JP2004357717A - EPITOPE OF CEDAR POLLEN ALLERGEN Cry j II - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein or a peptide containing at least one epitope of cedar pollen allergen Cry j II, especially a T cell epitope, useful for diagnosis, prevention and treatment of allergy to cedar pollen. <P>SOLUTION: A cDNA encoding the cedar pollen allergen Cry j II is cloned and the whole amino acid sequence of the Cry j II is determined. Moreover, an overlapping peptide over the full length of the amino acid sequence is synthesized and an overlapping peptide including the T cell epitope is determined using a Cry j II cell line derived from an allergic patient to cedar pollen. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a protein or peptide containing a cedar pollen allergen Cry j II epitope, particularly a T cell epitope, or a DNA encoding the protein or peptide, which is useful for diagnosis, prevention or treatment of cedar pollinosis.

  Cedar pollinosis is an allergic disease that is observed almost nationwide in early spring when cedar pollen scatters, and presents allergic symptoms accompanied by sneezing, nasal discharge, itchy eyes, and the like. The number of such cases has increased sharply since 1970, and currently less than 10% of the population, about 10 million, suffer from cedar pollinosis.

Allergic reactions that form allergic diseases are classified into four types, type I to type IV, by RGH Gell and RRA Coombs, and cedar pollinosis belongs to type I.
The pathogenesis of type I allergy is as follows.

A molecule that causes an allergic reaction is called an allergen (also referred to as an antigen in the present specification). In the case of pollen, this allergen is a protein antigen. When these foreign protein antigens enter the body, they are taken up by antigen-presenting cells (macrophages), broken down by proteolytic enzymes into peptide fragments, and are classified into major histocompatibility complex (MHC) class II molecules. (HLA class II molecules in humans) and presented on the cell membrane. Although HLA class II molecules show polymorphism, the CD4 ⁺ T cell receptor recognizes the antigen peptide bound to the HLA class II molecule together with the HLA class II molecule polymorphic part, and Activated. Activated CD4 ⁺ T cells differentiate into Th0 cells and Th1 / Th2 cells and produce various cytokines. At that time, the cytokine production pattern of each cell is different, Th1 produces IL-2 and IFNγ, Th2 produces IL-4, IL-5, IL-10, etc., and Th0 produces both cytokines.

  B cells, on the other hand, express IgM or IgD on the cell surface and are activated by taking up antigen into the cells. At this time, activated B cells are differentiated and proliferated to antibody-producing cells by the action of cytokines produced from Th2, and produce antigen-specific immunoglobulin E (IgE). The IgE thus produced is firmly bound to mast (mast) cells in the respiratory tract or nasal mucosa tissue or basophils in the blood via the IgE receptor, and sensitization is established.

  When the allergen enters the body again, one molecule of the allergen immediately binds to two or more molecules of IgE on mast cells and basophils to form a crosslinked structure. As a result, receptors bound to IgE molecules associate with each other, which triggers activation of various enzymes in the cell membrane and various chemical mediators such as histamine, prostaglandin, and leukotriene from cells. Released. These chemical mediators act locally on the nasal mucosa and respiratory tract, causing various allergic symptoms.

  An epitope recognized by a T cell is called a T cell epitope, and an epitope recognized by a B cell is called a B cell epitope.

  Since allergen epitopes are considered to be directly involved in the onset and exacerbation of type I allergy, identifying allergen epitopes is useful for diagnosis, prevention and treatment of type I allergy.

  The major allergen of cedar pollen was isolated and purified by Yasue et al. And named Sugi Basic Protein (SBP) (Yasueda, H., et al., J. Allergy Clin. Immunol. 71, 77-86, 1983). . This SBP has a molecular weight of 45-50 kDa and is currently called Cry j I according to WHO nomenclature. Furthermore, during the process of separation and purification of Cry j I, Cry j II having a molecular weight of 37 kDa and having a different antigenicity from Cry j I was separated (Taniai, M. et al. FEBS Letters 239, 329-332, 1988, Sakaguchi, M. et al. Allergy 45, 309-312, 1990).

  These results revealed that Cry j I and Cry j II were completely different proteins.However, it was reported that both Cry j I and Cry j II were reacted in cedar pollinosis patients. Was. That is, of 145 cedar pollinosis patient sera, 134 (92.4%) sera reacted with Cry j I and Cry j II, and 6 (4.1%) sera reacted only with Cry j I, Five (3.4%) sera were found to react only with Cry j II (1993 43rd Annual Meeting of the Japanese Society of Allergology, Hashimoto et al., Nippon University, Pre-Research, National Sagamihara Hospital, Hayashibara Kaken). That is, it was shown that both Cry j I and Cry j II are important for the onset of cedar pollinosis.

  For Cry j I, the cDNA encoding it has been cloned and a peptide containing a T cell epitope has been identified based on its deduced amino acid sequence (WO94 / 01560, "ALLERGENIC PROTEINS AND PEPTIDES FROM JAPANESE CEDAR POLLEN"). For Cry j II, 10 amino acid residues of Ala, Ile, Asn, Ile, Phe, Asn, Val, Glu, Lys and Tyr of the N-terminal amino acid sequence have been reported (Sakaguchi, M., et al. , Allergy 45, 309-312, 1990).

