JP4732040B2 - New allergen derived from Japanese cedar pollen - Google Patents

Description

  The present invention relates to a novel allergen protein derived from cedar pollen, and diagnosis, prevention and treatment of allergy using the same.

  In recent years, morbidity and mortality of allergic diseases have been increasing globally in accordance with changes in dietary habits and living environments. According to a private survey (present state and future prospects of new drug development, 1991 edition, Seed Planning Co., Ltd.), 1 in 3 people in Japan depend on typical IgE such as cedar pollinosis, atopic dermatitis, bronchial asthma It is said to show symptoms of type (I) allergic disease. This is the same as the result of the 2003 survey. According to the 2003 Health and Welfare Trends Survey on Allergy-like Symptoms (Ministry of Health, Labor and Welfare Minister's Secretariat Statistics Information Department) Of allergic symptoms in the eyes and nose, 35.9% had any symptoms in the past year. Allergic diseases are not directly related to life, but suddenly develop in the very young generation, and natural healing at an early stage cannot be expected. It is thought that it has had a great influence on social activities.

  Cedar pollinosis has been reported to affect 1 in 10 Japanese people, but the following epidemiological data are actually available. According to an epidemiological survey of approximately 17,000 people conducted in 1998 by Dorotaro Baba, Department of Otolaryngology, Dokkyo Medical University, the prevalence of Japanese cedar pollinosis was 18.1%. Furthermore, according to the Phase II Outcome Report on the Research on Responding to Consumers' Needs for Coordination of Science and Technology Agency's “Creative Research for Overcoming Cedar Pollen Allergy”, the epidemiological survey based on the objective diagnostic criteria conducted in 2001-2002 The prevalence of hay fever was 33.8% in urban areas (Shinagawa-ku, Tokyo) and 26.7% in rural areas (Makioka-cho, Yamanashi Prefecture). In particular, many patients suffer from allergic symptoms during the spring when pollen is scattered.

  The most effective way to treat hay fever is to avoid contact with allergens, but in patients who are sensitized or developed with allergens that are free and present throughout the living environment, side effects such as antihistamines may also occur. The reality is that we have to rely on temporary solutions with symptomatic treatments. For this reason, unless you continue to use it, it will repeat the onset, and you will be burdened financially and physically, and if you stop using it, you may be worried about worsening symptoms due to rebound. Yes.

  On the other hand, attempts have been made on desensitization therapy in which allergens, which are allergic causative agents, are repeatedly administered to patients to cure them. There is also a report that symptoms have been improved in over 90% of patients with desensitization treatment in ascidian asthma (see Non-Patent Document 1, for example), and it has been established as one of the standard treatment methods for allergies in the West. (For example, refer nonpatent literature 2). However, if there is a side effect such as anaphylactic shock if the antigen to be used is wrongly selected, an appropriate diagnosis for each patient is required.

  As the major allergens of Cryptomeria japonica pollen allergens, two molecular species having different antigenicity, Cry j 1 and Cry j 2, are known. Cry j 1 was reported by Yasue et al. (See, for example, Non-Patent Document 3; in this document, Cry j 1 was called SBP (Sugi basic protein)) and had a molecular weight of 45,000 to 50,000 daltons, etc. The protein has an electric point of about 9.0. Cry j 2 is a protein having a molecular weight of about 37,000 daltons and an isoelectric point of about 9.5 reported by Sakaguchi et al. (See, for example, Non-Patent Document 4). The nucleotide sequence of cDNA encoding Cry j 1 and the amino acid sequence of Cry j 1 are clarified (see, for example, Non-Patent Document 5), and the nucleotide sequence of cDNA encoding Cry j 2 and the amino acid sequence of Cry j 2 are clarified. (For example, see Non-Patent Document 6). These two major allergens frequently react with IgE in the serum of cedar pollinosis patients.

  In the desensitization therapy for Japanese cedar pollinosis, the main allergens Cry j 1 and Cry j 2 alone do not provide a sufficient therapeutic effect. For treatment, first, an accurate diagnosis is important, but at present, there are almost no diagnoses other than major allergens. Under such circumstances, in recent years, elucidation of detailed immunochemical properties of cedar pollen allergen proteins other than Cry j 1 and Cry j 2 has been desired.

  Several cedar pollen allergens other than Cry j 1 and Cry j 2 have been reported so far. Kawamoto et al. Revealed the cDNA and amino acid sequence of CJP-6, a cedar pollen-derived protein highly homologous to isoflavone reductase, and CJP-6 is a novel allergen recognized by Japanese cedar pollinosis IgE. (For example, see Non-Patent Document 7 and Patent Document 1). On the other hand, Fujimura et al. Isolated CJP-4, a protein derived from cedar pollen that shows high homology with chitinase, and revealed its cDNA sequence and amino acid sequence, and CJP-4 was recognized by IgE patients with cedar pollinosis. It was shown to be an allergen (see, for example, Non-Patent Document 8 and Patent Document 2 (referred to as CJP-16 in this document)). Obata et al. Revealed the cDNA sequence and amino acid sequence of CPA39, a protein derived from cedar pollen that shows high homology with β-1,3-glucanase, and the natural and recombinant forms of CPA39 were recognized by IgE patients with cedar pollinosis. It was shown that this is a novel allergen (Japanese Patent Application No. 2005-36507).

  Igawa et al. Found that a protein derived from Japanese cedar pollen having a molecular weight measured by SDS-polyacrylamide gel electrophoresis of 57,000-67,000 daltons and an isoelectric point of 7.0-9.0 contains a novel allergen ( For example, see Patent Document 3). The allergen was presumed to be pyruvate kinase based on the analysis of the amino acid sequence of the peptide fragment of the allergen by mass spectrometry (Ken Sawazaki, Research Promotion Promotion Department, Hiroshima Industrial Technology Promotion Organization)・ Strategic Rights Project ”3rd Research Promotion Meeting (3rd Proteome Project General Meeting) (Hiroshima), March 15, 2001). However, information specifying the molecular species of the allergen, such as the entire amino acid sequence of the allergen and the cDNA sequence encoding the allergen, has not been obtained.

