JPH06508994A - Allergenic proteins and peptides from Japanese cedar pollen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 11. A host transformed using the nucleic acid sequence according to claim 1.2.3 or 4. Purified Japanese cedar pollen allergen CrL produced in main cells 1■ or at least 1 of its antigenic fragments.

12. The Japanese cedar pollen allergen is immunoglobulin I specific for Japanese cedar pollen. Does not bind to gE or binds Japanese cedar pollen allergen to the immunoglobulin E If such binding occurs, the release of histamine from mast cells or basophils The purified Japanese cedar pollen allergen according to claim 11, characterized in that it does not cause .

13. The Japanese cedar pollen allergen is free from the purified original Japanese cedar pollen allergen. binds to immunoglobulin E to a substantially lower extent than when binding to immunoglobulin E; The purified Japanese cedar pollen allergen according to claim 11, wherein the purified Japanese cedar pollen allergen is

14. The purification date according to claim 11, wherein the host cell is E. coli. Japanese cedar pollen allergen or antigenic fragment thereof.

15 a) Codes for Japanese cedar pollen allergen Cry j I or its fragment A host cell transformed with the DNA sequence of the Japanese cedar pollen allergen C Mixing cells and medium containing ryj I or at least one fragment thereof b) purifying the mixture to produce substantially pure Japanese Japanese cedar pollen allerken Cry I, including cedar pollen allergen, or at least How to produce one such fragment.

16. Nucleic acid sequence encoding the whole or part of Japanese cedar pollen allergen CryjI Japanese cedar pollen allergen synthesized in host cells transformed using 1. A protein composition comprising a protein or at least one fragment thereof.

17. At least one of said fragments is an antigenic fragment. 17. The protein composition according to claim 16, characterized in that:

18. Chemically synthesized Japanese cedar pollen allergen Cry or at least A protein composition comprising one fragment thereof.

19, characterized in that the Cry↓I has the amino acid sequence of SEQ ID NO: 1 The protein composition according to claim 16 or 18.

20. Isolated antigenic fragment of allergen from Japanese cedar pollen.

21 The allergen from Japanese cedar pollen is Cry j I Antigenic fragment according to claim 2o.

22, that the antigenic fragment contains at least one T cell epitope; Antigenic fragment according to claim 2o or 21, characterized in:

23, that the antigenic fragment has minimal immunoglobulin E [activity]. 23. The antigenic fragment of claim 22, characterized in:

24. The antigenic fragment does not bind to Japanese cedar pollen-specific immunoglobulin. or if binding of the fragment to said immunoglobulin E occurs, such The binding is characterized by preventing the release of histamine from mast cells or basophil cells. 23. The antigenic fragment according to claim 22.

25 The antigenic fragment is immunized with the purified original Japanese cedar flower allergen. binds to said immunoglobulin E to a substantially lower extent than when binding to said immunoglobulin E; Antigenic fragment according to claim 2o, characterized in that:

26. The purified allergen or the antigenic fragment is transmitted to the Japanese cedar flower to which it has been administered. The allergic response to Japanese cedar pollen can be modified in powder-sensitive patients. The purified allele according to claim 11.20.21 or 22, characterized in that gene or antigenic fragment.

27. The purified allergen or the antigenic fragment is the patient's Japanese cedar pollen allergen. B-cell responses to cedar pollen antigens, T-cell responses to Japanese cedar pollen antigens in patients, or It is possible to modify both B cell and T cell responses to Japanese cedar pollen allergen. The purified allergen or antigenic flag according to claim 26, characterized in that gement.

28 Isolated antigenic fragment of Japanese cedar pollen allergen according to claim 20 A nucleic acid sequence encoding.

29. When administered to Japanese cedar pollen sensitive patients, the patient's response to Japanese cedar pollen allergen A modified Japanese cedar pollen allergen that reduces allergic responses.

30. The modified Japanese cedar pollen allergen is a modified p-yjI protein. The modified cedar pollen protein allergen according to claim 29.

31. When administered to Japanese cedar pollen sensitive patients, the patient's response to Japanese cedar pollen allergen Japanese cedar pollen allergen (with one modified protein) reduces allergic response. Ragment.

32, at least one modified fragment is a modified fragment of CryjI protein. at least one modification according to claim 31, characterized in that fragment.

゛゛33. Isolated protein immunologically related to Cryj I or a fragment thereof quality allergen or antigenic fragment thereof.

34, the protein allergen or antigenic fragment thereof is CryjI or its Claim 33, characterized in that it binds to an antibody specific to the fragment. An isolated protein allergen or antigenic fragment thereof.

35, the protein allergen or antigenic fragment thereof is Claim 33 is characterized in that it is capable of activating fragment-specific T cells. Isolated protein allergens or fragments thereof in their antigenic forms and pharmaceuticals as described Claim 36, characterized in that: The therapeutic composition described in.

38. The protein composition according to claim 16 or 18 in a therapeutically effective amount for mammals. Japanese wood pollen allergen or Japanese wood pollen allergen and immunization, including administering In mammals that are biologically susceptible to cross-reactive allergens, How to treat susceptibility to

39, for example, Japanese wood pollen allergen or cross-reactive with Japanese wood pollen allergen. Claims for use in therapy, such as treatment of patient sensitivity to allergens 19. The protein composition according to 16 or 18.

40, @A blood sample obtained from a mammal is transformed using the nucleic acid sequence according to claim 1. Purified Japanese pollen alleles produced in transformed host cells or chemically synthesized or antigenic fragments thereof, blood components and proteins or fragments thereof. of the mammal under suitable conditions for binding and determining the extent to which binding occurs. A method for detecting susceptibility to Japanese wood pollen allergen in species.

41. The extent to which binding occurs is determined by T cell function, T cell proliferation, B cell function, protein or Evaluation of the binding of fragments of 41. The method according to claim 40, characterized in that the method is determined by the value of the product.

42, produced in a host cell transformed using the nucleic acid sequence according to claim 1. purified or chemically synthesized Japanese wood pollen allergen Cry ↓ I or its antigenic fragments in an amount sufficient to cause an allergic response in the mammal. the Japanese pollen allergen or its antigenic fragment in the patient. including the step of determining whether an allergic response to the substance occurs. Methods for detecting mammalian susceptibility to powder allergens.

43. Japanese cedar pollen allergen>Cry j I or at least one antigenic molecule thereof A monoclonal antibody specifically reactive with the fragment.

Specification Background of the invention of allergenic proteins and peptides from Japanese wood pollen Genetically predisposed individuals, who make up approximately 10% of the population, are affected by the variety to which they are exposed. hypersensitivity (allergy) to antigens from environmental sources. Immediate and/or delayed Antigens that induce hypersensitivity are known as allergens. (King, T, P , Adv, Immuno 1.23: 77-105 (1976)). flower Anaphylaxis or atopy that induces symptoms of powdery mildew, asthma, and hives should be treated immediately. This is one form of seasonal allergy.

It contains a wide variety of materials such as grass, trees, weeds, animal dander, insects, food, pharmaceutical and chemical products, etc. It can be caused by various atopic allergens.

Antibodies associated with atopic allergies mainly belong to the IgH type of immunoglobulin. do. IgE binds to mast cells and basophils. Binds to mast cells or basophils When combined with a specific allergen, IgE cross-links on the cell surface. and produce the physiological effects of IgE-antigen interaction. These physiological effects include Histamine, serotonin, heparin, eosinophils and/or leucosin, among other substances Includes the release of chemotactic factors C4, D4 and E4 for triene, which causes contraction of bronchial smooth muscle for a period of time (Hood, L.E., et al., Immu. nol.

gy (2nd ed,), The Benjamin/CummingPubl Cumm1n Co, Inc, (1984))a These released substances are A medium that causes allergic symptoms caused by a combination of IgE and specific allergens A mediator. The effects of allergens are manifested through them. Their effects depend on the antigen. Depends on the route of entry into the body and the pattern of IgE deposition on mast cells or basophils. may be systemic or local in nature. Local manifestations are generally caused by the allergen entering the body. Occurs on the surface of the epithelium at locations where Systemic effects include anaphylaxis (anaphylaxis) -shock), which involves the IgE-basophil response to circulating (intravascular) antigens. This is the result.

Japanese wood (Sugi: Cryptomeria japonica) aponica)) Hay fever is one of the most serious allergic diseases in Japan. Ru. The number of people affected by this disease is increasing, in some regions up to 10% of the population. More than % are affected. Avoid allergens by administering Japanese pollen extract. Attempts have been made to treat Japanese wood pollinosis by sensitization. However, Japanese wood pollen extract Desensitization using can induce anaphylaxis if high doses are used. , use low dosages to avoid anaphylaxis and ensure tolerance to the extract. The disadvantage is that treatment must be continued for several years to recover.

The main allergen from Japanese wood pollen has been purified, and cedar basic protein (Sugi b) has been purified. It was named asic protein (SBP) or Cry-U. this The protein is a basic protein with a molecular weight of 4l-50kDa and a pi of 8.8. It has been reported. Allergens appear to have numerous isoforms, some of which are This may be due to different glycosylation (Yasueda et al. (198 3) The sequence of J, Aller acid and 16 internal amino acid sequences were determined (Ta A second allergen of niaiDa was also reported (Sakaguchi et al. Found.

In addition to desensitizing patients with Japanese cedar pollen allergy using low dosages of Japanese cedar pollen extract, 199 U.S. Patent No. 4 issued to Matsuhashi et al. ．． No. 939.239 contains a saccharide covalently bound to a Japanese cedar pollen allergen. A desensitizing explosive agent for desensitizing patients susceptible to Japanese cedar pollen is disclosed. This desensitization Explosive drugs enhance the production of IgG and IgM antibodies, but they are allergen-specific and anaphylactic. It has been reported that it reduces the production of IgE antibodies that are responsible for allergies and allergies. Ru. The allergen used in the desensitizing explosive is Asp-Asn-Pro-11e-As p-3er-X-Trp-Arg-Gly-Asp-3e r-Asn-Trp -NH2-terminal amino acid of -Ala-Gin-Asn-Arg-Met-Lys- sequence, where X is Ser, Cys, Thr or His (SEQ ID NO. 1) is preferred. Furthermore, Usui et al. (1990) Int. Arch, Allergy App+.

j 1) - The ability of the pullulan complex to induce the Althus reaction is significantly reduced, and the present It is reported that the cedar basic protein is about 1.000 times lower than the conventional cedar basic protein. Our results suggest that orchid complex is an excellent candidate for desensitization treatment against cedar pollinosis. .

The Cry J I allergen found in Cryptomeria japonica is Cupressus sempervirens ) was also found to be cross-reactive with allergens in pollen from other species of trees, including It was done. Panzani et al (Annals of Al lazy 5:26-30 (1986) on skin tests, RAST and RAST inhibition. In the pollen of Kupressus sempervirens and Cryptomeria japonica reported that cross-reactivity was detected between allergens. Yamamura (Mount ainCeder) (Juniperus sabinoides) The 50 kDa allergen isolated from P. inoides) has an NH2-terminal sequence. Gros having AspAsnPro Il eAsp (SEQ ID NO: 25) et al. (1978) Scand, J., 1mmuno], 8: 4 37-441), it is the first 5 NH,-terminus of the Cry I allergen. The sequence is identical to the number of amino acids. The Cry-jl allergen is also associated with the following species of trees: Qubresus Arisoni force was found to be allergenically cross-reactive. (Cupressus sarizonica), Cupressus macrocarba (Cupressus sarizonica) pressus macrocarpa), Jupels Bilginiana (J uniperus virginiana), Juniberus communis (Jun iperus communisL Tsuya orientalis (Thuya ori) entalis)’ & Chamaecyparis Ostusa (Chamaecypari) s obtusa).

Despite the attention that the Japanese cedar pollen allergen has received, there is no evidence that it is responsible for the negative effects it has on people. The definition or characterization of caries allergens is far from complete. Current desensitization treatments require high dosages Risk of anaphylaxis when administering large amounts of pollen extract or low doses of pollen Includes treatment with pollen extracts with a long desensitization period when the extract is administered.

Outline of the invention The present invention relates to Curvutomelia japonica pollen major allergen ycry jI and its Nucleic acid sequences encoding the fragments are presented. The present invention is based on Cry j I or Transformed with a nucleic acid sequence encoding a small number of fragments thereof Purified 9E II produced in host cells and a small amount (all one fragment thereof) Crypt and/or synthetically produced butthogonium j Nucleic acid sequence having fewer bases than the nucleic acid sequence encoding the entire amino acid sequence of I say. CryjI and its fragments for diagnosis, treatment and prevention of Japanese cedar pollinosis It is useful for The invention is more particularly described in the appended claims and as follows: The preferred embodiments are described in the description.

Brief description of the drawing Figure 1a shows 10mM sodium acetate (pH 5,0) and 15M NaCl. Figure 1b on equilibrated 5upe rdex75 (2,6x60cm) 5DS-PAGE (12,5%) analysis of fractions from the main peak shown in 8. show.