  The present invention provides a protein or peptide containing at least one epitope of the cedar pollen allergen Cry j II, particularly a T cell epitope, or a DNA encoding the protein or peptide, which is useful for diagnosis, prevention and treatment of cedar pollinosis. The purpose is to do.

The present inventors have set out to solve the above-mentioned problems.
(1) Elucidation of the entire amino acid sequence (primary structure) of Cry j II
(2) Preparation of overlapping peptide covering the entire amino acid sequence of Cry j II
(3) Establish a T cell line that specifically recognizes Cry j II allergens individually
(4) Establishment of antigen presenting cells (B cell line)
(5) The present invention was completed by identifying an overlapping peptide containing a T cell epitope. The epitope in the present invention is not limited to the T cell epitope, but the T cell epitope will be described in detail below. Each of these steps is described below.

(1) Elucidation of the entire amino acid sequence of Cry j II (i) Cloning of cDNA
a. RNA extraction
When extracting RNA, proteins are usually removed at an early stage. Therefore, as a general method, there are a phenol extraction method, a method using a guanidinium salt, a surfactant, and a protein denaturant such as urea.

  RNA extraction from cedar pollen can be performed by modifying the method of Breiteneder et al. (Int. Arch. Allergy Appl. Immunol. 87: 19-24, 1988).

Sugi pollen was suspended in 10 to 20 times the volume of extraction buffer (100 mM LiCl, 10 mM Na ₂ EDTA, 1% SDS, 20% mercaptoethanol, 100 mM Tris-HCl pH 9.0), and an equal amount of phenol and chloroform was added thereto. A mixed solution (phenol: chloroform: isoamyl alcohol = 24: 24: 1) is added and homogenized. Then, the mixture is centrifuged (10,000 g, 10 to 15 minutes) to separate into a phenol / chloroform layer and an aqueous layer. At this time, the denatured protein moves to the phenol / chloroform layer, and the nucleic acid moves to the aqueous layer. A mixed solution of phenol and chloroform is added to the aqueous layer, and the mixture is shaken to transfer impurities such as proteins remaining in the aqueous layer to the phenol / chloroform layer and removed. Such an operation is repeated twice.

Properties obtained RNA is extracted from the aqueous layer, a high concentration of LiCl (2 to 4 m) or CH ₃ COO Na (3M) is present when DNA and proteins remain in the supernatant, RNA except tRNA is to precipitate Use Add the same amount of 2-4M LiCl to the aqueous layer to precipitate the RNA. Next, this aqueous layer is dissolved in water, and 0.1 to 0.3 volume of cold ethanol (-20 ° C) is added to precipitate RNA (ethanol precipitation). Then, the precipitate is collected by centrifugation (10,000 g, 30 minutes) and dissolved in water to obtain a total RNA fraction.

b. mRNA preparation and cDNA synthesis
Since Cry j II mRNA has a poly (A) chain at the 3 ′ end, an oligo dT cellulose column (Clontech Laboratories Inc.) to which deoxythymidine (dT) of 12 to 18 bases is bound as a complementary ligand. , CA, USA). A buffer (3 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) is added to the cedar pollen RNA, and the mRNA is adsorbed to the column. The mRNA is eluted with 2-3 bed volumes of NaCl-free buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.4).

  Production of a cDNA library from the obtained mRNA can be performed using a commercially available cDNA library production kit (Amersham International plc., Buckinghamshare, England) using a phage as a vector.

c. Screening of Cry j II cDNA
Although 10 residues of the N-terminal amino acid of Cry j II are already known, Cry j II cDNA can be screened using a synthetic DNA having a nucleotide sequence deduced from this amino acid sequence as a probe. When synthesizing DNA to be used as a probe, it is desirable to design an oligonucleotide that hybridizes to a plurality of possible codon sequences, rather than synthesizing all oligonucleotides containing possible codons. The 5 ′ end of this synthetic oligonucleotide probe is labeled with [γ- ³² P] ATP and polynucleotide kinase, and a positive clone is screened from the cDNA library by plaque hybridization.

  A phage DNA is prepared from the obtained positive clone, the inserted cDNA fragment is separated, and subcloned into a plasmid such as pUC18. Oligonucleotide primers are synthesized as necessary, and the nucleotide sequence is determined by the Sanger method or the like, and clones are identified. SEQ ID NO: 5 shows the full length nucleotide sequence of the Cry j II cDNA isolated by the present inventors.

  The cDNA encoding Cry j II consists of a total of 1733 bp, and includes an open reading frame from a codon assumed to initiate translation (nucleotide ATG at positions 45 to 47) to a stop codon (nucleotide TAA at positions 1587 to 1589). , Encoding 514 amino acids. The nucleotide sequence of the open reading frame is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO: 1. The nucleotide sequence represented by SEQ ID NO: 3 may include polymorphism due to allelic variation between individuals and the resulting amino acid sequence variation. The nucleotide sequence of Cry j II having such a variation is considered. And amino acid sequences are also included in the present invention. The amino acid sequence encoded by the DNA sequence at positions 207 to 236 is Ala, Ile, Asn, Ile, Phe, Asn, Val, Glu, Lys or Tyr, and the N-terminal amino acid sequence of mature Cry j II (Sakaguchi, M. , et al., Allergy 45, 309-312, 1990). The N-terminal 54 amino acids are considered to be signal peptides because they are rich in hydrophobic amino acids found in other signal peptides and are not included in mature Cry j II.