  Nimura et al. Obtained three protein cDNAs (Cry j 3.1, Cry j 3.2, and Cry j 3.3) homologous to Jun a 3 known as an allergen of mountain cedar (Juniperus ashei) from a cedar pollen cDNA library. Isolation (see, for example, Non-Patent Document 9), and the subsequent isolation of Cry j 3.4, Cry j 3.5, and Cry j 3.6 cDNAs that are also homologous to Jun a 3 (see, for example, Non-Patent Document 9) 10). They showed that, of these six genes, only the Cry j 3.5 gene was highly expressed in developing male cones and pollen, suggesting that the protein encoded by the gene may have allergenic activity. (For example, refer nonpatent literature 11). On the other hand, Miyahara has also disclosed Cry j 3a, Cry j 3b, and Cry j 3c cDNA sequences having homology with Jun a 3 (see, for example, Patent Document 4). If the immunological characteristics of Cry j 3 protein encoded by these cDNAs, such as reactivity with the sera of Japanese cedar pollinosis, become clear, these Cry j 3 or some of these Cry j 3 It may be included in an important allergen for hay fever.

  In this way, despite the existence and importance of many cedar pollen allergens other than Cry j 1 and Cry j 2, their molecular species have been identified and their immunological properties have been evaluated. There are few things.

JP 2002-58487 A JP 2005-65536 A JP 2001-151797 A JP 2004-89103 A Shigeta S. et al., Arerugi, 39 (3), 313-21, 1990. Bousquet J, et al., J. Allergy Clin. Immunol. 102, 558-62, 1998. Yasueda, H. et al., J. Allergy Clin. Immunol. 71, 77-86, 1983. Sakaguchi, M. et al., Allergy, 45, 309-312, 1990. Sone, T. et al., Biochem. Biophys. Res. Commun., 199: 619-625, 1994. Komiyama, N. et al., Biochem. Biophys. Res. Commun., 201: 1021-1028, 1994. Kawamoto S. et al., Clin. Exp. Allergy, 32 (7), 1064-70, 2002. Fujimura T. et al, Clin. Exp. Allergy, 35 (2), 234-43, 2005. Futamura. N. et al., Biosci. Biotechnol. Biochem., 66, 2495-2500, 2002. Futamura. N. et al., 7th International Congress of Plant Molecular Biology (Barcelona), lecture number S10-87, June 25, 2003 Kenji Shinohara et al., Comprehensive research for overcoming Japanese cedar pollinosis (II report), 218-226, 2003

  In view of the prior art as described above, the present invention intends to provide novel allergens other than Cry j 1 and Cry j 2 related to cedar pollinosis, allergy diagnostic agents, preventive agents, therapeutic agents and the like using them. Is.

In order to achieve the above object, the present inventors have extensively searched for spots that react frequently with IgE antibodies in sera of cedar pollinosis patients by proteomic analysis of cedar pollen crude antigen. As a result, it was found for the first time that a protein (CPA63) having a molecular weight of 50,000 to 60,000 daltons and an isoelectric point of around 3.5 to 5.0 has high allergenicity. Furthermore, after intensive studies using various analytical techniques and genetic engineering techniques, the amino acid sequence, cDNA sequence, and immunological characteristics were clarified to complete the present invention.
That is, the present invention is as follows.

(1) A protein contained in cedar pollen, which shows a molecular weight of 50,000-60,000 daltons when measured by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of around 3.5-5.0 when measured by isoelectric focusing. A cedar pollen allergen characterized by showing.
(2) As a partial amino acid sequence
X-Val-Glu-Pro-Ala-Tyr-Asn-Ala-Met-Arg or Phe-Thr-Pro-XX-Ser-Asn-Ser-Arg (where X represents Ile or Leu) The cedar pollen allergen of 1).
(3) The cedar pollen allergen according to (1) or (2), which has the amino acid sequence set forth in SEQ ID NO: 2.
(4) Any one of (1) to (3) above, which is obtained from a cedar pollen crude antigen by a method such as affinity purification, ion exchange chromatography, gel filtration chromatography, centrifugation, concentration, or dialysis. Japanese cedar pollen allergen.
(5) A cedar pollen allergen protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or at least a part of the amino acid sequence.

(6) A nucleic acid molecule encoding the whole or at least a part of the amino acid sequence of the cedar pollen allergen protein according to any one of (1) to (4).
(7) A nucleic acid molecule having a base sequence encoding the amino acid sequence of the cedar pollen allergen protein of SEQ ID NO: 2 or at least a part of the amino acid sequence.
(8) A nucleic acid molecule having the base sequence set forth in SEQ ID NO: 1 encoding at least a part of the base sequence encoding cedar pollen allergen.
(9) The nucleic acid molecule according to (6) to (8), which is isolated from a cedar pollen or a cedar male flower.

(10) The protein according to any one of (1) to (5) or the amino acid sequence of at least a part thereof produced in a host cell transformed with the nucleic acid molecule according to (6) to (8) Containing protein.
(11) The protein according to any one of (1) to (5) prepared by a cell-free expression system or a protein comprising at least a part of the amino acid sequence.
(12) A protein comprising the protein according to any one of (1) to (5) or at least a part of the amino acid sequence prepared by chemical synthesis.
(13) The protein according to (5) or (10) to (12), which reacts with IgE in the serum of a cedar pollinosis patient.
(14) The T cell epitope of the cedar pollen protein according to any one of (1) to (5) and (10) to (12), wherein the T cell of a patient sensitized with cedar pollen can be expanded. peptide.

(15) A monoclonal antibody or a polyclonal antibody that specifically reacts with the cedar pollen protein of any one of (1) to (5) and (10) to (12) or at least one protein fragment thereof.
(16) A diagnostic agent for hay fever patients using the cedar pollen allergens of (1) to (4) and (10) to (12).
(15) A therapeutic agent for a desensitizing action using the cedar pollen protein according to (1) to (4) or (10) to (12).

  The novel cedar pollen allergen of the present invention can be used for diagnostics, prophylactics, and therapeutics for allergies, and the allergen protein and protein fragments thereof include Cry j 1, Cry j 2 and other By combining with a cedar pollen allergen, it can be used for diagnosis or desensitization treatment of cedar pollinosis.

