu Gly Thr Glu Thr Ile Asp Ile Ser Phe Val Lys Asn Gly Pro Leu Ser Arg Ala Asp Leu Asp Thr Asn Asp Thr Phe Ile Val Glu Asn Gly Ser Glu Gly Leu Trp Val Trp Val Gly Lys Lys Ala Thr Gln Lys Glu Arg Gln Ser Ala Ile Lys Tyr Ala Met Glu Leu Ile Asn Lys Lys Lys Tyr Pro Asn Asn Thr Pro Val Thr Lys Val Leu Glu Gly Asp Glu Ser Val Glu Phe Lys Ser Leu Phe Glu Ser Trp Gln Met Ser Glu Gln Glu Lys Ile Thr Ser Ala Arg Leu Phe Arg Val Ser Arg Asn Gly Ile Phe Lys Gln Val Ala Asn Tyr Glu Pro Asp Asp Leu Glu Glu Asp Ile Tyr His Asp Phe Gly Cys Tyr Gly. Has allergen activity. Does not induce an anaphylactic reaction.

(4) Recombinant mite allergen ma29 Color and properties: white Water-soluble: readily soluble Molecular weight: about 17,000. The amino acid sequence (SEQ ID NO: 4) includes the following. Lys Asp Asp Ile Glu Arg Met Val Lys Glu Ala Glu Ser Tyr Lys Glu Glu Asp Asp Lys Gln Arg Asp Arg Ile Ala Ala Lys Asn Ser Leu Glu Gly Tyr Ala Phe Gln Met Lys Ala Thr Leu Asp Glu Glu Ala Ile Lys Ser Lys Val Ser Glu Glu Asp Arg Lys Lys Ile Leu Asp Lys Val Asp Glu Val Leu Lys Trp Leu Asp Ala Asn Ala Leu Ala Glu Lys Asp Glu Phe Glu His Gln Arg Lys Glu Leu Glu Ser Val Cys Asn Pro Ile Ile Thr Lys Leu Tyr Gln Gln Ala Gly Gly Ala Gly Ala Gly Gly Met Pro Gly Gly Phe Pro Gly Gly Phe Pro Gly Thr Asp Gly Ser Gly Gly Gly Ala Ala Gly Gly Asp Gly Gly Lys Ser Gly Pro Thr Ile Glu Glu Val Asp Having antigenicity. Has allergen activity. Does not induce an anaphylactic reaction.

(5) Recombinant mite allergen ma44 Color and properties: white Water-soluble: readily soluble Molecular weight: about 33,000. The amino acid sequence (SEQ ID NO: 5) includes the following. Phe Phe Phe Val Ala Ala Lys Gln Gln Gln Gln Pro Ser Thr Lys Met Glu Ala Ile Lys Lys Lys Met Gln Ala Met Lys Leu Glu Lys Asp Asn Ala Ile Asp Arg Ala Glu Ile Ala Glu Gln Lys Ala Arg Asp Ala Asn Leu Arg Ala Glu Lys Ser Glu Glu Glu Val Arg Ala Leu Gln Lys Lys Ile Gln Gln Ile Glu Asn Glu Leu Asp Gln Val Gln Glu Gln Leu Ser Ala Ala Asn Thr Lys Leu Glu Glu Lys Glu Lys Ala Leu Gln Thr Ala Glu Gly Asp Val Ala Ala Leu Asn Arg Arg Ile Gln Leu Ile Glu Glu Asp Leu Glu Arg Ser Glu Glu Arg Leu Lys Ile Ala Thr Ala Lys Leu Glu Glu Ala Ser Gln Ser Ala Asp Glu Ser Glu Arg Met Arg Lys Met Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu Leu Asp Gln Thr Phe Ala Glu Leu Thr Gly Tyr Antigen Have sex. Has allergen activity. Does not induce an anaphylactic reaction.

(6) Recombinant mite allergen ma50 Color and properties: white Water-soluble: readily soluble Molecular weight: about 28,000. The amino acid sequence (SEQ ID NO: 6) includes the following. Phe Leu Thr Asn Asp Lys Arg Tyr Asn Phe Phe Phe Val Thr Met Lys Phe Leu Ile Tyr Ser Leu Ala Phe Ala Ala Met Ala Gly Leu Val Phe Gly Ser Glu Lys Arg Lys Lys Asn Pro Tyr Asp Val Ala Glu Ser Leu Ile Lys His Ala Asp Ala Leu Lys Glu Tyr Ala Glu Gln Glu Ile Gln Lys Tyr Gln Asn Asn Ser His Tyr His Leu Ala Ile Ile Arg Leu Glu His Glu Ile His Arg Val Glu Ala Ile Ala Asp Gln Leu Arg Asn Met Thr Lys Met Glu Asn Met Thr Glu His Tyr Met His His Phe His Gln Ile Glu Met Glu Leu Leu Ile Phe Glu Asn Ile Ile Glu Glu Glu Leu Tyr Tyr Leu Glu His Gly His His His His Asp His His Asp His Asp His His Asp His Met His Arg His Asp His Asp Asp Ser Asp Ile Ile Ala Gly Asp Asp Tyr Lys Asp Ala Leu Leu Lys Lys Ala Gln Gln Leu Ile Asp Glu Ala Glu Asp Ala Ile Lys Lys Tyr Gly Gln Gly Lys Asp Ile Asp Val Gln Val Ile Lys Ser Glu Val Val Ile Ile Lys Asn Leu Val Lys Gln Ile Asn Glu Ala Asn Asn Pro Gln Ala Leu Ala Val Leu Glu Ser Asn Leu Ala Gln His Glu Glu Ile Ile Arg Lys Trp Leu Asp Asp Ile Glu Pro His Thr Thr His Ala Pro Val Thr Ile Ala having antigenicity. Has allergen activity. Does not induce an anaphylactic reaction.

(7) Recombinant mite allergen ma113 Color and properties: white Water-soluble: readily soluble Molecular weight: about 38,000. The amino acid sequence (SEQ ID NO: 7) includes the following. Leu Gln Pro Asn Gly Pro Lys Gly Leu Arg Ser Pro Gly Ser Pro Asp Ser Gly Asn Leu Arg Lys Arg Lys Arg Arg Gly Gly Val Leu Gly Phe Lys Glu Leu Ala Ala Lys Ser Lys Leu Ser Ala Trp His Phe His Arg Val Phe Lys Ser Val Thr Gly Leu Thr Pro Lys Thr Tyr Gly Asp Lys Cys Trp Glu Tyr Ile Lys His Cys Arg Glu Asn Gly Glu Ser Phe Phe Phe Glu Gly Lys Gln Ala Asn Thr Asp Ser Gly Glu Val Ser Leu Ser Pro Pro Asn Met Cys Tyr Gln Thr Gln Thr Pro Ile Ser Ser Asn Thr Asp Ser His Asn Tyr Phe Asp Glu Ser Asp Asp Glu Lys Val Lys Lys Val Lys Leu Ser Pro Glu Val Glu Arg Asp Ile Ser Thr Lys Asp Ser Phe Asn Gly Glu Ser Ser Thr Pro Gly Asp Asp Cys Phe His Pro Asp Glu Asn Gly Leu Asp Asn Asn Leu Lys Asn Ile Gly Leu Gly Leu Ser Phe Ala Thr Lys Asp Leu Gly Ile Asn Pro Ser Ser Val Glu Asp Gly Leu Val Gln Pro Thr Cys Ser Phe Asp Phe Asn Leu Asn Asp Ala Thr Ile Pro Asn Val Asp Asp Phe Asp Asp Phe Ser Lys Val Pro Gln Phe Leu Asn Asp Asp Ser Ala Gly Phe Val Arg Ala Phe Ser Ala Pro Asp Leu Thr Lys Tyr Asn His Met Ser Ala Leu Ser Pro Glu Ser Asn Gly Asn Thr Asp Met Asn Lys Pro Lys Asp Ala Leu Gly Phe Asp Ser Tyr Leu Asp Thr Asp Ile Ser Asn Gln Glu Thr Asn Gly Asn Ala Leu Tyr Pro Val Val Asp Asp Val Pro Ser Leu Ser Gly Thr Gly Phe Met Pro Asn Thr Ser Ile Gln Asn Thr Ser Phe Leu Arg Pro Phe Glu Asp Ser Leu Asp Ile Gln Asp Thr Ala Glu Leu Ser Lys Phe Gly Tyr Asp Val Asp Ser Leu Ala Pro Ser Thr Pro Gly Leu Asp Met Lys Leu Asn Ser Arg Phe Ser Val Ala Gln Pro Asp Having antigenicity. Has allergen activity. Does not induce an anaphylactic reaction.

(8) Recombinant mite allergen ma114 Color and properties: white Water-soluble: readily soluble Molecular weight: about 18,000. The amino acid sequence (SEQ ID NO: 8) includes the following. Ser Thr Arg Trp Leu Thr His Glu Ser Asp Glu Glu Arg Trp Glu Gln Tyr Arg Leu Lys Gln Arg Met Pro Glu Asn Asp Phe Gly Pro Ile Ile Thr Gly His His Asp Asp Asp Asp Asp Asp Asp Val Asp Thr Pro Asn Glu Glu Glu Asn Arg Asp Lys Phe Ile Thr Ile Thr Asn Thr Asp Ser Phe Arg Arg Arg Thr Asn Tyr Ser Ser Ser Ser Asn Ser Asn Asn Asn Asn Asn Asn Asn Gly Lys Asp Tyr Gly His Gln His Gln His His His Pro Arg His His His His His Asn Pro Asn Lys Ser Asn Gly Asn Gly His Ala Phe Asn His Asn Asn Ala Ile Ile Ile Asn Asn Asn Asn Asn Asp Asp Ser Thr Ala Asn Leu Glu Gln Ile Tyr Asn Ser Lys Ser Asn Thr Thr Ser Arg Leu Antigen Have sex. Has allergen activity. Does not induce an anaphylactic reaction.

(9) Recombinant mite allergen ma115 Color and properties: white Water-soluble: readily soluble Molecular weight: about 32,000. The amino acid sequence (SEQ ID NO: 9) includes the following. Phe Ile Lys Ile Lys Asn Ser Ser Lys Met Lys Phe Val Leu Ala Ile Ala Ser Leu Leu Val Leu Ser Thr Val Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala Phe Asn Lys Asn Tyr Ala Thr Val Glu Glu Glu Glu Val Ala Arg Lys Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ala Asn Lys Gly Ala Ile Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Tyr Leu Met Ser Ala Glu Ala Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln Gln Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile Met Having antigenicity. Has allergen activity. Does not induce an anaphylactic reaction.

The mite body-derived gene of the present invention can be obtained by preparing mRNA from a live mite body and synthesizing cDNA by a conventional method using the mRNA as a template.
It encodes a protein having the above-mentioned amino acid sequence and having an allergen activity in the molecule, and specific examples of the DNA encoding the amino acid sequence include the following sequences (SEQ ID NO: 1 to 9) is exemplified.

For example, as ma10 (SEQ ID NO: 1), CTT GAA CAT CGA TCC ATC ACC GAT GAA GAA CGT ATG GAT GGT TTG GAA AAT CAA CTT AAA GAA GCC CGT ATG ATG GCC GAA GAT GCT GAT AGA AAA TAT GAT GAA GTT GCC CGT AAA TTG GCA ATG GTT GAA GCC GAT TTG GAA CGT GCT GAA GAA CGT GCC GAA ACC GGT GAA TGC AAA ATT GTT GAA CTC GAG GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA GTT AGC GAA GAG AAA GCT CAA CAA CGT GAA GAA GCC TAT GAA CAA CAG ATC CGT ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG GAA GAC GAA TTG GTC CAC GAA AAG GAA AAA TAC AAA TCC ATC TCC GAC GAA TTG GAC CAG ACA TTT GCC GGA ACT TAC TGG GTT ATT AAT AAT AAT TCT TTC ATA TTA CGC AGA CAG AAT TTT CGA TTA TTT TTA ACA ACA CCA CCA CCA CCA GCA ACA GCA ACA ACG AAA AAT CAC An array of

As ma3 (SEQ ID NO: 2), GTG ACC GCA TTG GAA TTA TTG TTG AAA GGT GAA ACT GAA GAC AAA ACT CGT CGC TAT GTT GCC GAA TTG ACT GCC GTT GGA TCG CCA AGC AAC AAA CAA GCT AAA GCA CAG ATC GAA GTG ACC AAA GGC GAA GAA TAC AAA ATC ACA TTG AAA TCA CCT GAA CAT GAA TTT AAC ACC GAA TTC ACC ATC CAT GCA GAC AAA AAC AAT TTG AAA ATG CAC ATG GAT TTC CCT AAC GTG TTC CAA GCT GAT CTG ACT GGA ACT TTC CAA CAT GAC AAG GAA AAC AAT GTG CGT AAA AAC CAA TTG AAT CTC CAA TAC AAA TTC GCT GGT GAT GAG AAA CCA CAC ACA GTG GAT TAT GAA AAC GAA TTT TCA TTC AAT TTG AAA CGA TCA TCA AAA GAC AAA AAC AGT GGG GTC GAT TAC CGA GCA AAA TAC ATG TCT AGC CAT TTT CCA ATC TTG AAC CAC AAA GTC AAC ATT CAA TTC AAA TAT CGA CCA TTC AAA GTT AAC GAA TTG AAT TTG GAA GGA GAA TTT GGT CGA GAA CTT CAA CAC AAA TTC CAA TTG ATG CGA AAC AGC CAG ATA GAA GTG GAA GAA GTG CGA CCA TTT AAG ATG CAT GGC AAT AGT GAC ATA AAA TTG ATG GCA AAC GAT CTG GAT ATT GAT TAT GAT CTC AAA TCT GAA TTC AAA TAT GAA AGC AAC AAA GGA ACC CCC ATT GAA TTG CAA TAC AAA ATT TCC GGT AAA GAT CGC TCG AAA AGA GCT GCC GAT TTG GGA GCC GAA GAT GTT GAA GGC GTT ATC GAT TAC AAA AAC AAT GGT TCG CCA ATT GAT TCT AAG ATG CAT GCT CAT CTC AAA ATG AAA GGC AAC AAC TAT GGC TAT GAT AGT GAA TTG AAA CAA ACA CAG CCA CAA CAA TAT GAA GGC AAA ATC ACA TTG TCC AAG AAT GAC AAG AAA ATC TTC ATC AAC CAC AAA TCA GAG ATG ACC AAA CCT ACC AAC ACT TTC CAT CTC AAA ACA GAT GCT GAT GTG TCG TAT TCG GAT TCG GAT ATG AAA AAA CAT TAT CAG ATG GAA sequence,

As ma15 (SEQ ID NO: 3), GAA TGG TAT CAT TTA TTT AAA AGG TGG TTA CGA AAG CGC TTT ACC AAA ATG ATT GAT GAA TTG AAG CCA TCA TTG TTG CAT GTT AAA GGC AAA AAA CGT CCA ATC GTT TAT GAA TGT GCT GAG ATT AGC TGG AAA GTT ATG AAT AAT GGT GAT GTA TTC ATT TTG CTT GTA CCG AAT TTT GTT TTC GTT TGG ACC GGT AAA CAT TCA AAT CGT ATG GAA CGT ACT ACT GCT ATT CGT GTG GCT AAT GAT TTG AAA TCT GAA TTG AAT CGT TTT AAA TTA TCA TCA ATC ATC TTG GAA GAT GGT AAA GAA GTT GAA CAA ACA TCC GGT GCT GAA TAT GAT GCA TTC AAT AAA GCA TTA TCA TTG GAT AAA AAA GAT ATT GAT CTT AAA CAA ATG CCA AAA GGC TAT GAT TAT GCT GCC AGT GAT AAA TCT TTT GAA TCA CAT GAA CGT TCA TTC GTT ACA TTG TAT AAA TGT TTC GAA GGT ACT GAA ACG ATT GAT ATT TCG TTT GTC AAA AAT GGA CCG TTA AGC CGT GCT GAT CTC GAT ACA AAT GAT ACA TTC ATT GTT GAA AAT GGT TCC GAA GGT TTA TGG GTA TGG GTT GGT AAG AAA GCC ACA CAA AAA GAA CGA CAA TCG GCC ATT AAA TAT GCA ATG GAA TTG ATT AAT AAG AAA AAA TAT CCA AAT AAT ACA CCG GTA ACC AAA GTA TTG G AA GGT GAT GAA AGT GTT GAA TTC AAA TCA TTA TTT GAA TCA TGG CAA ATG AGC GAA CAG GAA AAA ATT ACC AGT GCA CGA TTG TTT CGT GTT TCA CGA AAT GGT ATC TTT AAA CAG GTT GCC AAT TAT GAA CCA GAT GAT TTG GAA GAG GAT ATA TAT CAT GAT TTT GGA TGT TAT GGA sequence,

As ma29 (SEQ ID NO: 4), AAA GAC GAT ATT GAA CGT ATG GTC AAA GAG GCT GAA TCA TAT AAA GAA GAA GAT GAC AAA CAA CGT GAC CGT ATT GCA GCC AAA AAT TCA TTG GAA GGT TAT GCT TTC CAG ATG AAA GCC ACT TTG GAT GAA GAA GCA ATT AAA TCT AAA GTT TCT GAA GAA GAT CGT AAA AAA ATC CTG GAC AAA GTT GAT GAA GTG CTC AAA TGG TTG GAC GCT AAT GCT TTA GCA GAA AAA GAT GAA TTT GAA CAT CAA CGT AAA GAA TTG GAA AGT GTA TGC AAT CCG ATC ATT ACT AAA TTA TAT CAA CAA GCT GGT GGT GCA GGC GCT GGG GGT ATG CCA GGT GGA TTT CCA GGT GGT TTT CCC GGT ACT GAT GGT TCT GGT GGT GGT GCA GCC GGT GGT GAT GGT GGT AAA TCT GGG CCC ACC ATT GAA GAA GTA GAT sequence,

As ma44 (SEQ ID NO: 5), TTT TTT TTC GTT GCA GCT AAA CAA CAA CAA CAA CCA TCA ACA AAA ATG GAG GCC ATC AAG AAA AAA ATG CAG GCA ATG AAG CTC GAG AAA GAT AAT GCT ATC GAT CGA GCT GAA ATT GCC GAA CAA AAA GCC CGT GAT GCT AAT CTA CGT GCC GAA AAG TCT GAG GAA GAA GTT CGT GCA TTA CAG AAA AAA ATC CAA CAA ATT GAA AAT GAA TTG GAT CAG GTC CAA GAA CAA TTA TCG GCT GCC AAT ACA AAA TTG GAG GAA AAG GAA AAA GCC CTA CAG ACC GCT GAA GGT GAT GTT GCA GCA TTG AAT CGT CGT ATT CAA TTG ATT GAA GAA GAT TTG GAA CGA TCA GAA GAA CGA CTT AAG ATT GCT ACA GCC AAA TTG GAA GAG GCA TCA CAA TCT GCC GAT GAA CT GAA CGT ATG CGT AAA ATG CTT GAA CAT CGA TCC ATC ACC GAT GAA GAA CGT ATG GAT GGT TTG GAA AAT CAA CTT AAA GAA GCC CGT ATG ATG GCC GAA GAT GCT GAT AGA AAA TAT GAT GAA GTT GCC CGT AAA TTG GCA ATG GTT GAA GCC GAT TTG GAA CGT GCT GAA GAA CGT GCC GAA ACC GGT GAA TCG AAA ATT GTT GAA CTC GAA GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA GTT AGC GAA GAG AAA GCT CAA CAA CGT GAA GAA GCC TAT GAA CAA CAG ATC CGT ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG GAA GAC GAA TTG GTC CAC GAA AAG GAA AAA TAC AAA TCC ATC TCC GAC GAA TTG GAC CAG ACA TTT GCC GAA CTT ACT GGT TAT sequence,

