JP3149033B2 - Peptides in the mite major allergen Der f II - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a major allergen of mites
Peptides human anti Der f II IgE antibodies in the Der f II is represented by essential amino acid sequence for binding relates to DNA encoding this peptide.

[0002]

2. Description of the Related Art Many allergic diseases are sensitized to an antigen causing the disease, thereby producing an allergen-specific IgE antibody (reagin antibody) in serum and tissues and re-exposed to the antigen. As a result, it is considered that the antigen and the IgE antibody cause an antigen-antibody reaction on each tissue, and at that time, various symptoms occur.

[0003] As a method of fundamentally treating this allergic disease, there is desensitization therapy. This method is a method of repeatedly administering the causative antigen little by little and gradually increasing the dose according to the symptoms. This therapy is thought to produce a blocking antibody, suppress IgE antibody production, and reduce the amount of histamine released from mast cells, resulting in a therapeutic effect.

On the other hand, it has been revealed that allergic diseases such as bronchial asthma, pediatric asthma, and atopic dermatitis are mainly caused by allergies to ticks living in house dust. Major mite allergen proteins have been identified (Platts-Mil
ls) et al., The Journal of Allergy and
Clinical Immunology (J. Allergy Clin. Immuno
l.), 80, 755, 1987). Therefore, although the desensitization therapy for the above allergic diseases using this major allergen, particularly the site essential for the binding of IgE, is extremely effective, it requires a large amount of purified allergen and a large number of essential binding sites in the allergen. It is. A method for preparing a purified major mite allergen in large quantities has already been disclosed by Yuki et al. (Allergy (Japanese J. Allergology), 39, 557).
P. 1990). But anti- Der f II, a major tick allergen
The region of the IgE antibody binding essential region has not been identified so far, and its large-scale preparation has not been possible.

[0005]

SUMMARY OF THE INVENTION The present inventors have made various changes to the gene corresponding to the previously disclosed tick major allergen Der f II by genetic manipulation and expressed the same.
By examining the reactivity with allergic patient serum,
It has been found that it is possible to identify an essential site for binding an anti- Derf II IgE antibody and to prepare a large amount thereof.

Therefore, it is an object of the present invention to obtain a DNA sequence encoding an essential IgE binding site in a major mite allergen protein by using genetic engineering, and to obtain its DN.
An object of the present invention is to provide a method for expressing an essential region of IgE binding of a major mite allergen encoded by A and producing a large amount of an essential region where an IgE antibody binds to a target allergen.

[0007]

DISCLOSURE OF THE INVENTION The present invention relates to a DNA encoding an essential site to which an IgE antibody of an allergic patient to the major allergen of Dermatophagoides mite ( Der f II) binds and a peptide at the binding site. The gene encoding the major mite allergen Der f II and its preparation have already been disclosed by Yuki et al. (Allergy (Japanese J. Allergol)
ogy), 39, 557, 1990). Various methods can be used to identify the epitope site in the allergen protein.In the case of the present invention in which a gene encoding the protein can be used, the gene is modified and a suitable vector is used. A method of expressing in a host and searching by reaction between the expressed protein and patient serum is advantageous. Various procedures conventionally used can be used for this series of operations (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory).
or Laboratory), 1982).

The Der f II gene can be subjected to various modifications alone or after being inserted into an appropriate vector. In particular, a method in which the Der f II gene is modified while inserted into a cloning site of an expression vector such as pMAL-c is advantageous. . When this vector is used, the gene is expressed as a fusion protein with a maltose-binding protein, and its expression level is large, so detection is easy.

The Der f II gene can be obtained by the method disclosed by Yuki et al. (Allergy (Japanese J. Allergology), 39, 557
, 1990). The resulting plasmid containing the Der f II gene was modified with various restriction enzymes. First, digestion with the restriction enzyme KpnI was carried out, and the 3 ′ protruding end was cut with T4
Removed with polymerase. The resulting linear plasmid containing the Der f II gene was transformed into exonuclease II.
I deleted from the 5 'end. By adjusting the reaction time, a linear plasmid containing the Der f II gene having sequentially different degrees of deletion was obtained. The resulting plasmid
It was completely digested with Hin dIII, to obtain a fragment containing a deletion Der f II gene subjected to agarose gel electrophoresis. On the other hand, plasmid pMAL-c (New England Biolabs) was
Complete digestion with Stu I and Hin dIII. The previously prepared deletion Der f II gene was ligated using T4 ligase to obtain a circular plasmid. Escherichia coli was transformed with the resulting plasmids containing the Der f II gene having different degrees of deletion. All methods followed the protocol of New England Biolabs. After transformation, Der was performed in a colony immunoassay using an anti- Der f II polyclonal antibody.
Clones were selected that are producing the f II protein.