Figure 2 was separated by 5DS-PAGE, and Figure 3 was separated using mAB CBF2. FIG. 2 is a graph of purification using plasma from a pool of five allergic patients.

Figures 4a-b show two overlapping clones JC encoding CryjI. 71,6 and pUc19Jc91A are shown. Cry　j　I The complete cDNA sequence consists of 1312 nucleotides, including 66 nucleotides of 5' untranslated sequence. Cleotide, a 1122 nucleotide read starting at the start methionine codon frame, and 3' untranslated region.

Figures 4a-b also show the deduced amino acid sequence of Cry j I.

Figure 5a shows that the coating antigen (coating antigen) was derived from Japanese cedar pollen. FIG. 2 is a graphical representation of the results of IgE binding activity in the case of soluble pollen extract (SPE). Ru.

Figure 5b shows the IgE binding activity when the applied antigen is purified native CryjI. FIG.

Figure 6 shows the results when the applied antigen is soluble pollen extract (SPE) from Japanese cedar pollen. , competitive ELISA using pooled human plasma (PHP) from 15 patients ( It is a graph diagram of the results of competition ELISA).

Figure 7 shows that the applied antigen is soluble pollen extract (SPE) from Japanese cedar pollen, and the competition For each patient (indicated by patient number) when the antigen is purified native Cry j I. FIG. 2 is a graphical representation of the results of a competitive ELISA using plasma from

Figure 8a shows when the applied antigen is soluble pollen extract (SPE) from Japanese cedar pollen. Direct binding ELIS using plasma from each of 7 patients (indicated by patient number) FIG. 3 is a graphical representation of the results from A.

Figure 8b shows a modified version in which the coated antigen was denatured by boiling in the presence of the reducing agent DTT. from each of the seven patients (indicated by patient number) as a soluble pollen extract. FIG. 2 is a graphical representation of results from a direct binding ELISA using plasma.

Figure 9 shows that recombinant Cry j I (rCr j I) was applied to the wells and each patient was Figure 2 is a graphical representation of a direct ELISA assay for IgE binding in individuals.

Figure 10a shows that CBF2 (IgG) mAb was applied to the wells and used as a negative antigen standard. of 15 patients using PBS and when the antigen was purified recombinant Cryj I. Capture ELISA (capture ELISA) using human plasma pooled from individuals It is a graph diagram of the result of A).

Figure 10b shows that 20 μg/rr++ CBF2 (IgG>) was applied to the wells and S It is a graph figure of the result of A.

Figure 11 shows SPE from Japanese cedar pollen and purified original Cryj as added antigens. Using T and recombinant Cry j I, one patient with Japanese cedar pollen allergy was tested. FIG. 2 is a graphical representation of a histamine release analysis conducted.

Figure 12 shows that the antigen is the recombinant Cry I protein and the purified original Cry protein. ■Protein or recombinant 8mb a 1. If it is 1, then patient number #999? A graphical representation of the results of T cell proliferation analysis using the blood of these patients. Nucleic acid sequences are shown in Figures 4a and 4b. Preferably, it has the sequence shown (SEQ ID NO: 1). Cry shown in Figures 4a and 4b i The nucleic acid sequence encoding I (SEQ ID NO: 1) is from base 66 to base 128. Contains a 21 amino acid leader sequence. This leader sequence starts from base 129 to 1 cleaved from the mature protein encoded by up to 187. Cry j I's recommendation The constant amino acid sequence is also shown in Figures 4a and 4b (SEQ ID NO: 2). The nucleic acid sequence of the invention is It has an estimated molecular weight of 38.5 kDa, a pi of 7.8, and 5 N-linked glycans. Encodes a protein with a site capable of cosylation. Using these glycosylation sites presence will increase the molecular weight and affect the pi of the mature protein. Nucleic acid sequences of the present invention The deduced amino acid sequence for the mature protein encoded by the sequence Tan1ai The known NH,-terminal and internal amino acid sequences reported by are the same. Tan1ai et at,, as reported by the same as above. NH! - The end has the sequence shown in SEQ ID NO: 18. Tan1ai et al.

The internal sequence reported by , ibid., is the sequence GluAIaPheAsnVa IG IuAsnGlyAsnAlaThrProGlnLeuThrLys (sequence Number: 19). Sequence length characteristics are observed in the nucleic acid sequences of the present invention. for example One in the codon encoding amino acids 38.51 and 74 of SEQ ID NO: 1 Independent nucleotide substitutions (GGA for GAA, G for GCG, respectively) GGG’ for TG and GAG) G for E, V for A, and G for E), respectively. Further in Japan One cDNA clone derived from collected Cryptomeria japonica pollen Amino acid polymorphisms (Y for H) within the sequence can occur. Yet another silent A nucleotide substitution was detected. It is predicted that there is another type of sequence length type. One or more nucleotides (up to about 1% of the nucleotides) of a nucleic acid sequence Variations due to natural allelic variation are known to those skilled in the art. It will cost you money. Such nucleotide changes and resulting amino acid polymorphisms Any and all of the sexes are defined as proteins encoded by the present invention. Ru.

Fragments of nucleic acid sequences encoding fragments of CryjI are also included in the present invention. Within range. Fragments within the scope of the present invention include immunogenicity in mammals, particularly humans. response, e.g. stimulation of minimal amounts of [gE, binding of IgE, IgG and IgM antibodies. induction of production or T cell responses such as proliferation and/or lymphokine secretion and/or Encodes part of CryjI, which induces T cell anergy etc. Contains fragments.

The CryjI fragments described above are referred to herein as antigenic fragments. It will be done. Fragments within the scope of the invention include alleles cross-reactive with CryjI. Nucleic acids from other plant species used in screening proposals for the detection of Also included are fragments capable of forming hybrids. As used herein If the fragment of the nucleic acid sequence encoding Cry j I is Nucleotide sequence encoding the entire amino acid sequence of I and/or mature Cryjl Refers to a nucleotide sequence with fewer bases. In general, the flag of [2ヱ　1　I The nucleic acid sequence encoding the protein is selected from the bases encoding the mature protein; If the fragment is a leader sequence region of the nucleic acid sequence of the present invention, all or part of the fragment is It is preferable to choose from minutes. Nucleic acid sequences of the invention include linker sequences, modified restriction endonucleases, Crease site and cloning and expression of Cry j I or its fragments Alternatively, it can be obtained from plants that also contain other sequences useful for purification. but Applicants are commercially available Libtomeria japonica pollen from Cryj. It was discovered that mRNA encoding I could not be obtained. mRNA from pollen The lack of availability is due to problems associated with storing or transporting commercially available pollen. Dew. Applicants agree that new pollen and staminate cones Cryptomeria japonica is a well-known cedar species, and the plant material is derived from wild, The nucleic acid sequences described herein or elsewhere for gene isolation and cloning may be can be obtained using any suitable method. Nucleic acid sequences of the invention may be DNA or RNA. can be.

The present invention provides expression vectors and transformation fragments for the expression of the nucleic acid sequences of the present invention. Nucleic acids encoding ), yeast or mammalian cells, such as Chinese hamster ovary cells (CHO) can be expressed within. Suitable expression vectors, promoters, enhancers and other expression control elements are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 5 econd dition.

Col1d Spring Harbor Laboratory Press, Seen in Co1d Spring Harbor, New York (1989) can be issued. Other suitable expression vectors, promoters, enhancers and and other expression elements are known to those skilled in the art. mammal, yeast or insect Expression in cells involves partial or complete glycosylation and cleavage of the recombinant or lead to the formation of intrachain disulfide bonds. A vector suitable for expression in yeast is YepSecl (Baldari et al. (1987) Embo J, 6:229-234); pMFa (Kurjan and Herskow- 123) and pYES2 (Invitrogen Corporation, S an　Diego, CA). These vectors are freely available. Ru. Baculovirus and mammalian expression systems are also available. For example, in insect cells Baculovirus is commercially available (PharMingen, S. (Diego, CA), pMSG vector for expression in mammalian cells. is commercially available (Pharmacia, Piscataway, NJ).

For expression in E. coli, suitable expression vectors include pTRC (Aman et al. (1988) Gene Dan9:301-315); pGEX (Amrad Corp, Me 1bourne.

Au5tral ia): pMAL (N, E, Biolabs, Bever ly, MA); pRIT5 (Pharmacia, Piscataway, NJ); pET-1id (Novagen, Madson, WI) J ameel et al., (1990) J, Virol, 54: 3963- 3966; and pSEM (Knapp et al.

(1990) Bio TechniQues 8:280-281). . For example, pTRC and pET-1id lead to the expression of non-fusion proteins. p Using MALSpRIT5, pSEM and pGEX, maltose E binding protein (pMAL), protein A (pRIT5), truncated β- Galactosidase (PSEM) or glutathione S-transferase (p GEX) fused to the allergen. Cry I or its fragment When the component is expressed as a fusion protein, the carrier protein and Cry It is particularly advantageous to introduce an enzymatic cleavage site at the fusion junction between the fragments. It is. CryjI or its fragments are then enzymatically cleaved at the enzymatic site. biochemical purification using conventional methods for protein and peptide purification. It can be recovered from the fusion protein by manufacturing. Suitable enzymatic cleavage sites include blood Contains sites for coagulation factor Xa or thrombin and suitability for their cleavage. Enzymes and protocols can be found, for example, at Sigma Chemical Compa. Louis, St.

Commercially available from MO and N,E, Biolabs, Beverly, MA. Ru. Different vectors also have different promoter regions, allowing for constitutive expression or e.g. PTG induction (PRTC, Amann et al. 1.

-’ (1988) Same as above; pET-11d, Novagen, Madison .

Wl) or temperature induction ((pRIT5.Pharmacia, Piscata way, NJ) allows for inducible expression. Recombined Cry j I, It is expressed in different E. coli bacteria with different abilities to degrade the recombinantly expressed protein. It is also suitable to use the same method (for example, U.S. Pat. No. 4,758,512). in another case , modification of the nucleic acid sequence that does not affect the amino acid sequence of the expressed protein. It is advantageous to use codons that E. coli tends to use.

The host cell is calcium phosphate or calcium chloride coprecipitated, DEAE-dextra conventional methods such as ion-mediated transfection or electroporation. can be used to transform the nucleic acid sequences of the invention. host cell Suitable methods for the transformation of Sambrook et al. It can be found in the experimental book.

Nucleic acid sequences of the invention can also be synthesized using standard methods.

A host cell transformed with a DNA sequence encoding that fragment of cells containing the Japanese cedar pollen allergen or at least one fragment thereof and a culture medium; culture in a medium suitable for producing a mixture of soils, and purify the mixture to make it substantially pure. Japanese cedar pollen - allergen) Purified Japanese cedar pollen allergen 7Crヱjl or less ( A method for producing both of these fragments is also presented. dan E　1　I or less using an expression vector containing DNA encoding at least one of the fragments. The transformed host cells are cultured in a medium suitable for the host cells. Dan E 1 construction Proteins and peptides can be analyzed by ion exchange chromatography, gel filtration chromatography specific for Cryj1 or its fragments This technique involves the purification of peptides and proteins, including immunopurification using specific antibodies. can be purified from cell culture media, host cells, or both using known methods. . The terms isolated and purified are used interchangeably herein and are used interchangeably herein. cell material or culture medium if produced using alternative DNA methods, or chemically Peptides, proteins that, if synthesized, are substantially free of chemical precursors or other chemicals. Refers to white matter, protein fragments and nucleic acid sequences.

As another feature of the present invention, the whole or part of Japanese cedar pollen allergen) Cry j I synthesized in a host cell transformed with a DNA sequence encoding a or at least one chemically synthesized Japanese cedar pollen allergen. compositions containing fragments thereof, as well as transformations using the nucleic acid sequences of the invention. Purified Japanese cedar pollen allele produced in transformed host cells or chemically synthesized the protein or at least one antigenic fragment thereof. Show. In a preferred embodiment of the present invention, Cryj I protein is present in small amounts (both mature and mature). In a host cell transformed with a nucleic acid sequence encoding the Cry↓I protein produced.

Allergens from Japanese cedar pollen, preferably Cry-yellow, that elicit the desired antigenic response. 　■ Fragments (referred to as antigenic fragments in the text) are e.g. recombinant from the corresponding fragment of the nucleic acid sequence of the invention encoding a peptide chemically synthesized using methods known to those skilled in the art, produced by screening of peptides produced by chemical cleavage of allergens or by chemical cleavage of allergens. The allergen can be obtained by arbitrarily divide the desired length into fragments, preferably with no peptide overlap. into fragments of different lengths or preferably with overlapping lengths of desired length. Can be divided into fragments. Fragments are tested to determine their antigenicity. (e.g., the ability of the fragment to elicit an immune response). Japanese cedar pollen allergy For example, when using Cry↓I fragments for therapeutic purposes, activation, etc. capable of eliciting a T cell response (i.e. proliferation or lymphokine secretion) and Japanese cedar pollen allergen that can induce T-cell anergy and/or T-cell anergy. A fragment of Japanese cedar pollen having minimal IgE activation activity is particularly desirable. Also desirable. Additionally, for therapeutic purposes, purified Japanese cedar pollen allergens, e.g. y↓I and its fragments do not bind to IgE specific to Japanese cedar pollen, Or, if the original purified Japanese cedar pollen allergen binds to such IgE, Preferably, the IgE binds to a substantially lower extent. Refined Japanese cedar flower When powder allergens or fragments thereof bind to IgE, such binding More mediators (e.g. histamine) are not released by mast cells or basophils. preferable. The minimum IgE activation activity is activated by the original Cry↓I protein. IgE activation activity that is less than the amount of IgE produced.