As ma50 (SEQ ID NO: 6), TTT CTT ACA AAC GAT AAA CGA TAC AAT TTT TTT TTT GTA ACA ATG AAA TTC TTA ATC TAT TCA TTG GCA TTT GCC GCT ATG GCC GGT TTG GTT TTC GGT TCC GAA AAA CGA AAA AAG AAT CCA TAT GAT GTT GCC GAA TCA TTG ATT AAA CAT GCT GAT GCA TTG AAA GAA TAT GCC GAA CAG GAG ATA CAA AAA TAT CAG AAT AAT TCA CAT TAT CAT TTG GCC ATC ATA CGT TTG GAA CAT GAA ATT CAT CGT GTT GAA GCC ATT GCC GAT CAA CTT CGT AAT ATG ACT AAA ATG GAA AAT ATG ACC GAA CAT TAT ATG CAT CAT TTC CAT CAA ATT GAA ATG GAA CTA TTA ATT TTT GAA AAT ATT ATT GAA GAA GAA TTA TAT TAT TTA GAA CAT GGT CAC CAC CAT GAT CAT CAT GAT CAT GAT CAT CAT GAT CAT ATG CAT CGT CAT GAT CAT GAT GAT TCA GAC ATT ATC GCC GGT GAT GAT TAT AAA GAT GCT TTG TTG AAA AAA GCA CAA CAA CTT ATC GAT GAA GCT GAA GAT GCA ATA AAA AAA TAT GGA CAA GGT AAA GAT ATC GAT GTT CAG GTG ATA AAA TCC GAA GTT GTT ATA ATA AAA AAC TTG GTA AAA CAA ATC AAT GAA GCT AAT AAT CCA CAA GCA TTG GCC GTA TTG GAA TCA AAT CTA GCT CAA CAT GAG GAA A TT ATT CGT AAA TGG CTT GAT GAT ATT GAA CCA CAC ACT ACA CAT GCC CCA GTT ACT ATT GCT sequence,

As ma113 (SEQ ID NO: 7), TTA CAA CCC AAC GGA CCA AAG GGT TTG CGT TCT CCT GGA AGC CCT GAC AGC GGT AAT CTG CGT AAA CGT AAG AGA AGA GGA GGA GTT TTG GGT TTC AAG GAA TTG GCA GCG AAG TCT AAG TTA TCT GCC TGG CAT TTT CAT CGT GTC TTT AAG TCA GTG ACT GGA TTG ACT CCT AAG ACA TAT GGA GAT AAA TGT TGG GAA TAC ATT AAA CAT TGC AGA GAA AAT GGG GAG AGC TTC TTT TTC GAG GGT AAG CAA GCT AAC ACA GAC TCC GGA GAG GTC TCT CTT AGC CCA CCA AAT ATG TGC TAT CAG ACA CAG ACA CCT ATT AGC TCT AAT ACT GAT TCA CAT AAC TAT TTT GAT GAG TCG GAC GAT GAA AAA GTA AAG AAG GTT AAA TTG TCA CCA GAG GTG GAG CGG GGG ATT TCC ACC AAA GAT AGC TTC AAT GGC GAG TCA TCT ACT CCC GGT GAT GAT TGT TTT CAC CCT GAC GAA AAT GGA CTT GAT AAC AAT TTG AAG AAC ATT GGG CTA GGG CTG TCG TTC GCT ACT AAG GAT CTT GGC ATA AAT CCT AGT ACT CCT AGT GTT GAA GAT GGT TTG GTA CAG CCT ACA TGT TCT TTT GAC TTT AAT TTA AAT GAT GCT ACG ATA CCC AAT GTT GAC GAT TTC GAC GAT TTT TCA AAA GTT CCT CAA TTT TTA AAT GAT GAC TCT GCT GGG TTT GT G AGA GCG TTT TCT GCT CCT GAT TTG ACC AAG TAC AAC CAT ATG AGT GCA TTA AGT CCT GAA AGT AAT GGC AAC ACT GAC ATG AAC AAG CCC AAG GAT GCA TTA GGT TTC GAT TCC TAC CTT GAT ACA GAC ATT AGC AAC CAG GAA AAT GGG AAT GCT CTT TAT CCA GTT GTG GAT GAT GTT CCA TCA TTG TCT GGG ACA GGT TTT ATG CCA AAC ACT TCA ATT CAA AAT ACG TCT TTT TTA AGA CCA TTT GAA GAT AGC TTG GAT ATA CAA GAT ACT GCT GAA CTT TCA ATT GGC TAC GAT GTT GAT TCT CTC GCG CCT TCT ACA CCC GGA TTA GAT ATG AAG CTT AAT TCG AGG TTT AGT GTT GCG CAA CCT GAT sequence,

As ma114 (SEQ ID NO: 8), TCA ACA AGA TGG CTT ACA CAT GAA TCG GAC GAA GAA CGT TGG GAG CAA TAT AGA CTC AAA CAA AGA ATG CCA GAG AAT GAT TTT GGT CCG ATC ATT ACG GGT CAT CAT GAT GAT GAT GAT GAT GAT GAT GTC GAT ACA CCG AAT GAA GAA GAG AAT CGT GAT AAA TTC ATC ACA ATC ACC AAC ACT GAT TCA TTC AGA CGA CGT ACT AAT TAC AGC AGC AGC AGC AAC AGC AAC AAT AAT AAT AAC AAC AAT GGC AAG TAT GGC CAC CAG CAT CAG CAT CAC CAT CCC CGC CAT CAT CAT CAT CAC AAT CCG AAT AAA TCG AAT GGC AAT GGA CAT GCA TTT AAC CAC AAT AAT GCC ATC ATT ATC AAC AAC AAT AAT AAT GAT GAT TCG ACA GCC AAT TTG GAA CAG TAT AAC AGC AAA AGT AAC ACG ACC AGC CGT TTG sequence,

As ma115 (SEQ ID NO: 9), TTC ATC AAA ATT AAA AAT TCA TCA AAA ATG AAA TTC GTT TTG GCC ATT GCC TCT TTG TTG GTA TTG AGC ACT GTT TAT GCT CGT CCA GCT TCA ATC AAA ACT TTT GAA GAA TTC AAA AAA GCC TTC AAC AAA AAC TAT GCC ACC GTT GAA GAG GAA GAA GTT GCC CGT AAA AAC TTT TTG GAA TCA TTG AAA TAT GTT GAA GCT AAC AAA GGT GCC ATC AAC CAT TTG TCC GAT TTG TCA TTG GAT GAA TTC AAA AAC CGT TAT TTG ATG AGT GCT GAA GCT TTT GAA CAA CTC AAA ACT CAA TTC GAT TTG AAT GCC GAA ACA AGC GCT TGC CGT ATC AAT TCG GTT AAC GTT CCA TCG GAA TTG GAT TTA CGA TCA CTG CGA ACT GTC ACT CCA ATC CGT ATG CAA GGA TGT GGT TCA TGT TGG GCT TTC TCT GGT GTC GCC GCA ACT GAA TCA GCT TAT TTG GCC TAC CGT AAC ACG TCT TTG GAT CTT TCT GAA CAG GAA CTC GTC GAT TGC GCA TCT CAA CAC GGA TGT CAC GGC GAT ACA ATA CCA AGA GCA GAA TAC ATC CAA CAA AAT GGT GTC GTT GAA GAA AGA AGC TAT CCA TAC GTT GCA CGA GAA CAA CAA TGC CGA CGA CCA AAT TCG CAA CAT TAC GGT ATC TCA AAC TAC TGC CAA ATT TAT CCA CCA GAT GTG AA A CAA ATC CGT GAA GCT TTG ACT CAA ACA CAC ACA GCT ATT GCC GTC ATT ATT GGC ATT AAA GAT TTG AGA GCT TTC CAA CAT TAT GAT GGA CGA ACA ATC ATT CAA CAT GAC AAT GGT TAT CAA CCA AAC TAT CAT GCC GTC AAC ATT GTC GGT TAC GGA AGT ACA CAA GGC GTC GAT TAT TGG ATC GTA CGA AAC AGT TGG GAT ACT ACC TGG GGT GAT AGC GGA TAC GGA TAT TTC CAA GCC GGA AAC AAC CTC ATG ATG ATC GAA CAA TAT CCA TAT GTT GTA ATC ATG , With. These DNA sequences are
Known as the major mite allergen, Der p I, De
There is no homology other than ma115 compared to those reported for rp II, Der f I and Der f II.

The mite-derived gene of the present invention is ma1
0 means that the total base chain length of the cDNA is about 0.50 kbp
(Agarose electrophoresis), but in the actual expression,
Since it terminates at the preceding stop codon, the translated base chain length is 558 bp excluding the stop codon. As ma3, the total base chain length of cDNA is about 1.05 kbp (agarose electrophoresis method), and it has the restriction enzyme map shown in FIG. As ma15, the total base chain length of cDNA is about 1.66 kbp (agarose electrophoresis method), and it has the restriction enzyme map shown in FIG. As ma29, the total base chain length of cDNA is about 0.75 kbp (agarose electrophoresis method), and it has the restriction enzyme map shown in FIG. As ma44, the total base chain length of cDNA is about 0.99 kbp (agarose electrophoresis method). As ma50, the total base chain length of cDNA is about 0.90 kbp (agarose electrophoresis method), and FIG.
It has the restriction enzyme map shown in. The ma113 has a total base chain length of cDNA of about 1.26 kbp (agarose electrophoresis method) and has the restriction enzyme map shown in FIG. As ma114, the total base chain length of cDNA is about 0.62 kbp (agarose electrophoresis method), and it has the restriction enzyme map shown in FIG. ma
As for 115, the total base chain length of cDNA is about 1.04 kbp.
(Agarose electrophoresis method) and has the restriction enzyme map shown in FIG.

The expression vector of the present invention is an expression vector ligated so that the gene of the present invention can be expressed in transformed bacteria, yeast or mammalian cells. Vector D used for construction of expression vector
The NA is not particularly limited, and one widely used and easily available is used. For example, pUC18, pTV118 N (manufactured by Takara Shuzo), pUEX2 (manufactured by Amersham), pKK233-
2 (manufactured by Pharmacia), pMAM-neo (manufactured by Clontech) and the like.

The method for constructing the expression vector of the present invention is not particularly limited and can be carried out by a conventional method. For example, as shown in FIG. 1, cDNA of mite allergen integrated in λgt11 phage was digested with Not I, and pAK obtained by ligating to the Not I site of phagemid pBluescriptII SK (+) was digested with Not I. , Blunted with Klenow fragment and plasmid vector pUEX2 (Amersh
expression vector pAEX ligated to the Sma I site (manufactured by am)
Can be obtained.

The bacterium, yeast or mammalian cell transformed with the expression vector of the present invention is not particularly limited as long as it can express the gene of the present invention. Examples of bacteria include Escherichia coli and Bacillus subtilis. , Saccharomyces cerevisiae etc. as yeast, and mammalian cells as
Examples include Chinese hamster ovary (CHO) cells, monkey COS cells, mouse fibroblasts, and the like.

The method for producing the recombinant mite allergen of the present invention comprises the steps of culturing bacteria, yeast or mammalian cells transformed with an expression vector capable of expressing the gene of the present invention under conditions capable of expressing the gene. , Producing a recombinant mite allergen and then recovering the recombinant mite allergen. In order to obtain the cDNA encoding the recombinant mite allergen of the present invention from the mite body, poly (A) mR can be obtained from the total RNA fraction as described in detail in Examples below.
The NA fraction can be collected and cDNA can be synthesized using a commercially available cDNA synthesis kit or the like. Moreover, the following method is exemplified as a method for producing a recombinant mite allergen using an Escherichia coli transformant containing a fusion protein expression plasmid into which the mite allergen cDNA has been inserted.

First, an Escherichia coli transformant containing a plasmid for expressing a fusion protein with β-galactosidase in which the cDNA of mite allergen is inserted is shake-cultured by a conventional method to grow logarithmically, and temperature shift is carried out while maintaining this logarithmic growth phase. , Or by adding β-galactosidase inducer to induce synthesis of the fusion protein. The E. coli used as a host cell, for example E. coli JM105 (Pharmacia) is used, each strain transformed with the expression vector, a transformant of ma10 clone E. col
i JM 105 OL-10 (FERMP-13496), ma3 clone transformant was transformed into E. coli JM 105 OL-3 (FERMP-13812).
No.), a transformant of the ma15 clone was used as E. coli JM 105
The transformed strains of OL-15 (FERMP-13813) and ma29 clones were transformed into E. coli JM 105 OL-29 (FERMP-13814),
The ma44 clone transformant was transformed into E. coli JM 105 OL-44.
(FERMP-13815), a ma50 clone transformant was transformed into E. coli JM 105 OL-50 (FERMP-13817), ma1.
Transformants of 13 clones were transformed into E. coli JM 105 OL-113 (FE
RMP-13818), a transformant of ma114 clone
E. coli JM 105 OL-114 (FERMP-13819), ma11
Transformants of 5 clones were transformed into E. coli JM 105 OL-115 (FERM
P-13820).

After completion of the culture, the collected bacterial cells are suspended in a buffer solution containing serine / cysteine / aspartic acid / metalloprotease inhibitor and disrupted by ultrasonication.
Membrane-localized proteins in cell debris were identified as proteolytic enzyme inhibitors such as phenylmethanesulfonyl fluoride, monoiodoacetic acid, pepstatin A, ethylenediaminetetraacetic acid and sodium lauryl sulfate (SDS), Triton X-
Extract with a buffer containing a surfactant such as 100 and Nonidet P40. The mite allergen β-galactosidase fusion protein obtained from the extract or from the culture concentrate,
For example, gel filtration chromatography using Ultrogel AcA 44 (manufactured by IBF), affinity chromatography immobilized with anti-β-galactosidase antibody, affinity chromatography immobilized with anti-mite body antibody, anti-Derf I antibody-immobilized affinity chromatography and anti-Derf II. It is purified by antibody-immobilized affinity chromatography or the like. The anti-β-galactosidase antibody-immobilized carrier is
Anti-β-galactosidase antibody (5Prime → 3Prime) activated tresyl carrier (eg, Tresyl GM gel (Kurita industry), Tresyl Toyopearl (Tosoh), Tresyl sepharose (Pharmacia). ) And the like are used). The carrier for immobilizing anti-mite body antibody is the above-mentioned activated carrier to which a rabbit anti-mite body antibody is covalently bound.

The purified recombinant mite allergen fusion protein was digested with protease and subjected to ELISA and leukocyte histamine release test for patients with mite allergic disease (allergy, 3
7 , 725 (1988)), for example, gel filtration chromatography, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatofocusing, focus electrophoresis,
Known purification methods such as gel electrophoresis are fractionated individually or in combination, and the protein component derived from β-galactosidase is absorbed and removed from the active fraction through an anti-β-galactosidase antibody-immobilized affinity column. Further, the recombinant mite allergen fragment is purified using an anti-mite body antibody-immobilized affinity column, an anti-Derf I antibody-immobilized affinity chromatographic column, and an anti-Derf II antibody-immobilized affinity chromatographic column.

In addition, a digestion product of the product purified by the above-mentioned method with a proteolytic enzyme such as pronase, subtilisin, thermolysin, trypsin, or cyanogen bromide, 2-
Purification of mite allergen fragments from the decomposition products by chemical treatment with nitro-5-thiocyanobenzoic acid, hydroxylamine and the like can also be performed by the above methods alone or in combination.

In addition to the direct expression of the mite allergen protein, the method for producing the recombinant mite allergen of the present invention comprises:
There is also a method for producing a recombinant mite allergen by recovering what has been expressed as a fused recombinant mite allergen in which the mite allergen is fused with another protein via an intervening sequence, and then releasing the fused other mite allergen. Inclusive. As the other protein to be fused, those generally known to be a fusion protein such as β-galactosidase, glutathione-S-transferase and protein A are used.

Known methods can also be used for releasing other proteins. For example, when the other protein is β-galactosidase and a part of collagen or fibrinogen protein is present between the other protein and the mite allergen protein, A mite allergen protein is purified using collagenase or thrombin by the above methods alone or in combination.

The mite allergen fragment of the present invention is easily assembled by preparing a DNA encoding the amino acid sequence of the fragment by a conventional method, constructing an expression vector, and expressing in an appropriate host. It can be obtained as a mite allergen. Further, the mite allergen fragment is synthesized by a conventional solid-phase synthesis method, and as a synthetic mite allergen, alone or in an appropriate carrier, for example, human serum albumin, and ascidian antigen whose safety has been confirmed by intradermal administration. It can also be used by being combined with etc.

Further, a peptide having an epitope contained in a mite allergen derived from a mite body of the present invention or a peptide having an epitope which can be regarded as immunologically identical to the epitope is the following amino acid for ma10: Sequence (SEQ ID NO: 10), Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Se
Examples include r Leu Glu or a peptide containing the following amino acid sequence (SEQ ID NO: 11). Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu

The genes encoding these peptides include the following DNA sequences (SEQ ID NO: 10)
Containing GAG GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA or containing the following DNA sequence (SEQ ID NO: 11). ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG

Examples of the peptide having an epitope contained in the mite allergen derived from the mite body of the present invention or the peptide having an epitope which can be regarded as immunologically identical to the epitope include those described above, When used as an active ingredient in a mite allergic disease therapeutic agent or a mite allergic disease diagnostic agent, any of them can be used.

The therapeutic agent for mite allergic diseases of the present invention contains the above-mentioned purified recombinant mite allergen, mite allergen fragment or peptide having an epitope as an active ingredient, and is used as a therapeutic agent for various mite allergic diseases. . Here, the mite allergic disease refers to any allergic disease caused by a mite specific antigen, such as atopic bronchial asthma, allergic rhinitis, allergic conjunctivitis, and atopic dermatitis.

The method for preparing the therapeutic agent for mite allergic disease of the present invention is not particularly limited. For example, recombinant mite allergen purified by the above method, mite allergen fragment or epitope purified according to the above method The peptide having the above formula is dried and collected as a powder, and used as a therapeutic agent for hyposensitization against mite allergic disease. When the therapeutic agent for mite allergic disease of the present invention is used as a therapeutic agent for hyposensitization, the adjuvant or various additives generally used as they are or as necessary, for example, a stabilizer, an excipient, a solubilizing aid. It can be used as a compounding agent to which an agent, an emulsifying agent, a buffering agent, a soothing agent, a preservative, a coloring agent and the like are added by a conventional method. For example, powdered purified recombinant mite allergen is dissolved in physiological saline containing phenol and used as a stock solution of antigen for treating hyposensitization.

The therapeutic agent for mite allergic disease of the present invention can be administered by a usual administration route such as transdermal, oral, intradermal, subcutaneous, intramuscular,
It can be performed by an administration method such as intraperitoneal administration. Furthermore,
For example, it can be used as a transdermal or transmucosal drug such as troches, sublingual tablets, eye drops, nasal sprays, poultices, creams, lotions and the like. The dose and frequency of administration of the therapeutic agent for mite allergic disease of the present invention are appropriately selected so as to be in the range of about 20 μg or less per adult according to the administration route, symptoms, etc., and are administered about once a week. Further, the therapeutic agent for mite allergic disease of the present invention is useful as a preventive agent as well as a therapeutic agent for mite allergic disease. Further, it has no anaphylaxis-inducing action and can be safely used for the human body.