The transformed Escherichia coli was cultured on an L-broth agar medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% bactoaga, 50 μl).
g / ml ampicillin, pH 7.4), and the resulting colonies were inoculated into L broth liquid medium (L broth agar medium minus agar) as appropriate. After culturing at 37 ° C. for about 3 hours, IPTG (isopropyl-β-thiogalactopyranoside) was added to a concentration of 0.1 mM, and the culturing was continued for another 2 hours. After harvesting, the whole cell protein was subjected to SDS-polyacrylamide gel electrophoresis. The gel after electrophoresis was analyzed by Western blotting (Towbin, H.)
Proc. Natl. Acad. Sci. US Proceding of the National Academy of Sciences
A.), Volume 76, 4350, 1979) was transferred to a cellulose filter. The protein-transferred filter was blocked with 0.1% Tween 20 and reacted with the serum of an allergic patient. Then, a buffer solution (Tris-HCl buffer, 10 mM, pH 7.4, sodium chloride,
(MM, 150 mM, Tween 20, 0.05%). The washed filter was reacted with an anti-human IgE antibody labeled with peroxidase (ICN). Thereafter, the peroxidase-labeled antibody remaining without reacting was removed.
The filter was further reacted with hydrogen peroxide and the dye 4-chloro-1-naphthol.

As a result, when the deletion of the Der f II gene progressed to some extent, the Der f II protein did not react with IgE of allergic patients. The Der f II gene can be excised by complete digestion with the restriction enzyme Bam HI. Excised DNA
The fragments were prepared by the dideoxy method (Sanger et al., Journal of Molecular Biology (J. Mol. Bio).
l.) Vol. 162, p. 729, 1982) and the like. Allergies
The nucleotide sequences of the gene encoding the Der f II protein that binds IgE and the gene with the largest deletion and the gene encoding the Derf II protein that does not bind IgE to the allergic patient and the gene with the smallest deletion were determined. . The sequence of the non-overlapping part of the determined base sequences of the two strains, ie,
The nucleotide sequence deleted before and after losing the ability to bind to IgE is represented by the following formula I:
Shown in Formula I also shows the corresponding amino acids.

Formula I: GAT-CCA-TGC-ATC-ATC Asp-Pro-Cys-Ile-Ile The present DNA fragment and the DNA fragment containing the present DNA sequence are as follows:
It can be obtained from the Der f II gene by treatment with an appropriate restriction enzyme or exonuclease, but can also be synthesized using a DNA synthesizer. Thereafter, expression is carried out using a suitable vector and a suitable host, whereby the desired peptide can be produced. Alternatively, the desired peptide can be produced by chemical synthesis.

An epitope site of the mite allergen protein Der f II according to the present invention, prepared by chemical synthesis or genetic engineering, and a part thereof and a form in which they are fused with other peptides, proteins, etc. Peptides having the characteristics of the mite major allergen Der f II epitope can be used for the prevention, treatment or diagnosis of various allergic diseases caused by mites.

[0014]

According to the present invention, the IgE binding essential site of Dermatophagoides pteronyssin major allergen protein Derf II was determined by using genetic engineering, whereby a DNA fragment encoding a peptide essential for the binding essential site could be obtained. Therefore, the tick major allergen protein encoded by this DNA
It is possible to provide a method for expressing or chemically synthesizing an IgE-binding essential site and mass-producing the same, and using this IgE-binding essential site can be used for prevention, treatment or diagnosis of various allergic diseases.