The purified protein allergen from Japanese cedar pollen or its preferred antigenic fragment is , Japanese cedar pollen sensitive patients, or allergens cross-reactive with Japanese cedar pollen allergens , such as Tabresus sempervirens or Juniperus sabinoides ( When administered to patients allergic to allergens from pollen (discussed above), Japanese Modifying a patient's allergic response to cedar pollen or such cross-reactive allergens and preferably the patient's B-cell response, T-cell response to the allergen. or both B-cell and T-cell responses. As used herein Modification of allergic responses in patients susceptible to Japanese cedar pollen allergens is standard practice. Defined as non-responsiveness or symptomatic relief to the allergen as determined by standard clinical methods. can be defined (e.g. Varney et alBritish Med ical Journal, 302:265-269 (1990)).

Purified Cryj I protein or its fragment is a mammalian model of Japanese cedar pollen allergy. e.g. Tamura et al. (1986) Microbiol, rm munol, 30:883-896 or U.S. Pat. No. 4,939.239. or the mouse model disclosed in Chiba et al. (1999). 0) Int, Arch, AI　Preferably tested in Ierdel. protein or Initial screens for IgE binding to the fragment were performed in experimental animals or in humans. by scarification skin test or intradermal test on volunteers, or by RAST (RAST). radioallergosorbent test, est)), RAST inhibition, ELISA analysis, radioimmunoassay (RIA) or in an in vitro system such as histamine release (Example 7 and and 8).

The present invention has T cell activating activity and therefore contains at least one T cell epitope Antigenic fragments of are particularly desirable. T-cell epitopes are used in clinical allergy Involved in the initiation and persistence of immune responses to protein allergens that correspond to physical symptoms I think that the. These T-cell epitopes are linked to appropriate HLA on the surface of antigen-presenting cells. T helper cells by binding to molecules and stimulating related subpopulations of T cells It is thought that the initial event occurs at the level of . These events are associated with T cell proliferation, lymphogenesis, Caine secretion, local inflammatory response, recruitment of additional immune cells to the site, and antibody production leading to the subsequent activation of the B cell cascade. One of these antibody isoforms, I gE is basically important for the expression of allergic symptoms, and its production is dependent on the secreted phosphorus. Due to the nature of hokines, it occurs early in the cascade of events at the level of T helper cells. It will be done. T-cell epitopes are the basic elements or minimal molecules recognized by T-cell receptors. The epitope contains amino acids essential for receptor recognition. T cell epitome mimics the amino acid sequence of protein allergens and modifies the allergic response to protein allergens. Amino acid sequences that are within the scope of this invention.

A book containing at least one T-cell epitope and derived from a protein allergen Exposing a patient to a purified protein allergen of the invention or an antigenic fragment of the invention and a suitable T cell subpopulation is engineered by resistant or anergic cells, and they become unresponsive to protein allergens and stimulate the immune response upon such exposure. become uninvolved. The protein of the invention further comprises at least one T cell epitope. Administration of a naturally occurring protein allergen or antigenic fragment of the invention Altering the lymphokine secretion profile compared to exposure to the allergen or part thereof (e.g. cause a decrease in IL-4 and/or an increase in l-2) . Furthermore, exposure to such antigenic fragments or protein allergens is usually It affects the subpopulations of T cells involved in the response to allergens, making these T cells normally Keep flags away from areas exposed to allergens (e.g. nasal mucosa, skin and lungs). or to the site of therapeutic administration of protein allergens. This T cell subpopulation Redistribution of groups activates normal immune responses at sites normally exposed to allergens. It may improve or reduce the ability of the immune system of patients to reduce allergic symptoms. Possible/Purified Crx ↓ ■Protein and fragments or fragments derived therefrom (peptide) to Japanese cedar pollen allergen or cross-reactive protein allergen. Diagnosis and treatment for allergic reactions! and can be used in prophylactic methods. write Accordingly, the present invention provides a method for expressing (E) or at least one fragment thereof. Purified Japanese cedar pollen allergen produced in host cells transformed to cryj I or at least one fragment thereof, as well as a pharmaceutically acceptable The present invention provides therapeutic compositions that include a compatible carrier or diluent. The therapeutic composition of the present invention t’Lf:Cry j I or at least one fragment thereof; and a pharmaceutically acceptable carrier or diluent. present invention Administration of the therapeutic composition to the patient to be desensitized can be carried out using known methods. can. The Cry ↓ I protein or at least one fragment thereof may e.g. be administered to a patient in combination with a suitable diluent, carrier and/or adjuvant. I can do it. Pharmaceutically acceptable diluents include saline and buffered aqueous solutions. . Pharmaceutically acceptable carriers include polyethylene glycol (Wie at al. , (1981) Int, Arch, Allergy Ap, pl, Immuno L, Dan 4: 84-99) and liposomes (Strejan et al., ( 1984) J, Meuroimmunol Uni 27). T cell allele For the purpose of immunogenic responses, the therapeutic composition may be in non-immunogenic form, e.g. with an adjuvant. Preferably, it is administered in free form. Such compositions are generally administered by injection (subcutaneous, Administered by intravenous administration, oral administration, inhalation, transdermal application, or rectal administration. Main departure Akira's therapeutic compositions are designed to improve the patient's sensitivity to Japanese cedar pollen in patients with Japanese cedar pollen sensitivity. administered at a dosage and for a period effective to reduce allergic reactions (i.e., reduce allergic responses). Ru. The effective amount of the therapeutic composition will depend on the patient's degree of sensitivity to Japanese cedar pollen, the patient's age, age, sex, and weight, and whether the Cryj I protein or its fragment was present in the patient. will vary according to factors such as the ability to elicit an antigenic response.

Cry　j　　cDNA (or mRNA of the group from which it is transcribed) or its can be used to identify similar sequences in any type or type of plant. Therefore, it is possible to hybridize with Cry　j　cDNA or mRNA or a part thereof. Sequences with sufficient homology to form a lid, e.g. DNA from allergens such as Lens, Juniperus sabinoides, etc. It can be identified or "pull out" conditionally. sufficient homology (generally greater than 40%) and use the methods described herein to It can be further evaluated. High stringency conditions can be used instead of this In the case of the method, the DNA of the invention is used to plant other species of plants, preferably related families; Japanese cedar pollen in plants of the genus or species, such as Juniperus or Cupersus. Encoding a polypeptide having an amino acid sequence similar to allergen Cry↓I sequence and thus allergens in other species. Can be done. Thus, the present invention not only relates to DanE1　■, but also the DNA and hive of the present invention. It also includes other allergens encoded by the lid-forming DNA. Furthermore, the present invention for example, by antibody cross-reactivity or T cell cross-reactivity. Isolated allergenic protein or flag thereof that is immunologically related to the fragment isolated allergenic proteins or their fragments in case of antibody cross-reactivity. The agent can bind to antibodies specific for the proteins and peptides of the present invention, and In case of cell cross-reactivity, the isolated allergenic protein or its fragment is White matter or peptide-specific T cells can be activated.

The protein or peptide encoded by the cDNA of the invention can be, for example, "purified". It can be used as an allergen. Such a purified allergen is Japanese cedar pollen fever. useful in standardizing allergen extracts, which are important reagents for the diagnosis and treatment of . Furthermore, by using a peptide based on the CryjI nucleic acid sequence, standard Anti-peptide antisera or monoclonal antibodies can be produced using standard methods. Wear. These sera or monoclonal antibodies can be used to standardize allergen extracts. can be done.

Using the peptides and proteins of the invention, consistent and well-defined biological Active compositions can be prepared and used for therapeutic purposes (e.g. in Japanese cedar-sensitive patients). can be administered (to modify the allergic response to tree pollen). the The administration of such peptides or proteins can be used, for example, to cell response, T cell response to Dan2〈I　IaL//l/Ken, or both responses. be able to. Purified peptide is used for immunotherapy of Cryptomeria and Japonica allergy It can also be used to study therapeutic mechanisms and design modified derivatives or analogs useful in immunotherapy. I can do it.

Studies by other researchers have shown that high doses of allergens generally give the best results. (i.e., the best relief of symptoms). However, many people are allergic to Unable to tolerate high doses of allergen due to allergic reaction to stomach. have the same or enhanced therapeutic properties as their naturally occurring corresponding allergens, but Modified peptides or modified allergens with reduced side effects (especially anaphylakine reactions) Modifications of naturally occurring allergens can be engineered to produce Ru. These include, for example, the protein or peptide of the present invention (for example, the amino acid of CryjT). (all or part of the amino acid sequence), or modified proteins or peptides. or a protein or peptide analog. Improving solubility, treatment or prevention Enhancement of defense efficiency or stability (e.g., in vitro shelf life and in vivo Modifying the structure of the protein or peptide of the present invention for the purpose of (resistance to proteolysis) can do. amino acids to modify immunogenicity and/or reduce allergenicity. The amino acid sequence has been changed by amino acid substitution, deletion or addition, or the same purpose Modified proteins or peptides can be produced with added components for this purpose. example For example, amino acid residues essential for T cell epitope function can be determined using known methods. (eg, substitution of each residue and determination of the presence or absence of T cell activity). Required Residues that have been shown to be active (e.g., their presence is associated with T cell activity) can be modified. substitutions with other amino acids shown to enhance Residues that are not present can also be modified (for example, if their insertion enhances T cell activity by substitution with other amino acids that do not reduce binding to the associated MHC). protein Another example of modification of a protein or peptide is to change the cysteine residue to preferably alanine, Disulfide bond by substitution with phosphorus, threonine, leucine or glutamic acid The goal is to minimize dimerization. Other examples of modifications of the peptides of the invention include amino By chemical modification of the acid side chain or cyclization of the peptide.

The proteins or peptides of the present invention may be modified to enhance stability and/or reactivity. , polymorphism in the amino acid sequence of protein allergens resulting from natural allelic variation. One or more talents can also be inserted.

Further substitution or addition of D-amino acids, non-natural amino acids or non-amino acid analogs Modified proteins or peptides within the scope of the present invention can be produced by moreover The protein or peptide of the present invention was prepared by A, 5ehon and co-researchers (Wie et al. Modified using the polyethylene glycol (PEG) method of It is possible to produce proteins or peptides conjugated with Ru. Furthermore, PEG can be added during the chemical synthesis of the proteins or peptides of the present invention. can. Reduction/alkylation (T arr-Method of Protein Microcharacter rization, J, E, Si 1ver ed, HumanaPress, C11fton, NJ, pp155-194 (1986)); acylation (Ta rr, ibid): chemical coupling to a suitable support (Michell and Shiigi, eds, Selected Methods in Ce1lul ar　Immunology.

WHFreeman, San Francisco, CA (1980); No. 4.939.239; or mild formalin treatment (Marsh International Archives of Allergy and Applied Immunology.

41:199-215 (1971)).

to facilitate purification and possibly improve solubility of the proteins or peptides of the invention. For this purpose, a reporter group can be added to the peptide backbone.

For example, poly-histidine can be added to a peptide to immobilize metal ion affinity. Peptides can be purified chromatographically (H.

Chuli, E. et al., Bio/Technology, 6:1321 -1325 (1988)). If necessary, add a reporter group and the amino acid of the peptide. Introducing specific endoprotease cleavage sites between acid sequences and including unrelated sequences This can facilitate the isolation of peptides that are not present.

Adding functional groups to peptides to successfully desensitize patients to protein antigens or have a hydrophobic T cell epitope or hydrophobic epitope in the peptide containing no hydrophobic regions or containing no hydrophobic regions in the protein or peptide. Therefore, it is necessary to increase the solubility of the protein or peptide.

Possibly to aid in proper antigen processing of T-cell epitopes within the peptide. defined proteases between regions each containing at least one T cell epitope. Zeus-sensitive sites can be engineered recombinantly or synthetically. For example, KK Mata charges a pair of charged amino acids such as RR to a region within the peptide during recombinant construction of the peptide. It can be introduced in between. The resulting peptides contain cathepsins and/or other triglycerides. Becomes sensitive to homogeneous cleavage of psin and has one or more T cell epitopes Parts of the peptide can be generated that include. Furthermore, such charged amino acid residues The group improves the solubility of the peptide.

The peptide or protein of the present invention (e.g. Cryj I or a fragment thereof) using site-specific mutagenesis of the encoding DNA, as known in the art. The structure of a peptide or protein can be modified by methods. in such a way Among them, PCR using degenerate oligonucleotides (Hoe t a 1., G ene, E.E.: 51-59 (1989)) or total synthesis of mutant genes (Ho. stomsky, Z, et al.

