JPH07502648A - Cynodon dactylon species protein allergen - 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] 24. Isolated Bermuda grass 0) pollen tongue Park allergen and Eyu d I, or Claim 1.3.12.19 or at least one of the isolated antigenic molecules encoded by the nucleic acid sequence of 2o piece.

25, represented by amino acids 1 to 246 of Cyn d 1.18 shown in Figure 20. 26, comprising an amino acid sequence comprising: Bermuda grass pollen protein allergen An isolated antigenic fragment of.

27, Figure 20 1. Knee tCyn d1. CDI (7) 7mi/acid 1-24 6. Bermuda grass pollen protein with desired range 27 isolated antigenic fragments of quality allergens.

29 ρl shown in Fig. 20” 1. 2/3 (full length) amino acids 1-24 An isolated Berm buckwheat plant comprising the amino acid sequence represented by 4 uda grass) pollen protein allergen yndT0 30 Bermuda grass pollen according to claim 29 Isolated antigenic fragments of protein allergens.

31. The isolation of claim 24.26.28 or 30, which has T cell stimulating activity. antigenic fragments.

32, amino acids 5-246, amino acids 10-246, amino acids 20-33, amino acids amino acids 5-246, amino acids 10-246, amino acids 20-246, and amino acids Acid 25-246: Cyndl shown in Figure 20, C34 amino acid 5-244, amino acid amino acids 10-244, amino acids 20-244, and amino acids 25-244: the second CyndI shown in Figure 0.

It has an amino acid sequence selected from the group consisting of all 2/3 (full length) of 35 29. The isolated antigenic fragment of claim 28 having low immunoglobulin E stimulating activity. Piece.

36 Proteins of Cynodon dactylon species A substantial percentage of individuals susceptible to odor allergens does not bind to immunoglobulin E specific for the quality allergen, or If binding of the fragment to immunoglobulin E occurs, such binding is essential. Claim 24. does not result in the release of histamine from cells or basophil cells. 26.28 or 30 isolated antigenic fragments.

37 The allergen of the protein that induced the fragment binds to immunoglobulin E. Claim 28.32. binds to immunoglobulin E to a substantially lesser extent. 33 or 34 isolated antigenic fragments.

38. Individuals sensitive to Bermuda grass pollen When administered to the body, the pollen of Bermuda graSS Claims characterized by the allergic response of said individual to a protein allergen. 24 isolated protein allergens or isolated antigenic fragments.

39. Individuals susceptible to Bermuda grass pollen When administered to the body, the pollen of Bermuda graSS Claims characterized by the allergic response of said individual to a protein allergen. 27 isolated protein allergens.

40. Individuals susceptible to Bermuda grass pollen When administered to the body, the pollen of Bermuda graSS Claims characterized by the allergic response of said individual to a protein allergen. 28 isolated antigenic fragments.

41. Protein array of Bermuda grass pollen Response of an individual's B cells to rugen, Bermuda gras s) an individual's T cell response to pollen protein allergens, or Against the protein allergen of Bermuda grass pollen Claim 24 characterized by both a B cell response and a T cell response of an individual who Isolated protein allergens or isolated antigenic fragments.

42. Protein array of Bermuda grass pollen Response of an individual's B cells to rugen, Bermuda gras s) an individual's T cell response to pollen protein allergens, or Against protein allergens in Bermuda grass pollen Claim 27 characterized by both a B cell response and a T cell response in an individual who Isolated protein allergens.

43. Protein array of Bermuda grass pollen Response of an individual's B cells to rugen, Bermuda gras s) an individual's T cell response to pollen protein allergens, or Against protein allergens in Bermuda grass pollen Claim 33 characterized by both B cell responses and T cell responses of individuals who Isolated antigenic fragment.

44. Injecting pollen of Bermuda grass into susceptible individuals. When given, the protein of Bermuda grass pollen a modified human body that reduces the allergic response of said individual to the allergen of Dandi protein allergen.

45. Casting pollen of Bermuda grass on susceptible individuals. the allergic response of said individual to said protein allergen when given; Modification of at least one of the allergens of the protein of E.D.L. Fragment.

46. Greater than 73% homology with Edan dl or an isolated antigenic fragment thereof Isolated protein allergen with.

Isolated protein allergens that are epidemiologically cross-reactive.

48, T cells specific for Cyn d I or isolated antigenic fragments thereof Isolated protein allergens that can irritate.

49 Isolated Bermuda grass (D9 Eyu di or at least one isolated antigenic fragment thereof. and a pharmaceutically acceptable carrier or diluent.

50 The allergen of the above protein is shown in Fig. 20 t Cy nd d 1.　 The claimed amino acid sequence comprises the amino acid sequence represented by amino acids 1 to 246 of CD1. Range 49 therapeutic composition.

51, the allergen of the above protein is shown in FIG. 1cynd I.

Claim 4 having an amino acid sequence represented by 18 amino acids 1 to 246 9 therapeutic composition.

52. Cyndl, the protein allergen shown in FIG. 20.

Comprising the amino acid sequence represented by 2/3 (full length) amino acids 1 to 244 , the therapeutic composition of claim 49.

53. Administering to an individual a therapeutically effective amount of the therapeutic composition of claim 49. Bermuda grass pollen protein comprising or Bermuda grass pollen allergens. susceptibility in said individual to an allergen that is immunologically cross-reactive with the allergen; How to treat susceptibility.

54. Claim 50.5 of therapeutically effective amount for an individual, or the therapeutic composition of 52. of Bermudagrass, comprising administering a Pollen protein allergens, or Bermuda gra ss) against allergens that are immunologically cross-reactive with pollen allergens. Methods of treating susceptibility in individuals.

55. Blood samples obtained from individuals are treated with Bermudagrass. or the nucleic acid sequence of claim 1. produced in a host cell that has been transformed into a host cell, or chemically synthesized. antigenic fragments of and the association of blood components with said protein allergens or fragments thereof. combination, and the extent to which such combination occurs. determining the susceptibility of CyndI to a protein allergen. A method for detecting sex in said individual.

56. T cell function, antibodies present in blood and the above proteins or fragments thereof the degree to which combination occurs by evaluating the combination with or combinations thereof; 56. The method of claim 55, characterized in that:

57 produced in sufficient quantities to produce an allergic response in an individual. at least one antigenic fragment thereof, which has been synthesized or chemically synthesized. The pollen of Bermuda grass is administered to an individual. an allergic response in said individual to an allergen or antigenic fragment thereof. Bermuda gras s) A method for determining the susceptibility of an individual to pollen allergens.

A monoclonal antibody that is reactive with

59 a) encodes yndI or at least one antigenic fragment thereof The host cells transformed with the DNA sequence are cultured in an appropriate medium, and the Japanese cedar) pollen allergen Cyn~di or less (both produce a mixture of cells and medium containing one antigenic fragment thereof, and b) Purify said mixture to obtain substantially pure Cyndl, or at least one Cyndl or at least one thereof, comprising a step of producing a fragment thereof. How to produce fragments of.

60, a) recombinantly or synthetically producing a fragment of Cyn d I; b) producing a fragment of Cyn d I; In individuals susceptible to Bermuda grass pollen, B cells and testing said fragments for the ability to influence C and/or T cell responses; ) selecting suitable fragments containing the epitope recognized by the cell; Bermuda gr. When administered to individuals susceptible to pollen allergens of B. changing the symptoms of an individual's allergy to the pollen of Ermuda grass A method for designing antigenic fragments of Cyndl.

61, an isolated peptide of Cyn d I or an isolated fragment thereof, , the peptide or fragment thereof comprises at least one Cyndl, a single Cyndl isolated peptide or isolated fragment thereof.

62. An isolated peptide of Cyndl or an isolated fragment thereof, wherein The peptide or fragment thereof has at least one T cell epitope of CyndI. The peptide consists of amino acids 25 to cyndl and cDl shown in Figure 20. An isolated peptide or peptide of Cyndl having an amino acid sequence comprising 246 or isolated fragments thereof.

63, an isolated peptide of Cyn d I or an isolated fragment thereof, , the peptide or fragment thereof is an epitope of at least one T cell of Cyndl. The peptide comprises Cyn d1. as shown in FIG. 2/3 (full length) having an amino acid sequence comprising amino acids 25 to 244 of l isolated peptide or isolated fragment thereof.

Specification 7 Cynodon dactylon species actylon ) are pollen alleles in many regions of the world, especially in tropical and subtropical climates. It is an important source of gen. These allergens have been studied by a number of means. These methods include: IgE immunoblotting. (Ford D and Ba1do, B, A, J, Atlergy C1 in, Immunol.

79 Near 11-720 (1987); 5hen H, D, et al., C11n.

AIlergy 18:401-409 (1988), Karamuk 07 Tograph -(○rren, A and Dowd) eSS, Af r, Med, J, 5 1:586 (1977); Matthiesen et al., J.