  Cry j II encoded by the DNA sequence from position 207 to stop codons 1587 to 1589 consists of 460 amino acid residues from N-terminal Ala to C-terminal Pro and is considered to be a mature Cry j II. The nucleotide sequence corresponding to the mature Cry j II is shown in SEQ ID NO: 4, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO: 2. The theoretical molecular weight of Cry j II consisting of the amino acid sequence shown in SEQ ID NO: 2 is 50,444 Da. On the other hand, the native mature Cry j II shows a band at 45 KDa in SDS-polyacrylamide gel electrophoresis under reducing conditions (Sakaguchi, M., et al., Allergy 45, 309-312, 1990). This suggests that the C-terminal of Cry j II has been processed. In the amino acid sequence of mature Cry j II, there is Asn-X-Ser / Thr which may have an N-glycoside bond.

  DNA containing the full length of Cry j II or a part thereof is labeled with a fluorescent label, a radioactive label or an enzyme label to encode a biochemical test or a related protein or a protein containing a similar sequence. It can be used as a probe or primer for DNA screening. It can also be connected to an expression vector to express a protein or peptide containing at least one epitope.

(ii) Expression of recombinant Cry j II (rCry j II)
A recombinant protein or peptide containing an epitope of rCry j II or at least one Cry j II can be obtained by incorporating cDNA encoding each into an expression vector, introducing into E. coli, insect cells, yeast or mammals, and culturing. be able to. However, since expression systems using prokaryotic cells such as E. coli do not perform appropriate glycosylation, it is sometimes preferable to use eukaryotic cells such as yeast for rCry j II expression. is there.

  Examples of some expression systems of Cry j II are shown below.

a. Expression in E. coli
System using T7 phage promoter and RNA polymerase (FW Studier, AH Rosenberg, JJ Dunn, JW Dubendonff, "Methods in Enzymology", ed. By DDV Goeddel, vol. 185, p. 60, Academic Press, New York, 1990 ) Can be suitably used in the present invention since the expression success rate is extremely high. In this system, a recombinant plasmid having the gene of interest inserted into the multicloning site downstream of the T7 phage promoter is introduced into E. coli host BL21 (DE3) having the polymerase gene of T7 phage, and This is a system for expressing genes. For example, pGEMEX-1 (Promega) or the like can be used as an expression vector.

  In addition, systems for expressing a target protein by fusing it with a protein capable of large-scale expression are commercially available, and these systems can use an affinity column for purification, have high purification efficiency, and can be suitably used in the present invention. For example, when an expression vector pUEX (Amersham) having β-galactosidase as a fusion protein is used, rCry jII is obtained as a fusion protein with β-galactosidase, and can be efficiently purified with an affinity column. In addition, pGEX having glutathione S-transferase (Pharmacia) and pMAL using maltose binding protein (New England Biolabs, Berverly, MA), etc., have a blood coagulation factor Xa cleavage site introduced at the fusion site thereof. Cry j II can be isolated.

b. Expression in yeast A system using yeast as a host is capable of glycosylation of an expression product, which is advantageous for the expression of the glycoprotein Cry j II. For example, as a system for expressing a heterologous protein by yeast, a method using Pichia yeast as a host is known (JP-A-61-108383, JP-A-61-173781, and JP-A-63-44899). And JP-A-1-128790), and can be suitably used in the present invention. Other yeast expression systems are described in detail in D. Emr Scott, "Methods in Enzymology", ed. By DV Goeddel, vol. 185, p.231, Academic Press, New York (1990). Can be used in the invention.

c. Expression in insect cells In a system using insect cells as a host, the expression product can be glycosylated. A foreign gene expression system using a baculovirus is commercially available (PharMingen, San Diego, CA, USA) and can be suitably used in the present invention. This system is described in Luckow, VA et al., Trends in the Development of Baculovirus Expression Vector, Bio / Technology (September 11, 1987).

d. Expression in mammalian cells High expression can be achieved by incorporating the gene into an expression vector having a mammalian promoter (eg, metallothionein), virus promoter (eg, SV40 early promoter), and introducing the vector into mammalian cells.

(2) Synthesis of Overlapping Peptide In order to elucidate at a molecular level the Cry j II T cell epitope recognized by T cells of hay fever patients, based on the amino acid sequence encoded by the Cry j II cDNA described in SEQ ID NO: 2, Then, an overlapping peptide covering all 460 amino acid residues from Ala at the N-terminus to Pro at the C-terminus is prepared. These overlapping peptides can be easily synthesized by a commercially available automatic peptide synthesizer. From these overlapping peptides, a peptide containing at least one epitope is identified. In particular, in order to identify T cell epitopes, it is necessary to establish a T cell line that specifically recognizes Cry j II and responds to proliferation from peripheral blood lymphocytes of hay fever patients. In general, since T cell epitopes that react with each patient are different, it is desirable to establish a T cell line for each patient.