The diagnostic agent for mite allergic diseases of the present invention is used as a reagent for diagnosing intracutaneous reaction against mite allergic diseases and a titration reagent for diagnosing mite allergy. When used as an intradermal reaction diagnostic reagent, a recombinant mite allergen purified by the above method, a mite allergen fragment purified according to the above method or a peptide having an epitope can be prepared by a conventional method. The recombinant mite allergen is dried to give a powder, which is dissolved and diluted with physiological saline containing phenol before use. The method used as an intradermal reaction diagnostic reagent is used according to a conventional method.

When it is used as a titration reagent for diagnosing tick allergy, it is also prepared by a conventional method. For example, a recombinant mite allergen is appropriately dissolved in Hanks buffer and diluted to give a histamine-free titration reagent. To use. This method is usually performed by the following operating procedure. That is, a fixed amount of a blood cell suspension obtained by suspending a blood cell fraction obtained by centrifugation of the blood of a tick allergic disease patient and this blood in a buffer solution and titrating using a recombinant mite allergen as a titration reagent, an allergen The amount of histamine released from basophils (a type of white blood cell) upon stimulation is measured by HPLC [Allergy, 37 , 725 (1988)].

In this histamine release titration, the amount of histamine released from 50% of the maximum release amount (the inflection point of the titration curve) is determined. In this titration, (1) the allergen sensitivity of the patient is directly measured from the titrated value of the blood cell suspension. (2) The value (blood titration curve value) obtained by preliminarily reacting plasma with recombinant mite allergen and titrating the hemocyte suspension with the solution is usually titrated with recombinant mite allergen. Value obtained by (blood cell floating drop constant curve value)
Higher values are obtained. This is because there is an IgG antibody (blocking antibody) having an allergen neutralizing ability in plasma. Therefore, the blocking antibody titer can be obtained from the magnitude of the shift of the blood titration curve from the blood cell suspension titration curve. Accurate diagnosis of mite allergy is possible based on the allergen sensitivity and this blocking antibody titer. Moreover, this histamine free titration test is also useful as a monitor of the effect of desensitization treatment.

[0057]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. The reagents used in each example were commercially available reagents purchased from Nacalai Techs, Katayama Kogyo, Wako Pure Chemical Industries, Ltd. The genetic engineering reagents such as restriction enzymes used were those purchased from Takara Shuzo. In the examples, the rabbit anti-mite body crude antigen serum used was obtained by immunizing a rabbit with a crude mite body crude antigen, confirming an increase in antibody titer, and then collecting whole blood. Rabbit anti-Der f I serum and rabbit anti-Der f II serum were obtained from Dr. Yasuda of National Sagamihara Hospital.

Example 1 Extraction of mite total RNA and isolation of mite allergen Poly (A) mRNA: 6 g of live mite body obtained by culturing and propagating Dermatophagoides farinae by a conventional method was used to prepare 200 ml of 5.5 M guanidine isothiocyanate (Katayama Chemical Co., Ltd.). (Made) Grind with 10 g of quartz sand in a mortar and centrifuge (9000 rpm, 10 minutes, 4 ° C)
Then, the supernatant was inhaled with a 50 ml syringe equipped with an 18 G injection needle and the injection was repeated to partially cut the DNA. The supernatant obtained by centrifugation again was added to 16 ml of 17 ml cesium trifluoroacetate solution (Katayama Chemical Co., Ltd.).
At 85,000 × g for 24 hours (15 ℃, HITACHI
SCP55H swing PRS-27-2) Density gradient centrifugation was performed to collect the total RNA fraction that forms a pellet at the bottom of the tube. next,
Using a Fast Track mRNA isolation kit (manufactured by Invitrogen), poly (A) mRNA fraction was recovered from the obtained total RNA fraction. When the yield was measured by performing a spot test with an RNA solution dilution series of known concentration, it was about 20μ.
It was g.

Example 2 Synthesis of mite allergen cDNA: Next, this poly (A) mR
Using 5 μg of NA as a template, cDNA synthesis kit (Ph
(made by armacia) according to its manual
A of 13mer containing Not I sites at both ends
An EcoRI linker was connected.

Example 3 Construction of mite allergen cDNA library: This cD
1/10 amount of NA was 1 μg of EcoRI digested λgtll
(Stratagene Cloning System) and mixed with ethanol, then 200 units of T4 DNA lig
By adding ase and reacting at 12 ℃ for 15 hours, cD
NA was inserted into the EcoRI site of λgtll. From this reaction mixture, use the in vitro packaging kit (Gigapack PLUS, S
A cDNA library was prepared by using tratagene Cloning System) according to the manual. This library preparation operation was performed 7 times, and finally 6.6μ
133,000 pfu (plaqu) from g of poly (A) mRNA
A cDNA library of e forming unit) was obtained.

Example 4 Immunoscreening of mite allergen cDNA library: 1 of the cDNA libraries obtained from Example 3
36,000 recombinants were immunoscreened using rabbit anti-mite body crude antigen sera. The detailed protocol of the immunoscreening performed in this example is described below. Phage and host ( E.coli Y
1090, Stratagene Cloning System) was incubated at 37 ° C for 30 minutes and melted LB-soft agar
Was superposed on an Lb plate together with and incubated at 42 ° C. for 3 to 4 hours. After that, the upper part was covered with a nitrocellulose filter containing 10 mM of isopropyl-1-thio-β-D-galactoside and incubated at 37 ° C. for 3 hours to obtain a replica filter.

The filter was washed three times for 10 minutes with a solution (TBST) containing 0.05% Tween 20 in 10 mM Tris buffered saline (pH 7.6), and then 2% skim milk powder was added. Incubation was carried out for 1 hour or more at room temperature with TBS containing After this, TBS
After repeating 10 minutes of washing for 3 times with, the cells were incubated with TBST containing a 500-fold diluted rabbit antiserum (pretreated with E. coli lysate) at room temperature for 1 hour. Furthermore, 1 using TBS
After washing for 0 minutes three times, incubation was carried out for 1 hour or more at room temperature using peroxidase-labeled goat anti-rabbit IgG diluted 2000 times. next,
0.6 mg / ml of 3,3'-diaminobenzidine tetra
hydrochloride, 0.3% NiCl ₂ and 0.01% H ₂
Enzymatic reaction with TBS containing O ₂ causes black gray ~
Those that gave a black signal were collected as positive clones. As a result, 269 clones were positive in the primary screening.

Example 5 Mite allergen c by cross-hybridization
Classification of DNA: Cross-hybridization was carried out, assuming that some cDNAs incorporated in the rabbit anti-mite body crude antigen seropositive clones obtained by the immunoscreening of Example 4 may encode the same sequence. The mite allergen cDNA was classified by.

First, of the positive clones obtained in Example 4 above, the cDNAs incorporated in the first 20 positive clones obtained were excised from the λgtll phage by Not I digestion, and each of these cDNAs was probed. As a result, it was hybridized to the cDNA on the nylon membrane and roughly divided into groups.

Next, with respect to 46 positive clones other than the above 20 positive clones which had already been grouped, cDNA was excised from the λgtll phage at the Not I site to generate Not in pBluescript II SK (+) DNA. CD excised at Not I site after subcloning at I site
NA was spotted on the nylon membrane and hybridized to the cDNA on the nylon membrane by using the cDNA having the largest base chain length within the group as a probe among the cDNAs obtained in each of the groupings previously obtained. It was Where pBlue
After script II SK (+) DNA was subcloned into Not I site, digested at Not I site and subjected to agarose gel electrophoresis to obtain a fragment containing cDNA as GENE CLEAN.
It was extracted and purified using KIT (manufactured by Toyobo Co., Ltd.) to obtain a DNA for probe.

With respect to the remaining 134 clones, the lysate in which the phages were propagated by infecting E. coli Y1090 in a liquid medium was spotted on a nylon membrane with the cDNA inserted in the λgtll phage. Then, the DNA Labeling and
It was hybridized with a Detection Kit (Boehringer Mannheim) probe.

Hybridization Protocol:
Denatured cDNA fragments or λgtll phage lysates were spotted on nylon membranes. A denaturing solution (0.5N NaOH, 1.5M NaCl) was permeated for 5 minutes, and a neutral solution (1M Tris-HCl [p.
H8.0], 1.5 M NaCl) was permeated for 5 minutes, and then ventilation drying was performed for 1 hour or more. UVTransillumin
DNA was fixed with an ator for 3 minutes. The DNA-immobilized membrane was prehybridized with a hybridization buffer (60 ° C., 1 hour). Hybridization was performed with a hybridization buffer containing a labeled probe DNA (60 ° C., 6 hours or more). 2 x SSC /
It was washed twice with 0.1% SDS (room temperature, 5 minutes), and further washed twice with 0.1 × SSC / 0.1% SDS (68
(° C, 15 minutes) to obtain a hybridized membrane.

The resulting membrane was equilibrated with Buffer A for 1 minute and incubated with Buffer A containing 0.5% blocking reagent for 30 minutes. Anti-sheep digoxigenin Fab conjugated with alkaline phosphatase in buffer A after washing for 5 minutes in buffer A
Incubated with fragment. This was washed twice with buffer A for 15 minutes, equilibrated with buffer B for 1 minute, and then nitroblue tetrazolium salt, 5-bromo-4-chloro-3.
An immunoreaction was performed with buffer B containing -indolyl phosphate and toluidinium salt, and the reaction was stopped with TE (pH 8.0). As a result of the cross-hybridization carried out in this way, the gene of the present invention (ma10 clone, ma) which seems to encode a novel mite allergen.
3 clones, ma15 clones, ma29 clones, m
a44 clone, ma50 clone, ma113 clone, ma114 clone, ma115 clone) were obtained.

Example 6 Preparation of λgtll phage DNA: 200 μl of an overnight culture of E. coli Y1090 and λgtll phage lysate (10 ⁵ -1
100 μl of 0 ⁷ pfu) was incubated at 37 ° C. for 15 minutes. Add 5 ml of LB broth and shake at 37 ℃
After incubating for 6-8 hours at 4 ° C, 12,000
Centrifugation was performed at 0 rpm for 10 minutes. DNase
I and RNase A were added to a final concentration of 1 μg / ml and incubated at 37 ° C. for 30 minutes. Add equal volume of 20% PEG 6000 / 2M NaCl and add 4 ° C.
After cooling overnight at 4 ° C and 12,000 rpm for 10
Centrifuged for minutes. 350 μl TE (10 mM
Tris-HCl buffer (pH 7.5) and 1 mM
(Containing EDTA), proteinase K, SDS, and EDTA were added to a final concentration of 50 μg / ml, 0.
Add 5% and 12.5 mM and add 1 at 55 ° C.
After incubating for 5 minutes, it was extracted with a phenol / chloroform solution and precipitated with ethanol. Then T
It was dissolved in E to prepare a λgtll phage DNA solution.

Example 7 Cloning of mite allergen cDNA: SM (1 M Tris-HCl, pH 7.5, 0.1 M NaCl 10 mM MgSO 4) in which λgtll phage having each cDNA obtained in Example 5 inserted therein was suspended.
E. coli Y1090 cultured overnight with _4, 2% gelatin) solution
(Stratagene Cloning System) was incubated at 37 ° C for 30 minutes with shaking to infect the host with the phage, which was then LB agar medium (1% Bactotryptone, 0.5% Bactoye).
ast extract, 1.5% Bacto agar (Difco), 0.5%
Plates were plated on NaCl, pH 7.6) to form approximately 2,000 plaques per plate. After culturing at 42 ° C. for 3 hours, 10 mM isopropyl-1-thio-β-D-galactoside (IPTG) was dipped in advance to cover the surface of the medium with a dried nitrocellulose membrane (Hibond-C, Amersham). Β-induced and synthesized by culturing at ℃ for 3 hours
The galactosidase fusion protein was transcribed. This nitrocellulose membrane was added to TBS containing 10% Tris-
After blocking overnight in a solution of HCl, pH 8.0, 0.9% NaCl, rabbit anti-mite body crude antigen serum (100 times 2% BSA)
Diluted with TBS), rabbit anti-Derf I serum (1,200 times 2% B)
SA-containing TBS dilution), rabbit anti-DerfII serum (1,000-fold 2% BSA-containing TBS dilution), and peroxidase-conjugated goat anti-rabbit IgG antibody (Cappel, 2000-fold TBS dilution), each for 1 hour, Color development was performed in TBS solution containing 0.4 mg / ml diaminobenzidine tetrahydrochloride in the presence of 0.01% hydrogen peroxide. Rabbit anti-mite worm crude antigen serum was used for the whole mite breeding with saturated salt and PB.
It is an antiserum obtained by suspending mite worms with S, grinding the mite worms washed with PBS, and boosting the rabbits weekly for 10 weeks using this as an immunogen. Pick up the plaque that matches the position of the signal showing a dark brown color and SM
It was suspended in and stored as a positive clone.

Example 8 Preparation of plasmid DNA: 100-500 μl of the overnight culture of the transformant obtained in Example 7 was added to 7,000 r
Centrifuge at pm for 5 minutes to obtain 100 μl So
l. I (50 mM glucose, 25 mM Tris-H
Cl [pH 8.0], 10 mM EDTA),
200 μl Sol. II (0.2N NaOH, 1%
SDS) was added and cooled on ice for 5 minutes. Then 150
μl of ice-cold Sol. III (5M CH ₃ COOK, p
H4.8) was added and the mixture was allowed to stand on ice for 5 minutes to give 12,000.
Centrifugation was carried out at 4 ° C. for 10 minutes at rpm. The upper layer was extracted with a phenol / chloroform solution and precipitated with ethanol to obtain a plasmid DNA solution.

Example 9 Expression with the high expression vector pUEX2: pBluescript II SK was used as a probe for the first cross-hybridization (Example 5) to perform immunochemical analysis of transcripts of the mite allergen gene. (+) To Not I
It was cut out at the site and blunt-ended with a Klenow fragment, and then ligated to the Sma I site of the high expression vector pUEX2 so that the transcription direction and the frame would match, and a transformant of E. coli JM105 was obtained (FIG. 1). The obtained transformant was obtained by transforming the ma10 clone into E. coli JM 105 OL-10 (FERMP-1
3496), and a transformant of ma3 clone as E. coli JM 1
05 OL-3 (FERMP-13812), a ma15 clone transformant was transformed into E. coli JM 105 OL-15 (FERMP-13813).
No.), a transformant of the ma29 clone was used as E. coli JM 105
The transformed strains of OL-29 (FERMP-13814) and ma44 clone were transformed into E. coli JM 105 OL-44 (FERMP-13815),
The ma50 clone transformant was transformed into E. coli JM 105 OL-50.
(FERMP-13817), a transformant of ma113 clone was transformed into E. coli JM 105 OL-113 (FERMP-13818), ma.
A transformant of 114 clones was designated as E. coli JM 105 OL-114.
(FERMP-13819) and the transformant of ma115 clone were named E. coli JM 105 OL-115 (FERMP-13820). Using this transformant, cultivation at 28 ° C. and 4
After expression at 2 ° C, SDS-solubilized whole cells were used as a sample.

Example 10 Immunostaining of the expression product by SDS-PAGE and Western blot: The bacterial cell pellet was suspended in PB (pH 7.4) distilled water, and the final concentration of 62.5 mM Tris-HCl (pH 6. 8) 2% SDS and 2% 2-mercaptoethanol were added, heat-treated at 100 ° C. for 3 minutes, and then slab gel electrophoresis using 5-10% acrylamide gel was performed. Western blotting was performed by Immob using a semi-blotting apparatus (ATTO).
Ilon-P ^™ (manufactured by Millipore) or nitrocellulose filter (manufactured by Advantech) was transferred and immunostained according to the following protocol.

Western blotting protocol: The obtained transfer membrane was washed with TBST three times for 10 minutes, and then incubated with TBST containing 5% skim milk powder at room temperature for 1 hour or more. It was incubated with diluted primary antibody pretreated with E. coli lysate, then washed 3 times for 10 minutes with TBS, then incubated with diluted secondary antibody for 1 hour, then with 10% TBS.
Wash 3 times a minute. Next, 0.6 mg / ml of 3,
3'-diaminobenzidine tetrahydrochloride, 0.3% N
The enzyme reaction was carried out in TBS containing iCl ₂ and 0.01% H ₂ O _2, and a product in which a black gray to black signal was obtained was collected.

Example 11 Confirmation of expression by anti-β-galactosidase antibody: The obtained ma10 clone was expressed in the same manner as described above, subjected to SDS-PAGE, and then immunized by Western blot with the anti-β-galactosidase antibody. As a result of staining, β
-It was confirmed to be a fusion protein with galactosidase (Fig. 2). In addition, other clones (ma3 to ma)
115 clones) were similarly expressed and immunostained, and as a result, it was confirmed to be a fusion protein with β-galactosidase. The results are also shown in FIG.

Example 12 Confirmation of activity using rabbit anti-mite body crude antigen serum: ma1
0 clone was subjected to immunostaining by Western blot using rabbit anti-mite body crude antigen serum.
The result is as follows. Other clones (ma3 clone, ma15 clone, ma29 clone, ma44
Clone, ma50 clone, ma113 clone, m
The results shown in FIG. 3 were also obtained for 8 kinds of clones (a114 clone and ma115 clone). The results are also shown in FIG. Incidentally, as is clear from FIG.
The 113 clone, the ma114 clone, and the ma115 clone were not stained, but ma
Immunostaining was observed with rabbit anti-Derf I serum for 113 clones and ma115 clone, and with rabbit anti-Derf II serum for ma114 clone. From these, rabbit anti-mite body crude antigen serum and rabbit anti-mite
It was confirmed by immunostaining with Derf I serum and rabbit anti-Derf II serum that all clones retain the antigen activity.

Example 13 Immunostaining with pool serum of mite allergic patients: IgE immunostaining by Western blot using serum obtained by pooling 10 samples of mite allergic patients for each clone (9 clones of ma10 clone to ma115 clone). As a result, the same results as those obtained by immunostaining with rabbit anti-mite body crude antigen serum (Example 12) were obtained.

Example 14 Mite allergen gene (ma10 clone to ma115
Construction of restriction map of 9 kinds of clones): pA
Regarding the clones in which expression of transcripts was confirmed on EX,
Plasmid DNA was prepared with the cDNA inserted into pBluescript II SK (+) and digested with various restriction enzymes. The result of applying this to agarose gel electrophoresis was analyzed.
The results are shown in FIGS. Incidentally, the ma10 clone and the ma44 clone did not have a restriction enzyme cleavage site within the range of the restriction enzyme used.

Example 15 Mite allergen gene (ma10 clone to ma115
Determination of all nucleotide sequences of 9 kinds of clones): Based on the restriction enzyme map prepared in Example 14 above, a ma10 clone was prepared by the dioxy termination method,
ma3 clone, ma15 clone, ma29 clone, ma44 clone, ma50 clone, ma113
When the entire base sequences of the clone, ma114 clone, and ma115 clone were determined and the amino acid sequences were estimated, the base sequences and amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9 were obtained, respectively.

Example 16 Acquisition of deletion mutant: mite allergen gene (m
This Example and Example 17 were carried out for the purpose of analyzing the epitope of the transcript of the a10 clone).
That is, for ma10 clone, using cDNA inserted into pUEX2 using Exonuclease III (Takara Shuzo)
Deletion is performed from the downstream of the cDNA, and E. c.
A transformed strain of oli JM105 was obtained. The method followed the following protocol.