,Biochem,Biophys,Res,Comm,, 161:1056 -1063 (1989)). For enhanced bacterial expression, The method described above can be used in combination with other methods to encode the protein or peptide of the present invention. eukaryotic codons in a DNA construct that You can change the codons that tend to be used by the cell.

Using currently available structural information, we recommend administering sufficient doses to patients susceptible to Japanese cedar pollen. Cryj may alter a patient's allergic response to Japanese cedar pollen when administered. l peptides can be designed. For example, this can be done by examining the structure of Cry , produce the peptide (using an expression system, synthetically, or by other methods) and A suitable peptide containing an epitope recognized by B cells in pollen-sensitive patients This can be done by selecting. When referring to epitopes, epitopes receptors, especially immunoglobulins, histocompatibility antigens and T-cell receptors. epitope is the basic element or minimum unit of receptor recognition, and epitope is essential for receptor recognition. Contains amino acids. The amino acid sequence of the epitope is imitated, and the Amino acid sequences that can modulate and reduce allergic responses can also be used. Wear.

Here, Japanese cedar pollen allergen causes allergic reactions in Japanese cedar pollen sensitive patients. It is also possible to design reagents or drugs that can mask or inhibit the ability to induce It is possible. Such reagents can be used, for example, to bind to the relevant anti-IgH. together, thus preventing IgE-allergen binding and subsequent mast cell degranulation. It can be designed as follows. In other cases, such reagents interact with cellular components of the immune system. binding and suppressing allergic responses to Cryptomeria japonica pollen allergens. can cause desensitization. This example suppresses allergic responses to Japanese cedar pollen. suitable B and T cell epitopes based on the cDNA/protein structure of the present invention to control The present invention includes, but is not limited to, the use of peptide or modified products thereof.

This has been shown to be B and B in in vitro studies using blood components from patients susceptible to Japanese cedar pollen. Defining the structure of B and T cell epitope peptides that influence B and T cell function This can be done by

The protein, peptide or antibody of the present invention can be used for the detection and diagnosis of Japanese cedar pollen allergy. Can also be done. For example, this means that sensitivity to Japanese cedar pollen must be assessed. Cry blood or blood products obtained from a patient Isolated antigenic peptide of I or isolated EII protein and blood components ( e.g. antibodies, T cells, B cells) and peptides or proteins under conditions suitable for binding. This can be done by determining the extent to which such binding has occurred.

(ii) or culturing under conditions suitable for expression of at least one fragment; A method for producing Cry j I or a fragment thereof is also presented. expressed production The material is then recovered using known methods. In other cases Cry The components can be synthesized using known mechanical or chemical methods.

The DNA used in any aspect of the invention can be obtained as described herein. or otherwise of the sequences set forth herein. Any oligonucleotide sequence, in whole or in part, or functionally equivalent thereof. It can also be an equivalent. Such oligonucleotide sequences can be prepared using known methods. It can be produced chemically or enzymatically. Oligonucleotide sequence machine Functional equivalents include: 1) the sequence SEQ ID NO: 1 (or corresponding sequence part) or its fragments; hybridization with complementary oligonucleotides to which the fragment hybridizes. 2) A sequence complementary to SEQ ID NO: 1 (or a corresponding sequence) column part) and/or 3) Sequence number: 1 (or corresponding sequence part) A product with the same functional properties as the product to be coded (e.g. a polypeptide or It is a sequence encoding a peptide). Functional equivalents meet one or both criteria Whether it must be used depends on the application (e.g. If used only as a function, it is necessary to meet only the first or second criterion, When used to produce E.I. allergens, only the third criterion must be met. ).

The invention is further illustrated, but not limited, by the following examples.

Example 1 Purification of the original Japanese cedar pollen allergen (Cry j I) The following are the main allergens. This is an explanation of the research conducted to biochemically purify CryjI in its original form. Ru. Purification is a modification from the published method (Yasuda et al. l, , J, Al lergy Clin, Immunol E1 Near 7.1983 ).

100g Japanese cedar pollen (Hollister-8tier, 5po) obtained from Japan Kane, WA) was degreased 3 times in IL diethyl ether, and after filtration the flower was removed. The powder was collected and the ether was dried in vacuo.

The defatted pollen was treated with a final concentration of 50mM Tris-HCl, pH 7, 8, 0, 2M. NaC11 and protease inhibitor, soybean trypsin inhibitor (2μ g/ml L leupeptin (I μg/ml), pebustin A (1 μg/ml) and and phenylmethylsulfonyl fluoride (0.17 mg/m). Extraction was carried out in extraction buffer at 4°C overnight. Insoluble materials were extracted using 1.2 L of extraction buffer. , re-extracted overnight at 46C, combined extracts from both sleeves, and equilibrated with extraction buffer. Batch adsorption using an DE-52DEAE cellulose (dry weight 200g) Decolorized by

The decolorized material was then fractionated by ammonium sulfate precipitation at 80% saturation at 4°C. ), thereby removing many of the low molecular weight substances. Obtained partially purified Cry j I can be dialyzed in PBS buffer and used for T cell studies (see Example 6) , or further purified as described below.

The concentrated Cryj I material was then treated with protease inhibitors at 4°C. Both were dialyzed against 50mM Na acetate, pH 5.0. Non-binding substances (basic proteins) White matter) was then treated with 10 mM Na acetate containing protease inhibitors at 4°C. , a 50 ml 5uperdex 75 column (P 3 in 10 mM Na acetate, pH 5.0 by FPLC on Purification was performed at a flow rate of 0 ml/h.

The purified Cry ↓ ■ was further heated at 25°C using a 0-IM NaCl linear gradient. FPLC-5-Sepharose 16/10 column chromatography (Pharmacia). Cry j I eluted as the main peak. 2 gel filtration chromatography.

FPLC5uperdex 75 column (2゜6X60cm) (Pharmac ia, Piscataway, NJ) at a flow rate of 30 ml/hour at 25°C. ．． Dissolve in a downward flow of 1QmM NaAcetate% pH 5.0 containing 15M NaC+. I let go. Figure 1a shows 8 rows of chromadogs on gel filtration). 5DS-PA with silver staining Cry jI was separated into three bands by GE analysis (Figure 1b). In Figure 1b As shown, SDS PAGE (1 2.5%) analysis was performed under reducing conditions. The gel was silver stained from Bi'o-Rad. It was silver stained using Iroki. The samples in each row are as follows: 1 row, ovo Albumin (43,0OOkD), carbonic anhydrase (29,00 Prestained standard proteins containing α-lactoglobulin (18,400 kD) and α-lactoglobulin (18,400 kD) White matter (Gibco BRL); 2 rows, fraction 36; 3 rows, fraction 37: 4 rows, fraction 3 8; column 5, fraction 39; column 6, fraction 41.7 column, fraction 43: and column 8, fraction 44° All fractions are shown in Figure 1a.

These proteins were analyzed by Western protein analysis using the mouse monoclonal antibody CBF2. It was also analyzed by a kit (Fig. 2). As shown in Figure 2, the 5uperde shown in Figure 1 Fraction 36 (column 1)-1 fraction 39 (column 2) and fraction 43 (column 3) purified from x75 A 11 L aliquot of the column) was electrolyzed onto nitrocellulose by 5DS-PAGE. Cutroblotting and probing using mABCBF2. Vivid as a second antibody Using biotinIylated sheep anti-mouse Ig, conjugated anti- The body was revealed by 1251-strebavidin. Monoclonal CBF2 Dr.

D, Klapper (Chapel Hi II, N, Carolina) was induced against pig allergen Amb aI. AmbaI and Due to the homology between the AmbαI sequences, the large number of Antibodies were tested for reactivity with 9-yjl. The results showed that CBF2 was Recognizes denatured Cry jl as detected by Western blotting Indicates that t. .

Furthermore, Western blotting revealed Cry in the expected molecular weight range (Figure 2).

These results were consistent with findings from protein sequencing. Fraction 44 and fraction 39 ( When N-terminal sequencing is performed on Figure 1b), only the CryII sequence is detected. .

In short, three types of Cry 1 I isoforms with different molecular weights were purified from pollen extract. It was done. The molecular weight calculated by 5DS-PAGE is calculated under reducing and non-reducing conditions. Both ranged from 4O-35kD. The isoelectric points of these isoforms are approximately 9.5-8 ．． 6, and the average pl was 9.0. The N-terminal 20 amino acid sequence is The previously published Cry j I sequence is identical in these three bands. (Taniai et al., supra). The three isoforms are pooled Another purified subfraction of CryjI using the plasma of 15 allergic patients All monoclonal antibodies CB, as shown in allergic serum titers of Recognized by F2. They all bind to IgE in allergic patients (Fig. 3). Differences in molecular weight and isoelectric point may be due to post-translational modifications such as glycosylation, phosphorylation, etc. This may be due in part to oxidation or the lipid content may differ in these isoforms. Not possible. The possibility that these different isoforms are due to protease degradation is and that four different protease inhibitors were used during purification. Even though it is actually unlikely, it cannot be ruled out at present. Other possibilities are , due to winterism in genes or splicing changes in mRNA. However, in cDNA cloning research, only one major form of E11 protein has been identified. Only white matter was detected (see Example 4).

Can be used for purification of original Cry j I or recombinant Cry j I Another method is immunoaffinity chromatography.

This method is highly effective due to the specificity of the interaction between monoclonal antibodies and antigens. Provides selective protein purification. Production of Cryj I-reactive monoclonal antibody For experimental purposes, female Ba1bl/c mice were obtained from Jackson Labs. obtained. Each mouse was first emulsified in Freund's complete adjuvant. 00 μg of purified original EJI (as shown in Figure 1b) with 99% purity and low immunization was performed intraperitoneally using 54 days after the first injection in PBS. One additional intravenous injection of 10 gg of purified native Cry ↓ I was given. after 3 The pancreas was removed and cultured using myeloma line SP2.0 as described (CurrentP rotocols in Immunology, 1991. C.

Myeloma fusion (1igan et al, eds) ) was carried out. Cells were washed with 10% fetal bovine serum (HybrimaX) and hypoxanthine. and azaserine, and wells containing colonies of tzd bridomoma cells were treated with anti- A native binding ELISA was used to screen for antibody production.

Cells from positive wells were incubated with 10% fetal bovine serum (ybrima Clone at 3/10 cells/well in xanthine and select positive clones. It was subcloned one more time in hypoxanthine medium. second and third Capture ELISA (Example 7) was used for screening. (see also). This analysis (select clones that recognize the original protein) and therefore offer the advantage of being useful for immunoaffinity purification. . mAbs are therefore a useful tool in the purification of CryjI from pollen extracts. becomes. It can also be used for infinity chromatography. Generate more The monoclonal antibodies produced are also useful for diagnostic purposes. These and E11 Shows some specificity for different isoforms and thus the characterization of these isoforms It is also possible to induce different mAbs, which may be useful tools.

Example 2 Attempt to extract RNA from Japanese cedar pollen R from commercially available non-defatted Crypromelia japonica (Japanese cedar) pollen Many attempts were made to obtain NA (HollisterStier, 5eatt Le, WA), the sample was first dissolved by suspending it in 4M guanidine buffer, and the liquid Grind under nitrogen and pellet through 5.7 M cesium chloride by ultracentrifugation. Sambrook etal, Mo1ecular Cloning, AL aborat.

ry Manual, Col1d Spring Harbor Laborat ory Press, Cold Spring Harbor.

The method of New York (1989) was used. Different amounts of guanidine Various amounts (3, 5 and 10 g) of pollen were collected in Isis buffer (10 and 25 ml). tried. Centrifugation through cesium produces a viscous material at the bottom of the tube, from which RN It was not possible to recover the A pellet.

Ambrosi Artemisiifolia degreased using this method Obtaining RNA from a. artemisiifolia (pig) pollen I was able to do it (Greer Laboratories.

Lenior, NC), Crypromelia japonica before guanidine extraction Even if pollen is defatted with acetone, RNA cannot be obtained as determined by absorption in AHo. There wasn't.

Crypromeliae according to the method in Sambrook et al., supra. An attempt was made to extract RNA from japonica pollen using acid phenol. 4.5 pollen Grind in M guanidine solution, shear, and acidify by adding 2 M sodium acetate. The mixture was extracted with water-saturated phenol and chloroform. Pellet after precipitation was washed with 4M lithium chloride, 10mM) 15mM EDTA/1%SD Redissolved in S, extracted with chloroform, reprecipitated with NaCl and absolute ethanol. I let it happen. Using this method, we were able to extract Amprosia artemisiifolia. However, Cryptomeria japonica RNA could not be extracted.