Allergy C11n, Immunol, 81:266 (Ab) (198 8)'), and immunoelectrophoresis (Matthiesen et al., supra, 1988) .

Main allergens of Bermuda grass pollen Lugen has been identified as a protein with a molecular weight (MW) ranging from 30 to 34 kD. This protein has an affinity for Bermuda grass. 76% of allergic individuals bind IgE from serum and and Baldo (1987) supra; Hen et al. (1988) supra; and Cy nd I (Kahn and Marsh, (1986) Mo1.Im mnol, 23:1281-1288; Marsh et al. (1988) Ann, A. Lergy, 60: 499-504, Matthiesen et al., supra) . Cyndl is a member of the group 1 family of allergens (Kahn and Marsh, (1986) supra, these allergens are classified into a number of taxonomically related groups. Poaceae plants such as ryegrass (LolpI), Poapl ) and timothy (two ± DI) (Standring et al., 1987 1nt 1. Arch, Allergy and Applied, Immunol, 83: 96. -103; Esch and Klapper, (1987), J. Alle. rgy C11n, Immunol, 79189-495: Matthiese n and ri.

Wenstein (1991) CI in, Exp, AI Irgy, 21. 209-320. However, Berm The allergens of ucla grass) are limited to the allergens of other grasses. (March et al., supra; Berstein et al. (1999)). 76) J, AIlergy Chin, Immunol, 57:141-152 A number of studies have shown that Cyndl is a closely related group I homolog of grasses. (Matthiesen and Lowens te in (1991) supra.

The first 27 amino acids at the N-terminus of Cyndl were determined (Matthi esen et al., 1988, supra + Matthiesen et al. (1990) Eptop es of Atopic Allergens, Brussels SUCB I n5 titute of Allergu, 9-13; Singh et al., Mono graphs in AllergV, (1990), 28+101-120; Matthiesen and Lowenstein, (1991), supra).

Bermuda grass pollen allergen in the environment The presence of It continues to have a significant socio-economic impact on European society.

The available spectrum of drugs, including antihistamines and steroids, is Although it has led to improvements in the treatment of allergic diseases, there are disadvantages associated with long-term use. Has virtually no convenient side effects. Because of these problems, immunity to allergic diseases There has been a renewed interest in treatment.

Immunotherapy involves injecting allergen extracts to desensitize patients to allergic reactions. (Bousquet, J. and Michel, F.B., 1989) AIlergy and C11n 1mmo1. News 1 Near -10°Unfortunately, pollen preparations used as allergens are polyvalent. and the quality is poor. After all, crude extracts are often used in high concentrations and May induce potentially fatal systemic reactions including anaphylaxis . Synthetic peptides based on cloned genes, their fragments or allergen sequences The products expressed from Tide must be quality controlled, characterized and standardized. and does not optimally bind IgE, making it a safe medium for treatment. Provide quality.

Summary of the invention The present invention relates to Cynodon dactylon: z (Cynodon dactylon) species. A major protein allergen (Cynd I), or at least one of its provide a nucleic acid sequence encoding a fragment or a functional equivalent of such a nucleic acid sequence; . The present invention also provides expression vectors comprising such nucleic acid sequences and expression vectors comprising them. Provide transformed host cells. The present invention further provides isolated recombinant, The present invention provides chemically or synthetically produced Dandandan I or a fragment thereof. isolated Bermuda grass or its fragments ) to diagnose and treat sensitivity in individuals to pollen allergens. Useful.

Brief description of the drawing FIG. 1 shows the cDNA clone derived from the cDNA clone designated clone 2 (C2) and The nucleotide sequence encoding Edan dl and the predicted partial sequence of Edan dl The amino acid sequence is shown.

Figure 2 was derived from a cDNA clone designated clone 18 (C18). Partial nucleotide sequence encoding CyndI and predicted CyndI The partial amino acid sequence is shown.

FIG. 3 shows a comparison of the nucleic acid sequences of clones 2 and 18.

FIG. 4 shows a comparison of the deduced amino acid sequences of clones 2 and 18.

Figure 5 shows seven clones encoding Cyndl, clone 18 (C18), clone 22 (C22), clone 23 (C23), clone 2 (C2), clone clone 3 (C3), clone 21 (C21), and clone 33 (, C-3 -3) shows a comparison of the deduced amino acid sequences.

FIG. 6 shows the cytoplasm derived from the cDNA clone designated as clone 14a1. The partial nucleotide sequence encoding dI and the deduced partial nucleotide sequence of The amino acid sequence is shown.

FIG. 7 shows a partial DNA clone derived from the cDNA clone designated clone 14C1. and the partial nucleotide sequence encoding the deduced partial amino acid sequence. show.

Figure 8 shows CyndI predicted from the complex of clones 14a1 and 14c1. , 14 shows the partial amino acid sequence of Cyclll.

In FIG. 9, it is displayed as dandandan 118. The predicted total length of qend I The amino acid sequence is shown.

Figure 10 shows the predicted partial construction of cyyudan 1.2/3. The amino acid sequence is shown.

Figure 11a shows the main components of these proteins on Rotofor. 5DS-PA of protein fractions obtained by preparative isoelectric focusing (IEF) Separation by GE is shown.

Figure 11b shows a sample of isolated proteins screened with MAb 3.2. The stamp plot is shown.

No. 12al121 contains 10-13 rotofuses pooled from crude pollen extracts. 5DS-P of one protein fraction obtained by refractionation on Rotofor Separation is shown by AGE.

Figure 12b shows 15 to 20 rotophores pooled from the crude pollen extract. 5DS-PAGE of protein fractions obtained by refractionation on indicates separation.

Figure 13 shows the results of separation by 5DS-PAGE and Bermuc. Blobbing with IgE from the serum of individuals allergic to Figure 2 shows a Western plot of natural and dIa and dIb.

Figure 14 shows the transfer of MAb to cDNA from the Wataru DanEdan Iλgtll library. The binding of ID1.3A3.3C2 and 4D2 is shown. The number on the overlay is cD Corresponds to the number of NA clones.

Figure 15 shows CyndI derived from the cDNA clone designated clone 3. The partial nucleotide sequence encoding DanEdandanI and the predicted partial nucleotide sequence of The amino acid sequence is shown.

Figure 16 shows Cynd derived from the cDNA clone designated clone 22. The partial nucleotide sequence encoding Cyn dI and the deduced partial nucleotide sequence of Cyn dI The amino acid sequence is shown.

FIG. 17 shows a cDNA clone derived from the cDNA clone designated as clone 23 and Partial nucleotide sequence encoding Cyn dl and predicted E partial sequence of Cyn dl The amino acid sequence is shown.

Figure 18 shows Cynd derived from the full-length cDNA clone designated CDI. The nucleotide sequence and deduced amino acid sequence of 1 are shown.

Figure 19 shows Cyn derived from the cDNA clone designated KAT-391. The partial nucleotide and deduced amino acid sequence of dl is shown.

FIG. 20 shows Cyn d 1.18, Cyn d 1. CDI and quality prediction A comparison of the full-length amino acid sequences obtained is shown.

detailed description of the invention The present invention provides CyndI%, that is, Nucleic acid encoding the major allergen found in the pollen of Grass or a functional equivalent thereof. Cyndl is a closely related allele It appears to be from the Gen family. As defined herein, “allergen "family" refers to genes related in function and amino acid sequence but located at separate genetic loci. A protein encoded by a gene. Members of each family are nucleotides variation can occur at a given genetic locus and can have polymorphisms. Ru. Nucleic acid sequence polymorphisms can give rise to amino acid polymorphisms; This is not always the case when the nucleotide code encoding acids is degenerate. . Nucleic acid sequences encoding Cyndl vary from individual to individual due to natural allelic variation. It may vary among plants of Bermuda grass.

Appointment! and all such nucleotide variations and resulting amino acid polynucleotides. Formality is included within the scope of this invention.

A DNA clone derived from the cDNA clone designated as clone 2 and coated with Danwata I The partial nucleic acid sequence has the sequence shown in FIG. Copy the Cyndl shown in Figure 1. The coded partial nucleic acid sequence consists of 435 bases. 3'-untranslated region is base 436 and extends to base 662. Clone 2 (C2) The deduced partial amino acid sequence of the encoded Cyn dl is also shown in Figure 1. It is shown.

Figure 2 shows a second cDNA clone designated as clone 18 (C18). Partial nucleic acid sequences and deduced amino acid sequences are shown. Daneyu dl shown in Figure 2 The nucleic acid sequence encoding 600 amino acids encodes 200 deduced amino acids. It consists of nucleotides. The 3'-untranslated region begins at base 601 and and extend to base 775.

As shown in Figure 3, clone 2 and clone 18 (coding in A Although the sequences are clearly homologous, the 3'-untranslated region is very divergent. Ru. This suggests that clones 2 and 18 (and of the second I gene family) The ability to encode separate members.