(3) Establishment of T cell line
To establish a Cry j II antigen-specific T cell line, peripheral blood lymphocytes of a patient are usually cultured for about 7 days in the presence of the Cry j II antigen, and T cells are activated by antigen stimulation. The antigen-specific T cell line can be prepared by repeatedly culturing the modified T cells together with the antigen and the antigen-presenting cells for 7 days and stimulating the antigen several times. However, if the T cells are proliferating well in the presence of the growth factor IL-2, it may be desirable to only stimulate the antigen first. When a T cell line is antigen-stimulated several times, T cells having a high proliferation rate are selectively obtained, and when identifying a peptide containing a T cell epitope, a sufficient proliferation response may not be exhibited depending on the epitope.

  As an antigen to be used, a naturally-occurring Cry j II antigen is desirable in principle, but a mixture of recombinant Cry j II (rCry j II) or overlapping peptide is also preferable since only a very small amount can be extracted from cedar pollen. Can be used. rCry j II can be expressed and purified in Escherichia coli.

(4) Establishment of antigen-presenting cells (B-cell line) As antigen-presenting cells, those obtained by treating or irradiating peripheral blood lymphocytes of the same person as the T cell line with mitomycin C or irradiating the cells are desirable. However, because of the increased number of blood collections, Epstein-Barr virus (EBV) infecting and transforming its own B lymphocytes can continue to grow in vitro in lymphoblastoid cell lines (B cell lines). ), The B cell line may be used as an antigen presenting cell. A method for establishing a B cell line has already been established [Tissue Culture Techniques Second Edition, pp. 187-191, edited by The Histological Society of Japan (1988.8.10)].

(5) Identification of Overlapping Peptide Containing T Cell Epitope A peptide containing a T cell epitope recognized by a T cell line unique to each patient is identified as follows. The term "recognize" as used herein means that the T cell receptor specifically binds to an antigen epitope (including MHC molecules), and as a result, T cells are activated. It is observed by measuring lymphokine production and DNA synthesis using [ ³ H] thymidine incorporation as an index. That is, a T cell line and the same B cell line treated with mitomycin C were seeded on a 96-well flat-bottom plate, mixed and cultured with an overlapping peptide, and the [ ³ H] thymidine uptake (cpm) was measured using a liquid scintillation counter. Measure with At that time, the uptake of [ ³ H] thymidine differs in each culture system. For example, the amount of [ ³ H] thymidine incorporation (cpm) of the T cell line for each peptide is determined by the amount of the control to which no antigen is added. A peptide (stimulation index: SI) of 2 or more divided by the amount of [ ³ H] thymidine incorporation (cpm) is identified as a peptide containing a T cell epitope. Peptides containing the identified T cell epitopes are listed in FIG.

  The following can be considered for the thus-obtained peptide containing at least one T cell epitope of Cry j II of the present invention. From the results of peptide analysis (Chicz, RM et al .: J. Exp. Med., 178: 27-47, 1993), the length of the peptide presented as an antigen by binding to an HLA class II molecule is approximately 10 to Since it is considered that the peptide comprises 34 amino acid residues, the peptide containing the T cell epitope of the present invention also includes a peptide of such a length. Further, by modifying the peptide of the present invention such as amino acid substitution, deletion or addition, and measuring the proliferative response of a T cell line for each patient to these modified peptides, at least one of the Cry j II of the present invention. Since it is easy for those skilled in the art to easily produce a modified peptide having the same function immunologically as a peptide containing a T cell epitope, these modified peptides are also included in the present invention.

  Currently, the desensitizing agent used in desensitization therapy is a crude antigen extracted from cedar pollen and contains a large amount of polysaccharide. Lot-to-lot variation is considerable, and anaphylaxis rarely occurs when the lot is changed after initiating desensitization therapy. In addition, the therapeutic effect of desensitization has not improved so much since the start of desensitization treatment, and about 30% of patients are diagnosed as being highly effective by desensitization therapy.

Among the peptides containing the T cell epitope of the present invention, each peptide that reacts with a T cell line of more than half of hay fever patients is desensitized using a peptide obtained by mixing these peptides alone or a mixture of some of them. With therapy, desensitization may be possible in more than half of the treated patients. In addition, since the peptide used is a chemically synthesized peptide, it is considered that the possibility of causing side effects such as anaphylaxis is reduced. For example, FIG. 5 shows that the overlapping peptides recognized by the T cell lines established from 18 hay fever patients, respectively, are represented by the importance index [“mean stimulus coefficient” (“[ ³ H] thymidine incorporation (cpm) ”divided by“ [ ³ H] thymidine incorporation (cpm) when no antigen is added ”and the“ appearance frequency (%) ”(“ all T Of the “cell line” and the ratio of the “T cell line that recognized the test peptide as a T cell epitope” (%)), and the numbers 14, 17, 29, 38, The peptides of 48, 68, 70 and 71 have an average stimulation coefficient of about 3.9 or more and an importance index of more than 200, and are considered to be particularly effective for the treatment of desensitization.