[0015]

Next, the present invention will be described more specifically based on examples. These examples are merely illustrative of the invention and do not limit the scope of the invention. Example 1 Modification of the Der f II Expression Plasmid pFL11 The plasmid p, carrying the cDNA of the major mite allergen Derf II, already disclosed by Yuki et al.
FL11 (Allergy (Japanese J. Allergology, 39, 55
7 (1990), only the gene encoding Der f II was prepared. The outline is shown in FIG.

[0016] pFL11 can be those the expression plasmid PUEX1 (Amersham) was constructed based on, cDNA gene Der f II to the restriction enzyme Bam HI site via an adapter synthetic nucleotide (Amersham) Has been inserted. The pFL11 was completely digested with restriction enzymes Kpn I in the synthetic nucleotide adapters were separated digested DNA fragments were subjected to 0.7% agarose electrophoresis. Der f IIcDN
A Kpn I fragment of about 500 base pairs containing A completely was extracted and purified from the agarose gel after electrophoresis. Insect-derived mature body
Restriction enzyme Sa having a cleavage site just at the position of codon GAT corresponding to the N-terminal amino acid aspartic acid of Der f II
The Kpn I fragment was purified by u 3AI, was completely cut, using a DNA polymerase I Klenow (Klenow) fragment was blunted protruding ends of the cleavage site. Then, the restriction enzyme Nco I
In full cut, subjected to 5% polyacrylamide gel electrophoresis, the 63 bp Sau 3AI (filled) - the Nco I fragment was separated and purified. Similarly , after completely cutting the KpnI fragment with the restriction enzyme HinfI having a cleavage site immediately before the polyA sequence,
The ends were blunted. Then, after cutting with Nco I, 5
389% polyacrylamide gel electrophoresis.
Base pair Nco I- obtain a Hin fI (filled) fragment.

As an expression vector in E. coli, pMAL-c (New England Biolab) was used. By fusing a heterologous protein to the 3 'end of the maltose binding protein gene encoded by the expression plasmid, it is possible to induce and express a fusion protein of the maltose binding protein and the heterologous protein under the control of the lac promoter (Maina )
Et al., Gene, 74, 365, 1988). This pMAL
-c was completely digested with the restriction enzyme Stu I and treated with alkaline phosphatase. The obtained vector DNA fragment and 63
Base pair Sau 3AI (filled) -Nco I fragment and 389 base pair Nco I
-The Hin fI (filled) fragment is mixed, and a ligation reaction is performed using a DNA ligation kit (Takara Shuzo Co., Ltd.), and Escherichia coli TBl (New England Biolab,
Genotype ara , △ ( lac - proAB) , rpsL , (φ80 lacZ
ΔM15 ), hsdR ). The basic experimental methods for genetic manipulation (restriction enzyme treatment, modification enzyme treatment, etc.) used herein were based on the method of Maniatis et al. (Molecular Cloning, Cold Spring Harbor Press, 1982). For the transformation of E. coli, the electric pulse method (Bio-Rad, Gene
Pulsar). Plasmids were isolated from ampicillin-resistant colonies using the alkaline method (Birnboi
m) etc., Nucleic Acid Research (Nucl.
s Res.), 7, 1513, 1979). The resulting plasmid was completely digested with restriction enzymes Bam HI, Der
f II gene was confirmed that it is inserted. The strain thus obtained was designated as pMFL11-c / TB1.

[0018] 'with pMFL11-c Preparation said terminal region deleted gene, 5 of Der f II gene 5' of Example 2 Der f II gene produce plasmid series terminally deleted stepwise deleted (Fig. 2). This stepwise deletion results in exonuclease III
(ExonucleaseIII) and Mung Bean Nuclease (Mu
ng Bean Nuclease) (Henikoff) (Gene, 28, 351 (1984)).