Next, 4g of Cryptomeria japonica pollen was added to 10ml of extraction buffer (5QmM Tris, pH 9, 0, 0, 2M NaCL, 10mM Mg acetate and diluted to 01% Suspended in ethyl pyrocarbonate (DEPC) and placed in a mortar and on dry ice. Grind with a pestle, add 1% SDS, lQmMEDTA and 5% N-Laurylsa. It was transferred to a centrifuge tube with lucosin and the mixture was extracted five times with warm phenol. the last distance After the heart, the aqueous phase was collected and 2.5 volumes of absolute ethanol were added and the mixture was kept at 4°C. Incubated overnight. The pellet was collected by centrifugation and heated to 65°C. Resuspend in 1 ml of dH20, add 0.1 volume of 3M Na acetate and 2.0 volume of It was reprecipitated by adding ethanol. Absorption and gel electricity in A2-2 No detectable RNA was recovered in the pellet as judged by electrophoresis.

Finally, add 500 mg of Cryptomeria japonica pollen to a mortar on dry ice. and crushed with a pestle and crushed by Frankis and Mascarhenas ( 0.1% as previously described in Ann, Bot, Dan: 595-599. 0, 2M NaCl, 1mM EDTA, 1% SDS treated overnight with DEPC was suspended in 5 ml of 5QmM Tris pH 90 containing Phenol/Chlorophor After extraction 5 times with mu/isoamyl alcohol (mixed at 25:24:1) The material was precipitated from the aqueous phase using 0.1 volume of 3M Na-acetate and 2 volumes of ethanol. Let it stagnate. Pellets were collected by centrifugation, resuspended in dH20, and heated to 65°C. The precipitated material was solubilized. No further precipitation with lithium chloride It was. Detectable RN as determined by absorption at A26° and gel electrophoresis A was not recovered.

In short, it was not possible to recover RNA from commercially available pollen. RNA is preserved or whether it has degraded during transportation or if the proposal used in this example is an extant RNA. I don't know if it was not possible to recover the . However, RNA is a new cryprome. japonica pollen and male cone samples (see Example 3).

Example 3 Extraction of RNA from Japanese cedar pollen and male cones and cloning of Cry j I One Crypto at Arnold Arboretum (Boston, MA) Fresh pollen and male cone samples collected from Melia Noyaponi Chikara (Japanese cedar) trees were directly collected. It was later frozen on dry ice. Basically FrankiS Bnd Masca RNA was prepared from 500 mg of each sample as described by renhas, supra. Manufactured. The sample was ground on dry ice using a mortar and pestle, and 0.1% DEP 0,2M Nac 5 containing 1.1mM EDTA, 1% SDS treated overnight with C. ml of 5QmM) Lys pH 9.0. Phenol/Chloroform/I After 5 extractions with soamyl alcohol (mixed at 25:24:1), 0.1 volume of RNA was precipitated from the aqueous phase using 2M Na acetate and 2 volumes of ethanol. far Collect the pellet by heart, resuspend in dH20 and heat at 65°C for 5 minutes. Ta. Add 2 ml of 4M lithium chloride to the RNA samples and ink them overnight at 0°C. It was incubated. Collect RNA levels by centrifugation and re-incubate in 1 ml dH20. Suspended and again precipitated with 3M sodium acetate and ethanol overnight.

The final pellet was resuspended in 100 μm dH20 and stored at -80°C. .

Commercially available kit (cDNA DNA synthesis butterbur, BRL SG ai Thersburg, MD), Gubler and Hoffman (1983) Gene 25: 263-269. The first cD was obtained from 8 μg of flower heads and 4 μg of pollen RNA by T brining. NA was synthesized. Degenerate oligonucleotide CP-1 (which has the sequence 5'-GATA ATCCGATAGATAG-3', where T in position 3 can also be C and T in position 6 can also be C, and G in position 9 can be AST or C. can also be AftT or +tC at position 12, and T at position 15 is C. A at position 16 can also be T, and G at position 17 can be C. Using primer 11 number 3) and primers EDT and ED, which can be used with Attempts have been made to amplify the cDNA encoding Kounie 11. Primer ED Tf sequence 5'-GGAATTCTCTAGACTGCAGGTTTTTTT TTTTTTTT-3' (SEQ ID NO: 24). Primer ED is sequence 5 '-GGAATTCTCTAGACTGCAGGT-3' (SEQ ID NO: 23) has. CP-1 is the first 6 amino acids at the amino terminus of CT AsnProI 1eA, 5pSer, encodes amino acids 1-6 of SEQ ID NO: 1) A degenerate oligonucleotide sequence containing

EDT hybridizes with the poly-yao region of the gene. oligonucleotide lotus All Research Genetics, Inc, Huntsville, A Synthesized by L. Polymerase chain reaction (PCR) uses commercially available (GeneAmp DNA amplification kit, Perkin Elmer Cet US, Norwalk, CT), whereby a 10 μm sample containing dNTPs was used. 10x buffer with 1 μg CP-1 and 1 μg ED/EDT primer (ED: EDT (3:1 molar ratio), cDNA (20 μl first strand cDNA reaction mixture) (3-5 μm from 100 μm from The mixture was mixed and made up to 100 μm with distilled water.

The sample was manufactured using a programmable thermal controller (MJ Research, Inc.).

, Cambr., MA). The first five rounds of amplification are Denature for 1 min at 94°C, 1 min at 45°C, and 5 min of primer to template. and chain extension for 2 minutes at 70°C.

The final 20 rounds of amplification consisted of the above denaturation, 1.5 min annealing at 55°C. and extensions of the above. Then use 5% (5 μl) of this initial amplification and 1 μg of each CP-2 (which has the sequence 5'-GGGAATTCAATTGGGC GCAGAATGG-3', where T at position 11 can also be C; G at position 17 can also be A, T or C; G at position 20 can also be A T at position 23 can also be C, and G at position 24 can also be C. (SEQ ID NO: 4), nested primer (SEQ ID NO: 4), ) and the ED described above were used to perform a second amplification. Sequence 5 in primer CP-2 '-GGGAATTC-3' (bases 1-8 of SEQ ID NO: 4) is a cloning The EcoRI site added for this purpose is shown and the remaining degenerate oligonucleotide sequence is The sequence is [EjI amino acids 13-18 (AsnTrpAIaG]nAsnA rg, encodes amino acids 13-18 of SEQ ID NO: 1). multiple le) DNA band on 1% GTG agarose gel (FMC, Rodkpor) Sambrook et al. The 32p end-labeled probe CP-3 ( It did not hybridize with SEQ ID NO: 5). Therefore, the specific Cry DNA Hunt could not be selected and the method did not proceed. CP-3 is It has the sequence 5'-CTGCAGCCATTTTCIACATTAAA-3', and the position A at position 9 can also be G, T at position 12 can also be C, position 18 A in position 21 can also be G (SEQ ID NO:- 5). Inosine (1) G or A or T at position 15 to reduce degeneracy or used in place of C (Knoth et al. (1988) Nucl eic Ac1ds ReS, 16:10932). Arrangement in Bligh 7-CP-3 Row 5'-CTGCAG-3' (bases 1-6 of SEQ ID NO: 5) is the cloning sequence. The Pst I site added for the purpose is shown, and the remaining degenerate oligonucleotide sequence is , Amino acids PheAsnVa IGluAsn from the internal sequence of Cry j I The coding strand sequence encoding Gly (amino acids 327-332 of SEQ ID NO: 1) The corresponding non-coding strand sequence.

The first PCR was also carried out using the above CP-1 (SEQ ID NO: 3) and CP-3 (SEQ ID NO: 3). 5) on the first strand cDNA. The second PCR is CP-2 (SEQ ID NO. , 4) and CP-3' (SEQ ID NO: 5) using 5% of the first reactant. Ta. Multiple bands were observed again, all of which were Cry in the Southern plot. j　I could not be specifically identified and this procedure was not continued.

Commercially available from its thrifty 4 μg (pollen) or 8 μg (flower head) RNA kit (cDNA synthesis kit, BRL, Gaither sbu Double-stranded cDNA was synthesized using Rg, MD). Phenol extraction and After ethanol precipitation, T4 DNA polymerase (Promega, Madi cDNA was blunted using Rafnar et al., (1999). 991) J, Biol, Chem, λ9dan.

1229-1236; Frohman et al. (1990) Pr48-5 Modified Anchored PCR reaction (modified Anchored P Ethanol precipitated and self-annealed for use in CRreaction). AT (SEQ ID NO: 20) and AL (SEQ ID NO: 22) oligonucleotides connected to. Oligonucleotide AT has the sequence 5'-GGGTCTAGAGGTA CCGTCCGATCGATCATT-3' (SEQ ID NO: 20) (Rafna r et al.

Same as above). Oligonucleotide AL has the sequence 5°-AATGATCGAT GCT-3' (SEQ ID NO: 22) (Rafnar et al.

Same as above). The amino terminus of Cr　　■ was connected to the linked c　DNA (20 1 μg of each oligonucleotide AP (3 μl from a μl reaction) No. 21) and degenerate primer CP-7 (which has the sequence 5'-T TCATICGATTCTGGGCCC-3', where G at position 8 is T A in position 9 can also be G, and C in position 12 can be T. (G at position 15 can also be A, T or C) (SEQ ID NO: 6) was amplified using Boar (1) is G or G at position 6 to reduce degeneracy. Used in place of A or T or C (Knoth et al, supra). degeneracy Oligonucleotide CP-7 (SEQ ID NO: 6) is located at the amino terminus of Amino acids 14-20 from (TrpAIaG]nAsnArgMetLys( A non-coding sequence corresponding to the coding strand sequence encoding amino acids 14-20 of SEQ ID NO: 1) This is the code chain sequence. Oligonucleotide AP has the sequence 5'-GGGTCTAGAG GTACCGTCCG-3' (SEQ ID NO: U21).

The first PCR reaction was performed as described herein. 5% (5%) of this initial amplification μl) and then 1 μg of each AP (SEQ ID NO: 21) as described herein. ) and degenerate primer CP-8 (sequence number near) and internal assembly used for the second amplification using the incorporated Cry I oligonucleotide primer. Ta. Primer CP-8 has the sequence 5'-CCTGCAGCGATTCTGGGCC CAAATT-3', G in position 9 can also be T, A in position 10 is C in position 13 can also be T, G in position 16 can be A: , T or C, and A at position 23 can also be G (SEQ ID NO: ・7). Nucleotide 5'-CCTGCAG-3' (SEQ ID NO: Near base 1- 7) indicates a PstI restriction site added for cloning purposes] nAsnA rg, corresponding to the coding strand sequence encoding amino acids 13-18 of SEQ ID NO: 1). This is the non-coding strand sequence. The main product of amplification is ethidium bromide (EtBr) - Approximately 193 base pairs of DNA as visualized on a stained 3% GTG agarose gel It was a band.

Extraction with chloroform, phenol and chloroform in sequence and then 0.5 -20 using 75 volumes of ammonium acetate and 1.5 volumes of isopropanol The amplified DNA was recovered by precipitation at °C.

After precipitation and washing with 70% ethanol, the DNA was incubated with Xba in a 15 μm reaction. Simultaneously digested with I and Pst I, preparative 3% GTG NuSieve low melting point gel Electrophoresis was carried out through . DNA bands of appropriate size were visualized by EtBr staining, excised, and commercially available. Sequencing kits (Sequenase kits, U, S, Bioc chemicals, C1eveland.

○H) using the dideoxy chain termination method (Sanger et al.

(1977) Proc, Natl Acad Sci, USA 74:5463 -5476), appropriately digested Ml 3mp 18 connected inside. Initially, ligatable material could only be derived from male cone-derived RNA. So I thought. However, subsequent experiments showed that the ligatable material was derived from pollen-derived RNA. and male cone-derived RNA was shown to be recoverable from PCR products generated from It was.

The clone designated JC71,6 was found to contain a partial sequence of It was brought out. This is the disclosed NH2-terminal sequence of Cry j I (Tan iai et al, ibid.), and the truth of ejl It was confirmed as an authentic clone. The amino acid at position 7 is Cys was determined to be Cys, and was published in Yoshika patent no. 4.939,239. It matched the sequence disclosed in the specification. Amino acid numbering follows the mature protein sequence Amino acid 1 is disclosed as the NH2-terminus of Cry↓I based on (Taniai et al, supra) Corresponds to aspartic acid (A, sp).

The starting methionine is amino acid 21 with respect to the first amino acid of the mature protein. Found. The position of the initiating methionine is determined by the upstream in-frame stop codon (in-frame stop codon). me-stop codon) and the surrounding nonucleotide sequence is Lutc. ke et al. (1987) EMBOJ.