The present invention is described in detail below.
In Japanese cedar pollen allergy, the major antigens of Cry j 1 and Cry j 2 have been identified and studied in detail, but other allergens have not been fully studied. Therefore, the present inventors developed a cedar pollen crude antigen by two-dimensional electrophoresis with the aim of comprehensive analysis of allergens contained in cedar pollen, and then specific to cedar pollinosis patient serum IgE by immunoblotting method. Searched for spots that respond to. As a result, it was found that, besides Cry j 1 and Cry j 2 in the basic range, allergens that react with IgE exist not only in the basic range but also in the acidic range. Then, the amino acid sequence of the obtained positive spot was performed using MALDI-TOF MS, the partial amino acid sequence of the positive spot was determined, and it was named CPA63. When homology search was performed using the obtained amino acid sequence, there was no significant homology among sequences derived from other known plants, and the molecular species of this protein could not be inferred. . Next, when the EST database of Cryptomeria japonica (Japanese cedar) was searched using the obtained amino acid sequence, an EST (Accession number BP173910) having a matching translation sequence was found.

  In order to isolate the cedar pollen allergen (CPA63) gene of the present invention, a cedar cDNA library was constructed using total RNA purified from cedar pods. Oligonucleotide primers were designed based on the base sequence of EST (Accession number BP173910), and an unknown gene region was obtained by the RACE method. Thereafter, primers were designed to obtain the full-length gene based on the nucleotide sequence, and the full-length gene was obtained by PCR. The obtained sequence (SEQ ID NO: 1) contained a start codon and a stop codon, and its ORF was 1419 bases. This ORF encodes a protein of 472 amino acids, which revealed the entire amino acid sequence of the CPA63 protein (SEQ ID NO: 2).

  A homology search using the entire amino acid sequence of the resulting CPA63 protein reveals that this sequence has significant homology with aspartyl protease family proteins / nucleoid DNA-binding-like proteins from many plant species. became. As allergen proteins having homology with aspartyl protease, for example, Blag 2 derived from German cockroaches and Asp f 10 derived from Aspergillus fumigatus (fungi) have been reported. Aspartyl protease is a protease in which aspartic acid is present at the active center, and is an enzyme that catalyzes endo-type cleavage that cleaves peptide bonds containing hydrophobic amino acids. This aspartyl protease has been found, for example, in a wide variety of higher plant species. Aspartyl protease is sometimes called aspartyl proteinase, aspartic protease, aspartic proteinase, aspartate protease, etc., and may be called acidic protease, carboxyl protease, aspartate endopeptidase, or other names, but these are the same. It is well known to those skilled in the art that it is a name referring to something. Nakano et al. Purified nucleoid DNA binding protein from tobacco green cultured cells. The protein has a region containing a large amount of lysine at the N-terminus, and binds non-specifically to DNA. Furthermore, this protein also has an active region of aspartic protease and has been reported as a protein having two functions (Nakano, T., et al., Plant Cell, 9, 1673-1682, 1997). . It has also been reported that transformants that suppress the expression of this protein inhibit plant growth, which is an interesting protein involved in plant aging (Fumihiko Sato, Protein Nucleic Acid Enzyme, 45, 147-152, 2000).

A part of the amino acid sequence of CPA63 of the present invention may be any short part as long as all of the immunological properties of CPA63 are not lost. For example, even if the peptide has a length of about 10 to 20 amino acids in the amino acid sequence of CPA63, those having at least a part of the immunological properties of CPA63 are included in the present invention. For example, the local structure of an allergen molecule that determines the specificity of an antigen-antibody reaction in the course of allergic pathogenesis is called an epitope, and a part of the amino acid sequence of CPA63 of the present invention includes at least one epitope of CPA63. Amino acid sequences containing are included. The region that T cells specifically recognize is particularly called a T cell epitope, but generally the sequence length is about 10 to 20 amino acids.
The nucleic acid encoding at least a part of the CPA63 protein of the present invention includes a nucleic acid encoding an amino acid sequence of CPA63 having at least a part of the immunological properties of CPA63. For example, at least one of CPA63 Nucleic acids comprising a nucleic acid sequence encoding an amino acid sequence comprising an epitope are included in the present invention.
The immunological properties of CPA63 here include not only the immunological properties of CPA63 against humans but also the immunological properties of other animals such as dogs, cats, monkeys, rats, mice, rabbits, etc. .

The nucleic acid encoding the CPA63 encoding the CPA63 of the present invention or at least one fragment thereof can be known by those skilled in the art using the information of the amino acid sequence of CPA63 disclosed by the present invention and the nucleic acid sequence encoding the same. It can be easily prepared from natural samples and libraries using techniques. It can also be prepared using chemical nucleic acid synthesis techniques known to those skilled in the art.
The isolated natural CPA63 protein of the present invention was evaluated for its allergenicity by immunostaining. In one example in which the cedar pollen crude antigen (CJP) was subjected to two-dimensional electrophoresis and the specific IgE antibody titer of CPA63 was analyzed using cedar pollinosis patient serum, 47.5% of the patient serum showed a positive reaction.
The isolated natural CPA63 protein of the present invention and a protein fragment containing at least a part thereof are purified from cedar-derived samples, preferably from cedar pollen or cedar pollen extract, by affinity purification, ion exchange chromatography, gel filtration chromatography. It can be obtained using techniques known to those skilled in the art such as chromatography, centrifugation, concentration and dialysis.

  The protein containing CPA63 of the present invention and at least a part of the amino acid sequence thereof can be synthesized using any of various known protein expression systems. Such methods include, for example, E. coli expression systems, lactic acid bacteria expression systems, yeast expression systems, Neisseria gonorrhoeae expression systems, insect cell expression systems, animal cell expression systems, and cell-free expression systems. Those skilled in the art understand the advantages and disadvantages of each of these expression systems, so that those skilled in the art can select an appropriate expression system according to the purpose. For example, in expression systems that use prokaryotic cells such as E. coli, it may be better to use eukaryotic cells such as yeast to express recombinant CPA63 because appropriate modifications such as glycosylation are not performed. .