As shown in FIG. 4, the recommendations encoded by clone 2 and clone 18 The determined amino acid sequences have 88.2% homology (84.1% identity).

There are 22 amino acids in the 143 amino acid overlap deduced from the two clones. No acid differences exist, 6 of which are conservative substitutions and 16 are non-conservative substitutions. This is a replacement. The partial protein encoded by clone 18 is Ll at the carboxy terminus by a partial protein encoded by 2 amino acids r=tt length L) (Figure 4). Amino acid homology is determined by PCGENE ( Intelligenetics, Power IJ Fol. Demonstrated using the software contained within Nearzu ((Mountain View) ReIko.

Seven cDNA clones derived from the Cyndl library described in Example 1. A comparison of the predicted amino acid sequences W11 encoded by has been done. This one labeled C2, C3, C21, C22, C23 and C33 The longest clone encoded by one of the cDNA clones of Shown aligned with the deduced amino acid sequence encoded by C18 (C18). has been done. As shown in Figures 5 and 6, clone 18.22.23.2 The overlapping portion of the amino acid sequences encoded by 1 and 33 is are the same. As this suggests, clones 18.22.23.21 and 2 3 is an example of members of the same Cyndl gene family. However, clone 2 2 and 23 are shorter amino acids than clone 18 and have different 3'-untranslated It has a translation area (FIGS. 2, 16, and 17). This is clone 22 and and 23 represent separate members of the Cyndl gene family. a or they may represent differently spliced forms of the same family member. There is.

As shown in Figure 5, the estimated apertures encoded by clones 2 and 3 There are only five amino acids between the amino acid sequences. Therefore, clone 2 and 3 may represent polymorphisms of members of the Cyndl gene family; Members of the dl gene family are Cyndl genes to which clones 18.21 and 33 belong. Different from members of the child family. Clones 2 and 3 contain multiple members of the Cyndl gene family. Assuming that the form is expressed as a fact, it is expressed as Cyn d 1.2/3 shown in Figure 10. The predicted partial amino acid sequence of Edandi was encoded by clones 2 and 3. can be generated from the encoded amino acid sequence.

Figure 6 shows the nucleotide sequence of cDNA clone 14a1 and its deduced The amino acid sequence is shown. This clone was isolated from PCR as described in Example 2. and the amino acid sequence it encodes was partially duplicated by clone 18. Corresponds to the amino portion of the encoded Cyndl family member. clone 14 The 19 nucleotide oat between the 3' end of a1 and the 5' end of clone 18 ( - La Sobca (Exist.

Clone 14a1 was subjected to PCR (as described in Example 2) to create clone 1. Based on the non-coding strand sequence of 8 (using oligonucleotide primers) Amplified. encoded by nucleotides 41-431 of clone 14a1; It is assumed that methionine represents the first amino acid of translated protein/protein.

This is an in-frame termination at nucleotides 11-13 of clone 14a1. The first methionine encoded after the codon, which initiates protein translation. Methionine encoded by nucleotides 41-43 of clone 14a1 This shows that it does not occur at 5' of Surrounding the putative initiator methionine The nucleotide sequence for protein initiation in plants is the consensus nucleotide sequence for protein initiation in plants. Sequence 5'AACAATGGC-3' (Lutteke et al., 1987, EMB OJ, 6:43-48). 9th day of maturity I tongue / chestnut N - 22 amino acids before the start of the terminal (indicated by amino acid 11 in Figure 6) An acid leader sequence is present at the N-terminus (the first two 7 amino acids) has already been identified (Matthiesen et al., 1988, J. Allergy C11n, Immuno, 81:226 Shing et al., 19 90. Monogr, AI Iergy, 28:101-120; Matthi esen et al., 1991, J. Allergy C11n, Immunol, 88 Near 63-774).

Figure 7 shows the nucleotide sequence 7+1 of cDNA clone 14C1 and its predicted sequence. The amino acid sequence obtained is shown below. This clone 1, Matko, implementation flI 2 i2-kun The amino acid sequence isolated from PCR and encoding the self-containing is a member of the Cyndl family partially encoded by clone 18. corresponds to a portion. This clone is homologous to clone 14a1, but mature Has 1 amino acid difference from clone 14a1 in the synphosphatase sequence (The N-terminus of mature and dl tyrosine phosphatase is the amino acid in Figure 7. 1). Clone 14C1 encodes 4 additional amino acids 7 nucleotides different from clone 14a1, containing a 12 nucleotide insert It has a nucleotide difference in the leader sequence that encodes a amino acid.

The composite sequence of 14a1 and 14C1, including potential polymorphisms of these clones, is It is displayed at d1.14 shown in FIG.

The predicted full-length nucleotide sequence encoding dl is derived from the formula: , where is a nucleic acid sequence of 0 to 300 nucleotides; The column contains the nucleotides encoding the leader sequence of the Dandan dI protein. and can also include nucleotides of the 5'-untranslated region, where N is 60 A nucleic acid sequence consisting of a nucleotide force of up to 0 and a mature and Contains nucleotides encoding the mino-terminal portion, Y is clone 2, clone 18, Clone 3, Clone 22 or Clone 23 or mature Danedan dita of any polymorphic form of the nucleic acid sequences of those clones that encode proteins. X is a nucleic acid sequence of 0 to 600 nucleotides; The column contains the nucleotides of the 3' untranslated portion of cydandl. For example, printing is a clone. The nucleotide of clone 14a1 shown in FIG. 6 containing the 5'-untranslated region of 14a1 Clone 14 encoding the CyndI leader sequence as well as CyndI. represented by nucleotides a1 (nucleotides 41-106 shown in Figure 6) can include nucleic acid sequences. Ll is also the 5'-untranslated clone 14c1 Nucleotides 1-103 of clone 14c1 shown in FIG. 7 containing the region and C Nucleotides of clone 14c1 encoding the leader sequence of yndl (no. may contain the nucleic acid sequence represented by nucleotides 28 to 103) shown in Figure 7. Wear. L is also a clone that encodes only the leader sequence part of Cyndl. 14a1 (nucleotides 41 to 106 shown in FIG. 6) or Nucleus of clone 14c1 encoding only the leader sequence part of Cyndl otide (nucleotides 28-103 shown in Figure 7) or any polymorphism thereof can be a nucleic acid sequence comprising the form of One encodes mature Cyn d When generating a nucleic acid sequence to code, L+ is 0, X is 0, and the formula is simply NY. N preferably extracts the full-length or mature CyndT protein. A nucleic acid sequence that encodes a protein.

The total predicted value for Cyn d I 13 shown in Figure 9 The long amino acid sequence is the amino acid sequence shown in Figure 8, designated as Cyn d T, 14. By merging the sequence with amino acid residues 53 to 246 of clone 18 shown in FIG. can be generated. The predicted complex of the mature protein is in this case consists of amino acids 1 to 246 of Dandan d1.18 shown in Figure 9, and the translation It would have a predicted molecular weight of approximately 26.7 kDa without subsequent changes. Honmei As used in the specification, a “mature” and once dI protein is defined as a “mature” and once an I It does not contain the amino acid sequence of the protein leader. The items discussed herein Polymorphisms or potential polymorphisms are superscripted and subscripted in all applicable drawings. Indicated by

Full-length clones were generated using PCR as described in Example 3 and shown in Figure 18. occurred. Nucleotides 107-125 of clone 14a1 (Figure 6) and and oligonucleotides based on nucleotides 604 to 621 of clone 18 (Figure 2). Using the Otido primers, the clone shown in Figure 18 and designated clone CDI was used. A full-length clone was generated from PCR. Deduced amino acid of clone CDI The acid sequence was as described above, excluding the two amino acid sequences, as shown in Figure 9. , Cyndl 1.18 and the predicted members of the family of 6 Cyndl proteins. corresponds to the full-length amino acid sequence of the complex. Figure 18 and 20dl protein It is quality. 9yn d1. from the cDNA Δinsert of clone CD1. 2/3 The full-length amino acid sequence of Ike's predicted complex, indicated as (full length), is shown in Figure 20. show. A portion of this sequence corresponds to nucleotides 178-20 of clone 2 (Figure 1). 6 (identical to the corresponding nucleotide sequence of clone 3 (Figure 15)) and Nucleotides essentially identical to nucleotides 107-130 of clone 14a1 (Figure 6) Using nucleotide-based oligonucleotide primers, PcR'D) et al. It is estimated from the generated Cyn d T clone. Clone this clone Displayed as AT-39-1. Deduced amino acid sequence of clone KAT-39-1 The columns are the predicted partial amino acid sequence of Cyn d 1.2/3 shown in Figure 10. represents a partial amino acid sequence of Cyndl that overlaps with a portion of Cyndl. However, Therefore, the nucleic acid sequence and deduced amino acid sequence of clone KAT-39-1 were Combined with the nucleic acid sequence and deduced amino acid sequence of yn d 1.2/3 The composite array formed by this is Cyn d 1.2/3 shown in FIG. Nucleus of complex of predicted CyndI protein family members indicated (full length) Represents the acid sequence and deduced amino acid sequence. FIG. 20 shows Cynd 1. 1 8 Oyohidan ヱdandan 1. Amino acid sequence of composite sequence expressed as 2/3 (full length) , and Cyn d1. Full-length CDNA clone designated as CDI, CDI, Comparison of full-length amino acid sequences deduced from

The nucleic acid encoding the allele-luken of the Cyndl protein described above was ・Cynodon dactylon Nucleic acid that reacts with plants is here using the disclosed methods or other suitable techniques of gene isolation and cloning. You can get it.