  In addition, among the peptides containing at least one T cell epitope shown in FIG. 5 and clarified by the present inventors, a peptide having a common part with two kinds of peptides found to contain a B cell epitope described later Is not included. Therefore, the peptide of the present invention is considered not to stimulate B cell epitopes, and thus can be put to practical use as a desensitizer.

  It is also considered that the peptide containing the epitope of the T cell of the present invention can be orally administered to perform oral immune tolerance. Oral tolerance (oral desensitization) is a treatment currently under development, but results that show efficacy have begun to be reported. For example, it has been reported that oral administration of the T cell epitope of Myelin Basic Protein (peptide sequence 21-40, 71-90) to mice suppressed "experimental autoimmune Encephalomyelitis (abbreviated EAE)" [Shuichi Uenogawa] Tatsuhiro Hisatsune, Toshishi Yamura, Molecular Biology of Oral Tolerance, Protein Nucleic Acid Enzyme, 39, 2090-2101 (page 2098 right, lines 9-24) 1994]. From these examples, even in Japanese cedar pollinosis, if the identified T cell epitope peptide is orally administered as it is, or if it is orally administered by encapsulating it in some capsule so as not to be digested in the stomach, the immune tolerance state Could be The epitope peptide is orally administered before the time of the cedar pollen scattering, specifically in the period from December to January, to induce an immune tolerance state. In this state, even if the cedar pollen scatters and pollen adheres to the nasal mucosa, no symptoms are expected or the symptoms are expected to be reduced.

  Furthermore, a peptide containing a T cell epitope of the present invention, which is modified with amino acid substitution, deletion or addition, is synthesized to bind to an HLA class II molecule. Identify untransmitted analog peptides. When these peptides are used in patients as, for example, nasal drops, they naturally inhibit the natural T cell epitope, and thus are expected to prevent their onset.

  The epitope in the present invention includes a B cell epitope. Identification of the B cell epitope can be performed by a known method such as measurement of the reactivity between the overlapping peptide and the patient serum IgE antibody, and detection of inhibition of the binding of the overlapping peptide to the patient serum and the antigen (Japanese Patent Application Laid-open No. 6-69336). It is already known that monovalent B cell epitopes are useful for suppressing allergic reactions. This is thought to be due to the fact that monovalent B cell epitopes bind to the corresponding IgE molecules on mast cells or basophils and inhibit the formation of IgE molecule cross-links by multivalent epitopes. The present inventors synthesized overlapping peptides covering the entire amino acid sequence of Cry j II of the present invention, and measured the reaction between these peptides and serum IgE antibodies of cedar pollinosis patients by the enzyme antibody method. "Gln Cys Lys Trp Val Asn Gly Arg Glu Ile Cys (amino acid sequence 113-123)" and "Cys Thr Ser Ala Ser Ala Cys Gln Asn (amino acid sequence 293-301)" clearly contain B cell epitopes I made it. Such a peptide containing the B cell epitope of Cry j II is useful for diagnosis, prevention and treatment of cedar pollinosis.

  The peptide containing at least one epitope of Cry j II and the peptide containing a T cell epitope of the present invention are useful for diagnosis, prevention and treatment of cedar pollinosis.

  Furthermore, it is also possible to synthesize analog peptides that bind to HLA class molecules but do not transmit information to T cells, and use these peptides to prevent the onset of cedar pollinosis by inhibiting competition.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

<Collection of cedar pollen>
Japanese cedar pollen was collected from male flowers that bloomed on Japanese cedar branches cut down in February in Shizuoka and Kanagawa prefectures. Japanese cedar pollen for Cryj II antigenic purification was stored at -70 ° C, and Japanese cedar pollen for RNA preparation was rapidly frozen in liquid nitrogen and stored at -70 ° C.

<RNA extraction>
RNA was extracted from cedar pollen with modifications based on the method of Breiteneder et al. (Int. Arch. Allergy Appl. Immunol. 87: 19-24 1988).

1 g of the cryopreserved cedar pollen was suspended in 15 ml of an ice-cooled extraction buffer (100 mM LiCl, 10 mM Na ₂ EDTA, 1% SDS, 20% 2-mercaptoethanol, 100 mM Tris-HCl, pH 9.0), and further, 15 ml of Phenol: chloroform: isoamyl alcohol (24: 24: 1) was added. This suspension was transferred to a Teflon homogenizer, and homogenized for 20 to 30 strokes while rotating the Teflon pastel at the maximum speed with a motor. Thereafter, the aqueous layer and the organic layer were separated by centrifugation (10,000 g, 15 minutes) to obtain an aqueous layer. The same amount of phenol: chloroform: isoamyl alcohol was added to the aqueous layer, shaken for 5 minutes, and centrifuged (10,000 g, 15 minutes) to obtain an aqueous layer. The same operation was repeated twice, and once with 15 ml of chloroform: isoamyl alcohol (24: 1). The same amount of 4M LiCl was added to the obtained aqueous layer, and the mixture was allowed to stand at -20 ° C overnight. The frozen solution was thawed at room temperature, and a precipitate was obtained by centrifugation (20,000 g, 30 minutes). The precipitate was dissolved in a small amount of sterile distilled water, 0.3 volume of 3M CH ₃ COONa, pH 5.2 and 2.5 volumes of ethanol were added, and the mixture was allowed to stand at -20 ° C for 60 minutes. The precipitate collected by centrifugation (10,000 g, 30 minutes) was redissolved in sterile distilled water to obtain a total RNA fraction.