Protocol for preparation of deletion mutant plasmid DNA: pUEX2 (10 μ
g) was digested with Bam HI and Pst I, 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 m
After dissolving in 100 μl of a solution containing M MgCl ₂ and 10 mM mercaptoethanol, 270 units of exonuclease III was added, and 10 μl of the reaction mixture was collected at 120 or 200 seconds intervals while incubating at 25 ° C. Immediately cool it on ice, 100m
100 μl of 40 mM Na-acetate (pH 4.5) containing M NaCl, 2 mM ZnCl ₂ , 10% glycerol was added, incubated at 65 ° C. for 10 minutes, cooled to 37 ° C., and added with 5 units of Mung Bean nuclease. Incubated at 37 ° C for 60 minutes. It was then extracted with a phenol / chloroform solution and precipitated with ethanol. This is 0.1 mM EDT
A, 20 mM NaCl, 7 mM MgCl ₂ , 0.1
50 μl of 7 mM Tris-HC containing mMdNTPs
It was dissolved in 1 (pH 7.5). 2 units of this solution
The klenow fragment was added and incubated at 37 ° C for 30 minutes, followed by ethanol precipitation and 40 µl TE.
And ligated with T4 DNA ligase to prepare mutant plasmid DNA.

In FIG. 11, lane 1 shows the ma10 clone before the deletion, and lanes 2 to 12 show the deletion mutants. The larger the lane number, the shorter the base chain length of the deletion mutant. There is. The method for obtaining the deletion mutant strain in this example will be described in detail below.

Deletion mutant plasmid DNA was prepared according to the method described below using the plasmid vector into which the cDNA was inserted. This was used to transform E. coli JM105 to obtain a deletion mutant strain.

Example 17 Immunostaining of expression products of deletion mutants: Example 16
Among the deletion mutant strains obtained in 1., the ma10 clone was expressed in the same manner as in Example 9 (expression by the high expression vector pUEX2), subjected to SDS-PAGE, and then subjected to rabbit anti-tick body crude antigen. The results of immunostaining using serum and mite allergic patient pool serum are shown in FIG. 11.

The results of the immunostaining using rabbit anti-mite body crude antigen serum are shown in the column labeled "Anti-Dfb".
It was considered that the activity of the rabbit anti-mite worm crude antigen serum disappeared in two stages. In the figure, the activity is lane 1
The lanes that were almost the same as lanes were indicated by ++, the lanes having weak activity were indicated by +, and those in which the activity had disappeared were indicated by-. The results of immunostaining using pool serum of tick allergy patients
The column labeled "IgE" is shown. In the figure, the lane showing the activity is indicated by +, and the one in which the activity has disappeared is indicated by-.

From the results shown in FIG. 11, it is considered that at least a part of the epitope site is contained in the E1 and E2 portions, the activity is almost the same as that of lane 1, and the mutant strain having the minimum cDNA ( Lane 6), the primary sequence of E2, which is considered to contain at least a part of the epitope, by determining the nucleotide sequence of the cDNA of the mutant strain (lane 7) having no maximum activity for IgE and having the maximum cDNA chain length. Was determined (SEQ ID NO: 11). Similarly, although weak
By determining the nucleotide sequences of the cDNAs of the mutant strain having the minimum cDNA and having the activity against Anti-Dfb (lane 9) and the mutant strain having no activity and having the maximum cDNA chain length (lane 10). , The primary sequence of E1 which was thought to contain at least part of the epitope was determined (SEQ ID NO: 1
0).

Example 18 Human-derived 71 KD heat shock cognate protein (h
Homology between sc71) and recombinant mite allergen ma29 The same experiment as in Example 4 was carried out to obtain 273 clones that react with crude anti-mite body antigen antigen serum. The clones included 9 clones that reacted with the specific IgE antibody in the serum of mite allergic patients and the gene fragments of which did not hybridize with each other. As a result of homology search of the determined DNA base sequence and the deduced amino acid sequence, a gene showing homology to 70KD heat shock protein (hsp70) was found. This gene consisting of 752 bp can encode 145 amino acids (ma29, SEQ ID NO: 4), and its N-terminal residue has the highest homology (6).
Human derived 71KD heat shock cognate protein (hsc71) amino acid sequence (645%)
Corresponding to the 512th residue in (Fig. 12).

The E. coli gene of ma29. Although the binding of the patient serum-specific IgE antibody to the expression product in E. coli was inhibited by the rabbit anti-mite body crude antigen serum, this expression product did not cross-react with the so-called major antigens Der fI and Der fII. In addition, the IgE antibody specific to the gene product purified from patient serum specifically reacted with the ATP-binding 67KD component in the mite body extract. These results indicate that the indoor dust mite D. It strongly suggests that the hsp70 family derived from farinae may be one of the missed mite allergens.

Example 19 Purification of recombinant mite allergen Protein denatured in the presence of 2-mercaptoethanol was separated on 12.5% polyacrylamide and Coomassie
It was stained with Priliant Blue R-250. The result is shown in FIG. In the figure, the molecular weight is shown in kilodaltons on the left side, lane 1 is a molecular weight marker, and lane 2 is ma.
E. with 29 insert pGEX-2T. coli JM1
05 Transformant whole cell lysate (10 μl), lane 3
Shows a migration pattern of GST-ma29 (3 μg) purified by a glutathione-immobilized sepharose column, and Lane 4 shows a migration pattern of purified ma29 (3 μg), respectively.
This result indicates that ma29 was purified.

Example 20 IgE-Immunoblotting Df denatured in the absence of 2-mercaptoethanol.
b (20 μg / lane, panel A) and purified ma29 denatured in the presence of 2-mercaptoethanol (1 μg / lane).
Lanes, panels B and C) were separated on a 12.5% polyacrylamide gel and blotted to nitrocellulose film. To detect specific binding of ma29 etc. to human IgE on the blot, the blot was treated with biotin conjugated goat anti-human IgE antibody followed by peroxidase conjugated streptavidin.

The following were used as human sera. That is, in lanes 1 to 4, sera from individual patients with mite allergy are shown, and in lanes 5 and 6, Derfl or DerI
Pools of sera from 10 mite allergic patients sensitive (lane 5) or insensitive (lane 6) to fII were shown in lanes 7 and 8 as sera of allergic patients specific to cedar pollen and candida, respectively. High IgE concentrations (1,500 and 1,600 IU respectively)
/ Ml), non-atopic donor serum was used in lane 9, and serum-free was used in lane 10. In panel C, human serum was pretreated with Dfb (10 μg diluted with 100 μl serum) prior to incubation of the blot. The result is shown in FIG. The results show that in panel A only sera from mite allergic patients reacted, and in panel B it showed that patients reacted regardless of whether they were sensitive to Derfl or Derfl. Furthermore, it has shown that it reacted specifically with the serum of a mite allergic patient. The result of panel C is ma in Dfb
It is shown that those corresponding to 29 are included.

Example 21 Identification of native antigen bound to ma29-specific IgE: In FIG. 15, the molecular weight (in kilodaltons) is shown on the left. (A) Protein denatured in the absence of 2-mercaptoethanol was separated on a 12.5% polyacrylamide gel, and Coomassie Brilliant Bull-R-25
Stained with 0. (B) The protein blot was covered with an antibody specific for ma29 prepared from mite allergic serum. Then, specific binding between ma29 and IgE was detected by treatment with a biotin-conjugated goat anti-human IgE antibody and peroxidase-conjugated streptavidin.
In each panel, proteins were charged as follows.
Lane 1 contains the ATP-binding fraction from 10 mg of Dfb, lane 2 contains recombinant ma29 (2 μg), lane 3
Was charged with native DerfI (2 μg), and lane 4 was charged with native DerfII (2 μg). The results are shown in FIG. This result is ma
It shows that 29 native allergens are 67 KD proteins that bind to ATP.

Example 22 Fusion protein obtained by expressing as a fusion protein (GST
-Ma44) and purification of recombinant mite allergen ma44: SmaI of plasmid pGEX-2T (manufactured by Amersham).
The mite allergen ma44 gene was inserted into the site, and T-
4) Ligation with DNA ligase, and the resulting recombinant plasmid was transformed into E. coli JM105. This transformant was treated with streptomycin 30 μg / ml and ampicillin 10
6 at 37 ° C. in 2 × YT medium containing 0 μg / ml
The cells were cultured until the OD at 00 nm became 1.0. Then,
After adding IPTG to a final concentration of 1 mM, the cells were collected at 3 hours and washed once with PBS (pH 7.4).
The recollected cells were PBS containing 2 mM EDTA and 0.1% 2-mercaptoethanol (pH 7.
4) Ma which was dissolved by sonication in and fused with glutathione S-transferase (GST)
The insoluble fraction containing 44 was collected. Then SD for preparation
S-PAGE, 3.75 from 2 liters of culture
mg of fusion protein (GST-ma44) was obtained from the 67 KD band. The obtained fusion protein was treated with 150 mM N
Thrombin (1 μg / ml) in 50 mM Tris-HCl (pH 8.0) containing aCl and 2.5 mM CaCl _2.
500 μg fusion protein) and digested with ma44 to GST
To remove GST and then 50% (V
/ V) Glutathione-immobilized Sepharose column,
Further, ma44 was purified with a butyl-Sepharose column. The resulting purified ma44 showed a single band of 40KD on SDS-PAGE on a gel under reducing conditions.

Example 23 Intradermal reaction test with recombinant mite allergen ma44 The allergen activity of the recombinant mite allergen ma44 was assayed by an intradermal reaction test with respect to 9 mite allergic patients and 1 healthy subject. Before the intradermal reaction test, the amount of endotoxin contained in the recombinant mite allergen ma44 was measured using a Toxicolor System (LS-6 set, manufactured by Seikagaku Corporation), and the amount of endotoxin was 1 It was less than 0.0 EU / ml.

Example 24 Dot immunoblot test
test) 0.1, 0.01 of recombinant mite allergen ma44,
1 μl of the 0.001% solution was blotted on a nitrocellulose film (Hybond-C, Amersham) and the blocking solution (3% skim milk, 150 ml Na) was blotted.
Cl, 0.02% thimerosal, 0.05% Twee
10 mmol Tris-HCl buffer containing n20 [pH
7.5] solution) at room temperature for 1 hour. Then Ig
One of pool sera from patients with mite allergy for G staining
The 0-fold dilution was poured onto the sheets and incubated overnight in the cold.

This sheet was completely washed with TBS containing 0.05% Tween 20 (TBST), and then washed in a TBST solution containing a biotin-conjugated goat anti-human IgE antibody (manufactured by Zabindinsite) at room temperature for 1 hour. Reacted for hours. Then, it was immersed in a TBST solution containing streptavidin-peroxidase (manufactured by Tago) at room temperature for 1 hour. After washing thoroughly with TBST, the immunoreacted spots had 0.03% hydrogen peroxide and 0.03% hydrogen peroxide on the sheet.
Nickel chloride, 0.06% diaminobenzidine (DA
Detection was carried out by incubating with 50 mM Tris buffer (pH 7.6) containing B). After drying the sheet, Dfb, Derfl (dania allergen Derfl), using the colored spots as a control,
It was compared with Der fII (Dania allergen DerfII) and cedar (cedar pollen).

The results are shown in Table 1. In the table, +, ++ and ++ have a colored antigen concentration of 0.1%,
It shows 0.01% and 0.001%. As a result, it is shown to be a powerful allergen comparable to Der fI and Der fII.

[0098]

[Table 1]

[0099]

INDUSTRIAL APPLICABILITY The present invention has made it possible to provide a safe and effective recombinant mite allergen containing no anaphylaxis-inducing impurities as a therapeutic agent or diagnostic agent for mite allergic diseases.

[0100]

[Sequence Listing] SEQ ID NO: 1 Sequence Length: 558 Sequence Type: Nucleic Acid Number of Strands: Double Strands Topology: Linear Sequence Type: cDNA to mRNA Origin Organism Name: Dermatophagoides farinae Tissue Type: Parasite CTT GAA CAT CGA TCC ATC ACC GAT GAA GAA CGT ATG GAT GGT TTG GAA 48 Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu 1 5 10 15 AAT CAA CTT AAA GAA GCC CGT ATG ATG GCC GAA GAT GCT GAT AGA AAA 96 Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys 20 25 30 TAT GAT GAA GTT GCC CGT AAA TTG GCA ATG GTT GAA GCC GAT TTG GAA 144 Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu 35 40 45 CGT GCT GAA GAA CGT GCC GAA ACC GGT GAA TGC AAA ATT GTT GAA CTC 192 Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu 50 55 60 GAG GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA GTT 240 Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val 65 70 75 80 AGC GAA GAG AAA GCT CAA CAA CGT GAA GAA GCC TAT GAA CAA CAG ATC 288 Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile 85 90 95 CGT ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT 336 Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe 100 105 110 GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG GAA 384 Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu 115 120 125 GAC GAA TTG GTC CAC GAA AAG GAA AAA TAC AAA TCC ATC TCC GAC GAA 432 Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu 130 135 140 TTG GAC CAG ACA TTT GCC GGA ACT TAC TGG GTT ATT AAT AAT AAT TCT 480 Leu Asp Gln Thr Phe Ala Gly Thr Tyr Trp Val Ile Asn Asn Asn Asn Ser 145 150 155 160 TTC ATA TTA CGC AGA CAG AAT TTT CGA TTA TTT TTA ACA ACA CCA CCA 528 Phe Ile Leu Arg Arg Gln Asn Phe Arg Leu Phe Leu Thr Thr Pro Pro 165 170 175 CCA CCA GCA ACA GCA ACA ACG AAA AAT CAC 558 Pro Pro Ala Thr Ala Thr Thr Lys Asn His 180 185

SEQ ID NO: 2 Sequence length: 1047 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Insect body GTG ACC GCA TTG GAA TTA TTG TTG AAA GGT GAA ACT GAA GAC AAA ACT 48 Val Thr Ala Leu Glu Leu Leu Leu Lys Gly Glu Thr Glu Asp Lys Thr 1 5 10 15 CGT CGC TAT GTT GCC GAA TTG ACT GCC GTT GGA TCG CCA AGC AAC AAA 96 Arg Arg Tyr Val Ala Glu Leu Thr Ala Val Gly Ser Pro Ser Asn Lys 20 25 30 CAA GCT AAA GCA CAG ATC GAA GTG ACC AAA GGC GAA GAA TAC AAA ATC 144 Gln Ala Lys Ala Gln Ile Glu Val Thr Lys Gly Glu Glu Tyr Lys Ile 35 40 45 ACA TTG AAA TCA CCT GAA CAT GAA TTT AAC ACC GAA TTC ACC ATC CAT 192 Thr Leu Lys Ser Pro Glu His Glu Phe Asn Thr Glu Phe Thr Ile His 50 55 60 GCA GAC AAA AAC AAT TTG AAA ATG CAC ATG GAT TTC CCT AAC GTG TTC 240 Ala Asp Lys Asn Asn Leu Lys Met His Met Asp Phe Pro Asn Val Phe 65 70 75 80 CAA GCT GAT CTG ACT GGA ACT TTC CAA CAT GAC AAG G AA AAC AAT GTG 288 Gln Ala Asp Leu Thr Gly Thr Phe Gln His Asp Lys Glu Asn Asn Val 85 90 95 CGT AAA AAC CAA TTG AAT CTC CAA TAC AAA TTC GCT GGT GAT GAG AAA 336 Arg Lys Asn Gln Leu Asn Leu Gln Tyr Asn Leu Gln Tyr Lys Phe Ala Gly Asp Glu Lys 100 105 110 CCA CAC ACA GTG GAT TAT GAA AAC GAA TTT TCA TTC AAT TTG AAA CGA 384 Pro His Thr Val Asp Tyr Glu Asn Glu Phe Ser Phe Asn Leu Lys Arg 115 120 125 TCA TCA AAA GAC AAA AAC AGT GGG GTC GAT TAC CGA GCA AAA TAC ATG 432 Ser Ser Lys Asp Lys Asn Ser Gly Val Asp Tyr Arg Ala Lys Tyr Met 130 135 140 TCT AGC CAT TTT CCA ATC TTG AAC CAC AAA GTC AAC ATT CAA TTC AAA 480 Ser Ser His Phe Pro Ile Leu Asn His Lys Val Asn Ile Gln Phe Lys 145 150 155 160 TAT CGA CCA TTC AAA GTT AAC GAA TTG AAT TTG GAA GGA GAA TTT GGT 528 Tyr Arg Pro Phe Lys Val Asn Glu Leu Asn Leu Glu Gly Glu Phe Gly 165 170 175 CGA GAA CTT CAA CAC AAA TTC CAA TTG ATG CGA AAC AGC CAG ATA GAA 576 Arg Glu Leu Gln His Lys Phe Gln Leu Met Arg Asn Ser Gln Ile Glu 180 185 190 GTG GAA GAA GTG CGA CCA TTT AAG ATG CAT GGC AAT AGT GAC ATA AAA 624 Val Glu Glu Val Arg Pro Phe Lys Met His Gly Asn Ser Asp Ile Lys 195 200 205 TTG ATG GCA AAC GAT CTG GAT ATT GAT TAT GAT CTC AAA TCT GAA TTC 672 Leu Met Ala Asn Asp Leu Asp Ile Asp Tyr Asp Leu Lys Ser Glu Phe 210 215 220 AAA TAT GAA AGC AAC AAA GGA ACC CCC ATT GAA TTG CAA TAC AAA ATT 720 Lys Tyr Glu Ser Asn Lys Gly Thr Pro Ile Glu Leu Gln Tyr Lys Ile 225 230 235 240 TCC GGT AAA GAT CGC TCG AAA AGA GCT GCC GAT TTG GGA GCC GAA GAT 768 Ser Gly Lys Asp Arg Ser Lys Arg Ala Ala Asp Leu Gly Ala Glu Asp 245 250 255 GTT GAA GGC GTT ATC GAT TAC AAA AAC AAT GGT TCG CCA ATT GAT TCT 816 Val Glu Gly Val Ile Asp Tyr Lys Asn Asn Gly Ser Pro Ile Asp Ser 260 265 270 AAG ATG CAT GCT CAT CTC AAA ATG AAA GGC AAC AAC TAT GGC TAT GAT 864 Lys Met His Ala His Leu Lys Met Lys Gly Asn Asn Tyr Gly Tyr Asp 275 280 285 AGT GAA TTG AAA CAA ACA CAG CCA CAA CAA TAT GAA GGC AAA ATC ACA 912 Ser Glu Leu Lys Gln Thr Gln Pro Gln Gln Tyr Glu Gly Lys Ile Thr 290 295 300 TTG TCC AAG AAT GAC AAG AAA ATC TTC ATC AAC CAC AAA TCA GAG ATG 960 Leu Ser Lys Asn Asp Lys Lys Ile Phe Ile Asn His Lys Ser Glu Met 305 310 315 320 ACC AAA CCT ACC AAC ACT TTC CAT CTC AAA ACA GAT GCT GAT GTG TCG 1008 Thr Lys Pro Thr Asn Thr Phe His Leu Lys Thr Asp Ala Asp Val Ser 325 330 335 TAT TCG GAT TCG GAT ATG AAA AAA CAT TAT CAG ATG GAA 1047 Tyr Ser Asp Ser Asp Met Lys Lys His Tyr Gln Met Glu 340 345