Fragments of the nucleic acid sequence 1-T are also within the scope of the present invention. It is. Fragments within the scope of the invention may be used to induce an immune response in a mammal, preferably a human. , for example, stimulation of minimal amounts of IgE: induction of production of IgG and IgM antibodies, and induces T cell responses, e.g. induction of proliferation and/or secretion of lymphokines and / or encodes a portion of Cyndl that induces T cell anergy. Contains fragments. The aforementioned fragments of cydandl are referred to herein as antigenic fragments.

Fragments within the scope of the invention also detect allergens that are cross-reactive with Cyndl. Other plant species for use in screening protocols for fragments capable of hybridizing with nucleic acids from. Specification As used herein, a fragment of a nucleic acid sequence encoding CyndI is referred to as CyndI. dl and/or the entire amino acid sequence of mature CyndI. A nucleotide sequence with fewer bases than a nucleotide sequence is called a nucleotide sequence. In general, Cyndl The nucleic acid sequence encoding one or more fragments of the protein encodes the mature protein. In some cases, the leader of the nucleic acid sequences of the invention will be selected from the bases. - select all or a portion of one or more fragments from a sequence portion; desirable. Nucleic acid sequences of the invention may also include linker sequences, modified restriction endonucleases, Cloning, expression or It can contain other sequences useful for purification.

The present invention provides expression vectors and vectors transformed to express the nucleic acid sequences of the present invention. host cells. and a nucleic acid encoding dl, or at least one The fragments can be isolated from bacteria, such as Escherichia coli (E,c).

earthwork), insect cells (baculovirus), yeast, or mammalian cells, e.g. , can be expressed in Chinese hamster ovary cells (CHO). suitable Appropriate expression vectors, promoters, enhancer-1 and other expression regulatory elements include: Sambrook et al., Molecular Cloning: A Laborat ory Manual, 2nd edition, Co1d Spring Harbor La laboratory Press, Cold Sprinkle Harbor, New York (1989). Other suitable expression vectors, promoters Enhancer-1, enhancer-1 and other expression elements are known to those skilled in the art. mammals, Expression in yeast or insect cells allows partial or complete glycolysis of recombinant material. Leads to vine formation and chinma or chain. A suitable vector for expression in yeast is Y epSecl (Ba Idar et al. (1987) EMBOJ, 6:229- 234); pMFa (Kurjan and Herskowitz (1982) Cell 30:933 943); JRY88 (Schutz et al. (1 987) Gene 54°113-123) and pYES2 (in vitro Invitrogen Corporation San Diego, California). These vectors are freely available It is. Baculovirus and mammalian expression systems are also available.

For example, baculovirus systems are commercially available for expression in insect cells. (Pha rMingen, San Francisco, California) pMSG vectors are commercially available for expression in mammalian cells. (Pharmacja, New York, Bis.) Katanii).

For expression in Escherichia coli (E. coli), suitable expression vectors include, among others , pTRC (Amann et al. (1988) Gene 69: 301-315 : pGEX (Amrad Corporation) ion], Melbourne, Australia/L, N): pMAL (N, E, Bio pRIT5 (Biolabs, Beverly, Massachusetts); pRIT5 (Biolabs); pET-11d (Novagen [N Matteison, WI) Jammeel et al. (1990) J, Virol, 64:3963-3966: and psEM (Knapp et al. (1990) Bi.

Techniques 8:3963-3966). For example, I)T Use of Rc and pET-11d will lead to expression of the non-fusion protein.

The use of pMAL, pRIT5, pSEM and pGEX protein (pMAL), protein A (pRIT5), truncated β-galactocida (PSEM) or glutathione S-transferase (pGEX). This will lead to the expression of the fused allergen. Dan Eyu dI, part 1 or 2 or more When the fragment is expressed as a fusion protein, the carrier protein and Cynd It is particularly advantageous to introduce an enzymatic cleavage site into the fusion junction between L or a fragment thereof. It is advantageous. Cyndl or its fragments are then subjected to enzymatic cleavage and cleavage at the enzyme site. and by biochemical purification using common techniques for the purification of proteins and peptides. can be recovered from flavoproteins. A suitable enzymatic cleavage site is blood Includes sites for coagulation factor Xa or thrombin; Suitable enzymes and protocols for cleavage are available from, for example, Sigma Chemical Campa. Si gma Chemical Company, Missouri-St. Lewis and N.E., Biolabs, Bebe, Mass. It is commercially available from Lee. Different vectors can also be used, for example IPTG (PRTC, Amann et al. (1988) supra; pET-11d1 Novagen [Novagen] Madison, WI) or temperature induction (pRIT 5. Pharmacia, Piscataley, NJ) have different promoter regions, allowing constitutive or inducible expression with . It also has an altered ability to degrade recombinantly expressed proteins, Different Escherichia coli (E.

It may be appropriate to express recombinant Cyndl in a host (Ecoli). (eg, U.S. Pat. No. 4,758,512). Alternatively, Escherichia coli (E, co li) altering the nucleic acid sequence to use codons preferentially utilized by is advantageous, in which such nucleic acid modification changes the amino acid content of the expressed protein. It will not affect the acid sequence.

The host cells can be prepared using conventional techniques, e.g. calcium phosphate or calcium chloride. transfection, DEAE-dextran-mediated transfection, or electroporation. Inyeon can be used to transform the nucleic acid sequences of the invention to express them. Ru. Suitable methods for transformation of host cells are described by Samb r o o k et al., supra. , and other laboratory texts. Nucleic acid sequences of the invention can also be synthesized using standard techniques.

The present invention also provides step C].nd I or less (and one fragment thereof) culturing host cells transformed with the encoding DNA sequence in a suitable medium; A mixture of cells and a medium containing Cyndl or at least one fragment thereof producing and purifying said mixture; or at least one fragment thereof. The host cells transformed with the vector are cultured in a medium suitable for the host cells. C yndi proteins and peptides are available for purification of peptides and proteins. techniques known in the field, e.g. ion exchange chromatography. -, gel filtration chromatography, ultrafiltration, electrophoresis and Cyndl or Using immunopurification using antibodies specific for the fragment, cell culture, host It can be purified from cells or both. The terms isolated and purified , used interchangeably herein, and recombinant DNA technology, or chemical precursors. When produced by the body, it contains substantially no cellular material or culture medium, or Peptides and proteins that contain virtually no other chemicals when chemically synthesized proteins, protein fragments, and nucleic acid sequences. Therefore, the isolated Peptides can be produced by recombinant DNA technology or chemically synthesized. , and substantially free of cellular material, media, chemical precursors or other chemicals. stomach.

Another aspect of the invention provides that the DNA encoding all or a portion of cydandl synthesized in a host cell transformed with the sequence or chemically synthesized Cyndl or at least one fragment thereof and a nucleic acid sequence of the invention Produced in transformed host cells or chemically synthesized,! ll made Cyndl protein or at least one fragment thereof. do. produced in a host cell transformed with the Cyndl protein of the present invention. Ru.

For example, a fragment of Cyndl is , synthesized from the corresponding fragment of the nucleic acid sequence of the invention encoding a peptide, or chemically synthesized peptides using techniques known in the art. can be obtained by screening. allergen peptide Fragments (by selecting fragments of desired length with no peptide overlap) or over the desired length, which can be produced recombinantly or synthetically. can be obtained by selecting fragments that overlap. Fragments can be tested for antigenicity (e.g. For example, the ability of a fragment to elicit an immune response) can be determined. fragments like this are referred to herein as antigenic fragments. T cell responses, e.g. stimulation (i.e. proliferation or lymphokine secretion) and/or induce T cell proliferation or secretion of lymphokines). The allergenic fragment of the CyndI protein that can induce anergy is , especially desirable.