<Preparation of cedar pollen mRNA and cDNA synthesis>
After adding the same amount of binding buffer (3 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) using 1 mg of total RNA of cedar pollen as a starting material, a span column (CLONETECH Laboratories Inc. About 10 μg of mRNA was purified by eluting with an elution buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.4) (according to the protocol attached to CLONETECH Lab. Inc.). . Subsequently, about 4 μg of cDNA was synthesized from 5 μg of the purified mRNA using cDNA Synthesis System Plus (manufactured by Amersham International plc., Buckinghamshare, England) according to the attached protocol.

<Synthesis of oligonucleotide probe>
The amino acid sequence of 10 residues from the N-terminus of Cry j II is shown in FIG. 1A. The cDNA sequence predicted from this amino acid sequence is shown in FIG. 1B. Oligonucleotide probes (Oligo CJII) were synthesized as a mixture of 16 types in total, since two types of bases were used complementary to the sequence and at four positions (FIG. 1C). G: T base pairs are allowed to reduce species as a mixture.

<Cloning of Cry j II cDNA>
The cDNA library was prepared using a cDNA cloning system λgt10 (manufactured by Amersham International plc., Buckinghamshare, England) according to the attached protocol. 1 μg of the above cDNA was incorporated into λgt10 to prepare a cDNA library. Approximately 5,000 clones of approximately 500,000 libraries were spread on a single 150 mm diameter plate. As a probe for screening, the above oligonucleotide (Oligo CJII) was labeled with [γ- ³² P] ATP (7,000 Ci / mmol ICN Biochemicals, Inc.) using T4 polynucleotide kinase. The nitrocellulose filter on which the phage DNA was immobilized was washed with 5 × SSPE (1 × SSPE: 0.18 M NaCl, 10 mM sodium phosphate, 1 mM EDTA), 5 × FBP (1 × FBP: 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% % Polyvinylpyrrolidone), 0.3% SDS, and 100 μg / ml tRNA. Thereafter, the nitrocellulose filter was immersed in the newly prepared solution, a ³² P-labeled probe (Oligo CJII) was added, and hybridization was performed at 48 ° C. overnight. Thereafter, the filter was washed with a solution containing 6 × SSC (1 × SSC: 0.15 M NaCl, 0.015 M sodium citrate) and 0.1% SDS at room temperature of 30 ° C. and 48 ° C. for 5 minutes, and then subjected to autoradiography. Four strong signals were detected. One of the phage DNAs was extracted and digested with the restriction enzyme EcoRI, and it was found that a DNA fragment of about 1.7 Kbp was inserted. The inserted fragment was subcloned into pUC118, and a deletion mutant was prepared using a kilosequence deletion kit (Takara Shuzo) and used for determination of the entire nucleotide sequence. The nucleotide sequence was determined by performing a primer extension reaction using a synthetic primer and a dye-labeled dideoxy terminator, and reading it with an automatic sequencer (Model 370A, Applied Biosystems, Japan). The determined total nucleotide sequence of the cDNA is shown in SEQ ID NO: 5. In addition, the nucleotide sequence of only the open reading frame is represented by SEQ ID NO: 3 (the amino acid sequence encoded by the nucleotide sequence is represented by SEQ ID NO: 1), and the nucleotide sequence encoding mature Cry j II is represented by SEQ ID NO: 4 (the nucleotide sequence is encoded by SEQ ID NO: 1). The amino acid sequence is shown in SEQ ID NO: 2

<Expression of recombinant Cry j II in Escherichia coli>
The E. coli expression vector pGEMEX-1, commercially available from Promega, has a T7 promoter, T7 gene10 coding sequence and T7 terminator, and inserts an open reading frame into the multiple cloning site downstream of T7 gene10 to express T7 RNA polymerase. This is a vector that is highly expressed when introduced into Escherichia coli (eg, BL21 (DE3)). The Cry j II cDNA was digested with BamHI (an adapter linked to both ends of the cDNA contains a BamHI site), a cDNA fragment was cut out and inserted into the BamHI site of pGEMEX-1, to construct a Cry j II expression vector pEXCJII. pEXCII can express a fusion protein (T7 Cry jII, 73 kD) of a T7 gene10 expression product (23 kD) and a Cry j II protein (50 kD). A transformant in which pEXCII was introduced into Escherichia coli BL21 (DE3) was cultured, and T7 RNA polymerase was induced with IPTG to express Cry j II. The expressed E. coli cell extract was subjected to SDS polyacrylamide gel electrophoresis. BL21 (DE3) carrying pEXCII showed a band of about 73 kD, which was thought to be T7 Cry jII. However, these bands were not seen in the control BL21 (DE3) carrying the pGEMEX-1 or the parent strain BL21 (DE3).