  When expressing a recombinant protein, a secretory signal is used to improve its expression efficiency, to facilitate subsequent purification and detection, or to prevent the expressed protein from becoming insoluble in cells. The sequence of the portion is altered (for example, JP 2001-258565 and JP 2000-175686), or a tag sequence such as a GST tag or a histidine tag is added (for example, Terpe, K., Appl. Microbiol. Biotechnol., 60, 523-533, 2003), or expressed as a fusion protein with a fluorescent protein such as GFP or another protein or a partial sequence of another protein (eg Breitwieser, A. et al , Protein Eng., 15, 243-249, 2002, and WO2002 / 036785) are often performed by those skilled in the art. When expressing recombinant CPA63, those skilled in the art can use such a technique as necessary to prepare a protein containing CPA63 of the present invention and at least a part of the amino acid sequence thereof.

  As one example, the present inventors used a baculovirus-insect cell system to express recombinant CPA63 protein (rCPA63). RCPA63 was purified from the culture supernatant using HiTrap Chelating HP Columns and HiTrap Q FF columns. As a result, rCPA63 protein having a molecular weight of about 51,000 daltons was obtained as measured by SDS-polyacrylamide gel electrophoresis. As another example, the allergenicity of the obtained recombinant CPA63 was evaluated by ELISA. When CPA63-specific IgE antibody titer was analyzed using cedar pollinosis patient serum, about 33.3% patient serum was positive in this Example.

  Thus, the reaction with the serum IgE of cedar pollinosis patients was observed in both the natural CPA63 isolated from the cedar of the present invention and the CPA63 prepared by the recombinant technique of the present invention, and both these CPA63 were allergenic. It has been shown for the first time by the inventors. A person skilled in the art can appropriately select a method for obtaining the CPA63 of the present invention depending on the purpose.

  A protein comprising CPA63 of the present invention and at least a part of the amino acid sequence thereof may be chemically or physically mixed with one or more other substances as long as all of its immunological properties are not lost. It can also be used in a reversible or irreversible state by a general method. For example, glass, plastic, resin, inorganic substance, metal, semiconductor, fluorescent substance, radioactive substance, dye molecule, protein, nucleic acid, sugar, lipid, water-soluble polymer, water-soluble small molecule, biodegradable material, organic synthetic molecule, It can be used in a state of being bound to a biological molecule, a virus particle or the like. In addition, any shape may be used, for example, plate shape, bead shape, cube shape, membrane shape, capsule shape, gel shape, crystal shape, liquid crystal shape, fiber shape, hollow fiber shape, porous shape, solution It can be a molecule, nanoparticle, colloid, composite of these, or a more complex shape.

  The new cedar pollen allergen CPA63 obtained in the present invention can be used as a diagnostic reagent for cedar pollinosis. In addition, it is possible to provide information on cross-sensitization with allergens (eg, Blag 2 or Asp f 10) that are homologous to aspartyl protease from the diagnosis results.

  In addition, since the entire amino acid sequence of the CPA63 protein has been clarified by the present invention, it has become possible to identify the T cell epitope site of the CPA63 protein. Therefore, those T cell epitope peptides have applications in cedar pollinosis immunotherapy. The protein containing at least a part of the amino acid sequence of CPA63 of the present invention includes a protein containing such a TPA epitope peptide of CPA63.

  Hyposensitization is one of the treatments for allergies, but side effects such as anaphylaxis are also considered, so in recent treatments, T cells are specifically recognized rather than administering allergens to patients. Peptide vaccines that administer peptides consisting only of the minimum region of allergens, that is, T cell epitopes, have attracted attention.

  A method for identifying a T cell epitope of an allergen protein has already been established. For example, T cell epitopes of Cry j 1 and Cry j 2 which are main antigens have been reported so far (for example, Hashiguchi Shuhei, Nippon Linyi, 54, 2233-2242, 1996, JP 7-118295 A) And JP-A-8-47392). Attempts to link them have also been disclosed (for example, Sone, T. et al., J. Immunol., 161, 448-457, 1998, and JP-A-10-259198). The T cell epitope peptide of CPA63 protein can also be obtained by such a known method.

  The T cell epitope peptide of CPA63 protein obtained in the present invention can be used alone or in combination with or combined with the T cell epitope peptide of Cry j 1, Cry j 2 and other cedar pollen allergens. Can be used for immunotherapy.

  Furthermore, the present invention can provide monoclonal antibodies and polyclonal antibodies that specifically react with the CPA63 protein, or a protein fragment thereof. Methods for producing monoclonal antibodies and polyclonal antibodies that specifically react with a protein or protein fragment thereof are known to those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited to the following Example.
<Example 1> Proteomic analysis of cedar pollen crude antigen Preparation of cedar pollen crude antigen 3.0 L of extraction buffer (20 mM PBS + 3 mM EDTA pH 7.6) was added to 80 g of Japanese cedar pollen (collected in Toyomachi, Kure City, Hiroshima Prefecture). And stirred at 4 ° C. for 4 hours. Thereafter, ammonium sulfate was added to the supernatant obtained by centrifugation (7,000 rpm, 30 minutes) so that the final concentration became 80% saturation, and the mixture was stirred at 4 ° C. overnight. Next, the precipitate was collected by centrifugation (7,000 rpm, 30 minutes) and dialyzed overnight against milliQ water. Thereafter, the supernatant obtained by centrifugation (10,000 rpm, 30 minutes) was freeze-dried to obtain a cedar pollen crude antigen (CJP).

Two-dimensional electrophoresis of crude Japanese cedar pollen antigen Suspended with Japanese cedar pollen crude antigen (CJP) 200 mg, suspended in 4 ml of PBS + 60 mg of dithiothreitol (DTT), and then added 2 ml of trichloroacetic acid adjusted to 60% with PBS And then left on ice for 90 minutes. Thereafter, centrifugation (3,500 rpm, 15 minutes) was performed, and the precipitate was collected. The precipitate was suspended by adding 10 ml of cold acetone and washed. Further, after centrifugation (3,500 rpm, 20 minutes), the precipitate was dried by speed back. 1 ml of Lysis Buffer (8 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, 1% DTT, 0.8% Ampholine) was added to the precipitate, suspended, and completely dissolved by sonication. . Thereafter, centrifugation (18,000 rpm, 20 minutes) was performed, and the supernatant was used as a sample for two-dimensional electrophoresis.