6:43-48 and the consensus sequence of plants containing the starting methionine and 78% Supported because they are homologous.

oligonucleotide CP-9 (which has the sequence 5'-ATGGATTCCCCCT) TGCTTA-3′) (SEQ ID NO: 8) and AP (SEQ ID NO: 21) The cDNA was cloned from the ligated cDNA by using oligonucleotide CP-9 (SEQ ID NO: 8) is a Cry j from the leader sequence of DanE↓I. Amino acid of I ~) etAsp3erProCysLeu (SEQ ID NO: 1) encodes the amino acid 2l-16) and partially into Cry j I clone JC76,1. Based on the nucleotide sequence determined for

The second PCR reaction consisted of 1 μg each of AP (SEQ ID NO: 21) and CPie (this This is the sequence 5'-GGGAATTCGATAATCCCATAGACAGC-3' (SEQ ID NO: 9) and the incorporated primers were used to perform the initial amplification mix. It was carried out for 5% of the compound. Nucleotide sequence of primer CP-10 5'-GG GAATTC-3' (bases 1-8 of SEQ ID NO: 9) was used for cloning purposes. Added EcoRI restriction site is shown. The remaining oligonucleotide sequences are Amino acids 1-6 of Ejl (AspAsnProI 1eAspSer) (sequence Sequence number: 1 encodes amino acids 1-6), and partially cloned JC Based on the nucleotide sequence determined for 76,1. amplified DNA production The product was purified as described above, precipitated and then digested with Eco R1 and Xba I. and electrophoresed through a preparative 1% low melting gel. Cut out the main DNA band, M13mp19 and pUC19 were ligated for sequencing. In this case as well, it is possible to connect Potential materials include cDNA generated from pollen-derived RNA and male cone-derived RNA. were recovered. 2 of digested pUc19Jc91a and pUc19Jc91d One clone was selected for full-length sequencing. Then they have the same sequence It was discovered that

Commercially available kits (Sequenase kits (U, S.

The dideoxy linkage was DNA was sequenced by the chain termination method (Sanger et al, supra). N 113 forward and reverse primer r) (N, E, Biolabs, BeverlL MA) and internal sequencing Fixed primers CP-13 (SEQ ID NO: 10), CP-14 (SEQ ID NO: 11) ), CP-15 (SEQ ID NO: 12), CP-16 (SEQ ID NO: 13), C P-18 (SEQ ID NO: 15), CP-19 (SEQ ID NO: 16) and CP-2 Both mirrors were fully sequenced using 0 (SEQ ID NO: 17). CP-13 has sequence 5 '-ATGCCTATGTACATTGC-3' (SEQ ID NO: 10) .

CP-13 (SEQ ID NO: 10) is amino acids 82-87 (Met ProM82-87 (1eA1a, amino acids 82-87 of SEQ ID NO: 1) do. CP-14 has the sequence 5'-GCAATGTACATAGGCAT-3' (SEQ ID NO: 11), and has the non-coding strand sequence of CP-13 (SEQ ID NO: 10). handle. CP-15 has the sequence 5'-TCCAATTCTTCTGATGGT-3 ' (SEQ ID NO: 12), which consists of amino acids 169-174 of 9-yjl. (SerAsnSerSerAspGIy, amino acid 169- of SEQ ID NO: 1 174). CP-16 has the sequence 5'-TTTTGTCAATTGAG It has GAGT-3' (SEQ ID NO: 13), and amino acid 335 of Cry↓I -340 (ThrProGlnLeuThrLys, amino acid of SEQ ID NO: 1 The non-coding strand sequence corresponds to the coding strand sequence encoding 335-340).

CP-18 has the sequence 5'-TAGCAAACTCCAGTCGAAGT-3' (sequence sequence number: 15), which is the fourth nucleotide of CP-18 (SEQ ID NO: 15). Cry j except that the nucleotide is synthesized as C instead of the correct nucleotide T Amino acids 181-185 of I (ThrSerThrGIyValThr, sequence Substantially corresponds to the coding strand sequence encoding amino acids 181-186 of sequence no. This is the non-coding strand sequence. Sequence 5'-TAGCTCTCATTTGGTGC- 3' (SEQ ID NO: 16) is amino acid 27 of Cry↓I. 0-275 (AlaProAsnGluSerTyr, amino acid of SEQ ID NO. 1 The non-coding strand sequence corresponds to the coding strand sequence encoding acid 270-275). . CP-20 has the sequence 5'-TATCGAATTer, amino acid 2 of SEQ ID NO: 1 51-256).

The sequenced DNA has the sequence shown in Figures 4a and 4b (SEQ ID NO: 1) was found. This consists of two overlapping clones, JC71,6 and pUC. This is a composite sequence from 19J91A. Cry I The complete cDNA sequence consists of 66 nucleotides of 5' untranslated sequence, starting methionine. 1, which includes an open reading frame of 1122 nucleotides starting with a don and a 3' untranslated region. Contains 312 nucleotides. 3' untranslated region 25 nucleotides 5' polyyao region have a common polyadenylation signal sequence. The position of the starting methionine is upstream within the frame. 78% salt due to the presence of a stop codon and with plant consensus sequences containing the start methionine Groups 62-70)). The open reading frame covers a protein of 374 amino acids. The first 21 amino acids contain a leader sequence that is cleaved from the protein. nothing. The amino terminus of the mature protein is based on the published NH2-terminal sequence (Taniai et al. (1988) ibid.), and purified original Cryj I directly. Identification was made by comparison with sequences determined by contiguous amino acid analysis. 353 amino The deduced amino acid sequence of the mature protein contains the first 20 amino acids with respect to the NH2-terminus. The published CryjI contains amino acids and 16 consecutive internal amino acids. It has complete sequence identity with the protein sequence (Taniai et al, supra).

The mature protein contains an N-linked glycosylation site corresponding to the consensus sequence N-X-5/T. Also includes 5 possible sites.

Example 4 Extraction of RNA from Japanese cedar pollen collected in Japan Fresh pollen collected from rear japonica (Japanese cedar) trees is immediately placed on dry ice. Frozen above. Basically Frankis and Mascarenhas A RN from 500 mg of pollen as described in nn, Bot, Dan: 595-599 A was prepared. The sample was ground on dry ice using a mortar and pestle to give a concentration of 0.1%. 0, 2M NaC], 1mM EDTA, 1% SDS treated with DEPC overnight. r! in 5 ml of 50 mM Tris pH 9.0 containing r! It got cloudy. Phenol/chlorophore After 5 extractions with lum/isoamyl alcohol (mixed at 25:24:1), 0. RNA was precipitated from the aqueous phase using 1 volume of 3M Na-acetate and 2 volumes of ethanol. I set it. The pellet was collected by centrifugation, resuspended in dH20, and heated to 65°C for 5 minutes. It was hot. Add to 2 ml of 4M lithium chloride”’L RN A sample and hold them at 9° Incubated overnight at C.

The RNA pellet was collected by centrifugation, resuspended in 1 ml dH,O, and reconstituted with 3M of sodium acetate and ethanol overnight. 1 final pellet Resuspended in 00 μl of dH,O and stored at -80°C.

Gubler and Hoffman (1983) Gene 25263-2 cDNA synthesis system kit (B Double-stranded cDNA was synthesized from 8 μg of pollen RNA using RL). polymerase Chain reaction (PCR) was performed using the GeneAmp DNA amplification kit (Perkin Elm er Cetus), thereby adding 10 μm of 10x buffer containing dNTPs. 10,100 pl each of sense oligonucleotide and anti-sense oligonucleotide Oligonucleotide, (10 μl in 400 μm double-stranded cDNA reaction mixture) , 0.5. Mix with czl's Amplitaq DNA polymerase and add with distilled water. The volume was 100 μl.

The sample was purchased from MJ Research, Inc. (Cambridge).

Amplification was performed using a programmable thermal controller from MA). First 5 rounds of amplification Denaturation of template at 94°C for 1 minute, primer template for 1 minute at 45°C. and chain extension for 1 minute at 72°C. The last 20 ra of amplification denaturation as described above, annealing for 1 minute at 55°C and extension as described above. .

The following seven types of Cr S-↓ ■ primer pairs were used to amplify double-stranded cDNA. : CP-9 (SEQ ID NO: 8), CP-17 (SEQ ID NO: 14), CP-10 ( SEQ ID NO: 9), CP-17 (SEQ ID NO: 14), CP-10 (SEQ ID NO: 9) ) and CP-16 (SEQ ID NO: 13), CP-10 (SEQ ID NO: 9) and CP- 19 (SEQ ID NO.

16), CP-10 (SEQ ID NO: 9) and CP-18 (SEQ ID NO: 215), CP-13 (SEQ ID NO: 10) and CP-17 (SEQ ID NO: 14), and C P-13 (SEQ ID NO: 10) and CP-19 (SEQ ID NO: 16). CP-17 ( SEQ ID NO: 14) is the sequence 5'-CCTGCAGAAGCTTCATCAACAA CGTTTAGA-3', amino acid 5KRC* (amino acid of SEQ ID NO: 1) 350-353 and a stop codon) corresponding to the coding strand sequence encoding Corresponds to chain sequence. Nucleotide sequence 5'-CCTGCAGAAGCTT-3' (SEQ ID NO: 14 bases 1-13) is Pst added for the purpose of cloning I and HindIII restriction sites are shown. Nucleotide sequence 5'-TCA-3' (SEQ ID NO: 14 bases 13-15) corresponds to the non-coding strand sequence of the stop codon. Ru. Amplification as seen on an ethidium-bromide (EtBr)-stained agarose gel. all gave products of the expected size. Two of these primer pairs are used for amplification. The product was cloned into pUc19 for full-length sequencing. C Duplex c using P-10 (SEQ ID NO: 9) and CP-16 (SEQ ID NO: 13) The PCR reaction performed on the DNA gave a band of approximately 1.1 kb, which was identified as JC1. It was called 30. Separate first strand cDNA reactions were performed as described above using 8 μg of pollen RNA. The oligonucleotide primer CP-10 (SEQ ID NO: 9) and CP -17 (SEQ ID NO: 14). This amplification was performed using the amino terminal of the mature protein. A full length cDNA from the end to the stop codon was given and designated as JC135.

The amplified DNA was sequentially extracted with chloroform, phenol and chloroform, Rear end: 5 volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol It was recovered by precipitation at -20°C. Precipitation and 70% ethanol After washing with a 15 μm reaction tube, the DNA was blunted with T4 polymerase and , in the case of JC130 (7), digest with Eco RI, or in the case of JC135 Simultaneously digested with Eco RI and Pst I, preparative 1% 5eaPlaque low melting Electrophoresis was performed through a spot gel (FMC). The size is more suitable for EtBr staining. DNA bands were visualized and excised using a commercially available sequencing kit (Se quenase kit, U, S, Biochemicals, C1evelan dideoxy chain termination method (Sanger et al., (19 77) Proc, Nat 1. Acad, Sci, USA74: 5463- 5476), appropriately digested pUC1 for dideoxy DNA sequencing. Connected to 9.

Chain measurement is M13 forward and reverse ply 7- (N, E, Biolabs, Beverl y, MA) and internal sequencing primer CP-13 (SEQ ID NO: 10) , CP-15 (SEQ ID NO: 12), CP-16 (SEQ ID NO: 13), CP- 18 (SEQ ID NO: 15), CP-19 (SEQ ID NO: 16) and CP-20 (SEQ ID NO: 16) Sequencing was performed using column number: 17). By sequencing, the amplified JC130 Two clones (JC130a and.

JC130b) and amplified JC135 (7) 1m (7)ri o-:/ (J CI35g) was found to be a Cry j I clone. Clone J Nucleotide and deduced amino acid sequences of C130a and JC130g and one nucleus It was found that there were differences in leotide. Clone JCI30b is sequence number 1 had a T at nucleotide position 306 instead of the previously described C. This Nucleo The change is from Tyr to H at amino acid 60 of the mature Cryj I protein. resulting in a putative amino acid change to is. This polymorphism is still independent of the independently induced P Not confirmed by CR cloning or by direct amino acid sequencing. deer Primary nucleotide and amino acid sequences Such polymorphism in the column is expected.

Example 5 9JC91a was digested with XbaI, precipitated, and blunted using T4 polymerase. It became. Bam Hl linker (N, E, Biolabs.

Beverly, MA) was blunt-end ligated into pUc19Jc91a overnight and NAC S ion exchange mini column (BRL, Gai Thersburg).

Excess linker was removed by filtration through MD). Then the ligated cDNA was simultaneously digested with Eco R1 and Bam Hl. This digested product is 1%5eaP Cryj by electrophoresis through an Iaque low melting point agarose gel. I insert (from the nucleotide encoding the amino terminus of the mature protein to the stop codon) (extending through) was isolated. Then 6 histides immediately 3' of the ATG start codon (His6) followed by a unique Eco R1 end. Expression vector p modified to be followed by a nuclease restriction site and appropriately digested. ET-11d (Novagen, Madison, WT: Jameel eta (1990) J. Virol, 64+3963-3966). The pieces were connected. Second EcoRI endonuclease restriction site in the vector with adjacent C1a I and Hind JII endonuclease restriction sites. was removed in advance by digestion with Eco R1 and Hind III, and then Smoothed and connected. Histidine (HiSs) sequence is N i” chelate column (Hochuli et al.

Added for purification. Using recombinant clones, co-coding T7 polymerase Isopropyl-β-D-thiogalactopyranoside (IPT G) - E. coli harboring a plasmid with an inducible promoter (Escher Ichia coli strain BL21-DE3 was transformed. Using IPTG Upon induction, a large amount of T7 polymerase is expressed, which has a T7 promoter. It is necessary for the expression of recombinant protein in pET-1id. clone pET -1idΔHRhis, JC91a, d uses CP-14 (SEQ ID NO: 11) Dideoxy sequencing (Sanger et al., supra) determined the expression It was confirmed that this is a Cry j I clone in the correct reading frame. .