<Reactivity of fusion protein of Cry j II and T7 gene10 (T7Cry j II) with serum of Japanese cedar pollinosis patient>
The extract of Escherichia coli expressing T7 Cry j II was subjected to SDS polyacrylamide gel electrophoresis, and then subjected to Western blotting on a Millipore PVDF membrane to obtain serum of five patients with cedar pollinosis and three healthy persons. Was studied for its reactivity. As a control, BL21 containing pGEMEX-1 and an extract of BL21 expressing a fusion protein of T7 gene10 and Cry j I (T7 Cry j I) and natural Cry j I purified from cedar pollen were simultaneously blotted and reacted. Was examined. As shown in FIG. 2, two patient sera reacted with T7 Cry j II. Both sera of the two patients reacted with T7 Cry j II and native Cry j I, but did not react with BL21 extract containing pGEMEX-1 or T7 Cry j I. From these results, it was confirmed that T7Cry j II has an antigenicity that reacts with IgE in the serum of cedar pollinosis patients.

<Synthesis of overlapping peptides>
The overpeptide was synthesized using Peptide Synthesizer PSSM-8 (manufactured by Shimadzu Corporation). Based on the primary structure of Cry j II shown in SEQ ID NO: 2, 90 types of 15-mer overlapping peptides including 10 residues overlapping portion, starting from Ala at the N-terminal 55th position to Pro at the C-terminal Was synthesized. FIGS. 3 to 6 show all overlapping peptides synthesized using the one-letter code of amino acids.

<Establishment of B cell line>
Peripheral blood lymphocytes (1 × 10 ⁶ ) obtained by Ficoll-Paque specific gravity centrifugation were incubated with about 1 × 10 ⁶ PFU (plaque forming units) of Epstein-Barr virus (EBV) for 1 hour at 37 ° C. To infect cells. When the virus-infected cells are transferred to a 24-well culture plate and cultured for about 2 weeks in the presence of 100 ng / ml cyclosporin A, B cell colonies appear. At this point they were split in half and transferred to new wells. When this operation is sequentially repeated and subculture is performed, B cells capable of self-proliferation may appear. The cells in the wells containing the self-proliferating B cells were expanded, and after confirming the proliferation, the cells were transferred to a 25 cm ² culture flask, cultured for 30 to 50 days, and transformed by EBV (EBV-transformed). A B cell line was obtained. Some of the B cell lines were stored frozen.

<Establishment of Cry j II antigen-specific T cell line>
Lymphocytes were isolated from peripheral blood of 18 patients with cedar pollinosis by Ficoll-paque specific gravity centrifugation, and stored in liquid nitrogen until use. Peripheral blood lymphocytes (4 × 10 ⁶ ) of cedar pollinosis patients were suspended in 2 ml of RPMI-1640 supplemented with 20% of autologous plasma, and expressed and purified in 10 μg / ml Escherichia coli. The cells were cultured for 7-8 days on a 24-well culture plate together with the II antigen. At the time when T cells activated by the Cry j II antigen stimulation (blasting reaction, blastogenesis) were confirmed under a microscope, 5 Unit / ml of IL-2 was added, and the cells were cultured overnight. From the next day, the cells were cultured for 9 days with RPMI-1640 supplemented with 20 Unit / ml IL-2 and 20% human AB type serum (commercially available) while changing the culture medium every day. At this point, a portion of the expanded T cell line that specifically recognizes the Cry j II antigen was cryopreserved. Further, the T cell line was cultured in the above culture solution for 4 days to obtain cells for epitope identification.

<Identification of overpeptide containing T cell epitope>
T cell lines established from 18 hay fever patients were cultured with cedar pollen allergen overlapping peptides, respectively, and peptides containing Cry j II antigen-specific T cell epitopes were identified.

Cultured B cell lines established from the same patient as the T cell line were treated with 50 μg / ml mitomycin C for 30 minutes and the cells were washed four times with RPMI-1640. After inoculating the B cells into a 96-well flat-bottomed plate (5 × 10 ⁴ / well), Cry j II (25 μg / ml final concentration) or each overlapping peptide (final concentration 0.5 μM ) Was added to each well and incubated for about 60-90 minutes. A T cell line (2 × 10 ⁴ / well) was inoculated in each well, and after culturing for 48 hours, 0.5 μl / Ci [ ³ H] thymidine was added to the well, and the cells were further cultured for 16 hours. The cells were collected on a glass filter using a cell harvester, dried, and the count (cpm) of [ ³ H] thymidine incorporated into the cells was measured with a liquid scintillation counter.

The measurement was performed in triplicate culture, results, ^[3 H] thymidine uptake of T cell lines by overlapping peptides stimulate (cpm), ^[3 H] thymidine uptake without the addition of antigen (control) (cpm) The overlap peptide having a value of 2 or more, which was calculated by a stimulation index (SI) which was a value divided by, was identified as an overpeptide containing a T cell epitope. FIGS. 7 and 8 show peptides containing at least one T cell epitope recognized by at least one Cry j II antigen-specific T cell line established from 18 hay fever patients. FIG. 9 shows “average stimulation coefficients” (average stimulation coefficients obtained by a plurality of experiments), “appearance frequency (%)”, and “importance index” for all overlapping peptides.