Proteins that do not bind to immunoglobulin E (IgE) or induce fragments A fragment of Cyndl that binds to IgE to a substantially lesser extent than the quality allergen , is also particularly desirable. The major complications of standard immunotherapy are systemic reactions, e.g. For example, it is anaphylaquinone. Immunoglobulin E is present in mast cells or basophils. Antigen binding and cross-reactivity to IgE on cells and mesoieters (e.g. The release of histamine, serotonin, niotunophils, chemotactic factors) It is a mesoieter of the nafilaquinone reaction. In this way, anaphyrakin (′ 1. Is it possible to avoid this by using a fragment that does not bind to IgE? If the fragment binds to IgE, such binding may occur in mast cells or basophils. This results in the release of mesoieters (eg, histamine, etc.) from the cells. moreover, Fragments with minimal IgE stimulating activity are particularly desirable for therapeutic efficacy. most Small IgEililIm activity was observed in whole Bermuda grass. ) less IgE production than that stimulated by the protein allergen. Call stimulus activity. Preferred fragments of the invention include, but are not limited to: Unspecified・Amino acids 5 to 246.10 of Wataru 1.18 shown in Figure 20 Fragments derived from 246.20-246 and 25-246; shown in Figure 20 Cyndl, cDl amino acids 5-246.10-246.20-246 and 25-246, and the fragment d1.2/3 shown in FIG. full length) amino acids 5-244.10-244.20-244 and 25-244 Fragment derived from.

≧Wataru] and preferred antigenic fragments thereof are Bermuda g. rass) pollen to individuals susceptible to the allergen. can modify the allergic response and preferably to the allergen altering an individual's B cell, T cell response, or both B cell and T cell response; be able to. When using it here, use Bermuda (Gyoginba). sensitized to pollen protein allergens, such as Cyndl. Modification of the allergic response of susceptible individuals is accomplished using standard clinical procedures (see, e.g., V Arney et al., Br1tish Medical Journal 1302: 265-269)), non-responsiveness or symptomatic reduction, e.g., induced by the pollen of Bermuda graSS Defined as a decrease in asthma symptoms. In this specification, reduction of furrow symptoms is mentioned. After a treatment regimen using the tannoseri peptides of the present invention, Involves a reduction in symptoms in an individual's allergic response to an allergen. this Reduction in symptoms can be determined subjectively (i.e., the patient is less sensitive to the allergen). (feel more comfortable when exposed to) or clinically, e.g. can be determined using a test. IgE to Cyndl or its fragments Initial screening for binding in laboratory animals or human volunteers by scratch test or sarcastic skin test or by inv. itro-based, for example, RAST (Ran-or-allergosorbent test [radio allergosorbent test]), RAST prevention, ELI by SA assay, radioimmunoassay (RIA), or histamine release. It can be implemented.

A book having progeny cell stimulatory activity and consisting of at least one T cell epitope Antigenic fragments of the invention are desirable for Blocking or perpetuating the immune response to protein allergens that cause symptoms I believe it is related to the sequel. These T cell epitopes are expressed on the surface of antigen-presenting cells. bind to appropriate HLA molecules on the surface and stimulate relevant T cell subpopulations. thought to trigger early events at the level of T helper cells by It will be done. These events include T cell proliferation, lymphokine secretion, local inflammatory responses, recruitment of additional immune cells to the site and recruitment of B cells leading to antibody production. Leads to activation of the skate. One of these antibodies, IgE, is is of fundamental importance to the development of allergic symptoms, and its production is important for the occurrence of the event. In a cascade, at the level of T helper cells, secreted lymphokine affected initially by the nature of the process. ■Cellular epitopes are T cell receptors The basic element of recognition by Consists of amino acids essential for receptor recognition and protein amino acids. (A) can be adjacent and/or non-adjacent to the acid sequence (A). mimics the amino acid sequence of the tope and is highly effective against allergies to protein allergens. Amino acid sequences that alter response are within the scope of this invention.

The Cyn dI or antigen of the present invention consisting of only one T cell epitope Exposure of the patient to the protein allergen causes the patient to become non-responsive to the protein allergen. To avoid stimulation of the immune response during such exposures, appropriate T-cells should be used. A subpopulation of cells can be made resistant, or some cells can be allergic. difference Furthermore, the Cyndl or Cyndl of the present invention consisting of a small number of Administration of antigenic fragments (e.g., naturally occurring protein allergens or To a part! The ability to alter the profile of lymphokine secretion compared to dew (e.g., resulting in a decrease in IL-4 and/or an increase in IL-2).

Additionally, exposure to Cyncll or such antigenic fragments (usually associated with allergens) can influence subpopulations of T cells that participate in the response to These Tm cells are located at one or more sites of normal exposure to allergens (e.g. (e.g., nasal mucosa, skin, and lungs) therapeutic administration of one or more fragments is attracted towards the area of This redistribution of T cell subpopulations The ability of an individual's immune system to stimulate a normal response at the site of normal exposure to To reduce or to reduce power.

Cyndl and fragments or portions (peptides) derived therefrom are Diagnosis of allergic response to Bermuda grass pollen, Can be used in methods of treatment and prevention. Thus, the present invention isolated and at least one fragment thereof and a pharmaceutically acceptable The present invention provides a therapeutic composition comprising a carrier or diluent that can be used as a carrier or diluent. 〔end　I or At least one fragment thereof is preferably a protein allergen or a protein allergen thereof. produced in cells transformed to express the fragment, or synthetically produced. Manufactured in Administration of the therapeutic compositions of the invention to the individual to be desensitized can be performed using known techniques. It can be carried out using techniques. Cyndl or a fragment thereof, e.g. be administered to an individual in combination with appropriate diluents, carriers and/or adjuvants. I can do it. Pharmaceutically acceptable diluents include physiological saline or aqueous buffers. includes. A pharmaceutically acceptable carrier is polyethylene glycol (Wie (1981) Int! , Arch, Allergy and Applied, Immun.

1.64:84-99) and liposomes (Strejan et al. (1984) J, Neuroimmunol, 7:27). Induces T cell anergy Therapeutic compositions are preferably administered in non-immunogenic form, e.g. It contains no adjuvant. Such compositions are generally administered by injection (subcutaneously or (e.g. intravenously), orally, by inhalation, percutaneously or rectally. will be administered more frequently. The therapeutic composition of the present invention is a therapeutic composition of the present invention. In the treatment regimen for individuals susceptible to the pollen of the grass Decrease the susceptibility of individuals to Bermuda grass pollen. i.e., at an effective dose and over a period of time to reduce the allergic response). administered. An effective amount of the therapeutic composition includes a factor such as Berber The degree of sensitivity of individuals to pollen of Muda grass, age, sex, and The weight of each individual, and the pollen content of Bermuda grass. Allergens or fragments thereof vary according to their ability to elicit an antigenic response in an individual. There will be.

cDNA encoding Dandan dI (or mRNA transcribed from it) or its identify similar sequences in any variety or type of plant using a portion of Thus, the protein allergen cDNA or mRNA or a portion thereof identify sequences with sufficient homology to hybridize to, or Alternatively, it can be used to "single out". For example, the cDNA of the present invention are temperate grasses, such as ryegrass, sagebrush, timothy, and kamoga. DNA1 from Bahia and other grasses, such as Bahia grass. hybridize under low stringency conditions to DNA from can be used. have sufficient homology (generally greater than 40%) Sequences can be selected for further evaluation using the methods described here. . Alternatively, high stringency conditions can be used. this method The DNA of the invention can be used to plant other types of plants, preferably related families, In the genus or species, it has an amino acid sequence similar to the amino acid sequence of CyndI. Sequences encoding polypeptides can be identified that encode polypeptides that Allergens in can be identified. Thus, the present invention (Bermuda grass) allergen Cyndl as well as Katsuma encoded by a DNA that hybridizes to the DNA of the present invention Includes other allergens. The present invention is based on the following features: um) eliminate allergens or their fragments of proteins from the genus These include, for example, allergens or fragments thereof that are cross-reactive with antibodies. Cyndl or protein allergens or fragments thereof of the present invention or by other immunoassays that can bind to antibodies specific for the fragment, or or isolated antigenic proteins or fragments thereof may be used as proteins or fragments of the present invention. and an array of T cell cross-antibiotics that can stimulate T cells specific for peptides and peptides. or fragments thereof.