Expression of the recombinant protein was confirmed in the first small culture (50ml). the law of nature O-:/]) Overnight culture of ET-11dΔHRh i ssJ C91a, d 50 ml of a medium containing ampicillin (200 μg/ml) (Brain Heart Infusion Media, DifcO) and Ago Grow until o=1.0 and then induce with IPTG (1mM, final concentration) for 2 hours. Ta. 1 ml aliquots of bacteria were collected before and after induction and pelleted by centrifugation. 50mM Tris HCI, pH 6, 8, 2mM EDTA, 1% SDS, 1 % β-mercaptoethanol, 10% glycerol, 0.25% bromophenol LeBleu (Studier et al., (1990) Methods in Boil the pellet for 5 minutes in Enzymology 1 Dandan: 6O-89) Crude cell lysates were prepared by: Recombinant protein expression was performed using Sambr Coomassie loaded with 40 μm coarse lysate according to ook et al., supra. - with an expected molecular weight of approximately 38 kDa on blue-stained 5DS-PAGE gels. visualized as a band. Negative standard is plasmid with CryjI Crude lysates and plasmid-free bacterium from uninduced bacteria containing Includes induction lysade from the rear.

Thereafter, the pET-11dΔHRhissJC91a, d clone was transformed into the recombinant protein. were grown on a large scale for quality expression and purification. 2 ml containing recombinant plasmid Cultured bacteria were grown for 8 hours and grown on solid medium (e.g. 200 ag/m I). LB medium containing ampicillin (G i b c o-BRL.

6 Petri plates containing 1.5% agarose in Gaithersburg, MD). It was inoculated in a line on a dish (100 x 15 mm), grown overnight to a high density, and then uncapped. 9L liquid medium (Brain Heart) containing picillin (200U/ml) Infusion media, DifcO). Culture A6 ．． Grow until 0 becomes 1.0, add 1PTG (final concentration 1mM), and then add 2 The culture was grown for hours.

Collect bacteria by centrifugation (7,930xg, 10 minutes) and transfer to 90ml of 6M gel. Shake vigorously for 1 hour in Anidine-HCl, 0.1M NatHPOn, pH 8.0. It dissolved. Remove insoluble materials by centrifugation (11,000 x g, 10 minutes, 4°C). I left. The pH of the lysate was adjusted to pH 8.0 and 6M guanidine HCI, 1 80ml nickel NTA equilibrated with 00mM Na, HPO4, pH 8.0 Lysade was applied to an agarose column (Qiagen). Place the column in a 6M group. Anidine HCI, 100mM Na, HPO, 10mM Tris-HCL p H8.0, then 8M urea, 1100n1 Na@HPOa, pH 8.0, and finally 8M urea, 100mM sodium acetate, 10mM Tris-H. Cl.

Washing was performed at pH 6 and pH 3 in sequence. The column is heated until the passing liquid reaches A, 8°≦0.05. Washed with each buffer.

Sodium, eluted with 10mM Tris-HCl, pH 4,5, 10ml were collected in aliquots. The protein concentration of each portion is AH,1 and the pooled Determined by peak fraction. An aliquot of the collected recombinant protein was transferred to Sambro Analyzed on 5DS-PAGE according to the method of Ok et al., supra.

The first 9L sample JCpET-1 was analyzed by Coomassie blue stained SDS to PAGE gel. Shimadzu FlyingSpot 5cann er, Shjmadzu Scientific Instruments, I 30 mg of Cry as determined by nc., Braintree, MA) It gave about 78% purity. A second 9L sample prepared similarly, JC pET-2 gave 41 mg of Cryjl with approximately 77% purity.

Example 6 Cry I - T cell research for Japanese cedar pollen allergy patients using cedar pollen major antigen research T cell response to cedar pollen antigen peptide Peripheral blood mononuclear cells (PBMCs) exhibiting clinical symptoms of seasonal rhinitis and Japanese cedar pollen. 60 from Japanese cedar pollen allergy patients with positive MAST and skin tests for rnl peripheral blood was purified by lymphocyte separation medium (LSM) centrifugation. complete medium ( RPMI-1640, 2mM L-glutamine, 100U/ml penicillin/s Treptomycin, 5X10-'M2-mercaptoethanol, and 5% 2X10' in bulk culture of 10mM HEPES supplemented with activated human AB serum. PBL/ml was converted to 20 μg/ml partially purified native Cryj I (Fig. 75% purity containing three bands similar to the three hands of 2) and humidified 5%C. Long-term T-cell line by activation for 7 days at 37°C in O2 incubator and selected yjI-reactive T cells. The amount of this initial immunization antigen is determined to be optimal for T cell activation from most cedar pollen allergy patients. . Viable cells were purified by LSM centrifugation and added to 7 ml of 5 units of recombinant human IL-2 and In complete medium supplemented with 5 units of recombinant human IL-4/ml, cells no longer The cells were cultured for up to 3 weeks until they did not respond to lymphokines and appeared to be "quiescent." after that Recombinant Cryj I (rCryj IL-purified original Cryjl or group Replacement Amb a1. 'l　(r, Amb　al, 1) of T cell proliferation Assessed ability. For analysis, duplicate or triplicate cells were plated in 96-well round bottom plates. Separate 2xlO' resting cells in 200μl complete medium in double wells for 4x1 0' autologous Epstein-Barr virus (EBV)-transformed B cell 2 in the presence of cells (prepared as described below) (gamma irradiated at 25,0 OORADS). −50 μg/ml r-tan I.

Reconstitute for 2-4 days using purified original Amb.jl or rAmb.a 1.1. Lived. Optimal incubation was found to be 3 days. The latter The wells were given 1 μCi of tritiated thymidine for 16-20 hours. inserted card Collect the samples onto a glass fiber filter mat for liquid scintillation counting. It was processed. Figure 12 shows the original Cry j I purified from recombinant Cry j L. and showing the effect of varying antigen dosage in assays using recombinant Ambal, l. vinegar. Figure for use as antigen-presenting cells (EBV) - Preparation of transformed B cells Autologous EBV-transformed cell lines were γ-irradiated at 25,000 Rad and They were used as antigen-presenting cells in the secondary proliferation analysis and secondary mass activation. these cells The system is also used as a standard in immunofluorescence flow cytometry analysis. this Their EBV-transformed cell line was transferred to a 12 x 75 mm polypropylene round bottom Falc on snap cap tube (Becton Dickinson LAbs Ware, Link.

In Park, NJ) at 37°C for 6 hours, 1 μg/ml of phorbo 1 ml of B-5 in the presence of 12-myristate 13-acetate (PMA) 9/8 Marmoset cell line (ATCC CRL1612, American Type　Culture　Co11ection.

5 x 106 P Formed by incubating BL. These cells were then divided into 10% RPMI-164 as described above except supplemented with heat-inactivated fetal bovine serum of Dilute to 1.25X10' cells/ml in 0.0 and no visible colonies are detected. were cultured in 200 μl aliquots in flat-bottomed culture plates. After that, They were transferred to larger wells until the cell line was established.

Example 7 Both direct and competitive ELISA analyzes were performed to examine significance.

For direct ELISA analysis, soluble pollen extract (SPE) or is purified original Cry j I (analyzed at 90% purity by protein sequencing) were applied to the wells and the binding of human IgE antibodies to these antigens was analyzed. Ta. Isomorphs from 15 patients with Japanese cedar pollen MAST scores of 2.5 or greater Pooled human plasma and two individual patient plasma samples were analyzed for this analysis. compared with. Figure 5 shows the reactivity results of binding with these two antigens. of the bond The overall pattern is that for competitive analysis, Japanese cedar pollen SPE was added to ELISA wells. and measured IgE binding in allergic patients. What is wrong with these analyses? The source of lugic IgE was pooled plasma (designated PHP) from 15 patients. or each of the 7 from patients with a Japanese cedar MAST score of 2.5 or greater. This was a plasma sample. Pooled human plasma samples were subjected to Japanese cedar pollen SPE and Compare with the related allergen source, Dokumugi SPE. Figure 6 shows pooled human A graph of the results of competitive EL and ISA using plasma is shown. During Japanese cedar pollen SPE The concentration of protein present is greater than that of purified native CryjI at each competitive point. About 170 times higher. From this analysis, it was found that the purified original Cry j I was Japanese cedar pollen. Very high in IgE binding to the full range of proteins present in soluble pollen extracts. It is clear that they will compete well. This is most anti-Cry Ig This means that the reactivity of E is directed toward purified original Cry j I. negative mark The SPE does not show specific competitive reactivity, and the SPE in the competing solution is completely transferred to the coated wells. can be removed. We use this analysis to evaluate IgE responses within allergic populations. repeated for each patient as a measure of range. Figure 7 shows this result, where In this case, purified native Crjl was used to compete for binding to SPE. patient show different dosage responses to Japanese cedar pollen SPH, but not to Japanese cedar pollen SPE. IgE binding in each of the seven patients was competed with purified native CryjI. The results show that it was possible. The meaning of these data is In some cases, IgE reactivity is mainly directed towards Cry I, but among patients This reactivity is variable. Previous findings as a whole (Yasueda et am, (1988) ibid). .

The reactivity of IgE from patients with cedar pollen allergy to pollen proteins indicates that these proteins It decreases dramatically if it is denatured. One method for analyzing this property is the direct bond E This is a LISA method, in which case the applied antigen is Japanese cedar pollen SPE or reducing agent D. This is a modified Japanese cedar pollen SPE modified by boiling in the presence of TT. this are then tested for IgE binding reactivity using plasma from allergic patients. Figure 8 a shows direct binding analysis to SPE using seven respective plasma samples. Figure 8b The results of binding using modified SPE showed that the reactivity was significantly lower after this treatment. It shows that it is going down. Determine the extent of CryjI binding to ELISA wells. In order to determine the rabbit polyclonal Cry j-1 was detected using antiserum. These proteins are Cry-engineered. It has a high degree of sequence identity (46%) to I, and this antiserum was used in a cross-reactive antibody detection system. It can be used as Ultimately, these data suggest that after denaturation of SPH, IgE This indicates a significant loss of reactivity.

Example 8 IgE reactivity and histamine release analysis Recombinant expressed in bacteria and subsequently purified (as described in Example 5) The Cry j I protein (rCry j I) was investigated for IgE reactivity. child The first method applied to the test was a direct ELISA, where wells were filled with recombinant Cr. yjI was applied and analyzed for IgE binding for each patient. Figure 9 shows this direct EL It is a graph diagram of ISA. The only positive signal in this data set was the rabbit Polyclonal anti-Amba I & II (Rabbit anti-Amb a　I & II) and E reactivity was not shown.

Other analytical methods applied to examine IgE reactivity to recombinant CryjI was a capture ELISA. The basis of this analysis is a defined antibody, in this case CB F2 is used to bind the antigen and bind the antibody to other epitope sites. That is. The format of this capture ELISA consists of: 1) applying MAb CBF2 to the wells; , 2) Add antigen or PBS (as one negative standard) and mix with the applied MAb. 3) standard antibody anti-Amba I&II (Fig. 10b) or human allergic plasma (Fig. 10b) as a detection antibody; 4) anti- The objective is to evaluate the detection of body binding. Figures 10a and 10b are the results of these analyses. This is a graph of For IgE analysis, pooled human plasma (15 patients) was used. Ta. The conclusion from these results is that this analytical method can detect - No specific binding to CrΣjI was demonstrated. However, fig. As evidenced by the standard antibody binding curve shown in Figure 10b, rCry jl be captured. - Lack of IgE binding to CryjI is due to carbohydrate or other post-translational modifications. (because there is no decoration and/or).

Japanese cedar pollen SPE, purified original Tantan I and rCr1 I as antigens In addition, a histamine release analysis was performed on one Japanese cedar pollen allergic patient.

This assay is a measure of IgE reactivity through mediator release of human basophils. . The results of this analysis, shown in Figure 11, show that purified native Cr over a wide concentration range Indicating strong histamine release for both yj-I and Japanese cedar pollen SPH. Ru. The only point where there is measurable histamine release in the case of CryjI is at 50μ This is the point of highest concentration in g/ml. Possible regarding this release by rCry I Two possible explanations are: 1) a very low proportion of anti-Crヱヱr　IgE or 2) Due to low abundance bacterial contaminants observed only at the highest antigen concentrations. This is the specific release that occurs. So far, this result has only been shown in one patient. It is only shown as follows. Furthermore, the data presented are for one protein at each protein concentration. This is from the data points.

Since the E. coli-expressed substance has T cell reactivity (Example 6), the recombinant 1-yoriR Representation: This -q-yjl protein can be used for immunotherapy, but It does not seem to bind to IgE from Tomeria japonica atob, and test tube prevent the release of histamine from such atopic mast cells and basophils within . Expression of r and ejl, which can bind IgE, can be expressed in yeast, insects ( virus) or mammalian cells (e.g. CHO, human and mouse) was completed. rCry I, which can actively bind IgE, makes recombinant substances a diagnostic target. This is important when you use it for a specific purpose.