Amino acid sequence of 10 residues from N-terminal of cedar pollen allergen Cry j II (A). DNA sequence (B) predicted from the amino acid sequence of 10 residues from the N-terminus of cedar pollen allergen Cry j II. DNA sequence of a probe for screening cDNA encoding cedar pollen allergen Cry j II (C). FIG. 3 shows the results of identifying the antigenicity of T7 Cry j II by Western blotting using sera of two cedar pollinosis patients. Lane 1 is BL21 (DE3) retaining pMGEMEX-1 (negative control), lane 2 is BL21 (DE3) expressing T7 Cry j I, lane 3 is BL21 (DE3) expressing T7 Cry j II, lane 4 Indicates Cry j I purified from cedar pollen. A and B are the same except for the patients from which the serum is derived. 1 shows overlapping peptides covering the entire amino acid sequence of Cry j II. 1 shows overlapping peptides covering the entire amino acid sequence of Cry j II. 1 shows overlapping peptides covering the entire amino acid sequence of Cry j II. 1 shows overlapping peptides covering the entire amino acid sequence of Cry j II. 2 shows a peptide comprising at least one T cell epitope of Cry j II. 2 shows a peptide comprising at least one T cell epitope of Cry j II. [Fig. 3] Fig. 3 shows the importance index of overlapping peptides recognized by T cell lines specific to Cry j II allergens established from 18 cedar pollinosis patients.

Claims (4)

SEQ ID NO: 6 (peptide number 1), SEQ ID NO: 7 (peptide number 4), SEQ ID NO: 8 (peptide number 5), SEQ ID NO: 9 (peptide number 6), SEQ ID NO: 10 (peptide number 7), SEQ ID NO: 11 (peptide No. 8), SEQ ID No. 12 (peptide No. 9), SEQ ID No. 13 (peptide No. 10), SEQ ID No. 14 (peptide No. 14), SEQ ID No. 15 (peptide No. 16), SEQ ID No. 16 (peptide No. 17), sequence No. 17 (peptide No. 18), SEQ ID No. 18 (peptide No. 25), SEQ ID No. 19 (peptide No. 26), SEQ ID No. 20 (peptide No. 27), SEQ ID No. 21 (peptide No. 28), SEQ ID No. 22 (peptide No. 29), SEQ ID NO: 23 (peptide No. 30), SEQ ID NO: 24 (peptide No. 31), SEQ ID NO: 25 (peptide No. 32), SEQ ID NO: 26 (peptide No. 33), SEQ ID NO: 27 (peptide No. 34), SEQ ID NO: 28 (Peptide No. 37), SEQ ID No. 29 (Peptide No. 38), SEQ ID No. 30 (Peptide No. 41), SEQ ID No. 31 (Peptide No. 42) SEQ ID NO: 32 (peptide number 44), SEQ ID NO: 33 (peptide number 47), SEQ ID NO: 34 (peptide number 48), SEQ ID NO: 35 (peptide number 49), SEQ ID NO: 36 (peptide number 50), SEQ ID NO: 37 ( Peptide No. 51), SEQ ID No. 38 (Peptide No. 52), SEQ ID No. 39 (Peptide No. 53), SEQ ID No. 40 (Peptide No. 54), SEQ ID No. 41 (Peptide No. 60), SEQ ID No. 42 (Peptide No. 61), SEQ ID NO: 43 (peptide No.62), SEQ ID NO: 44 (peptide No.63), SEQ ID NO: 45 (peptide No.64), SEQ ID NO: 46 (peptide No.65), SEQ ID NO: 47 (peptide No.66), SEQ ID NO: 48 (peptide No. 68), SEQ ID No. 49 (peptide No. 69), SEQ ID No. 50 (peptide No. 70), SEQ ID No. 51 (peptide No. 71), SEQ ID No. 52 (peptide No. 72), SEQ ID No. 53 (peptide No. 73), sequence No. 54 (peptide No. 74), SEQ ID No. 55 (peptide No. 78), SEQ ID No. 56 (peptide No. 79), SEQ ID No. 57 (peptide De No. 86), and peptides chosen from the group consisting of SEQ ID NO: 58 (Peptide No. 87);
Alternatively, a peptide in which amino acids have been substituted, deleted and / or added in the above peptide, and the peptide has an average stimulation coefficient of 2 or more.   SEQ ID NO: 14 (peptide No. 14), SEQ ID NO: 16 (peptide No. 17), SEQ ID NO: 22 (peptide No. 29), SEQ ID NO: 28 (peptide No. 37), SEQ ID NO: 29 (peptide No. 38), SEQ ID NO: 34 (peptide No. 48), SEQ ID No. 48 (peptide No. 68), SEQ ID No. 49 (peptide No. 69), SEQ ID No. 50 (peptide No. 70), and selected from the group consisting of SEQ ID No. 51 (peptide No. 71), cedar pollen allergen 2. The peptide according to claim 1, comprising at least one T cell epitope of Cry j II.   A DNA encoding the peptide according to claim 1 or 2.   An agent for preventing or treating cedar pollinosis, comprising the peptide according to claim 1 or 2 or a mixture thereof as an active ingredient.

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