The protein or peptide encoded by the cDNA of the present invention is, for example, , "purified" allergens can be used. Such purified a The allergen is sensitive to the pollen of Bermuda graSS. In the standardization of allergen extracts, which are key reagents for the diagnosis and treatment of It is useful. Furthermore, proteins based on the nucleic acid sequence of dan dl or those By using fragments of anti-peptide antisera, polyclonal antibodies or Monoclonal antibodies can be produced by Keyaki's method. These serum or Polyclonal or monoclonal antibodies can be used to standardize allergen extracts. or natural or recombinant protein allergens that can be used for It can be used in the purification of

By using its antigenic fragments, a consistent, well-defined composition and and biologically active preparations that can be made and administered for therapeutic purposes. (for example, those sensitive to the pollen of Bermuda graSS) to modify allergic responses. The administration of such peptides or proteins For example, B cell response to Cyndl, T cell response to Cyndl You can change the response or both responses. Additionally, using isolated peptides It is used to relieve allergic reactions to Bermuda grass pollen. Modified derivatives useful in studying the mechanism of epidemic treatment and in immunotherapy or analogs can be designed.

increase solubility, improve therapeutic or prophylactic efficacy, or stability (e.g. ex vi vo shelf life and resistance to in vivo proteolytic degradation) The structure of Cyndl or its fragments can be modified to enhance the Wear. If the amino acid sequence is changed, for example, by amino acid substitution or deletion, reduced genicity and/or reduced allergenicity, or for the same purpose. Producing modified and modified dT or modified fragments thereof with the addition of certain components can be done. For example, amino acid residues essential for the function of a T cell epitope. groups using known techniques (e.g., substitution of each residue and presence or absence of T cell reactivity). (current measurements). Modify residues shown to be essential (e.g. other agents whose presence has been shown to enhance T cell reactivity) substitutions with amino acids) as well as altering residues that are not required for T cell reactivity. (e.g., its incorporation enhances T cell reactivity, but its incorporation into related MHC (by substitution with other amino acids that do not reduce binding). Stability and/or Cyndl or a fragment thereof to enhance reactivity. Proteins modified to have one or more polymorphisms obtained from mutant forms of natural alleles can be incorporated into the amino acid sequence of a quality allergen. Furthermore, D-ami substitutions or additions of amino acids, non-natural amino acids or non-amino acid analogs to the present invention. Modified proteins or fragments within a range can be produced. moreover, Cyndl or its fragments were investigated by A, Sehon and collaborative research. (Wie et al., supra) by the polyethylene glycol (PEG) method. , can produce peptides conjugated with PEG. rhoyndl or its disconnection Modification of fragments can also be achieved by reduction/alkylation (Tarr: Methods of Pr otein Microcharacterization SJ, E, 5iIv erEi, Humana Press, Lifton, New Jersey, pp. 155-194 (1986)) near acylation (Tarr, supra) 7 esterification ( Tarr, supra), chemical coupling to a suitable carrier (Michell and Edited by Shiigi, 5 selected Methods in Ce1lular Immunology SWH Freeman, Sun Franci, California Scott (1981); U.S. Pat. No. 4,939,239), or mild formali Processing (Marsh, International Archives of Allergy and Applied Immunology 41:1 99-215 (1971)).

and site-directed mutagenesis of DNA encoding Edandl or its fragments. can be used to modify the structure. Such methods include PCR (Ho et al. Gene 77: 51-59 (1989)) or mutated genes. Overall synthesis (Hostoms ky, Z, et al., Biochem, old op. hys, Res, Comm, 161:1056-1063 (1989) ) can be included. Alternatives to the aforementioned methods to enhance bacterial expression Used in combination with Can be exchanged.

Using currently available structural information, Bermuda gra ss) pollen in a sufficient amount to an individual susceptible to pollen, z<(Be rmuda grass) that alters the individual response to pollen. You can design petit de. This, for example, inspects the structure of DanEdan dl and individuals susceptible to Bermucla grass pollen. Tests for the ability to influence B cell and/or T cell responses in the body to produce the desired peptide (through an expression system or synthetically) and by cells. Performed by selecting appropriate or T-cell epitopes that are recognized be able to. The protein, peptide or antibody of the present invention can also be used as Detects the sensitivity of Bermuda grass pollen to allergens. or can be used to diagnose. For example, this could be done like this: against the pollen of Bermuda grass that can be Blood or blood products from individuals to be evaluated for susceptibility to Cyr5dl isolated antigenic fragments, or isolated antigenic fragments, and components in blood ( For example, antibodies, T cells, B cells) and one or more fragments or proteins. combine under suitable conditions for binding and determine the extent to which such binding occurs. decide.

Currently, there are also individuals who are sensitive to Bermuda grass pollen. Blocking or inhibiting the ability of Miedan dI to trigger an allergic response in the body It is possible to design agents or drugs that can. like this The factors bind, for example, to the anti-Cynd I-IgE with which they are associated, thus to prevent IgE-allergen binding and subsequent mast cell degranulation. It can be designed in any way. Alternatively, such factors may be linked to immune system cells. It binds to the components of the vacuole and acts against the pollen of Bermuda grass. can suppress or desensitize allergic responses. This non-limiting Examples include suitable and T cell production based on the cDNA/protein constructs of the invention. Using epitopic peptides or modified versions thereof, rmuda grass) by suppressing the allergic response to pollen. Ru. This is because individuals are sensitive to Bermuda grass pollen. In in VitrO studies using blood components from the body, B and T cells By determining the peptide structure of T cell epitopes that influence the function of It can be implemented by

In charge of this invention! The DNA used in this assay is as described herein. The resulting cDNA can be or as expressed herein. The function that has all or part of the array! oligonucleotide sequence, or can be functional equivalents thereof. Such an oligonucleotide sequence , can be produced chemically or mechanically by known techniques. Oligonuku A functional equivalent of a leotide sequence is one in which the sequence (or corresponding portion of the sequence) is hybridizing complementary oligonucleotide, complementary to a nucleic acid sequence can hybridize to a sequence (or a corresponding part of a sequence) encoded by the event and/or array (or corresponding part of the array) products that have the same functional properties as the product being treated (e.g., a polypeptide or peptide). Criteria for one or both functional equivalents must be satisfied, but it will depend on the use (for example, If it is used only as an oligoprobe, it only satisfies the first criterion. When it needs to be used for cyndl production, it uses the second Only the criteria need to be met. ) The invention is further illustrated by the following examples.

Example 1 Cyndl isolated pollen extraction for protein sequencing and MAb production preparation of things Bermuda grass pollen is collected from the Greer Laboratory. Greer Laboratories, Reno, North Carolina, USA Yes, I bought it. Soluble protein loaded on Rotofor To prepare the texture, 5 g Bermuda grass of pollen was incubated at 4°C with 10ml of 10mM phosphate buffered saline (PBS). Extracted three times by shaking for 1 hour. After each extraction, centrifuge ( 250 rpm, 10 minutes) and the supernatant was collected. After 3 extractions, supernatant The liquid was pooled and passed through a 3 mm Whatman filter. It was filtered.

Preparative isoelectric focusing (IEF) Rotofor (Biorad, California) IEF for preparation in Sutchmond, State) was prepared according to Egan et al. (1988) Analyze t, Biochem, 172.488-494. simply Specifically, 5 ml of a solution of ampholyte (Bio-lyteSpH range 3-10; 4o%) was added to the pollen extract and the volume was adjusted to 5Qml with distilled water. child The mixture was loaded into a Rotofor cell and heated to 4°C and and a constant power of 12W. After 4 hours, collect 20 fractions and The pH was determined. Fractions containing the protein of interest were isolated after 5DS-PAGE. Identified with MAb 3.2 on Munoplot. This MAb was purified Lo]p Raised against I, but included Bermudagrass S was found to cross-react with group 1 homologues from nine other grasses. (Kahn and Marsh, 1986, Mo1. Immnol, 23. 1281-1288). Pool both portions containing the protein of interest, and Rotophoresis was performed under the same conditions as above, except that the samples were subjected to a 25 hour tofor). The pH of each fraction was determined.

5DS-PAGE and Western Bronoting Rotofor ) fractions were purified under reducing conditions with a 10-15% gradient of 5DS- Resolved by electrophoresis on a polyacrylamide gel. The conditions for electrophoresis are , essentially Singh and Knox, Int, Arches Appl, Imm Un, 78:300-304 (1985). Ta. Low molecular weight (MW) from Ph7-macia (Pha rma ci a) Molecular weight (MW) was determined using a standard. Ta on polyacrylamide gel Protein with Cooma's Brilliant Blue Visualization was performed using Brlliant Blue) R250.

Proteins were prepared by Towbin et al. (1979): Proc, Natl.

Acad, Sci, U, S, A, 76.4350-4354: 120 Nitrocellulose (Schleicher and Schleicher) overnight at mA and 4 °C. [5chleicher & 5chuell], 045 μm).

After transferring the proteins, powdered milk [TBS (Tris-buffered saline: 1 10% in 50mM NaCl/10mM Tris HCL pH 7,5) Western blot incubation blocks non-specific binding sites did.

In dividing the fractions obtained by preparative IEF by 5DS-PAGE, Temperature can be absorbed into fractions 10-20 in the pH range of 6-10. revealed. These fractions contain the 31-32 kD protein bound to MAb3.2. Contains protein. of both fractions 10-13 (32kD) bound to MAb3.2. The protein has a slightly higher MW than that in both fractions 15-20 (31 kD). (Figures 11a to 11b). Intermediate fraction 14 bound MAb3.2 Fractions 10-13 of Figure 11a containing both Tan Cyndla were pooled and However, it was found that it was superior to protein components 13 to 20 on both sides (first discovery). However, the protein profile of fractions 1 to 12 was superior to that in immunoplot analysis. Western blots were inked in MAb 3.2 or serum from allergic individuals. was developed. MAb 3.2 in PBS containing 0.5% BSA : Diluted to 1000. Binding of MAbs was performed using Singh and Knox (19 85) Peroxidase-labeled anti-mouse Ig antibodies as described above (Dakopatts Corporation n), Carpinchilia, California, USA). and visualization by subsequent addition of enzyme substrate. Human serum with 0.5% BSA Diluted to 1.4 in PBS containing 150mM. 1257 standards for IgH binding Discrimination anti-human 1gE (Kalestad) (PBS/ Incubation of the plots in (diluted to 16 in BSA) and subsequent and Ib due to their ability to bind IgH from the serum of allergic individuals. (Fig. 13). Both fractions were collected from Bermuda g. rass) from the serum of allergic individuals (7) bound to IgE.