After pMFL11-c was digested with the restriction enzyme Stu I to make it linear, the protruding end of the Kpn I cleavage site was blunted using a DNA blunting kit (Takara Shuzo Co., Ltd.).
5 μg of this linear DNA was added to 105 μl of ExoIII buffer (50 μl).
mM Tris-HCl, pH 8.0, 100 mM sodium chloride, 5 mM magnesium chloride, 10 mM 2-mercaptoethanol), and add 1 μl of 180 units of exonuclease III.
The reaction was started at ℃. Sample 7.5 μl every 20 seconds and stop the reaction with 100 μl Mung Bean nuclease buffer (40 mM sodium acetate pH 4.5, 10
0 mM sodium chloride, 2 mM zinc chloride, and 10% (v / v) glycerin). After treatment with Mung Bean nuclease, end repair was performed with DNA polymerase I Klenow fragment.
For this series of operations, a deletion kit for kilo sequence (Takara Shuzo Co., Ltd.) was used. The resulting linear DNA, restriction enzyme Hin was completely digested with dIII, subjected to 10% polyacrylamide gel electrophoresis, 5 'fragment comprising sequentially from Der f II gene introduced deletion ends were separated and purified .

Meanwhile, the plasmid pMAL-c Stu I and Hin dI
Complete digestion with II. This DNA fragment and the previously prepared deletion
The Der f II gene was ligated to a circular DNA using a DNA ligation kit. Using this reaction solution, Escherichia coli TB1 was transformed. If the codon reading frame of the maltose binding protein gene and the deleted Der f II gene encoded by the plasmid carried by the transformant match, expression of the fusion protein in which the N-terminus of the Der f II protein has been deleted can be performed. Conceivable. Therefore, colony immunoassay (Agricultural and Biological chemistry (Agric. B
iol. Chem.) 55, 1233, 1991), a strain which reacts with a mouse-derived anti- Der f II antibody, that is, produces a Der f II fusion protein was selected. Positive strain was placed in L liquid medium (1
% Bactotripton (Tryptone, manufactured by Difco), 0.5
% Yeast extract (manufactured by Difco), 0.5% sodium chloride), and after culturing, the plasmid was extracted, completely digested with restriction enzymes Bam HI and Cla I, and subjected to polyacrylamide gel electrophoresis. , Der
The degree of fII gene deletion was examined. In this way, Der
5 'end of the f II gene 0 to 200 bp, sequentially deleted in which holds a plasmid and each plasmid N-terminal deletion Der f II fusion protein 100 different E. coli TB1 expressing, was obtained.

[0021] Example 3 N-terminal deletion Der f II fused N-terminal deletion Der f II fusion protein IgE antibodies protein 100 kinds of transformants obtained in detecting a second embodiment of the IgE antibody binding capacity of expressing Binding ability was determined by Western blotting (Proc. Natl. Acad. Sci. USA, 69, 1409).
Page, 1972).

The resulting transformant was inoculated into an L liquid medium containing 100 μg / ml ampicillin, and cultured with shaking at 37 ° C.
_{When the 660} nm reached 0.4, IPTG (isopropyl-β-thiogalactopyranoside) was added to 0.1 mM, and the cells were further cultured for 2 hours. After the cells after culturing are collected by centrifugation, sample buffer (10% (v / v) glycerin, 5% (v / v) 2-mercaptoethanol, 3%
(w / v) SDS (sodium dodecyl sulfate), 62 mM Tris-
(PH 6.8) and heat-treated at 100 ° C. for 5 minutes to extract whole cell proteins. The obtained protein sample was separated by SDS-acrylamide gel electrophoresis (electrophoresis apparatus manufactured by Tefco, gel concentration: 8%). From the gel after electrophoresis, nitrocellulose (Bio-Rad) or synthetic polymer (Millipore, Immobilon)
The proteins were electrotransferred onto a membrane filter of the same. This transfer was carried out using a semi-dry type protein transfer device (Ato Co., Horizon Blot). The membrane after transfer is treated with PB containing 0.1% Tween 20 as a blocking operation to prevent non-specific adsorption of the antibody.
S buffer (Dulbecco, etc., Journal of Experimental Medicine (J. Exp.Med.), 99
Vol., P. 167, 1954) for 1 hour. As a primary antibody, serum from a patient allergic to Dermatophagoides pteronyssinus (the serum of one patient with a high IgE content in the RAST test or the pooled serum mixed with the sera of several allergic patients) was used. The membrane was immersed in a solution obtained by diluting the serum 4 times with a PBS buffer and shaken overnight. Then, 0.05% Tween 20
The antibody was washed three times with a PBS buffer solution containing, and the non-adsorbed primary antibody was washed away. As the secondary antibody, a peroxidase-labeled anti-human IgE antibody (manufactured by ICN) was used, diluted with PBS (400-fold), adsorbed (4 hours), and washed as in the case of the primary antibody. Coloring reagent chloronaphthol (4-chloro
-1-naphthol) at a concentration of 0.5 mg / ml in PBS buffer,
After a hydrogen peroxide solution was added to a final concentration of 0.05%, the membrane was immersed to perform color development.