The dry strip of pI range 3-10 was swollen with Lysis buffer overnight, then the CJP sample was applied to the strip and the first dimension (isoelectric point) was run.
A 9-18% polyacrylamide gradient gel was prepared, and the gel after isoelectric focusing was set thereon. After the low melting point agarose was overlaid and solidified from the top of the gel, electrophoresis of the second dimension (molecular weight) was performed overnight at 80 V.
After the second dimensional electrophoresis was completed, the protein was detected by staining the gel. An example of the result is shown in FIG. In the crude cedar pollen antigen, many proteins other than the main antigens Cry j 1 and Cry j 2 were confirmed.

Western blotting The protein after two-dimensional electrophoresis was transferred to a membrane under a condition of 60 V for about 6 hours using a blotting kit (Hoefer DALT). Thereafter, the membrane was washed with PBST (0.1% Tween20 / PBS) and shaken overnight with a blocking solution (5% skim milk, 1% BSA / PBST). Thereafter, the cells were washed with PBST and incubated in the serum of a cedar pollinosis patient diluted 10-fold with a blocking solution while shaking for 4 hours. After washing, anti-human IgE-biotin label (Biosource) diluted 2,500 times with blocking solution was added, and incubated for 2 hours with shaking. After washing with PBST, the cells were incubated with streptavidin-HRP label (ZYMED) diluted 2,500 times with blocking solution for 1 hour. Thereafter, the plate was washed with PBST, incubated with ECL Western blotting detection reagents (Amersham Pharmacia Biotech) for 5 minutes, and exposed to X-ray film to detect positive spots.
As a result of Western blot analysis, it was found that there were many spots showing positive reaction in addition to Cry j 1 and Cry j 2 in the crude cedar pollen antigen. Of the 12 cedar pollinosis patient serum IgE surveys, spots with particularly strong positive reactions are shown as a box in FIG.

  Among them, the amino acid sequence of CPA63 protein that showed a reaction frequency of about 50% or more was performed using MALDI-TOF MS. As a result, the sequences of X-Val-Glu-Pro-Ala-Tyr-Asn-Ala-Met-Arg and Phe-Thr-Pro-XX-Ser-Asn-Ser-Arg (X represents Ile or Leu) Obtained. When homology search was performed using this sequence, there was no known homologous sequence derived from other plants, and the molecular species of the protein could not be inferred. Next, when the EST database of Cryptomeria japonica (Japanese cedar) was searched using the sequence of X-Val-Glu-Pro-Ala-Tyr-Asn-Ala-Met-Arg, EST (Accession with matching translation sequence) was searched. number BP173910). Therefore, in order to further determine the sequence of the unknown gene region and obtain the full-length gene of CPA63, the method of Example 2 was employed.

<Example 2> Determination of nucleotide sequence and amino acid sequence of CPA63 gene Purification of cedar cocoon total RNA 5 g of cedar cocoon crushed in liquid nitrogen was placed in a 50 ml centrifuge tube, and Concert ^TM Plant RNA Reagent ( 25 ml of Invitrogen) was added and suspended. The tube was left at room temperature for 5 minutes with the centrifuge tube lying on its side. This was centrifuged (2,600 × g, 5 minutes, 4 ° C.). The supernatant was filtered with a cell strainer (BD Falcon) having a mesh size of 100 μm. 2 ml of 5 M sodium chloride was added to 10 ml of the filtered supernatant and suspended. 6 ml of chloroform was added to 10 ml of the filtered supernatant and suspended. This was centrifuged (2,600 × g, 30 minutes, 4 ° C.). To the supernatant, 0.9 times the amount of isopropanol as the supernatant was added and suspended. This was allowed to stand at room temperature for 10 minutes. Next, this was centrifuged (2,600 × g, 30 minutes, 4 ° C.). The supernatant was removed, and 10 ml of 75% ethanol was added to the precipitated total RNA and lightly suspended. This was centrifuged (2,600 × g, 5 minutes, 4 ° C.). The supernatant was removed, and the centrifuge tube was opened and left at room temperature for 15 minutes to dry the total RNA. To this, 300 μl of RNase free water was added to dissolve the total RNA. The obtained total RNA solution was stored at -80 ° C.

Purification and concentration of cedar cocoon mRNA Oligotex ^™ -dT30 <Super> mRNA Purification kit (TaKaRa) was used to purify mRNA from cedar cocoon total RNA. 150 μl of a total RNA solution containing 250 μg of cedar pod total RNA was prepared. To this, 150 μl of 2 × Binding buffer and 15 μl of Oligotex ^™ -dT30 <Super> were added and suspended. The mixture was incubated at 70 ° C. for 3 minutes and allowed to stand at room temperature for 10 minutes. This was centrifuged (15,000 rpm, 5 minutes, room temperature). The supernatant was removed, and 350 μl of Wash buffer was added to suspended Oligotex ^™ -dT30 <Super> and suspended. This was transferred to a spin column set cup and centrifuged (15,000 rpm, 30 seconds, room temperature). The cup was transferred to a new spin column centrifuge tube. 350 μl of Wash buffer was added to Oligotex ^™ -dT30 <Super> in the cup and suspended. This was centrifuged (15,000 rpm, 30 seconds, room temperature). The cup was transferred to a new spin column centrifuge tube. (1) 30 μl of RNase free water at 70 ° C. was added to Oligotex ^™ -dT30 <Super> in the cup, suspended, and centrifuged (15,000 rpm, 30 seconds, room temperature) to recover the mRNA solution. The operation of (1) was performed twice more. A series of mRNA purification operations were further performed 5 times to obtain about 500 μl or more of mRNA solution.