Sequencing of NH2-terminal amino acids As mentioned above, the protein Cyn Ia and Ib are sea urchin, separate and transfer buffer (Ward et al., 1990) (3[cyclohexyl 10mM CAP8 10% meth Polyvinylidene difluoride (PVDF) membrane (PVDF) was prepared using alcohol (pH 11,0). Rebore [MiIl 1pore], Bedford, Massachusetts, USA) electrotransferred onto the surface and then stained with Coomane Brilliant Blue R250. Visualize by mixing with methanol and acetic acid (50:10:40, v/v). /v) and washed thoroughly with deionized water. Cyndla of both and the NH2-terminal amino acid sequence of the Cynd Ib protein was determined by Ward et al. (1990).

Cyndl protein isolated by Rotofor was also , purified by reverse-phase HPLC, and purified by NH,-terminal amino acid of the 31 kD protein. The amino acid sequence was determined.

The two Cyndl components have a small amino acid sequence in their NH2-terminal region. and homology between Dankodan dl and Lolpl from ryegrass. exists (Table 1).

Table 1: Cyndl isoallergen and LolpI NH2-terminal sequences Determined after transfer to -1-PVDF.

*Determined after HPLC purification.

'Matthiesen et al., 1991, supra.

2 Matthiesen et al., 1991, European Congress A11erty C11n, Immun○], Conference, France Paris, May 1992, Abs trac t.

3Cottam et al., 1986, Biochem, J, 234.305-310. 1. Griffith et al., 1991, FEBS Letters, 279.21 0-215; Perez et al. (1990), J. Biol, Chem, 265 :16210-16215゜ MAb production and screening Ba1b/Cv mice were mixed with 50 μg of Cyn dI (of-7t-[R.

Biorad, California Anti-Cyndl MAb was obtained by intraperitoneal immunization with Ta. RIBI (RIBI Immunochem, Montana, USA) (Hamilton, State) was used as an adjuvant in the first of four immunizations.

The remaining intraperitoneal immunizations were in saline. hybridoma fusion and Growth is essentially as described by Harlow and Lane, 1988, Antibody s:A Laboratory Manual, Cold Spring Ha It was as described in Rbor Laboratory. single cell Cloning was by limiting dilution. Hypuritoma-producing anti-Cyndi antibody is Identified by ELISA assay. CAPS buffer (6,67mM NaCO 30 μg of Kyodai annua (335 mM NaHCO3 pH 9,6) Bermuda grass) pollen extract was added to the ELI SA plate overnight. coated. The wells were then filled with TPBS (P containing 0.1% Tween 20). BS) three times and PBS containing 1% BSA (PBS/BSA ) for 30 minutes. Add 100μI of primary antibody to each well and Incubate for 60 minutes, then wash as above) and β-ga1 in labeled anti-mouse Ig (1/250 dilution in PBS/BSA, 60 min) Incubated. After washing, 200 μm of 4-methylumbeta of the fluorescent substrate was added. Referyl-B-D-galactonodide (MUG) was added to each well and 37 Incubated for 30 minutes at °C. The plate is then fluorocarbonated. f1uorocount 96 fluorometer (Furman [P harmacia]).

Antibodies that were positive by this method were designated as 3A2.4D2, ID1 and 3C2. Bermuda g. rass) pollen protein binding to Cyndl on Western plot. I tested it.

cDNA library and immunological screening essentially Grid as described in N and Michael S (1984). Greer Laboratories, North Carolina, USA Bermuda grass purchased from Lenoil, Waraina state. Poly(A+) mRNA was isolated from the pollen of A. The cDNA was purified by Pharmasore (P h　a　rma　c　a) cDNA synthesis using known techniques; and cloned into the EcoR1 site of lambgoo gt11 of the vector. P The rated cDNA library was transferred to a nitrocellulose film impregnated with IPTG. Recombinant tags from phage plaques are obtained by overlaying with filters. Proteins were transferred to nitrocellulose filters. Then these filters was inked in a mixture of anti-Cyndl MAbs.

Binding of MAbs to recombinant proteins was visualized as described above. anti and purification did.

Isolation of cDNA clones The cDNA of Bermuda grass pollen was as described above. First, we first developed an anti-Cyndl hybridoma containing mainly MAb 3.2. A mixture of clear fluids was screened and 3o positive cDNAs were plaque purified. Manufactured. These clones were then treated with anti-Cyn dI MAb3A2.4D2 ．． Binding to 3C2 and ID1 was tested. 1st round screening All clones selected after testing included recombinant fusions specific for MAb3A2. produced protein. Binding of clones to MAbs is shown in Figure 14 and in Table This is summarized in 2. The cDNA clone isolated here is suitable for the recombinant fusion protein. Based on the binding of MAb shown as It will be done. MAbl, Dl has much higher background binding than other MAbs. and made the combination much more subjective.

Table 2 Binding of monoclonal antibodies Nucleotide and amino acid sequences of cDNA clones Clone 2.3.18 ．． 21, 22, 23 and 33 (see Table 2), based on their antibody affinities. selected for further study. Clones 2.3.18.21.22.23 and and 33 were isolated from the phage, and pGEM-42 (Promega [Prom e　g　a]) or Bluescript (Strata The vector was subcloned into the Stratagenel vector. T7 Polymerase (Pharmacia) was used to terminate the chain (SarIger et al., Pr. oc.

Natl, Acad, Sci. (1977), 74:5460-5463). The DNA sequence was determined by double-stranded sequencing performed by of these clones The nucleotide sequence and deduced amino acid sequence are shown in Figure 1 (clone 2) and Figure 2. Figure (clone 18), Figure 15 (clone 3), Figure 16 (clone 22) and and FIG. 17 (clone 23).

All sequenced clones were specifically identified in the open reading frame (OR In F), homology with each other is shown. In addition, all clones sequenced and Lo l p I, i.e., the significant Nucleotide sequence homology exists. However, the sequenced clones Three groups based on nucleotide and predicted amino acid sequence homology The one with the most similar sequence to clone 2 (i.e. clone 3) ), those with the most similar sequences to clone 18 (i.e., clones 21 and and 33), and those with the most similar sequence to clone 22 (i.e., clone 23). The predicted The amino acid sequence was compared to the deduced amino acid sequence of Lolpl (Perez et al., 1991, supra; Griffith et al., 1991, supra) (Figure 6). Lo 67% between lpI and clone 18.

There is 72% amino acid homology between Ipl and clone 2. clone 2 and 18 deduced amino acid sequences are 83% identical to each other (87% homologous). gender).

Example 2 Cloning two mA c D N A of the 5' end of Cynd I, approximately 4μ g pollen RNA (Grill Laboratories, Lenoy, North Carolina, USA) cDNA Synthesis System Plus Kit (BRL, Bermuda, Maryland, USA). Synthesized using Sesda). After phenol extraction and ethanol precipitation, cDNA A with T4 DNA polymerase (Promega, Madison, WI, USA) blunt-ended and ethanol precipitated, self-annealed, toT, 5°- GGGTC-AGAGGTACCGTCCGA-GATCATr-3', YO U AL, 5'-p-AATGA CGA DING GCT-3°, i.e. change Anchored PCR (Marsh et al., 1986: Roux and Dhanarajan, 1990: Rafnar et al., 1991) coupled to oligonucleotides, for use in reactions. 1μg oligonu Creotide AP, 5' GGGTCT, AG, AGG Ding ACCGTCCG-3' , and CD-5・5'-GATGTC+CrCGTAGTTCTT-3, sub ; that is, the non-codin of Cyndl corresponding to amino acid (11KNYE) 11 A linker was attached to each of the oligonucleotide primers based on the sequence. From the cDNA (5 μm from a 20 μm reaction), the amino terminus of CyndT was inserted. The encoding cDNA was amplified. 10 μM 10x various dNTP-containing buffers, 1 μg of each primer, cDNA, 0.5 μl of Ambrituff (Amp II Gene Amp (G) in a reaction mixture containing DNA polymerase (taq) ene Amp) DNA Amplification Kit (Perkin Elmer Setus [Perki Elmer Cetus, Norwalk, CT, USA). , MJ Research, Inc. In a programmable controller from Cambridge, Sat. - Perform next polymerase chain reaction (PCR) and make 100 μm with distilled water. Ta.