SEQ ID NO: 3 Sequence length: 879 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Insect body GAA TGG TAT CAT TTA TTT AAA AGG TGG TTA CGA AAG CGC TTT ACC AAA 48 Glu Trp Tyr His Leu Phe Lys Arg Trp Leu Arg Lys Arg Phe Thr Lys 1 5 10 15 ATG ATT GAT GAA TTG AAG CCA TCA TTG TTG CAT GTT AAA GGC AAA AAA 96 Met Ile Asp Glu Leu Lys Pro Ser Leu Leu His Val Lys Gly Lys Lys 20 25 30 CGT CCA ATC GTT TAT GAA TGT GCT GAG ATT AGC TGG AAA GTT ATG AAT 144 Arg Pro Ile Val Tyr Glu Cys Ala Glu Ile Ser Trp Lys Val Met Asn 35 40 45 AAT GGT GAT GTA TTC ATT TTG CTT GTA CCG AAT TTT GTT TTC GTT TGG 192 Asn Gly Asp Val Phe Ile Leu Leu Val Pro Asn Phe Val Phe Val Trp 50 55 60 ACC GGT AAA CAT TCA AAT CGT ATG GAA CGT ACT ACT GCT ATT CGT GTG 240 Thr Gly Lys His Ser Asn Arg Met Glu Arg Thr Thr Ala Ile Arg Val 65 70 75 80 GCT AAT GAT TTG AAA TCT GAA TTG AAT CGT TTT AAA TTA TCA TCA ATC 288 Ala Asn Asp Leu Lys Ser Glu Leu Asn Arg Phe Lys Leu Ser Ser Val 85 90 95 ATC TTG GAA GAT GGT AAA GAA GTT GAA CAA ACA TCC GGT GCT GAA TAT 336 Ile Leu Glu Asp Gly Lys Glu Val Glu Gln Thr Ser Gly Ala Glu Tyr 100 105 110 GAT GCA TTC AAT AAA GCA TTA TCA TTG GAT AAA AAA GAT ATT GAT CTT 384 Asp Ala Phe Asn Lys Ala Leu Ser Leu Asp Lys Lys Asp Ile Asp Leu 115 120 125 AAA CAA ATG CCA AAA GGC TAT GAT TAT GCT GCC AGT GAT AAA TCT TTT 432 Lys Gln Met Pro Lys Gly Tyr Asp Tyr Ala Ala Ser Asp Lys Ser Phe 130 135 140 GAA TCA CAT GAA CGT TCA TTC GTT ACA TTG TAT AAA TGT TTC GAA GGT 480 Glu Ser His Glu Arg Ser Phe Val Thr Leu Tyr Lys Cys Phe Glu Gly 145 150 155 160 ACT GAA ACG ATT GAT ATT TCG TTT GTC AAA AAT GGA CCG TTA AGC CGT 528 Thr Glu Thr Ile Asp Ile Ser Phe Val Lys Asn Gly Pro Leu Ser Arg 165 170 175 GCT GAT CTC GAT ACA AAT GAT ACA TTC ATT GTT GAA AAT GGT TCC GAA 576 Ala Asp Leu Asp Thr Asn Asp Thr Phe Ile Val Glu Asn Gly Ser Glu 180 185 190 GGT TTA TGG GTA TGG GTT GGT AAG AAA GCC AC A CAA AAA GAA CGA CAA 624 Gly Leu Trp Val Trp Val Gly Lys Lys Ala Thr Gln Lys Glu Arg Gln 195 200 205 TCG GCC ATT AAA TAT GCA ATG GAA TTG ATT AAT AAG AAA AAA TAT CCA 672 Ser Ala Ile Lys Tyr Ala Met Glu Leu Ile Asn Lys Lys Lys Tyr Pro 210 215 220 AAT AAT ACA CCG GTA ACC AAA GTA TTG GAA GGT GAT GAA AGT GTT GAA 720 Asn Asn Thr Pro Val Thr Lys Val Leu Glu Gly Asp Glu Ser Val Glu 225 230 235 240 TTC AAA TCA TTA TTT GAA TCA TGG CAA ATG AGC GAA CAG GAA AAA ATT 768 Phe Lys Ser Leu Phe Glu Ser Trp Gln Met Ser Glu Gln Glu Lys Ile 245 250 255 ACC AGT GCA CGA TTG TTT CGT GTT TCA CGA AAT GGT ATC TTT AAA CAG 816 Thr Ser Ala Arg Leu Phe Arg Val Ser Arg Asn Gly Ile Phe Lys Gln 260 265 270 GTT GCC AAT TAT GAA CCA GAT GAT TTG GAA GAG GAT ATA TAT CAT GAT 864 Val Ala Asn Tyr Glu Pro Asp Asp Leu Glu Glu Asp Ile Tyr His Asp 275 280 285 TTT GGA TGT TAT GGA 879 Phe Gly Cys Tyr Gly 290

SEQ ID NO: 4 Sequence length: 435 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Parasite AAA GAC GAT ATT GAA CGT ATG GTC AAA GAG GCT GAA TCA TAT AAA GAA 48 Lys Asp Asp Ile Glu Arg Met Val Lys Glu Ala Glu Ser Tyr Lys Glu 1 5 10 15 GAA GAT GAC AAA CAA CGT GAC CGT ATT GCA GCC AAA AAT TCA TTG GAA 96 Glu Asp Asp Lys Gln Arg Asp Arg Ile Ala Ala Lys Asn Ser Leu Glu 20 25 30 GGT TAT GCT TTC CAG ATG AAA GCC ACT TTG GAT GAA GAA GCA ATT AAA 144 Gly Tyr Ala Phe Gln Met Lys Ala Thr Leu Asp Glu Glu Ala Ile Lys 35 40 45 TCT AAA GTT TCT GAA GAA GAT CGT AAA AAA ATC CTG GAC AAA GTT GAT 192 Ser Lys Val Ser Glu Glu Asp Arg Lys Lys Ile Leu Asp Lys Val Asp 50 55 60 GAA GTG CTC AAA TGG TTG GAC GCT AAT GCT TTA GCA GAA AAA GAT GAA 240 Glu Val Leu Lys Trp Leu Asp Ala Asn Ala Leu Ala Glu Lys Asp Glu 65 70 75 80 TTT GAA CAT CAA CGT AAA GAA TTG GAA AGT GTA TGC AAT CCG ATC ATT 288 Phe Glu His Gln Arg Lys Glu Leu Glu Ser Val Cys Asn Pro Ile Ile 85 90 95 ACT AAA TTA TAT CAA CAA GCT GGT GGT GCA GGC GCT GGG GGT ATG CCA 336 Thr Lys Leu Tyr Gln Gln Ala Gly Gly Ala Gly Ala Gly Gly Met Pro 100 105 110 GGT GGA TTT CCA GGT GGT TTT CCC GGT ACT GAT GGT TCT GGT GGT GGT 384 Gly Gly Phe Pro Gly Gly Phe Pro Gly Thr Asp Gly Ser Gly Gly Gly 115 120 125 GCA GCC GGT GGT GAT GGT GGT AAA TCT GGG CCC ACC ATT GAA GAA GTA 432 Ala Ala Gly Gly Asp Gly Gly Lys Ser Gly Pro Thr Ile Glu Glu Val 130 135 140 GAT 435 Asp

SEQ ID NO: 5 Sequence length: 897 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Parasite TTT TTT TTC GTT GCA GCT AAA CAA CAA CAA CAA CCA TCA ACA AAA ATG 48 Phe Phe Phe Val Ala Ala Lys Gln Gln Gln Gln Pro Ser Thr Lys Met 1 5 10 15 GAG GCC ATC AAG AAA AAA ATG CAG GCA ATG AAG CTC GAG AAA GAT AAT 96 Glu Ala Ile Lys Lys Lys Met Gln Ala Met Lys Leu Glu Lys Asp Asn 20 25 30 GCT ATC GAT CGA GCT GAA ATT GCC GAA CAA AAA GCC CGT GAT GCT AAT 144 Ala Ile Asp Arg Ala Glu Ile Ala Glu Gln Lys Ala Arg Asp Ala Asn 35 40 45 CTA CGT GCC GAA AAG TCT GAG GAA GAA GTT CGT GCA TTA CAG AAA AAA 192 Leu Arg Ala Glu Lys Ser Glu Glu Glu Val Arg Ala Leu Gln Lys Lys 50 55 60 ATC CAA CAA ATT GAA AAT GAA TTG GAT CAG GTC CAA GAA CAA TTA TCG 240 Ile Gln Gln Ile Glu Asn Glu Leu Asp Gln Val Gln Glu Gln Leu Ser 65 70 75 80 GCT GCC AAT ACA AAA TTG GAG GAA AAG GAA AAA GCC CTA CAG ACC GCT 288 Ala Ala Asn Thr Lys Leu Glu Glu Lys Glu Lys Ala Leu Gln Thr Ala 85 90 95 GAA GGT GAT GTT GCA GCA TTG AAT CGT CGT ATT CAA TTG ATT GAA GAA 336 Glu Gly Asp Val Ala Ala Leu Asn Arg Arg Ile Gln Leu Ile Glu Glu 100 105 110 GAT TTG GAA CGA TCA GAA GAA CGA CTT AAG ATT GCT ACA GCC AAA TTG 384 Asp Leu Glu Arg Ser Glu Glu Arg Leu Lys Ile Ala Thr Ala Lys Leu 115 120 125 GAA GAG GCA TCA CAA TCT GCC GAT GAA TCT GAA CGT ATG CGT AAA ATG 432 Glu Glu Ala Ser Gln Ser Ala Asp Glu Ser Glu Arg Met Arg Lys Met 130 135 140 CTT GAA CAT CGA TCC ATC ACC GAT GAA GAA CGT ATG GAT GGT TTG GAA 480 Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu 145 150 155 160 AAT CAA CTT AAA GAA GCC CGT ATG ATG GCC GAA GAT GCT GAT AGA AAA 528 Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys 165 170 175 TAT GAT GAA GTT GCC CGT AAA TTG GCA ATG GTT GAA GCC GAT TTG GAA 576 Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu 180 185 190 CGT GCT GAA GAA CGT GCC GAA ACC GGT GAA TCG AAA ATT GTT GAA CTC 624 Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu 195 200 205 GAA GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA GTT 672 Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val 210 215 220 AGC GAA GAG AAA GCT CAA CAA CGT GAA GAA GCC TAT GAA CAA CAG ATC 720 Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile 225 230 235 240 CGT ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT 768 Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe 245 250 255 GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG GAA 816 Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu 260 265 270 GAC GAA TTG GTC CAC GAA AAG GAA AAA TAC AAA TCC ATC TCC GAC GAA 864 Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu 275 280 285 TTG GAC CAG ACA TTT GCC GAA CTT ACT GGT TAT 897 Leu Asp Gln Thr Phe Ala Glu Leu Thr Gly Tyr 290 295

SEQ ID NO: 6 Sequence length: 762 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Parasite TTT CTT ACA AAC GAT AAA CGA TAC AAT TTT TTT TTT GTA ACA ATG AAA 48 Phe Leu Thr Asn Asp Lys Arg Tyr Asn Phe Phe Phe Val Thr Met Lys 1 5 10 15 TTC TTA ATC TAT TCA TTG GCA TTT GCC GCT ATG GCC GGT TTG GTT TTC 96 Phe Leu Ile Tyr Ser Leu Ala Phe Ala Ala Met Ala Gly Leu Val Phe 20 25 30 GGT TCC GAA AAA CGA AAA AAG AAT CCA TAT GAT GTT GCC GAA TCA TTG 144 Gly Ser Glu Lys Arg Lys Lys Asn Pro Tyr Asp Val Ala Glu Ser Leu 35 40 45 ATT AAA CAT GCT GAT GCA TTG AAA GAA TAT GCC GAA CAG GAG ATA CAA 192 Ile Lys His Ala Asp Ala Leu Lys Glu Tyr Ala Glu Gln Glu Ile Gln 50 55 60 AAA TAT CAG AAT AAT TCA CAT TAT CAT TTG GCC ATC ATA CGT TTG GAA 240 Lys Tyr Gln Asn Asn Ser His Tyr His Leu Ala Ile Ile Arg Leu Glu 65 70 75 80 CAT GAA ATT CAT CGT GTT GAA GCC ATT GCC GAT CAA CTT CGT AAT ATG 288 His Glu Ile His Arg Val Glu Ala Ile Ala Asp Gln Leu Arg Asn Met 85 90 95 ACT AAA ATG GAA AAT ATG ACC GAA CAT TAT ATG CAT CAT TTC CAT CAA 336 Thr Lys Met Glu Asn Met Thr Glu His Tyr Met His His Phe His Gln 100 105 110 ATT GAA ATG GAA CTA TTA ATT TTT GAA AAT ATT ATT GAA GAA GAA TTA 384 Ile Glu Met Glu Leu Leu Ile Phe Glu Asn Ile Ile Glu Glu Glu Leu 115 120 125 TAT TAT TTA GAA CAT GGT CAC CAC CAT CAT GAT CAT CAT GAT CAT GAT 432 Tyr Tyr Leu Glu His Gly His His His His Asp His His Asp His Asp 130 135 140 CAT CAT GAT CAT ATG CAT CGT CAT GAT CAT GAT GAT TCA GAC ATT ATC 480 His His Asp His Met His Arg His Asp His Asp Asp Ser Asp Ile Ile 145 150 155 160 GCC GGT GAT GAT TAT AAA GAT GCT TTG TTG AAA AAA GCA CAA CAA CTT 528 Ala Gly Asp Asp Tyr Lys Asp Ala Leu Leu Lys Lys Ala Gln Gln Leu 165 170 175 ATC GAT GAA GCT GAA GAT GCA ATA AAA AAA TAT GGA CAA GGT AAA GAT 576 Ile Asp Glu Ala Glu Asp Ala Ile Lys Lys Tyr Gly Gln Gly Lys Asp 180 185 190 ATC GAT GTT CAG GTG ATA AAA TCC GAA GTT GTT ATA ATA AAA AAC TTG 624 Ile Asp Val Gln Val Ile Lys Ser Glu Val Val Ile Ile Lys Asn Leu 195 200 205 GTA AAA CAA ATC AAT GAA GCT AAT AAT CCA CAA GCA TTG GCC GTA TTG 672 Val Lys Gln Ile Asn Glu Ala Asn Asn Pro Gln Ala Leu Ala Val Leu 210 215 220 GAA TCA AAT CTA GCT CAA CAT GAG GAA ATT ATT CGT AAA TGG CTT GAT 720 Glu Ser Asn Leu Ala Gln His Glu Glu Ile Ile Arg Lys Trp Leu Asp 225 230 235 240 GAT ATT GAA CCA CAC ACT ACA CAT GCC CCA GTT ACT ATT GCT 762 Asp Ile Glu Pro His Thr Thr His Ala Pro Val Thr Ile Ala 245 250

SEQ ID NO: 7 Sequence length: 1080 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Parasite TTA CAA CCC AAC GGA CCA AAG GGT TTG CGT TCT CCT GGA AGC CCT GAC 48 Leu Gln Pro Asn Gly Pro Lys Gly Leu Arg Ser Pro Gly Ser Pro Asp 1 5 10 15 AGC GGT AAT CTG CGT AAA CGT AAG AGA AGA GGA GGA GTT TTG GGT TTC 96 Ser Gly Asn Leu Arg Lys Arg Lys Arg Arg Gly Gly Val Leu Gly Phe 20 25 30 AAG GAA TTG GCA GCG AAG TCT AAG TTA TCT GCC TGG CAT TTT CAT CGT 144 Lys Glu Leu Ala Ala Lys Ser Lys Leu Ser Ala Trp His Phe His Arg 35 40 45 GTC TTT AAG TCA GTG ACT GGA TTG ACT CCT AAG ACA TAT GGA GAT AAA 192 Val Phe Lys Ser Val Thr Gly Leu Thr Pro Lys Thr Tyr Gly Asp Lys 50 55 60 TGT TGG GAA TAC ATT AAA CAT TGC AGA GAA AAT GGG GAG AGC TTC TTT 240 Cys Trp Glu Tyr Ile Lys His Cys Arg Glu Asn Gly Glu Ser Phe Phe 65 70 75 80 TTC GAG GGT AAG CAA GCT AAC ACA GAC TCC GGA GAG G TC TCT CTT AGC 288 Phe Glu Gly Lys Gln Ala Asn Thr Asp Ser Gly Glu Val Ser Leu Ser 85 90 95 CCA CCA AAT ATG TGC TAT CAG ACA CAG ACA CCT ATT AGC TCT AAT ACT 336 Pro Pro Asn Met Cys Tyr Gln Thr Gln Thr Pro Ile Ser Ser Asn Thr 100 105 110 GAT TCA CAT AAC TAT TTT GAT GAG TCG GAC GAT GAA AAA GTA AAG AAG 384 Asp Ser His Asn Tyr Phe Asp Glu Ser Asp Asp Glu Lys Val Lys Lys 115 120 125 GTT AAA TTG TCA CCA GAG GTG GAG CGG GAC ATT TCC ACC AAA GAT AGC 432 Val Lys Leu Ser Pro Glu Val Glu Arg Asp Ile Ser Thr Lys Asp Ser 130 135 140 TTC AAT GGC GAG TCA TCT ACT CCC GGT GAT GAT TGT TTT CAC CCT GAC 480 Phe Asn Gly Glu Ser Ser Thr Pro Gly Asp Asp Cys Phe His Pro Asp 145 150 155 160 GAA AAT GGA CTT GAT AAC AAT TTG AAG AAC ATT GGG CTA GGG CTG TCG 528 Glu Asn Gly Leu Asp Asn Asn Leu Lys Asn Ile Gly Leu Gly Leu Ser 165 170 175 TTC GCT ACT AAG GAT CTT GGC ATA AAT CCT AGT AGT GTT GAA GAT GGT 576 Phe Ala Thr Lys Asp Leu Gly Ile Asn Pro Ser Ser Val Glu Asp Gly 180 185 190 TTG GTA CAG CCT ACA TGT TCT TTT GAC TTT AAT TTA AAT GAT GCT ACG 624 Leu Val Gln Pro Thr Cys Ser Phe Asp Phe Asn Leu Asn Asp Ala Thr 195 200 205 ATA CCC AAT GTT GAC GAT TTC GAC GAT TTT TCA AAA GTT CCT CAA TTT 672 Ile Pro Asn Val Asp Asp Phe Asp Asp Phe Ser Lys Val Pro Gln Phe 210 215 220 TTA AAT GAT GAC TCT GCT GGG TTT GTG AGA GCG TTT TCT GCT CCT GAT 720 Leu Asn Asp Asp Ser Ala Gly Phe Val Arg Ala Phe Ser Ala Pro Asp 225 230 235 240 TTG ACC AAG TAC AAC CAT ATG AGT GCA TTA AGT CCT GAA AGT AAT GGC 768 Leu Thr Lys Tyr Asn His Met Ser Ala Leu Ser Pro Glu Ser Asn Gly 245 250 255 AAC ACT GAC ATG AAC AAG CCC AAG GAT GCA TTA GGT TTC GAT TCC TAC 816 Asn Thr Asp Met Asn Lys Pro Lys Asp Ala Leu Gly Phe Asp Ser Tyr 260 265 270 CTT GAT ACA GAC ATT AGC AAC CAG GAA ACT AAT GGG AAT GCT CTT TAT 864 Leu Asp Thr Asp Ile Ser Asn Gln Glu Thr Asn Gly Asn Ala Leu Tyr 275 280 285 CCA GTT GTG GAT GAT GTT CCA TCA TTG TCT GGG ACA GGT TTT ATG CCA 912 Pro Val Val Asp Asp Val Pro Ser Leu Ser Gly Thr Gly Phe Met Pro 290 295 300 AAC ACT TCA ATT CAA AAT ACG TCT TTT TTA AGA CCA TTT GAA GAT AGC 960 Asn Thr Ser Ile Gln Asn Thr Ser Phe Leu Arg Pro Phe Glu Asp Ser 305 310 315 320 TTG GAT ATA CAA GAT ACT GCT GAA CTT TCA AAG TTT GGC TAC GAT GTT 1008 Leu Asp Ile Gln Asp Thr Ala Glu Leu Ser Lys Phe Gly Tyr Asp Val 325 330 335 GAT TCT CTC GCG CCT TCT ACA CCC GGA TTA GAT ATG AAG CTT AAT TCG 1056 Asp Ser Leu Ala Pro Ser Thr Pro Gly Leu Asp Met Lys Leu Asn Ser 340 345 350 AGG TTT AGT GTT GCG CAA CCT GAT 1080 Arg Phe Ser Val Ala Gln Pro Asp 355 360