  16.5 μl of 3 M sodium acetate (pH 5.2) was added to 500 μl of the mRNA solution and suspended. 3 μl of Ethachinmate (Nippon Gene) was added to this and suspended. 500 μl of isopropanol was added to this and suspended. This was centrifuged (15,000 rpm, 10 minutes, 4 ° C.). The supernatant was removed, and 1 ml of 75% ethanol was added to the precipitated mRNA and suspended briefly. This was centrifuged (15,000 rpm, 5 minutes, 4 ° C.). The supernatant was removed, and the mRNA was dried by allowing to stand at room temperature for 15 minutes with the microtube lid open. To this was added 10 μl of RNase free water to dissolve the mRNA. The obtained mRNA solution was stored at −80 ° C.

Synthesis of Japanese cedar cDNA The BD SMART ^™ RACE cDNA Amplification Kit (BD Biosciences) was used for the synthesis of 5′-RACE and 3′-RACE cDNA from Japanese cedar mRNA. For 5'-RACE cDNA synthesis, 1 μg of 1 μg / μl mRNA solution, 1 μl of 5′-CDS primer, 1 μl of BD SMART II A oligo, and RNase free water in a 0.5 ml microtube. 2 μl was added and suspended. On the other hand, for the synthesis of 3'-RACE cDNA, 1 µg of 1 µg / µl mRNA solution, 1 µl of 3'-CDS primer A, and 3 µl of RNase free water were suspended in a 0.5 ml microtube. did. The mixture was incubated at 70 ° C. for 2 minutes and left on ice for 2 minutes. Each reaction solution was suspended by adding 2 μl of 5 × First-Strand Buffer, 1 μl of 20 mM dithiothreitol, 1 μl of dNTP mix, and 1 μl of BD PowerScript Reverse Transcriptase. Incubated for 90 minutes at 42 ° C. 250 μl of Tricine-EDTA Buffer was added to the reaction solution and suspended. This was incubated at 72 ° C. for 7 minutes. The obtained cDNA solution was stored at −30 ° C.

Determination of CPA63 cDNA sequence CPA63 amino acid sequence estimated using mass spectrometry (X-Val or Val-X) -Glu-Pro-Ala-Tyr-Asn-Ala-Met-Arg (where X is Ile or Leu The EST database of GenBank was searched using a searcher. As a result, Cryptomeria japonica EST (Accession number BP173910) having a translation sequence of Leu-Val-Glu-Pro-Ala-Tyr-Asn-Ala-Met-Arg was found. Based on the nucleotide sequence of BP173910, 5'-RACE primer 5'-CTC CAC CAG CCT AGT GAT GAC TGT TCC-3 'and 3'-RACE primer 5 '-CGG CGA CGT GGA GTT TCC TCT CAT C-3' was prepared. Using each of the prepared primers and Universal Primer A Mix (primer attached to BD SMART ^™ RACE cDNA Amplification Kit), 5′-RACE and 3′-RACE were performed using cedar cDNA as a template. For this RACE reaction, BD Advantage ^™ 2 PCR Kit (BD Biosciences) was used. 5'-RACE and 3'-RACE products are purified using QIAquick PCR Purification Kit (QIAGEN) and ligated to pGEM-T Easy Vector (Promega) using DNA Ligation Kit ver. 2.1 (TaKaRa) Competent high E. coli DH5α (TOYOBO) was transformed. A plasmid was prepared from the obtained transformant using QIAprep Spin Miniprep Kit (QIAGEN). Using the prepared plasmid as a template, a cycle reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

  The reaction solution was purified by ethanol precipitation, and the nucleotide sequences of 5′-RACE product and 3′-RACE product were analyzed using DNA sequencer 3100-Avant (Applied Biosystems). The amino acid sequence deduced from the base sequence of the 5'-RACE product matches the other amino acid sequence Phe-Thr-Pro-Leu-Leu-Ser-Asn-Ser-Arg of CPA63 deduced using mass spectrometry There was a sequence to do. This was an event that further supported that this gene is the CPA63 gene. To obtain the full-length gene of CPA63, based on the obtained nucleotide sequence, primers 5'-GGA TCA TTG CCT GTG CTT CTC ATA GAC G-3 'and 5'-TTA TAG TGA AGC AGA GTG GAC GTT GG -3 'was produced. Using these primers, PCR was performed using 5′-RACE cedar cDNA as a template to amplify the full-length gene of CPA63. For this PCR reaction, KOD-Plus-DNA Polymerase (TOYOBO) was used. The obtained full-length gene of CPA63 was purified and ligated to pUC118 DNA HincII / BAP (TaKaRa), and then transformed into E. coli DH5α. A plasmid was prepared from the transformant, and the base sequence of the full-length gene of CPA63 was analyzed. As a result, the base sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the full-length cDNA of CPA63 were obtained. The full-length cDNA of CPA63 consisted of 1525 bp bases, and its ORF encoded 472 amino acids.

<Example 3> Homology search for CPA63 gene and protein
Homology searches were performed on the CPA63 gene and protein sequences obtained by FASTA and BLAST searches using the GenBank database. FIG. 2 shows the results of comparing the homology of the amino acid sequence of the cedar pollen allergen CPA63 (SEQ ID NO: 2) with the aspartyl protease family protein / nucleoid DNA-binding-like protein derived from other plants.

  In FIG. 2, ARATHA1 and ARATHA2 are the amino acid sequences of nucleoid DNA-binding-like proteins derived from Arabidopsis thaliana (GenBank Accession AAM65914 and AAM66983, respectively). ORYSAT is the amino acid sequence of nucleoid DNA-binding-like protein derived from Oryza sativa (rice) (GenBank Accession BAD31106). POPTRI is the amino acid sequence of putative aspartyl protease family protein / nucleoid DNA-binding-like protein derived from Populus trichocarpa (black cottonwood) It is. MALDOM1 is the amino acid sequence of putative aspartyl protease family protein / nucleoid DNA-binding-like protein from Malus x domestica (apple) (GenBank Accession CN861156, CN884543, CN884846, CN887971, CN900689, CN909749, CN911584, CN939698, CN947159, and CO898345) (ORF translation sequence obtained). MALDOM2 is an amino acid sequence of putative aspartyl protease family protein / nucleoid DNA-binding-like protein from Malus x domestica (apple) (GenBank Accession CN878811, CN888988, CN890192, CN890327, CN900189, CN900844, CN938123, CN943706, CN946109, and CO752714 (ORF translation sequence obtained). CITSIN was derived from putative aspartyl protease family protein / nucleoid DNA-binding-like protein (GenBank Accession CF509569, CF835209, CK934020, CK934803, CV714459, CV885520, CX045648, CX051916, CX072584, and CX072585 derived from Citrus sinensis (orange) Translated sequence).