- Although the invention has been described with reference to its preferred embodiments, other embodiments may be used with the same result. can be given. Changes and modifications to this invention will be apparent to those skilled in the art. and all such modifications and equivalents are included within the scope of the appended claims and are hereby incorporated by reference. shall comply with the true spirit and scope of the invention.

Sequence list Array number = 1 Array length +1337 Sequence type: Nucleic acid Number of strands: 1 strand Topology: linear Sequence type: cDNA to mRNA origin Biological core: Cryptomeria japonica ca) Characteristics of arrays Symbols/CDS representing characteristics Location: 55. ．． 1187 Characteristics of arrays Symbol representing characteristics: mat peptide 129. ．． 1187 array 40...,. . 0,.

Sequence number: 2 Array length: 374 sequence type diamino acid topology, linear Sequence type: protein array Tyr　C1y　Enter 1a　T:,=　Enr9　person sp　Enter rg　P:o　Lau 7:p Ile Ila? he Sir Gly λ5■ Pha, iy v and L Ciu? =o VaL Has? =o　C;! n Enter sp Niriichi Enter 12 people 1a Lau Thr La■ 140! 4S! S O" 55λ': 9T-person 1aτ1; λ dew+'l :ia", :pmill)jpH4sλ1n511':?haSarA!n5e r! OO265 54: に、X:′I2.5X′ILe”? F4? Ha in! n1risl:ic L’,,’l-’w’a1M4:;,,iuL&’J(a NiAHih 190 Yu95:! 00 Sequence number 3 Array length 17 type of sequence, nucleic acid Number of strands/1 strand Topology: linear array GAYAAYCCNA THGAYWS sequence number, 4 length of array, 25 Sequence type/nucleic acid Number of strands, 1 strand Topology: linear array GGGAATTCAA YTGGGCNCARAAYSG Sequence number: 5 length of array, 23 Sequence type: Nucleic acid Number of strands: 1 strand Topology: linear Characteristics of arrays Symbol representing characteristics: modified base location 15 Other information: /mod base=i array CTGCAGCCRT TYTCNACRTT RAA sequence number: 6 Array length = 20 Sequence type: Nucleic acid Number of strands: 1 strand Topology: linear Characteristics of arrays Symbol representing characteristics: modified base Location: 6 Other information: /mod base=i array TTCATNCKRT TYTGNGCCCA sequence number near Array length 25 Sequence type Nucleic acid Number of strands: 1 strand Tropology: Straight array CCTGCAGCKRTTYTGNGCCCAAARTT Sequence number = 8 Array length = 18 Sequence type: Nucleic acid Number of strands, 1 strand Tropology: linear array ATGGATTCCCCTTGCTTA Sequence number = 9 Array length: 26 Sequence type: Nucleic acid Number of strands = 1 strand Tropology: linear array GGGAATTCGA TAATCCCATA GACAGC sequence number = 10 Array length: 17 Sequence type: Nucleic acid Number of strands, 1 strand Tropology, II linear row of shapes ATGCCTATGT ACATTGC Sequence number: 17 Array length: 18 Sequence type: Nucleic acid Number of strands = 1 strand Tropology: linear array TATGCAATTG GTGGGAGT Sequence number: 18 Array length: 20 Sequence type/amino acid Tropology: Straight Sequence type: Peptide Fragment type two N-terminal fragment origins Organism name: Cryptomeria Japonica ca) Characteristics of arrays Symbols representing characteristics: Mod i f i ed-s i t e existence position near Other characteristics/notes = “The amino acid at position 7 is Ser, Cys, Thr or HiS” array Sequence number: 19 Array length: 16 sequence type diamino acid Topology: l[chain-like Sequence type: Peptide Fragment type: middle fragment origin Biological color: Cryptomeria Japonica ca) array Sequence number, 20 Array length: 30 Sequence type: Nucleic acid Number of strands: 1 strand Topolon - straight-shaped array GGGTCTAGAG GTACCGTCCG ATCGATCATT fraction number Column index 5-cGrCATCTG CTri2TAATCA TeTAGCCGT^TqA Stop AMTTCTIOσ-TE Gopework　ωoqt　o.

1/dilution of PHP Rabbit anti-Ambal and dilution of 4Y: 4% Protein concentration, μg/ml international search report 1*lon++++-5lApdle+1m1lc PCT/US 9210 5661 International Search Report PCT/US 92105661 S^　62104 Continuation of front page (51) Int, C1,5 identification symbol Internal reference number Cl2P 21102 C8214-48GOIN 33153 Q 8310-2J331577 B 8310-2J //(C12P 21102 C12R1:19) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), AU, CA , J.P., K.R. I (72) Inventor: Bond, Julian, F, Massachusetts, United States 0 2188 Waymouth Commercial Street 294

Claims (1)

[Claims] 1. Japanese cedar pollen allergen CryjI or at least one antigenic fragment thereof A nucleic acid sequence encoding an agent, or a functional equivalent of the nucleic acid sequence. 2. The nucleic acid sequence has a nucleotide sequence of bases 66-1187 of SEQ ID NO: 1. Nucleic acid sequence according to claim 1, characterized in that: 3. The nucleic acid sequence has the nucleotide sequence of bases 129-1187 of SEQ ID NO: 1. Nucleic acid sequence according to claim 1, characterized in that: 4. The nucleic acid sequence essentially comprises at least one coding portion of the nucleic acid sequence of SEQ ID NO: 1. Nucleic acid sequence according to claim 1, characterized in that it comprises a fragment of . 5. Japanese cedar pollen allergen CryjI or at least one antigenic fragment thereof an expression vector containing a nucleic acid sequence encoding a target, or a functional equivalent of the nucleic acid sequence; Tar. 6. The nucleic acid sequence has a nucleotide sequence of bases 66-1187 of SEQ ID NO: 1. The expression vector according to claim 5, characterized in that: 7. The nucleic acid sequence has the nucleotide sequence of bases 129-1187 of SEQ ID NO: 1. The expression vector according to claim 5, characterized in that: 8. The nucleic acid sequence essentially comprises at least one coding portion of the nucleic acid sequence of SEQ ID NO: 1. The expression vector according to claim 5, characterized in that it contains a fragment of . 9. A protein encoded by the nucleic acid sequence according to claim 1, 2, 3 or 4 or is a host cell transformed to express the peptide. 10. The host according to claim 9, characterized in that the host cell is E. coli. cell. 11. A host transformed using the nucleic acid sequence according to claim 1, 2, 3 or 4. Purified Japanese cedar pollen allergen Cryj1 or at least one produced in main cells its antigenic fragment. 12. The Japanese cedar pollen allergen is immunoglobulin I specific for Japanese cedar pollen. Does not bind to gE or binds Japanese cedar pollen allergen to the immunoglobulin E If such binding occurs, the release of histamine from mast cells or basophils The purified Japanese cedar pollen allergen according to claim 11, characterized in that it does not cause hmm. 13. The Japanese cedar pollen allergen is free from the purified original Japanese cedar pollen allergen. binds to immunoglobulin E to a substantially lower extent than when binding to immunoglobulin E; The purified Japanese cedar pollen allergen according to claim 11, characterized in that the purified Japanese cedar pollen allergen is 14. Purification according to claim 11, characterized in that the host cell is E. coli. Japanese cedar pollen allergen or antigenic fragment thereof. 15a) D encoding the Japanese cedar pollen allergen CryjI or a fragment thereof A host cell transformed using the NA sequence was transformed with the Japanese cedar pollen allergen Cryj. For the production of a mixture of cells and culture medium containing I or at least one fragment thereof b) purifying the mixture to obtain a substantially pure Japanese cedar pollen allele; producing CryjI or at least one fragment thereof. , the production of the Japanese cedar pollen allergen CryjI or at least one fragment thereof Birth law. 16. A nucleic acid sequence encoding the whole or part of the Japanese cedar pollen allergen CryjI Japanese cedar pollen allergen CryjI synthesized in host cells transformed with or a protein composition comprising at least one fragment thereof. 17. that at least one of said fragments is an antigenic fragment; 17. A protein composition according to claim 16, characterized in that: 18. Chemically synthesized Japanese cedar pollen allergen CryjI or at least one A protein composition comprising that fragment of. 19. The CryjI is characterized in that it has the amino acid sequence of SEQ ID NO: 1. The protein composition according to claim 16 or 18. 20. Isolated antigenic fragments of allergens from Japanese cedar pollen. 21. Claims characterized in that the allergen from Japanese cedar pollen is CryjI. Antigenic fragment according to claim 20. 22. that the antigenic fragment contains at least one T cell epitope; Antigenic fragment according to claim 20 or 21, characterized in that: 23. that the antigenic fragment has minimal immunoglobulin E-stimulating activity; 23. Antigenic fragment according to claim 22, characterized in that: 24. The antigenic fragment does not bind to Japanese cedar pollen-specific immunoglobulin. or if binding of the fragment to said immunoglobulin E occurs, such The binding is characterized by preventing the release of histamine from mast cells or basophil cells. 23. The antigenic fragment of claim 22. 25. The antigenic fragment is purified from the original Japanese cedar pollen allergen. binds to said immunoglobulin E to a substantially lower extent than when binding to said immunoglobulin E; Antigenic fragment according to claim 20, characterized in that: 26. The purified allergen or the antigenic fragment is transmitted to the Japanese cedar flower to which it is administered. The allergic response to Japanese cedar pollen can be modified in powder-sensitive patients. The purified allele according to claim 11, 20, 21 or 22, characterized in that or antigenic fragment. 27. The purified allergen or the antigenic fragment is the patient's Japanese cedar pollen allergen. B-cell responses to cedar pollen antigens, T-cell responses to Japanese cedar pollen antigens in patients, or It is possible to modify both B cell and T cell responses to Japanese cedar pollen allergen. The purified allergen or antigenic protein according to claim 26, characterized in that Ragment. 28. Isolated antigenic fragment of Japanese cedar pollen allergen according to claim 20 A nucleic acid sequence encoding. 29. When administered to Japanese cedar pollen sensitive patients, the patient's response to Japanese cedar pollen allergen A modified Japanese cedar pollen allergen that reduces allergic responses. 30. The modified Japanese cedar pollen allergen is a modified CryjI protein. The modified cedar pollen protein allergen according to claim 29. 31. When administered to Japanese cedar pollen sensitive patients, the patient's response to Japanese cedar pollen allergen at least one modified version of a Japanese cedar pollen allergen that reduces allergic response; Ragment. 32. At least one modified fragment is a modified fragment of CryjI protein. at least one modified fan according to claim 31, characterized in that Ragment. 33. Isolated protein alleles immunologically related to CryjI or fragments thereof gen or antigenic fragment thereof. 34. If the protein allergen or antigenic fragment thereof is CryjI or its fragment Claim 33, characterized in that it binds to an antibody specific for the fragment. An isolated protein allergen or antigenic fragment thereof. 35. If the protein allergen or antigenic fragment thereof is CryjI or its fragment Claim 33, characterized in that it is capable of activating T cells specific to the fragment. An isolated protein allergen or antigenic fragment thereof as described. 36. Purified Japanese cedar pollen allergen CryjI or at least one fragment thereof A therapeutic composition comprising a pharmaceutically acceptable carrier or diluent. 37. CryjI has a sequence of amino acids 1-353 of SEQ ID NO: 1. 37. The therapeutic composition of claim 36, characterized in that: 38. The protein composition according to claim 16 or 18 in a therapeutically effective amount for mammals. Japanese cedar pollen allergen or Japanese cedar pollen allergen and immunization, including administering In mammals that are biologically susceptible to cross-reactive allergens, How to treat susceptibility to 39. For example, Japanese cedar pollen allergen or cross-reactive with Japanese cedar pollen allergen. Claims for use in therapy, such as treatment of patient sensitivity to allergens 19. The protein composition according to 16 or 18. 40. A blood sample obtained from a mammal is transformed using the nucleic acid sequence according to claim 1. Purified Japanese cedar pollen allele produced in transformed host cells or chemically synthesized or antigenic fragments thereof, blood components and proteins or fragments thereof. of the mammal under suitable conditions for binding and determining the extent to which binding occurs. A method for detecting susceptibility to Japanese cedar pollen allergen in species. 41. The extent to which binding occurs can be determined by T cell function, T cell proliferation, B cell function, protein or Evaluation of the binding of fragments of 41. The method according to claim 40, characterized in that the method is determined by the value of the product. 42. produced in a host cell transformed using the nucleic acid sequence according to claim 1. CryjI, a chemically synthesized purified Japanese cedar pollen allergen, or its anti-allergen the mammal in an amount sufficient to cause an allergic response in the mammal. and administering the Japanese cedar pollen allergen or antigenic fragment thereof in the patient. Japanese cedar pollen, including the step of determining whether an allergic response to Methods for detecting mammalian susceptibility to allergens. 43. Japanese cedar pollen allergen CryjI or at least one antigenic flag thereof A monoclonal antibody specifically reactive with ment.