SEQ ID NO: 8 Sequence length: 447 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Parasite TCA ACA AGA TGG CTT ACA CAT GAA TCG GAC GAA GAA CGT TGG GAG CAA 48 Ser Thr Arg Trp Leu Thr His Glu Ser Asp Glu Glu Arg Trp Glu Gln 1 5 10 15 TAT AGA CTC AAA CAA AGA ATG CCA GAG AAT GAT TTT GGT CCG ATC ATT 96 Tyr Arg Leu Lys Gln Arg Met Pro Glu Asn Asp Phe Gly Pro Ile Ile 20 25 30 ACG GGT CAT CAT GAT GAT GAT GAT GAT GAT GAT GTC GAT ACA CCG AAT 144 Thr Gly His His Asp Asp Asp Asp Asp Asp Asp Val Asp Thr Pro Asn 35 40 45 GAA GAA GAG AAT CGT GAT AAA TTC ATC ACA ATC ACC AAC ACT GAT TCA 192 Glu Glu Glu Asn Arg Asp Lys Phe Ile Thr Ile Thr Asn Thr Asp Ser 50 55 60 TTC AGA CGA CGT ACT AAT TAC AGC AGC AGC AGC AAC AGC AAC AAT AAT 240 Phe Arg Arg Arg Thr Asn Tyr Ser Ser Ser Ser Asn Ser Asn Asn Asn 65 70 75 80 AAT AAC AAC AAT GGC AAG GAT TAT GGC CAC CAG CAT CAG CAT CAC CAT 288 Asn Asn Asn Asn Gly Lys Asp Tyr Gly His Gln His Gln His His 85 90 95 CCC CGC CAT CAT CAT CAT CAC AAT CCG AAT AAA TCG AAT GGC AAT GGA 336 Pro Arg His His His His Asn Pro Asn Lys Ser Asn Gly Asn Gly 100 105 110 CAT GCA TTT AAC CAC AAT AAT GCC ATC ATT ATC AAC AAC AAT AAT AAT 384 His Ala Phe Asn His Asn Asn Ala Ile Ile Ile Asn Asn Asn Asn Asn 115 120 125 GAT GAT TCG ACA GCC AAT TTG GAA CAG ATT TAT AAC AGC AAA AGT AAC 432 Asp Asp Ser Thr Ala Asn Leu Glu Gln Ile Tyr Asn Ser Lys Ser Asn 130 135 140 ACG ACC AGC CGT TTG 447 Thr Thr Ser Arg Leu 145

SEQ ID NO: 9 Sequence length: 990 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Parasite TTC ATC AAA ATT AAA AAT TCA TCA AAA ATG AAA TTC GTT TTG GCC ATT 48 Phe Ile Lys Ile Lys Asn Ser Ser Lys Met Lys Phe Val Leu Ala Ile 1 5 10 15 GCC TCT TTG TTG GTA TTG AGC ACT GTT TAT GCT CGT CCA GCT TCA ATC 96 Ala Ser Leu Leu Val Leu Ser Thr Val Tyr Ala Arg Pro Ala Ser Ile 20 25 30 AAA ACT TTT GAA GAA TTC AAA AAA GCC TTC AAC AAA AAC TAT GCC ACC 144 Lys Thr Phe Glu Glu Phe Lys Lys Ala Phe Asn Lys Asn Tyr Ala Thr 35 40 45 GTT GAA GAG GAA GAA GTT GCC CGT AAA AAC TTT TTG GAA TCA TTG AAA 192 Val Glu Glu Glu Glu Val Ala Arg Lys Asn Phe Leu Glu Ser Leu Lys 50 55 60 TAT GTT GAA GCT AAC AAA GGT GCC ATC AAC CAT TTG TCC GAT TTG TCA 240 Tyr Val Glu Ala Asn Lys Gly Ala Ile Asn His Leu Ser Asp Leu Ser 65 70 75 80 TTG GAT GAA TTC AAA AAC CGT TAT TTG ATG AGT GCT GAA GCT TTT GAA 288 Leu Asp Glu Phe Lys Asn Arg Tyr Leu Met Ser Ala Glu Ala Phe Glu 85 90 95 CAA CTC AAA ACT CAA TTC GAT TTG AAT GCC GAA ACA AGC GCT TGC CGT 336 Gln Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Ser Ala Cys Arg 100 105 110 ATC AAT TCG GTT AAC GTT CCA TCG GAA TTG GAT TTA CGA TCA CTG CGA 384 Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg 115 120 125 ACT GTC ACT CCA ATC CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT 432 Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala 130 135 140 TTC TCT GGT GTC GCC GCA ACT GAA TCA GCT TAT TTG GCC TAC CGT AAC 480 Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn 145 150 155 160 ACG TCT TTG GAT CTT TCT GAA CAG GAA CTC GTC GAT TGC GCA TCT CAA 528 Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Cys Ala Ser Gln 165 170 175 CAC GGA TGT CAC GGC GAT ACA ATA CCA AGA GGC ATC GAA TAC ATC CAA 576 His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln 180 185 190 CAA AAT GGT GTC GTT GAA GAA AGA AGC TAT CCA TAC GTT GCA CGA GAA 624 Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu 195 200 205 CAA CAA TGC CGA CGA CCA AAT TCG CAA CAT TAC GGT ATC TCA AAC TAC 672 Gln Gln Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr 210 215 220 TGC CAA ATT TAT CCA CCA GAT GTG AAA CAA ATC CGT GAA GCT TTG ACT 720 Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr 225 230 235 240 CAA ACA CAC ACA GCT ATT GCC GTC ATT ATT GGC ATT AAA GAT TTG AGA 768 Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg 245 250 255 GCT TTC CAA CAT TAT GAT GGA CGA ACA ATC ATT CAA CAT GAC AAT GGT 816 Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly 260 265 270 TAT CAA CCA AAC TAT CAT GCC GTC AAC ATT GTC GGT TAC GGA AGT ACA 864 Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr 275 280 285 CAA GGC GTC GAT TAT TGG ATC GTA CGA AAC AGT TGG GAT ACT ACC TGG 912 Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp 290 295 300 GGT GAT AGC GGA TAC GGA TAT TT C CAA GCC GGA AAC AAC CTC ATG ATG 960 Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Met 305 310 315 320 ATC GAA CAA TAT CCA TAT GTT GTA ATC ATG 990 Ile Glu Gln Tyr Pro Tyr Val Val Ile Met 325 330

SEQ ID NO: 10 Sequence length: 45 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Insect body GAG GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA 45 Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu 1 5 10 15

SEQ ID NO: 11 Sequence length: 90 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Dermatophagoides farinae Tissue type: Parasite ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT GCT 48 Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala 1 5 10 15 GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG 90 Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu 20 25

[Brief description of drawings]

FIG. 1 shows the construction of recombinant plasmid pAEX.

FIG. 2 shows the results of immunostaining by Western blot using anti-β-galactosidase antibody after subjecting the mite allergen fusion protein expressed in Example 11 to SDS-PAGE. In the figure, lanes ma10 to ma115 are for immunostaining of the fusion protein, and lane C is for a molecular weight marker (β-
Galactosidase: about 116,000).

FIG. 3 shows the results of performing immunostaining by Western blotting for each clone (ma10 clone to ma115 clone) in Example 12, using a rabbit anti-mite body crude antigen serum.

FIG. 4 is a view showing a restriction enzyme map of cDNA encoding a mite allergen (ma3 clone).

FIG. 5 is a diagram showing a restriction enzyme map of cDNA encoding a mite allergen (ma15 clone).

FIG. 6 is a view showing a restriction enzyme map of cDNA encoding a mite allergen (ma29 clone).

FIG. 7 is a diagram showing a restriction enzyme map of cDNA encoding mite allergen (ma50 clone).

FIG. 8 shows a restriction map of cDNA encoding mite allergen (ma113 clone).

FIG. 9 is a diagram showing a restriction enzyme map of cDNA encoding a mite allergen (ma114 clone).

FIG. 10 is a view showing a restriction enzyme map of cDNA encoding a mite allergen (ma115 clone).

[Fig. 11] Fig. 11 is a diagram showing the presence or absence of antigen activity related to the deletion mutant of the ma10 clone in Example 17.

FIG. 12: Mite allergen ma29 and human-derived 71KD
It is a figure which shows the homology in the amino acid sequence of heat shock cognate protein (hsc71).

FIG. 13 The mite allergens ma29 and GST-ma
It is a figure which shows the electrophoresis pattern of 29.

FIG. 14 shows specific binding of mite allergen ma29 to human IgE.

FIG. 15 shows the identification of native antigens that bind IgE specific for the mite allergen ma29.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // C12P 21/02 C 9282-4B C07K 99:00

Claims (62)