CPA63 is an Aspartic proteinase derived from Nepenthes gracilis (Athauda SB, et al., Biochem. J., 381, 295-306, 2004) and 34.9% at the amino acid level, 50.9% at the DNA level, and Nicotiana tabacum (Nakano, T et al., Plant Cell, 9, 1673-1682, 1997) showed 35.5% identity at the amino acid level and 48.3% identity at the DNA level (Fig. 3, * indicates matching amino acid residues). Group).
As described above, it was confirmed that CPA63 has high homology with other types of aspartyl protease family protein / nucleoid DNA-binding-like protein. On the other hand, aspartyl protease has been reported as an allergen so far, for example, Blag 2 from German cockroaches (Pollart SM, J. Allergy Clin. Immunol., 87, 511-21, 1991) and Aspergillus fumigatus (fungus) derived Asp f 10 (Crameri R., Int. Arch. Allergy Immunol., 115, 99-114, 1998). Therefore, CPA63 of the present invention can be used as a means that can provide information on cross-reactivity with allergens other than cedar pollen.

<Example 4> Expression of recombinant CPA63 protein (rCPA63) Recombinant CPA63 protein (rCPA63) was expressed in a baculovirus-insect cell system. Using the cDNA encoding CPA63 as a template, amplification was performed by PCR using DNA amplification enzyme KOD-plus- to obtain a CPA63 gene fragment having BamH I and EcoR I sites at both ends. This amplified product was subjected to restriction enzyme treatment with BamH I and EcoR I, and similarly inserted into insect cell expression vector pVL1393 treated with BamH I and EcoR I to obtain CPA63 / pVL1393. The sequence of the inserted CPA63 gene was confirmed by DNA sequencer 3100-Avant (Applied Biosystems). CPA63 / pVL1393 was cotransfected into insect cultured cells Sf9 together with baculovirus DNA (Sapphire ^™ DNA (Orbigen)) to prepare a recombinant baculovirus having the CPA63 gene. CPA63 gene-specific PCR was performed using the recombinant baculovirus genomic DNA as a template to confirm that the CPA63 gene was integrated into the viral genome. Next, this recombinant baculovirus was inoculated into insect cultured cells Sf9 and then cultured at 27 ° C. for 4 days to express the rCPA63 protein. The rCPA63 protein was expressed as a histidine tag fusion protein in which polyhistidine (6 × histidine residue) was added to the C-terminus. The culture solution was centrifuged at 3,000 rpm and 4 ° C. for 10 minutes, and then the supernatant was collected. The supernatant was collected and subjected to SDS-PAGE, followed by silver staining and Western blot analysis to confirm the expression of rCPA63 (FIGS. 4 and 5). Western blotting uses the Penta-His Antibody (QIAGEN) antibody as the primary antibody and the HRP-labeled Anti-mouse IgG (Seikagaku) antibody as the secondary antibody, and luminescence is obtained with the ECL Plus Western Blotting Detection System (Amersham Biosciences). RCPA63 was detected. 4 and FIG. 5, lane 1 shows a molecular weight marker, and lane 2 shows a culture supernatant.

<Example 5> Purification of recombinant CPA63 protein (rCPA63) The rCPA63 protein expressed in Example 4 was purified from the culture supernatant of insect cells. Purification using a His column was performed using the histidine tag added to the C-terminus of rCPA63. The culture supernatant was applied to a HiTrap Chelating HP column (Amersham Biosciences) and adsorbed on the column, and the protein adsorbed on the column nonspecifically was washed with PBS buffer. Thereafter, the concentration of imidazole in PBS buffer was gradient from 10 mM to 500 mM, and rCPA63 was eluted from the column. The obtained fraction was analyzed by SDS-PAGE / Western blotting to confirm the eluted fraction of rCPA63 (lane 2 in FIG. 4). Thereafter, the solution containing rCPA63 was concentrated by a stirring ultra holder (ADVANTEC). The concentrated solution was applied to a HiTrap Desalting column (Amersham Biosciences), and the PBS buffer solution was replaced with 20 mM Tris-HCl buffer solution. The solution was applied to an anion exchange column HiTrap Q FF column (Amersham Biosciences) to adsorb rCPA63 onto the column. Thereafter, the NaCl concentration was gradient from 20 mM to 1 M, and rCPA63 was eluted from the column. The obtained fraction was analyzed by SDS-PAGE / Western blotting to confirm the eluted fraction of rCPA63. Thereafter, the solution containing rCPA63 was concentrated by a stirring type ultra holder.

The purification process of rCPA63 protein is shown in FIG. 4 and FIG. Lanes 1 in FIGS. 4 and 5 are molecular weight markers. Insect cell culture supernatant (Fig. 4, lane 2), fraction after application to HiTrap Chelating HP column (Fig. 4, lane 3), and rCPA63 protein purified by HiTrap Q FF column (Fig. 4, lane 4) as samples Used to perform SDS-PAGE. After electrophoresis using a separation gel having a polyacrylamide concentration of 12.5%, proteins were stained with a silver staining solution (Daiichi Chemical Industry). As a result, a single band having a molecular weight of about 51,000 daltons (lane 4 in FIG. 4) was confirmed. Further, the result of Western blotting using the Anti-Penta His antibody (FIG. 5, lane 4) confirmed that the protein was rCPA63. Lane 2 in FIG. 5 shows the culture supernatant, and lane 3 shows rCPA63 after purification by His column.
Further, the obtained purified rCPA63 was also subjected to Western blot analysis using anti-crude cedar pollen protein rabbit antiserum prepared by immunizing rabbits with crude extract of cedar pollen as a primary antibody. As the secondary antibody, an anti-Rabbit Ig antibody labeled with HRP (Amersham Biosciences) was used, and immunostaining was performed by emitting light with ECL Plus Western Blotting Detection System (Amersham Biosciences). As a result (FIG. 6), a colored band derived from rCPA63 was confirmed at 51,000 daltons, indicating that rCPA63 reacts with anti-crude cedar pollen protein rabbit antiserum as well as natural cedar pollen-derived protein (FIG. 6). 6 lane 3). In FIG. 6, lane 1 shows a molecular weight marker, lane 2 shows Cry j 2, and lane 4 shows BSA.