By this method, the fusion protein expressing pMFL11-c / TB1 before the deletion operation clearly reacted with the IgE antibody in the patient's serum. On the other hand, the N-terminal-deleted Der f II fusion protein reacted with the IgE antibody while the degree of deletion was small, but lost its reactivity after deletion to a certain extent. Where IgE
Select the strain with the least deletion among the plasmids encoding the fusion protein that does not react with the antibody and the strain with the most deletion among the plasmids encoding the fusion protein that reacts with the IgE antibody, and attempt to determine their nucleotide sequences. Was.

Example 4 Determining the nucleotide sequence of the deleted plasmid Among the plasmids encoding the fusion protein which does not react with the IgE antibody,
The nucleotide sequence of the plasmid with the largest deletion among the plasmids encoding the fusion protein reacting with the antibody was determined.

The resulting plasmid was completely digested with Bam HI, subjected to 1.5% agarose gel electrophoresis to separate and purify a DNA cleavage including Der f II gene. Obtained DNA
The digestion was digested to completion with Bam HI and sequenced by Sanger (Science 214, 1205-1210).
P. 1981 (Messing (Mes.
sing), etc.
Escherichia coli host MV1184 (Takara Shuzo, genotype ara ,
thi , strA , △ ( lac − proAB ), (φ80 lacZ △ M15),
△ (srl − recA ) 306 :: Tn10 (tet ^r ) F ′ (proAB ⁺ , lacI ^q Z
ΔDM15, traD 36)) was transformed. The single-stranded DNA of the recombinant plasmid was isolated and purified, and the nucleotide sequence was determined according to the method of Sanger. A fluorescent automatic DNA sequencer (Applied Biosystems, Model 373A) was used for sequencing. As a result, it was found that when the deletion plasmid deleted the base sequence represented by the above formula I in the Der f II gene, the IgE binding ability was lost. This means that before and after the translated protein loses its ability to bind IgE,
This suggests that only the amino acids shown in Formula I have been deleted.

[Brief description of the drawings]

FIG. 1 shows a major mite allergen Der f II in Example 1.
FIG. 4 is a diagram showing steps until a plasmid pMFL11-c for constructing a plasmid pMFL11-c for searching for an epitope site by modifying the plasmid pFL11 holding the cDNA of FIG.

FIG. 2 is a diagram showing a step of preparing a plasmid series in which the 5 ′ end of the Der f II gene is deleted stepwise using pMFL11-c in Example 2.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yasushi Okumura 2-13-1 Omorikita, Ota-ku, Tokyo Asahi Breweries Co., Ltd. Applied Technology Laboratory (72) Inventor Tetsusuke Sugiyama 3-chome, Minamihachiman, Ichikawa-shi, Chiba No. 3 in Torii Pharmaceutical Co., Ltd. (72) Inventor Hiroshi Yamakawa 3-14-3 Minamihachiman, Ichikawa-shi, Chiba Prefecture Torii Pharmaceutical Co., Ltd. (56) References JP-A-3-254683 (JP, A) J.I. Allergy Clin. Im munol. , Vol. 83, No. 6, (1989), p. 1055-1067 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/12 C07K 7/06 BIOSIS (DIALOG) CA (STN) MEDLINE (STN) WPIDS (STN)

Claims (2)

(57) [Claims] 1. The major allergen of Dermatophagoides pteronyssinus ( Der f II)
Essential following formula I to a human anti Der f IIIgE antibodies in binds: peptide represented by the amino acid sequence of Asp-Pro-Cys-Ile- Ile. Wherein the following formula II: DNA encoding the amino acid sequence of claim 1, wherein represented by the nucleotide sequence of GAT-CCA-TGC-ATC- ATC.