[Claims] 1. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 1) obtained by expression of a gene derived from a mite body. Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu Leu Asp Gln Thr Phe Ala Gly Thr Tyr Trp Val Ile Asn Asn Asn Ser Phe Ile Leu Arg Arg Gln Asn Phe Arg Leu Phe Leu Thr Thr Pro Pro Pro Pro Ala Thr Ala Thr Thr Lys Asn His 2. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 2) obtained by expressing a gene derived from a mite body. Val Thr Ala Leu Glu Leu Leu Leu Lys Gly Glu Thr Glu Asp Lys Thr Arg Arg Tyr Val Ala Glu Leu Thr Ala Val Gly Ser Pro Ser Asn Lys Gln Ala Lys Ala Gln Ile Glu Val Thr Lys Gly Glu Glu Tyr Lys Ile Thr Leu Lys Ser Pro Glu His Glu Phe Asn Thr Glu Phe Thr Ile His Ala Asp Lys Asn Asn Leu Lys Met His Met Asp Phe Pro Asn Val Phe Gln Ala Asp Leu Thr Gly Thr Phe Gln His Asp Lys Glu Asn Asn Val Arg Lys Asn Gln Leu Asn Leu Gln Tyr Lys Phe Ala Gly Asp Glu Lys Pro His Thr Val Asp Tyr Glu Asn Glu Phe Ser Phe Asn Leu Lys Arg Ser Ser Lys Asp Lys Asn Ser Gly Val Asp Tyr Arg Ala Lys Tyr Met Ser Ser His Phe Pro Ile Leu Asn His Lys Val Asn Ile Gln Phe Lys Tyr Arg Pro Phe Lys Val Asn Glu Leu Asn Leu Glu Gly Glu Phe Gly Arg Glu Leu Gln His Lys Phe Gln Leu Met Arg Asn Ser Gln Ile Glu Val Glu Glu Val Arg Pro Phe Lys Met His Gly Asn Ser Asp Ile Lys Leu Met Ala Asn Asp Leu Asp Ile Asp Tyr Asp Leu Lys Ser Glu Phe Lys Tyr Glu Ser Asn Lys Gly Thr Pro Ile Glu Leu Gln Tyr Lys Ile Ser Gly Lys Asp Arg Ser Lys Arg Ala Ala Asp Leu Gly Ala Glu Asp Val Glu Gly Val Ile Asp Tyr Lys Asn Asn Gly Ser Pro Ile Asp Ser Lys Met His Ala His Leu Lys Met Lys Gly Asn Asn Tyr Gly Tyr Asp Ser Glu Leu Lys Gln Thr Gln Pro Gln Gln Tyr Glu Gly Lys Ile Thr Leu Ser Lys Asn Asp Lys Lys Ile Phe Ile Asn His Lys Ser Glu Met Thr Lys Pro Thr Asn Thr Phe His Leu Lys Thr Asp Ala Asp Val Ser Tyr Ser Asp Ser Asp Met Lys Lys His Tyr Gln Met Glu 3. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 3) obtained by expressing a gene derived from a mite body. Glu Trp Tyr His Leu Phe Lys Arg Trp Leu Arg Lys Arg Phe Thr Lys Met Ile Asp Glu Leu Lys Pro Ser Leu Leu His Val Lys Gly Lys Lys Arg Pro Ile Val Tyr Glu Cys Ala Glu Ile Ser Trp Lys Val Met Asn Asn Gly Asp Val Phe Ile Leu Leu Val Pro Asn Phe Val Phe Val Trp Thr Gly Lys His Ser Asn Arg Met Glu Arg Thr Thr Ala Ile Arg Val Ala Asn Asp Leu Lys Ser Glu Leu Asn Arg Phe Lys Leu Ser Ser Val Ile Leu Glu Asp Gly Lys Glu Val Glu Gln Thr Ser Gly Ala Glu Tyr Asp Ala Phe Asn Lys Ala Leu Ser Leu Asp Lys Lys Asp Ile Asp Leu Lys Gln Met Pro Lys Gly Tyr Asp Tyr Ala Ala Ser Asp Lys Ser Phe Glu Ser His Glu Arg Ser Phe Val Thr Leu Tyr Lys Cys Phe Glu Gly Thr Glu Thr Ile Asp Ile Ser Phe Val Lys Asn Gly Pro Leu Ser Arg Ala Asp Leu Asp Thr Asn Asp Thr Phe Ile Val Glu Asn Gly Ser Glu Gly Leu Trp Val Trp Val Gly Lys Lys Ala Thr Gln Lys Glu Arg Gln Ser Ala Ile Lys Tyr Ala Met Glu Leu Ile Asn Lys Lys Lys Tyr Pro Asn Asn Thr Pro Val Thr Lys Val Leu Glu Gly Asp Glu Ser Val Glu Phe Lys Ser Leu Phe Glu Ser Trp Gln Met Ser Glu Gln Glu Lys Ile Thr Ser Ala Arg Leu Phe Arg Val Ser Arg Asn Gly Ile Phe Lys Gln Val Ala Asn Tyr Glu Pro Asp Asp Leu Glu Glu Asp Ile Tyr His Asp Phe Gly Cys Tyr Gly 4. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 4) obtained by expressing a gene derived from a mite body. Lys Asp Asp Ile Glu Arg Met Val Lys Glu Ala Glu Ser Tyr Lys Glu Glu Asp Asp Lys Gln Arg Asp Arg Ile Ala Ala Lys Asn Ser Leu Glu Gly Tyr Ala Phe Gln Met Lys Ala Thr Leu Asp Glu Glu Ala Ile Lys Ser Lys Val Ser Glu Glu Asp Arg Lys Lys Ile Leu Asp Lys Val Asp Glu Val Leu Lys Trp Leu Asp Ala Asn Ala Leu Ala Glu Lys Asp Glu Phe Glu His Gln Arg Lys Glu Leu Glu Ser Val Cys Asn Pro Ile Ile Thr Lys Leu Tyr Gln Gln Ala Gly Gly Ala Gly Ala Gly Gly Met Pro Gly Gly Phe Pro Gly Gly Phe Pro Gly Thr Asp Gly Ser Gly Gly Gly Ala Ala Gly Gly Asp Gly Gly Lys Ser Gly Pro Thr Ile Glu Glu Val Asp 5. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 5) obtained by expressing a gene derived from a mite body. Phe Phe Phe Val Ala Ala Lys Gln Gln Gln Gln Pro Ser Thr Lys Met Glu Ala Ile Lys Lys Lys Met Gln Ala Met Lys Leu Glu Lys Asp Asn Ala Ile Asp Arg Ala Glu Ile Ala Glu Gln Lys Ala Arg Asp Ala Asn Leu Arg Ala Glu Lys Ser Glu Glu Glu Val Arg Ala Leu Gln Lys Lys Ile Gln Gln Ile Glu Asn Glu Leu Asp Gln Val Gln Glu Gln Leu Ser Ala Ala Asn Thr Lys Leu Glu Glu Lys Glu Lys Ala Leu Gln Thr Ala Glu Gly Asp Val Ala Ala Leu Asn Arg Arg Ile Gln Leu Ile Glu Glu Asp Leu Glu Arg Ser Glu Glu Arg Leu Lys Ile Ala Thr Ala Lys Leu Glu Glu Ala Ser Gln Ser Ala Asp Glu Ser Glu Arg Met Arg Lys Met Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu Leu Asp Gln Thr Phe Ala Glu Leu Thr Gly Tyr 6. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 6) obtained by expressing a gene derived from a mite body. Phe Leu Thr Asn Asp Lys Arg Tyr Asn Phe Phe Phe Val Thr Met Lys Phe Leu Ile Tyr Ser Leu Ala Phe Ala Ala Met Ala Gly Leu Val Phe Gly Ser Glu Lys Arg Lys Lys Asn Pro Tyr Asp Val Ala Glu Ser Leu Ile Lys His Ala Asp Ala Leu Lys Glu Tyr Ala Glu Gln Glu Ile Gln Lys Tyr Gln Asn Asn Ser His Tyr His Leu Ala Ile Ile Arg Leu Glu His Glu Ile His Arg Val Glu Ala Ile Ala Asp Gln Leu Arg Asn Met Thr Lys Met Glu Asn Met Thr Glu His Tyr Met His His Phe His Gln Ile Glu Met Glu Leu Leu Ile Phe Glu Asn Ile Ile Glu Glu Glu Leu Tyr Tyr Leu Glu His Gly His His His His Asp His His Asp His Asp His His Asp His Met His Arg His Asp His Asp Asp Ser Asp Ile Ile Ala Gly Asp Asp Tyr Lys Asp Ala Leu Leu Lys Lys Ala Gln Gln Leu Ile Asp Glu Ala Glu Asp Ala Ile Lys Lys Tyr Gly Gln Gly Lys Asp Ile Asp Val Gln Val Ile Lys Ser Glu Val Val Ile Ile Lys Asn Leu Val Lys Gln Ile Asn Glu Ala Asn Asn Pro Gln Ala Leu Ala Val Leu Glu Ser Asn Leu Ala Gln His Glu Glu Ile Ile Arg Lys Trp Leu Asp Asp Ile Glu Pro His Thr Thr His Ala Pro Val Thr Ile Ala 7. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 7) obtained by expressing a gene derived from a mite body. Leu Gln Pro Asn Gly Pro Lys Gly Leu Arg Ser Pro Gly Ser Pro Asp Ser Gly Asn Leu Arg Lys Arg Lys Arg Arg Gly Gly Val Leu Gly Phe Lys Glu Leu Ala Ala Lys Ser Lys Leu Ser Ala Trp His Phe His Arg Val Phe Lys Ser Val Thr Gly Leu Thr Pro Lys Thr Tyr Gly Asp Lys Cys Trp Glu Tyr Ile Lys His Cys Arg Glu Asn Gly Glu Ser Phe Phe Phe Glu Gly Lys Gln Ala Asn Thr Asp Ser Gly Glu Val Ser Leu Ser Pro Pro Asn Met Cys Tyr Gln Thr Gln Thr Pro Ile Ser Ser Asn Thr Asp Ser His Asn Tyr Phe Asp Glu Ser Asp Asp Glu Lys Val Lys Lys Val Lys Leu Ser Pro Glu Val Glu Arg Asp Ile Ser Thr Lys Asp Ser Phe Asn Gly Glu Ser Ser Thr Pro Gly Asp Asp Cys Phe His Pro Asp Glu Asn Gly Leu Asp Asn Asn Leu Lys Asn Ile Gly Leu Gly Leu Ser Phe Ala Thr Lys Asp Leu Gly Ile Asn Pro Ser Ser Val Glu Asp Gly Leu Val Gln Pro Thr Cys Ser Phe Asp Phe Asn Leu Asn Asp Ala Thr Ile Pro Asn Val Asp Asp Phe Asp Asp Phe Ser Lys Val Pro Gln Phe Leu Asn Asp Asp Ser Ala Gly Phe Val Arg Ala Phe Ser Ala Pro Asp Leu Thr Lys Tyr Asn His Met Ser Ala Leu Ser Pro Glu Ser Asn Gly Asn Thr Asp Met Asn Lys Pro Lys Asp Ala Leu Gly Phe Asp Ser Tyr Leu Asp Thr Asp Ile Ser Asn Gln Glu Thr Asn Gly Asn Ala Leu Tyr Pro Val Val Asp Asp Val Pro Ser Leu Ser Gly Thr Gly Phe Met Pro Asn Thr Ser Ile Gln Asn Thr Ser Phe Leu Arg Pro Phe Glu Asp Ser Leu Asp Ile Gln Asp Thr Ala Glu Leu Ser Lys Phe Gly Tyr Asp Val Asp Ser Leu Ala Pro Ser Thr Pro Gly Leu Asp Met Lys Leu Asn Ser Arg Phe Ser Val Ala Gln Pro Asp 8. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 8) obtained by expressing a gene derived from a mite body. Ser Thr Arg Trp Leu Thr His Glu Ser Asp Glu Glu Arg Trp Glu Gln Tyr Arg Leu Lys Gln Arg Met Pro Glu Asn Asp Phe Gly Pro Ile Ile Thr Gly His His Asp Asp Asp Asp Asp Asp Asp Val Asp Thr Pro Asn Glu Glu Glu Asn Arg Asp Lys Phe Ile Thr Ile Thr Asn Thr Asp Ser Phe Arg Arg Arg Thr Asn Tyr Ser Ser Ser Ser Asn Ser Asn Asn Asn Asn Asn Asn Asn Gly Lys Asp Tyr Gly His Gln His Gln His His His Pro Arg His His His His His Asn Pro Asn Lys Ser Asn Gly Asn Gly His Ala Phe Asn His Asn Asn Ala Ile Ile Ile Asn Asn Asn Asn Asn Asp Asp Ser Thr Ala Asn Leu Glu Gln Ile Tyr Asn Ser Lys Ser Asn Thr Thr Ser Arg Leu 9. A recombinant mite allergen containing the following amino acid sequence (SEQ ID NO: 9) obtained by expressing a gene derived from a mite body. Phe Ile Lys Ile Lys Asn Ser Ser Lys Met Lys Phe Val Leu Ala Ile Ala Ser Leu Leu Val Leu Ser Thr Val Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala Phe Asn Lys Asn Tyr Ala Thr Val Glu Glu Glu Glu Val Ala Arg Lys Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ala Asn Lys Gly Ala Ile Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Tyr Leu Met Ser Ala Glu Ala Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln Gln Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Iet Gle Glu Gln Tyr Pro Tyr Val Val Ile Met 10. The gene according to any one of claims 1 to 9 is a gene derived from the body of Dermatophagoides farinae.
The recombinant mite allergen according to any one of the above. 11. The recombinant mite allergen according to claim 1, which has a molecular weight of about 19,000 (SDS-PAGE). 12. The recombinant mite allergen according to claim 2, which has a molecular weight of about 34,000 (SDS-PAGE). 13. The recombinant mite allergen according to claim 3, which has a molecular weight of about 22,000 (SDS-PAGE). 14. The recombinant mite allergen according to claim 4, which has a molecular weight of about 17,000 (SDS-PAGE). 15. The recombinant mite allergen according to claim 5, which has a molecular weight of about 33,000 (SDS-PAGE). 16. The recombinant mite allergen according to claim 6, which has a molecular weight of about 28,000 (SDS-PAGE). 17. The recombinant mite allergen according to claim 7, which has a molecular weight of about 38,000 (SDS-PAGE). 18. The recombinant mite allergen according to claim 8, which has a molecular weight of about 18,000 (SDS-PAGE). 19. The recombinant mite allergen according to claim 9, which has a molecular weight of about 32,000 (SDS-PAGE). 20. The recombinant mite allergen according to claim 1, which has a total base chain length of about 0.50 kbp (agarose electrophoresis). 21. The recombinant mite according to claim 2, which has a total base chain length of about 1.05 kbp (agarose electrophoresis method) and is obtained by expression of a gene having the restriction map of FIG. Allergen. 22. The recombinant mite allergen according to claim 3, which has a total base chain length of about 1.66 kbp agarose electrophoresis) and is obtained by expression of a gene having the restriction map of FIG. . 23. The recombinant mite according to claim 4, which has a total base chain length of about 0.75 kbp (agarose electrophoresis) and is obtained by expressing a gene having the restriction enzyme map of FIG. Allergen. 24. The recombinant mite allergen according to claim 5, which has a total base chain length of about 0.99 kbp agarose electrophoresis. 25. The recombinant mite according to claim 6, which has a total base chain length of about 0.90 kbp (agarose electrophoresis) and is obtained by expression of a gene having the restriction enzyme map of FIG. 7. Allergen. 26. The recombinant mite according to claim 7, which has a total base chain length of about 1.26 kbp (agarose electrophoresis method) and is obtained by expressing a gene having a restriction enzyme map shown in FIG. Allergen. 27. The recombinant mite according to claim 8, which has a total base chain length of about 0.62 kbp (agarose electrophoresis) and is obtained by expression of a gene having the restriction map of FIG. Allergen. 28. The total base chain length is about 1.04 kbp (agarose electrophoresis method) and is obtained by expression of a gene having the restriction map of FIG.
The recombinant mite allergen described. 29. A fused recombinant mite allergen, wherein the recombinant mite allergen according to any one of claims 1 to 9 is fused with another protein. 30. The fusion recombinant mite allergen according to claim 29, wherein the other protein according to claim 29 is β-galactosidase. 31. A mite body-derived gene encoding a protein having an allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 1) in a molecule. Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu Leu Asp Gln Thr Phe Ala Gly Thr Tyr Trp Val Ile Asn Asn Asn Ser Phe Ile Leu Arg Arg Gln Asn Phe Arg Leu Phe Leu Thr Thr Pro Pro Pro Pro Ala Thr Ala Thr Thr Lys Asn His 32. A mite body-derived gene encoding a protein having the allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 2) in the molecule. Val Thr Ala Leu Glu Leu Leu Leu Lys Gly Glu Thr Glu Asp Lys Thr Arg Arg Tyr Val Ala Glu Leu Thr Ala Val Gly Ser Pro Ser Asn Lys Gln Ala Lys Ala Gln Ile Glu Val Thr Lys Gly Glu Glu Tyr Lys Ile Thr Leu Lys Ser Pro Glu His Glu Phe Asn Thr Glu Phe Thr Ile His Ala Asp Lys Asn Asn Leu Lys Met His Met Asp Phe Pro Asn Val Phe Gln Ala Asp Leu Thr Gly Thr Phe Gln His Asp Lys Glu Asn Asn Val Arg Lys Asn Gln Leu Asn Leu Gln Tyr Lys Phe Ala Gly Asp Glu Lys Pro His Thr Val Asp Tyr Glu Asn Glu Phe Ser Phe Asn Leu Lys Arg Ser Ser Lys Asp Lys Asn Ser Gly Val Asp Tyr Arg Ala Lys Tyr Met Ser Ser His Phe Pro Ile Leu Asn His Lys Val Asn Ile Gln Phe Lys Tyr Arg Pro Phe Lys Val Asn Glu Leu Asn Leu Glu Gly Glu Phe Gly Arg Glu Leu Gln His Lys Phe Gln Leu Met Arg Asn Ser Gln Ile Glu Val Glu Glu Val Arg Pro Phe Lys Met His Gly Asn Ser Asp Ile Lys Leu Met Ala Asn Asp Leu Asp Ile Asp Tyr Asp Leu Lys Ser Glu Phe Lys Tyr Glu Ser Asn Lys Gly Thr Pro Ile Glu Leu Gln Tyr Lys Ile Ser Gly Lys Asp Arg Ser Lys Arg Ala Ala Asp Leu Gly Ala Glu Asp Val Glu Gly Val Ile Asp Tyr Lys Asn Asn Gly Ser Pro Ile Asp Ser Lys Met His Ala His Leu Lys Met Lys Gly Asn Asn Tyr Gly Tyr Asp Ser Glu Leu Lys Gln Thr Gln Pro Gln Gln Tyr Glu Gly Lys Ile Thr Leu Ser Lys Asn Asp Lys Lys Ile Phe Ile Asn His Lys Ser Glu Met Thr Lys Pro Thr Asn Thr Phe His Leu Lys Thr Asp Ala Asp Val Ser Tyr Ser Asp Ser Asp Met Lys Lys His Tyr Gln Met Glu 33. A mite body-derived gene encoding a protein having the allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 3) in the molecule. Glu Trp Tyr His Leu Phe Lys Arg Trp Leu Arg Lys Arg Phe Thr Lys Met Ile Asp Glu Leu Lys Pro Ser Leu Leu His Val Lys Gly Lys Lys Arg Pro Ile Val Tyr Glu Cys Ala Glu Ile Ser Trp Lys Val Met Asn Asn Gly Asp Val Phe Ile Leu Leu Val Pro Asn Phe Val Phe Val Trp Thr Gly Lys His Ser Asn Arg Met Glu Arg Thr Thr Ala Ile Arg Val Ala Asn Asp Leu Lys Ser Glu Leu Asn Arg Phe Lys Leu Ser Ser Val Ile Leu Glu Asp Gly Lys Glu Val Glu Gln Thr Ser Gly Ala Glu Tyr Asp Ala Phe Asn Lys Ala Leu Ser Leu Asp Lys Lys Asp Ile Asp Leu Lys Gln Met Pro Lys Gly Tyr Asp Tyr Ala Ala Ser Asp Lys Ser Phe Glu Ser His Glu Arg Ser Phe Val Thr Leu Tyr Lys Cys Phe Glu Gly Thr Glu Thr Ile Asp Ile Ser Phe Val Lys Asn Gly Pro Leu Ser Arg Ala Asp Leu Asp Thr Asn Asp Thr Phe Ile Val Glu Asn Gly Ser Glu Gly Leu Trp Val Trp Val Gly Lys Lys Ala Thr Gln Lys Glu Arg Gln Ser Ala Ile Lys Tyr Ala Met Glu Leu Ile Asn Lys Lys Lys Tyr Pro Asn Asn Thr Pro Val Thr Lys Val Leu Glu Gly Asp Glu Ser Val Glu Phe Lys Ser Leu Phe Glu Ser Trp Gln Met Ser Glu Gln Glu Lys Ile Thr Ser Ala Arg Leu Phe Arg Val Ser Arg Asn Gly Ile Phe Lys Gln Val Ala Asn Tyr Glu Pro Asp Asp Leu Glu Glu Asp Ile Tyr His Asp Phe Gly Cys Tyr Gly 34. A mite body-derived gene encoding a protein having an allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 4) in a molecule. Lys Asp Asp Ile Glu Arg Met Val Lys Glu Ala Glu Ser Tyr Lys Glu Glu Asp Asp Lys Gln Arg Asp Arg Ile Ala Ala Lys Asn Ser Leu Glu Gly Tyr Ala Phe Gln Met Lys Ala Thr Leu Asp Glu Glu Ala Ile Lys Ser Lys Val Ser Glu Glu Asp Arg Lys Lys Ile Leu Asp Lys Val Asp Glu Val Leu Lys Trp Leu Asp Ala Asn Ala Leu Ala Glu Lys Asp Glu Phe Glu His Gln Arg Lys Glu Leu Glu Ser Val Cys Asn Pro Ile Ile Thr Lys Leu Tyr Gln Gln Ala Gly Gly Ala Gly Ala Gly Gly Met Pro Gly Gly Phe Pro Gly Gly Phe Pro Gly Thr Asp Gly Ser Gly Gly Gly Ala Ala Gly Gly Asp Gly Gly Lys Ser Gly Pro Thr Ile Glu Glu Val Asp 35. A mite body-derived gene encoding a protein having an allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 5) in a molecule. Phe Phe Phe Val Ala Ala Lys Gln Gln Gln Gln Pro Ser Thr Lys Met Glu Ala Ile Lys Lys Lys Met Gln Ala Met Lys Leu Glu Lys Asp Asn Ala Ile Asp Arg Ala Glu Ile Ala Glu Gln Lys Ala Arg Asp Ala Asn Leu Arg Ala Glu Lys Ser Glu Glu Glu Val Arg Ala Leu Gln Lys Lys Ile Gln Gln Ile Glu Asn Glu Leu Asp Gln Val Gln Glu Gln Leu Ser Ala Ala Asn Thr Lys Leu Glu Glu Lys Glu Lys Ala Leu Gln Thr Ala Glu Gly Asp Val Ala Ala Leu Asn Arg Arg Ile Gln Leu Ile Glu Glu Asp Leu Glu Arg Ser Glu Glu Arg Leu Lys Ile Ala Thr Ala Lys Leu Glu Glu Ala Ser Gln Ser Ala Asp Glu Ser Glu Arg Met Arg Lys Met Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu Asn Gln Leu Lys Glu Ala Arg Met Met Ala Glu Asp Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile Arg Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val His Glu Lys Glu Lys Tyr Lys Ser Ile Ser Asp Glu Leu Asp Gln Thr Phe Ala Glu Leu Thr Gly Tyr 36. A mite body-derived