<Example 6> Allergenic nature of native CPA63 protein (nCPA63) nCPA63 isolated from a cedar pollen crude antigen was examined for its reaction with cedar pollinosis patient serum. In the same manner as in Example 1, crude cedar pollen antigen was prepared and two-dimensional electrophoresis was performed to separate nCPA63. For each of the 40 cedar pollinosis patient sera, Western blotting was performed in the same manner as in Example 1 to examine the reaction with nCPA63. As a comparative example, the reaction with known major allergens Cry j 1 and Cry j 2 was also examined. The results are shown in Table 1. nCPA63 tested positive in 19 (47.5%) of 40 sera from Japanese cedar pollinosis patients. Moreover, the reaction situation of nCPA63 was different from the reaction situation of Cry j 1 and Cry j 2. From this, it was shown that nCPA63 is a novel cedar pollen allergen that reacts with sera of cedar pollinosis patients at high frequency and has immunological properties different from those of Cry j 1 and Cry j 2.

<Example 7> Allergenicity of recombinant CPA63 protein (rCPA63) The binding ability of the rCPA63 protein expressed and purified in Example 4 and Example 5 to IgE derived from the sera of cedar pollinosis patients was measured by ELISA. First, 50 μl of an antigen solution (10 μg / ml) diluted with 100 mM sodium bicarbonate buffer (pH 9.6) was applied to the well of a microtiter plate. In addition, human IgE standard was first diluted to 200 ng / ml, 50 μl of the double dilution series was added to each well, and left at room temperature for 2 hours. After washing with PBST, 200 μl of blocking buffer [PBS (pH 7.4), 0.5% Tween20, 3% skim milk, 1% BSA] was applied and allowed to stand at 4 ° C. overnight. After washing with PBST, 50 μl of sera of cedar allergic patients and healthy subjects diluted 10-fold with blocking buffer was applied and allowed to stand at 4 ° C. for 4 hours. After washing with PBST, 50 μl of anti-human IgE EPSILON CHAIN BIOTIN CONJUGATE (Biosource International, Inc.) diluted 1,000-fold with blocking buffer was applied and allowed to stand at room temperature for 2 hours. After washing with PBST, 50 μl of alkaline phosphatase-labeled Streptavidin diluted 1,000-fold with blocking buffer was applied and allowed to stand at room temperature for 1 hour. After thorough washing, 50 μl of AttoPhos ^™ was added, and the fluorescence intensity was measured with CytoFluor ^™ II (PerSeptive Biosyatems).

  FIG. 7 shows the results of analysis of rCPA63-specific IgE antibody titers using cedar pollinosis patient serum 30 samples (RAST Score ≧ 3) and healthy subject serum 6 samples (RAST Score = 0). As a reference, a value obtained by adding three times the standard deviation to the average value of six healthy subjects is indicated by a broken line. A specimen showing an IgE value equal to or higher than the value of the broken line was evaluated as positive for the rCPA63 with a risk rate of 5% or less. As a result, 10 patients (P3, 11, 16, 17, 19, 22, 23, 26, 28, 30) of 30 patients were positive for IgE reaction against rCPA63.

SEQ ID NO: 1-Description of artificial sequence: cDNA prepared from amino acid sequence of CPA63

It is a figure which shows the two-dimensional electrophoresis of a cedar pollen crude antigen (CJP). It is a figure which shows the homology of a cedar pollen allergen CPA63 and aspartyl protease family protein / nucleoid DNA-binding-like protein derived from another plant. It is a figure which shows the homology of a cedar pollen allergen CPA63 and aspartyl protease family protein / nucleoid DNA-binding-like protein derived from another plant. It is the figure which analyzed the purification process of recombinant CPA63 allergen by SDS-PAGE. It is the figure which analyzed the purification process of recombinant CPA63 allergen by Western blot. It is the figure which immunostained the recombinant CPA63 allergen using the antibody which immunized the protein derived from a cedar pollen. RCPA63-specific IgE using the value obtained by adding 3 times the standard deviation to the mean value of 30 samples (P1-P30) of cedar pollinosis patients (RAST Score ≧ 3) and 6 samples of healthy subjects (* and dashed line) It is a figure which shows the result of having analyzed the antibody titer.

Claims (11)

It is a protein contained in cedar pollen and has a molecular weight of 50,000 to 60,000 daltons when measured by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 3.5 to when measured by isoelectric focusing. A cedar pollen allergen protein, which is 5.0 and comprises the amino acid sequence set forth in SEQ ID NO: 2.   The cedar pollen allergen according to claim 1, obtained from a crude cedar pollen antigen by a method selected from the group consisting of affinity purification, ion exchange chromatography, gel filtration chromatography, centrifugation, concentration and dialysis. protein.   A nucleic acid molecule having a base sequence encoding the amino acid sequence of the cedar pollen allergen protein described in SEQ ID NO: 2.   A nucleic acid molecule comprising the base sequence set forth in SEQ ID NO: 1 encoding cedar pollen allergen protein.   The nucleic acid molecule according to claim 3 or 4 isolated from a cedar pollen or a cedar male flower.   The cedar pollen allergen protein according to claim 1 or 2, produced in a host cell transformed with the nucleic acid molecule according to claim 3 or 4.   The cedar pollen allergen protein according to claim 1 or 2, prepared by a cell-free expression system.   The cedar pollen allergen protein according to claim 1 or 2, prepared by chemical synthesis.   The cedar pollen allergen protein according to any one of claims 1, 2, and 6 to 8, which reacts with IgE in the serum of a cedar pollinosis patient.   A monoclonal antibody or a polyclonal antibody that specifically reacts with the cedar pollen allergen protein according to any one of claims 1, 2, and 6-8. The diagnostic for hay fever patients containing the cedar pollen allergen protein of any one of Claims 1, 2, and 6-8.