gene encoding a protein having the allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 6) in the molecule. Phe Leu Thr Asn Asp Lys Arg Tyr Asn Phe Phe Phe Val Thr Met Lys Phe Leu Ile Tyr Ser Leu Ala Phe Ala Ala Met Ala Gly Leu Val Phe Gly Ser Glu Lys Arg Lys Lys Asn Pro Tyr Asp Val Ala Glu Ser Leu Ile Lys His Ala Asp Ala Leu Lys Glu Tyr Ala Glu Gln Glu Ile Gln Lys Tyr Gln Asn Asn Ser His Tyr His Leu Ala Ile Ile Arg Leu Glu His Glu Ile His Arg Val Glu Ala Ile Ala Asp Gln Leu Arg Asn Met Thr Lys Met Glu Asn Met Thr Glu His Tyr Met His His Phe His Gln Ile Glu Met Glu Leu Leu Ile Phe Glu Asn Ile Ile Glu Glu Glu Leu Tyr Tyr Leu Glu His Gly His His His His Asp His His Asp His Asp His His Asp His Met His Arg His Asp His Asp Asp Ser Asp Ile Ile Ala Gly Asp Asp Tyr Lys Asp Ala Leu Leu Lys Lys Ala Gln Gln Leu Ile Asp Glu Ala Glu Asp Ala Ile Lys Lys Tyr Gly Gln Gly Lys Asp Ile Asp Val Gln Val Ile Lys Ser Glu Val Val Ile Ile Lys Asn Leu Val Lys Gln Ile Asn Glu Ala Asn Asn Pro Gln Ala Leu Ala Val Leu Glu Ser Asn Leu Ala Gln His Glu Glu Ile Ile Arg Lys Trp Leu Asp Asp Ile Glu Pro His Thr Thr His Ala Pro Val Thr Ile Ala 37. A mite body-derived gene encoding a protein having an allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 7) in the molecule. Leu Gln Pro Asn Gly Pro Lys Gly Leu Arg Ser Pro Gly Ser Pro Asp Ser Gly Asn Leu Arg Lys Arg Lys Arg Arg Gly Gly Val Leu Gly Phe Lys Glu Leu Ala Ala Lys Ser Lys Leu Ser Ala Trp His Phe His Arg Val Phe Lys Ser Val Thr Gly Leu Thr Pro Lys Thr Tyr Gly Asp Lys Cys Trp Glu Tyr Ile Lys His Cys Arg Glu Asn Gly Glu Ser Phe Phe Phe Glu Gly Lys Gln Ala Asn Thr Asp Ser Gly Glu Val Ser Leu Ser Pro Pro Asn Met Cys Tyr Gln Thr Gln Thr Pro Ile Ser Ser Asn Thr Asp Ser His Asn Tyr Phe Asp Glu Ser Asp Asp Glu Lys Val Lys Lys Val Lys Leu Ser Pro Glu Val Glu Arg Asp Ile Ser Thr Lys Asp Ser Phe Asn Gly Glu Ser Ser Thr Pro Gly Asp Asp Cys Phe His Pro Asp Glu Asn Gly Leu Asp Asn Asn Leu Lys Asn Ile Gly Leu Gly Leu Ser Phe Ala Thr Lys Asp Leu Gly Ile Asn Pro Ser Ser Val Glu Asp Gly Leu Val Gln Pro Thr Cys Ser Phe Asp Phe Asn Leu Asn Asp Ala Thr Ile Pro Asn Val Asp Asp Phe Asp Asp Phe Ser Lys Val Pro Gln Phe Leu Asn Asp Asp Ser Ala Gly Phe Val Arg Ala Phe Ser Ala Pro Asp Leu Thr Lys Tyr Asn His Met Ser Ala Leu Ser Pro Glu Ser Asn Gly Asn Thr Asp Met Asn Lys Pro Lys Asp Ala Leu Gly Phe Asp Ser Tyr Leu Asp Thr Asp Ile Ser Asn Gln Glu Thr Asn Gly Asn Ala Leu Tyr Pro Val Val Asp Asp Val Pro Ser Leu Ser Gly Thr Gly Phe Met Pro Asn Thr Ser Ile Gln Asn Thr Ser Phe Leu Arg Pro Phe Glu Asp Ser Leu Asp Ile Gln Asp Thr Ala Glu Leu Ser Lys Phe Gly Tyr Asp Val Asp Ser Leu Ala Pro Ser Thr Pro Gly Leu Asp Met Lys Leu Asn Ser Arg Phe Ser Val Ala Gln Pro Asp 38. A mite body-derived gene encoding a protein having the allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 8) in the molecule. Ser Thr Arg Trp Leu Thr His Glu Ser Asp Glu Glu Arg Trp Glu Gln Tyr Arg Leu Lys Gln Arg Met Pro Glu Asn Asp Phe Gly Pro Ile Ile Thr Gly His His Asp Asp Asp Asp Asp Asp Asp Val Asp Thr Pro Asn Glu Glu Glu Asn Arg Asp Lys Phe Ile Thr Ile Thr Asn Thr Asp Ser Phe Arg Arg Arg Thr Asn Tyr Ser Ser Ser Ser Asn Ser Asn Asn Asn Asn Asn Asn Asn Gly Lys Asp Tyr Gly His Gln His Gln His His His Pro Arg His His His His His Asn Pro Asn Lys Ser Asn Gly Asn Gly His Ala Phe Asn His Asn Asn Ala Ile Ile Ile Asn Asn Asn Asn Asn Asp Asp Ser Thr Ala Asn Leu Glu Gln Ile Tyr Asn Ser Lys Ser Asn Thr Thr Ser Arg Leu 39. A mite body-derived gene encoding a protein having an allergen activity, which comprises the following amino acid sequence (SEQ ID NO: 9) in the molecule. Phe Ile Lys Ile Lys Asn Ser Ser Lys Met Lys Phe Val Leu Ala Ile Ala Ser Leu Leu Val Leu Ser Thr Val Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala Phe Asn Lys Asn Tyr Ala Thr Val Glu Glu Glu Glu Val Ala Arg Lys Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ala Asn Lys Gly Ala Ile Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Tyr Leu Met Ser Ala Glu Ala Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln Gln Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Iet Gle Glu Gln Tyr Pro Tyr Val Val Ile Met 40. The mite body-derived gene according to claim 31, which comprises the following DNA sequence (SEQ ID NO: 1). CTT GAA CAT CGA TCC ATC ACC GAT GAA GAA CGT ATG GAT GGT TTG GAA AAT CAA CTT AAA GAA GCC CGT ATG ATG GCC GAA GAT GCT GAT AGA AAA TAT GAT GAA GTT GCC CGT AAA TTG GCA ATG GTT GAA GCC GAT TTG GAA CGT GCT GAA GAA CGT GCC GAA ACC GGT GAA TGC AAA ATT GTT GAA CTC GAG GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA GTT AGC GAA GAG AAA GCT CAA CAA CGT GAA GAA GCC TAT GAA CAA CAG ATC CGT ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG GAA GAC GAA TTG GTC CAC GAA AAG GAA AAA TAC AAA TCC ATC TCC GAC GAA TTG GAC CAG ACA TTT GCC GGA ACT TAC TGG GTT ATT AAT AAT AAT TCT TTC ATA TTA CGC AGA CAG AAT TTT CGA TTA TTT TTA ACA ACA CCA CCA CCA CCA GCA ACA GCA ACA ACG AAA AAT CAC 41. A mite body-derived gene according to claim 32, which comprises the following DNA sequence (SEQ ID NO: 2): GTG ACC GCA TTG GAA TTA TTG TTG AAA GGT GAA ACT GAA GAC AAA ACT CGT CGC TAT GTT GCC GAA TTG ACT GCC GTT GGA TCG CCA AGC AAC AAA CAA GCT AAA GCA CAG ATC GAA GTG ACC AAA GGC GAA GAA TAC AAA ATC ACA TTG AAA TCA CCT GAA CAT GAA TTT AAC ACC GAA TTC ACC ATC CAT GCA GAC AAA AAC AAT TTG AAA ATG CAC ATG GAT TTC CCT AAC GTG TTC CAA GCT GAT CTG ACT GGA ACT TTC CAA CAT GAC AAG GAA AAC AAT GTG CGT AAA AAC CAA TTG AAT CTC CAA TAC AAA TTC GCT GGT GAT GAG AAA CCA CAC ACA GTG GAT TAT GAA AAC GAA TTT TCA TTC AAT TTG AAA CGA TCA TCA AAA GAC AAA AAC AGT GGG GTC GAT TAC CGA GCA AAA TAC ATG TCT AGC CAT TTT CCA ATC TTG AAC CAC AAA GTC AAC ATT CAA TTC AAA TAT CGA CCA TTC AAA GTT AAC GAA TTG AAT TTG GAA GGA GAA TTT GGT CGA GAA CTT CAA CAC AAA TTC CAA TTG ATG CGA AAC AGC CAG ATA GAA GTG GAA GAA GTG CGA CCA TTT AAG ATG CAT GGC AAT AGT GAC ATA AAA TTG ATG GCA AAC GAT CTG GAT ATT GAT TAT GAT CTC AAA TCT GAA TTC AAA TAT GAA AGC AAC AAA GGA ACC CCC ATT GAA TTG CAA TAC AAA ATT TCC GGT AAA GAT CGC TCG AAA AGA GCT GCC GAT TTG GGA GCC GAA GAT GTT GAA GGC GTT ATC GAT TAC AAA AAC AAT GGT TCG CCA ATT GAT TCT AAG ATG CAT GCT CAT CTC AAA ATG AAA GGC AAC AAC TAT GGC TAT GAT AGT GAA TTG AAA CAA ACA CAG CCA CAA CAA TAT GAA GGC AAA ATC ACA TTG TCC AAG AAT GAC AAG AAA ATC TTC ATC AAC CAC AAA TCA GAG ATG ACC AAA CCT ACC AAC ACT TTC CAT CTC AAA ACA GAT GCT GAT GTG TCG TAT TCG GAT TCG GAT ATG AAA AAA CAT TAT CAG ATG GAA 42. The mite body-derived gene according to claim 33, which comprises the following DNA sequence (SEQ ID NO: 3): GAA TGG TAT CAT TTA TTT AAA AGG TGG TTA CGA AAG CGC TTT ACC AAA ATG ATT GAT GAA TTG AAG CCA TCA TTG TTG CAT GTT AAA GGC AAA AAA CGT CCA ATC GTT TAT GAA TGT GCT GAG ATT AGC TGG AAA GTT ATG AAT AAT GGT GAT GTA TTC ATT TTG CTT GTA CCG AAT TTT GTT TTC GTT TGG ACC GGT AAA CAT TCA AAT CGT ATG GAA CGT ACT ACT GCT ATT CGT GTG GCT AAT GAT TTG AAA TCT GAA TTG AAT CGT TTT AAA TTA TCA TCA ATC ATC TTG GAA GAT GGT AAA GAA GTT GAA CAA ACA TCC GGT GCT GAA TAT GAT GCA TTC AAT AAA GCA TTA TCA TTG GAT AAA AAA GAT ATT GAT CTT AAA CAA ATG CCA AAA GGC TAT GAT TAT GCT GCC AGT GAT AAA TCT TTT GAA TCA CAT GAA CGT TCA TTC GTT ACA TTG TAT AAA TGT TTC GAA GGT ACT GAA ACG ATT GAT ATT TCG TTT GTC AAA AAT GGA CCG TTA AGC CGT GCT GAT CTC GAT ACA AAT GAT ACA TTC ATT GTT GAA AAT GGT TCC GAA GGT TTA TGG GTA TGG GTT GGT AAG AAA GCC ACA CAA AAA GAA CGA CAA TCG GCC ATT AAA TAT GCA ATG GAA TTG ATT AAT AAG AAA AAA TAT CCA AAT AAT ACA CCG GTA ACC AAA GTA TTG GAA GGT GAT GAA AGT GTT GAA TTC AAA TCA TTA TTT GAA TCA TGG CAA ATG AGC GAA CAG GAA AAA ATT ACC AGT GCA CGA TTG TTT CGT GTT TCA CGA AAT GGT ATC TTT AAA CAG GTT GCC AAT TAT GAA CCA GAT GAT TTG GAA GAG GAT ATA TAT CAT GAT TTT GGA TGT TAT GGA 43. The mite body-derived gene according to claim 34, which comprises the following DNA sequence (SEQ ID NO: 4): AAA GAC GAT ATT GAA CGT ATG GTC AAA GAG GCT GAA TCA TAT AAA GAA GAA GAT GAC AAA CAA CGT GAC CGT ATT GCA GCC AAA AAT TCA TTG GAA GGT TAT GCT TTC CAG ATG AAA GCC ACT TTG GAT GAA GAA GCA ATT AAA TCT AAA GTT TCT GAA GAA GAT CGT AAA AAA ATC CTG GAC AAA GTT GAT GAA GTG CTC AAA TGG TTG GAC GCT AAT GCT TTA GCA GAA AAA GAT GAA TTT GAA CAT CAA CGT AAA GAA TTG GAA AGT GTA TGC AAT CCG ATC ATT ACT AAA TTA TAT CAA CAA GCT GGT GGT GCA GGC GCT GGG GGT ATG CCA GGT GGA TTT CCA GGT GGT TTT CCC GGT ACT GAT GGT TCT GGT GGT GGT GCA GCC GGT GGT GAT GGT GGT AAA TCT GGG CCC ACC ATT GAA GAA GTA GAT 44. The mite body-derived gene according to claim 35, which comprises the following DNA sequence (SEQ ID NO: 5): TTT TTT TTC GTT GCA GCT AAA CAA CAA CAA CAA CCA TCA ACA AAA ATG GAG GCC ATC AAG AAA AAA ATG CAG GCA ATG AAG CTC GAG AAA GAT AAT GCT ATC GAT CGA GCT GAA ATT GCC GAA CAA AAA GCC CGT GAT GCT AAT CTA CGT GCC GAA AAG TCT GAG GAA GAA GTT CGT GCA TTA CAG AAA AAA ATC CAA CAA ATT GAA AAT GAA TTG GAT CAG GTC CAA GAA CAA TTA TCG GCT GCC AAT ACA AAA TTG GAG GAA AAG GAA AAA GCC CTA CAG ACC GCT GAA GGT GAT GTT GCA GCA TTG AAT CGT CGT ATT CAA TTG ATT GAA GAA GAT TTG GAA CGA TCA GAA GAA CGA CTT AAG ATT GCT ACA GCC AAA TTG GAA GAG GCA TCA CAA TCT GCC GAT GAA TCT GAA CGT ATG CGT AAA ATG CTT GAA CAT CGA TCC ATC ACC GAT GAA GAA CGT ATG GAT GGT TTG GAA AAT CAA CTT AAA GAA GCC CGT ATG ATG GCC GAA GAT GCT GAT AGA AAA TAT GAT GAA GTT GCC CGT AAA TTG GCA ATG GTT GAA GCC GAT TTG GAA CGT GCT GAA GAA CGT GCC GAA ACC GGT GAA TCG AAA ATT GTT GAA CTC GAA GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TCA TTG GAA GTT AGC GAA GAG AAA GCT CAA CAA CGT GAA GAA GCC TAT GAA CAA CAG ATC CGT ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG GAA GAC GAA TTG GTC CAC GAA AAG GAA AAA TAC AAA TCC ATC TCC GAC GAA TTG GAC CAG ACA TTT GCC GAA CTT ACT GGT TAT 45. The mite body-derived gene according to claim 36, which comprises the following DNA sequence (SEQ ID NO: 6): TTT CTT ACA AAC GAT AAA CGA TAC AAT TTT TTT TTT GTA ACA ATG AAA TTC TTA ATC TAT TCA TTG GCA TTT GCC GCT ATG GCC GGT TTG GTT TTC GGT TCC GAA AAA CGA AAA AAG AAT CCA TAT GAT GTT GCC GAA TCA TTG ATT AAA CAT GCT GAT GCA TTG AAA GAA TAT GCC GAA CAG GAG ATA CAA AAA TAT CAG AAT AAT TCA CAT TAT CAT TTG GCC ATC ATA CGT TTG GAA CAT GAA ATT CAT CGT GTT GAA GCC ATT GCC GAT CAA CTT CGT AAT ATG ACT AAA ATG GAA AAT ATG ACC GAA CAT TAT ATG CAT CAT TTC CAT CAA ATT GAA ATG GAA CTA TTA ATT TTT GAA AAT ATT ATT GAA GAA GAA TTA TAT TAT TTA GAA CAT GGT CAC CAC CAT CAT GAT CAT CAT GAT CAT GAT CAT CAT GAT CAT ATG CGT CAT GAT CAT GAT GAT TCA GAC ATT ATC GCC GGT GAT GAT TAT AAA GAT GCT TTG TTG AAA AAA GCA CAA CAA CTT ATC GAT GAA GCT GAA GAT GCA ATA AAA AAA TAT GGA CAA GGT AAA GAT ATC GAT GTT CAG GTG ATA AAA TCC GAA GTT GTT ATA ATA AAA AAC TTG GTA AAA CAA ATC AAT GAA GCT AAT AAT CCA CAA GCA TTG GCC GTA TTG GAA TCA AAT CTA GCT CAA CAT GAG GAA ATT ATT CGT AAA TGG CTT GAT GAT ATT GAA CCA CAC ACT ACA CAT GCC CCA GTT ACT ATT GCT 46. The mite body-derived gene according to claim 37, which comprises the following DNA sequence (SEQ ID NO: 7): TTA CAA CCC AAC GGA CCA AAG GGT TTG CGT TCT CCT GGA AGC CCT GAC AGC GGT AAT CTG CGT AAA CGT AAG AGA AGA GGA GGA GTT TTG GGT TTC AAG GAA TTG GCA GCG AAG TCT AAG TTA TCT GCC TGG CAT TTT CAT CGT GT AAG TCA GTG ACT GGA TTG ACT CCT AAG ACA TAT GGA GAT AAA TGT TGG GAA TAC ATT AAA CAT TGC AGA GAA AAT GGG GAG AGC TTC TTT TTC GAG GGT AAG CAA GCT AAC ACA GAC TCC GGA GAG GTC TCT CTT AGC CCA CCA AAT ATG TGC TAT CAG ACA CAG ACA CCT ATT AGC TCT AAT ACT GAT TCA CAT AAC TAT TTT GAT GAG TCG GAC GAT GAA AAA GTA AAG AAG GTT AAA TTG TCA CCA GAG GTG GAG CGG GAC ATT TCC ACC AAA GAT AGC TTC AAT GGC GAG TCA TCT ACT CCC GGT GAT GAT TGT TTT CAC CCT GAC GAA AAT GGA CTT GAT AAC AAT TTG AAG AAC ATT GGG CTA GGG CTG TCG TTC GCT ACT AAG GAT CTT GGC ATA AAT CCT AGT AGT GTT GAA GAT GGT TTG GTA CAG CCT TCA TGT TCT GAC TTT AAT TTA AAT GAT GCT ACG ATA CCC AAT GTT GAC GAT TTC GAC GAT TTT TCA AAA GTT CCT CAA TTT TTA AAT GAT GAC TCT GCT GGG TTT GTG AGA GCG TTT TCT GCT CCT GAT TTG ACC AAG TAC AAC CAT ATG AGT GCA TTA AGT CCT GAA AGT AAT GGC AAC ACT GAC ATG AAC AAG CCC AAG GAT GCA TTA GGT TTC GAT TCC TAC CTT GAT ACA GAC ATT AGC AAC CAG GAA ACT AAT GGG AAT GCT CTT TAT CCA GTT GTG GAT GAT GTT CCA TCA ATC TTG GCT GGT TTT ATG CCA AAC ACT TCA ATT CAA AAT ACG TCT TTT TTA AGA CCA TTT GAA GAT AGC TTG GAT ATA CAA GAT ACT GCT GAA CTT TCA AAG TTT GGC TAC GAT GTT GAT TCT CTC GCG CCT TCT ACA CCC GGA TTA GTT ATG ATG AAT TCG AGG TTT AGT GTT GCG CAA CCT GAT 47. The mite body-derived gene according to claim 38, which comprises the following DNA sequence (SEQ ID NO: 8). TCA ACA AGA TGG CTT ACA CAT GAA TCG GAC GAA GAA CGT TGG GAG CAA TAT AGA CTC AAA CAA AGA ATG CCA GAG AAT GAT TTT GGT CCG ATC ATT ACG GGT CAT CAT GAT GAT GAT GAT GAT GAT GAT GTC GAT ACA CCG AAT GAA GAG AAT CGT GAT AAA TTC ATC ACA ATC ACC AAC ACT GAT TCA TTC AGA CGA CGT ACT AAT TAC AGC AGC AGC AGC AAC AGC AAC AAT AAT AAT AAC AAC AAT GGC AAG GAT TAT GGC CAC CAG CAT CAG CAT CAC CAT CCC CGC CAT CAT CAT CAC AAT CCG AAT AAA TCG AAT GGC AAT GGA CAT GCA TTT AAC CAC AAT AAT GCC ATC ATT ATC AAC AAC AAT AAT AAT GAT GAT TCG ACA GCC AAT TTG GAA CAG ATT TAT AAC AGC AAA AGT AAC ACG ACC AGC CGT TTG 48. A mite body-derived gene according to claim 39, which comprises the following DNA sequence (SEQ ID NO: 9): TTC ATC AAA ATT AAA AAT TCA TCA AAA ATG AAA TTC GTT TTG GCC ATT GCC TCT TTG TTG GTA TTG AGC ACT GTT TAT GCT CGT CCA GCT TCA ATC AAA ACT TTT GAA GAA TTC AAA AAA GCC TTC AAC AAA AAC TAT GCC ACC GTT GAA GAG GAA GAA GTT GCC CGT AAA AAC TTT TTG GAA TCA TTG AAA TAT GTT GAA GCT AAC AAA GGT GCC ATC AAC CAT TTG TCC GAT TTG TCA TTG GAT GAA TTC AAA AAC CGT TAT TTG ATG AGT GCT GAA GCT TTT GAA CAA CTC AAA ACT CAA TTC GAT TTG AAT GCC GAA ACA AGC GCT TGC CGT ATC AAT TCG GTT AAC GTT CCA TCG GAA TTG GAT TTA CGA TCA CTG CGA ACT GTC ACT CCA ATC CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GCA GTC GCC ACT GAA TCA GCT TAT TTG GCC TAC CGT AAC ACG TCT TTG GAT CTT TCT GAA CAG GAA CTC GTC GAT TGC GCA TCT CAA CAC GGA TGT CAC GGC GAT ACA ATA CCA AGA GGC ATC GAA TAC ATC CAA CAA AAT GGT GTC GTT GAA GAA AGC TAT CCA TAC GTT GCA CGA GAA CAA CAA TGC CGA CGA CCA AAT TCG CAA CAT TAC GGT ATC TCA AAC TAC TGC CAA ATT TAT CCA CCA GAT GTG AAA CAA ATC CGT GAA GCT TTG ACT CAA ACA CAC ACA GCT ATT GATT GTCATT GGC ATT AAA GAT TTG AGA GCT TTC CAA CAT TAT GAT GGA CGA ACA ATC ATT CAA CAT GAC AAT GGT TAT CAA CCA AAC TAT CAT GCC GTC AAC ATT GTC GGT TAC GGA AGT ACA CAA GGC GTC GAT TAT TGG ATC GTA CGA AAC AGT GAT ACT ACC TGG GGT GAT AGC GGA TAC GGA TAT TTC CAA GCC GGA AAC AAC CTC ATG ATG ATC GAA CAA TAT CCA TAT GTT GTA ATC ATG 49. In the base sequence shown in SEQ ID NO: 1, at least about 190 bp to about 24 from upstream.
0 bp, about 290 bp to about 380 bp or about 190 b
A mite allergen fragment containing the amino acid sequence encoded within the region from p to about 380 bp. 50. A peptide having an epitope contained in a mite allergen derived from a mite body. 51. A peptide having an epitope that can be regarded as immunologically identical to the epitope contained in a mite allergen derived mite allergen. 52. The following amino acid sequence (SEQ ID NO: 1)
52. The peptide according to claim 50 or 51 containing 0). Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Se
r Leu Glu 53. The following amino acid sequence (SEQ ID NO: 1)
52. The peptide according to claim 50 or 51 containing 1). Ile Met Thr Ala Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu 54. A gene encoding the peptide according to claim 52 or 53. 55. The following DNA sequence (SEQ ID NO: 10)
55. The gene according to claim 54, which comprises: GAG GAA GAA TTA CGT GTT GTC GGT AAC AAT CTC AAA TC
A TTG GAA 56. The following DNA sequence (SEQ ID NO: 11)
55. The gene according to claim 54, which comprises: ATA ATG ACG GCT AAA CTT AAA GAA GCC GAA GCA CGT GCC GAA TTT GCT GAA CGT TCG GTA CAA AAA CTC CAG AAA GAA GTC GAT CGT TTG 57. An expression vector capable of expressing the gene according to any one of claims 31 to 48 in transformed bacteria, yeast or mammalian cells. 58. A bacterium, yeast or mammalian cell transformed with the expression vector of claim 57. 59. The bacterium, yeast or mammalian cell according to claim 58 is cultured under conditions capable of expressing the gene to produce a recombinant mite allergen, and then the recombinant mite allergen is recovered. A method for producing a recombinant mite allergen, which is characterized. 60. The bacterium, yeast or mammalian cell according to claim 58 is cultured under conditions capable of expressing the gene to produce a fused recombinant mite allergen, and the fused recombinant mite allergen is recovered. Next, a method for producing a recombinant mite allergen, which comprises releasing other fused proteins. 61. A therapeutic agent for mite allergic diseases, which comprises the recombinant mite allergen according to any one of claims 1 to 28 as an active ingredient. 62. A tick allergy disease diagnostic agent comprising the recombinant mite allergen according to any one of claims 1 to 28 as an active ingredient.