25 rounds of amplification consisted of 1 min denaturation at 94°C, 1.5 min at 65°C. Annealing of primers to template for 2 minutes and chaining for 2 minutes at 72°C. It consisted of an extension. Next, 5% (5 μm) of this secondary tank width was injected with 1 μg of each C. D-4,5'-GGGGATCCGAGGCCOT-CCTrGAAG-3°, Regarding CD-5 based on the non-coding chain sequence corresponding to the amino acids IFKDGL Cynd I oligonucleotide primers and AP was used as above in secondary amplification using . all oligonuks Reotide is manufactured by Research Genetics Inc. ch Genetics, Inc.

) (Huntshill, Alabama). oligonucleotide primers -AP, AT, and eight were previously described (Rafnar et al., 1991:j vlorgenstern et al., 1991: Rogers et al., 1991). CD Bam) IT system for cloning purposes by adding the first 8 nucleotides of -4 I created a limited area.

The amplified DNA was subjected to sequential chloroform, phenol, and chloroform extractions. 0.5 volume of 75 ammonium acetate or and 15 volumes of isopropanol.

After precipitation and washing with 70% ethanol, the DNA was incubated with Xb in a 15 μM reaction mixture. Digested simultaneously with al and BamHT and preparative 3% Nubrak (Sea Plaque's low melting point agarose gel (FMC Corporation, USA) (Rockland, N.C.).

DNA bands of appropriate size are visualized by ethidium bromide (EtBr) staining. oxidation, excision, and sequenase (S eq u e n a se y) using (U, S Biochemicals, Riebrand, Ohio, USA) Deoxy DNA sequencing (Sanger et al. (1977), Proc, Natl. , Acad, Sci, USA, 74:5460-5463). It bound into M13mp19. Two clones 14a1 and 14C1 A completely sequenced and in-frame image obtained from this It was discovered that it contains the methionine of Ninoator. 14C1 sequence nucleus Methionine encoded by otides 28-30 (Figure 6) is the most preferred represents the start codon. This is because the surrounding sequences are the common plant sequence 5'-A, ACAATGGC-3' (Lutcke et al. (1987) EMBO J6143-48) and an in-frame termination just upstream. This is because codons exist. 14cl (Figure 7) has two potential inflation The methionine-containing part of the system is encoded by nucleotides 27-29. The most commonly used methionine is vinyl/ether methionine. Because surrounding The sequence contains a methionine encoded by nucleotides 42-45. Census plant sequence 5'-AACAA engineering GGC-3' (Lutcke et al., supra. (78% vs. 56% match). Furthermore, the corresponding array Nucleotides 27-29 are identical to those of clone 14a1. clone 1 The sequences of both 4a1 and clone 14c1 are the longest CyndI clone, all That is, it had a 17 nucleotide overlap with clone 18. mature C Amino terminus of yndI, NH2-A encoded by clone 14a1 IGDKPGF'NTTATGNKWLEAKATFYG and claw: /1 NH2-AIGDKPGPPNITATGSKV/encoded by 4C1 LEAKATFYG- is a combination of two proteins previously announced for Quality sequence NH2-AMGDKPGP? ITATYGDKWLDAKATFYG( Matthiesen et al. 1988. Previous t' Matthiesen La, 19 90. ! at 711 Matthiesen et al., 1991. Previous 1%> Oyo hNH2-A]GDKPGPKffATY? ? KWLEAKAT(Singh) La, 1990. (cited above).

It was possible to identify it by comparing it with As a result, clone 14a] and 14c1 have leader sequences of 22 and 26 amino acids, respectively. It was shown that When these leader sequences are cleaved, the yndl protein becomes A mature form will be obtained. As shown in Figure 9, the arrangement of Cyn d 1.14 By joining clone 18 with Cy in their hyperply, Cy Potential full-length amino acid sequence of Cyndl designated as nd 1.18 (Fig. 9 ) was created. In both cases, the track formation suspension of Dankotan di is 26.7kDa. It is predicted to be 246 amino acids with a calculated molecular weight of .

Example 3 Chomczynski and Acchi (1987) Analyt, Bi ochem, 162:156-159. Use pollen of Cynodon dactylon. RNA was isolated. 9 ml guanidinium thiocyanate buffer (0.05% Tris HCI [pH 7,0], 0.055 volume β-mercaptoethanol, 0 Pollen was harvested in liquid nitrogen using 1% by volume sodium lauroylsarconone. Shattered. The pollen solution was then shaken with phenol (10 ml) for 10 min. , then add 10 ml of chloroform isoamyl alcohol, and this The mixture was shaken for an additional 20 minutes. 7. Add this mixture. Centrifuge for 25 minutes in OOOXg and the aqueous phase was collected.

The aqueous phase was re-extracted with phenyl chloroform isoamyl alcohol 2524]. , then 2. until the interface is clear. It was centrifuged in OOOXg. Then add the aqueous phase to 3 m 1 of CsCl xenoylon (5,7M EDTA1 density = 1.71 g/ml) Decant into a Quickknoll ultracentrifuge tube placed in a Beckman Ti7010-tar (Beckman L8-70 ultra-long distance) Shinki, Beckman Instruments Centrifuged (20 hours, 40,000 r) in Fullerton, CA) prn, 20'C), after centrifugation, the RNA in the pellet was placed in 0005% SDS. Re-vf! extracted with phenol/chloroform and odor-208C. and allowed to settle overnight.

Pharmacia mRNA purified according to manufacturer's instructions. Poly A″R\, was isolated using a kit manufactured by the company.

Add 0.8 μg of mRNA to 700C and add 0.5 μg of oligo-dT primer (Factor). - Mana, New 7 Yazii Piscataei) The first cDNA was prepared. After cooling the mRNA on ice, 5x first strand binding and and 25 U of RNasein ribonuclease inhibitor were added. Then this mixture was heated to 42°C for 1 hour. Final reaction conditions were 50 µm with a final volume of 25 µm. M Tris HCL pH 8, 3, 50mM KCI, 10mM MgCl2.0 ．． 5mM spermidine, 10mM DTT, 4mM sodium pyrophosphate, 1mM each dATP, dCTP, dGTP and TTP, 25U RN synthesis Bonuclease inhibitor and 15 μAMV reverse transcriptase/μg RNA (Promega CDNA Synthesis Kit, Promega, Madison, Wis.).

The cDNA sequence encoding Cyndl was derived from Parkin-Elmer Setus (P Erkin-Elmer Cetus) gene amplification kit (U, S, Bioke Michaels, OH (Rebrand) was used for amplification. 5 μl (25%) The first antibody cDNA synthesis product was mixed with 10x buffer and 2mM MgCh, 50% mM KCI, 10 mM Tris-HCI, 1 μg of oligonucleotide Immer CD15'N.

5', GGGA, TrcGCcA'rc (T+GCG-Ac-Act-cCA (i-3" 1 μg O+J conucleotide primer CDL3"B I8 ．． 5'-CCCTGCAGA Ding G-GAGGATCATCGTCTC-3' 0 ．． 2mM dNTP and 25 units of Taq DNA polymerase (Furman , Piscataley, New York, Y., 7). CD15'N Add nucleotides 1-8 to create an EcoR1 target for cloning purposes. The clone in Figure 6 that encodes the nuclease restriction site (GDKP) corresponding to nucleotides 107-125 of 14a1 (Table 1, Figure 8 and Figure 8). Figure 9). Add nucleotides 1 to 8 of CD13'B18 to start cloning A Pstl endonuclease restriction site was created for the target, but at nucleotide 9. ~26 is a non-coding sequence complementary to nucleotides 604-621 of clone 18. This corresponds to the leading strand sequence (Figure 2).

Perkin-L for PCR? -・Seth (Perkin-ElmerCetus) Thermal Cycler (Perkin-Elmer, 'D Now, NE) oak), followed by 5 cycles of denaturation (94°C, 1 min) and annealing. (45°C, 11,5 min) and extended (72°C, 3 min) and continued (20 s. cycle denaturation (94°C, 1 min), annealing (55°C, 15 min) and extension. (72°C for 13 minutes). Final extension reaction was 10 min at 72°C. It was carried out for a period of time. The amplified product was subjected to phenol extraction, chloroform extraction, and then 0.5 volumes of 7.5 M ammonium acetate and 1.5 volumes of isochloride at 0°C. Recovered by precipitation using propatool. The reaction product is converted into DNA polymerase. blunt-ended with the Klenow fragment of and Bluescript (BIuescript), which was modeled on Hinclr ) cloned into a vector. Clone CDI was cloned using the nodeoxy chain termination method ( Sanger, supra), as described in Example 1, and The nucleotide sequence and deduced amino acid sequence of Cyndl shown in Figure 18 are It was discovered that it contains

Example 4 Double-stranded cDNA was prepared and subjected to a primary PCR reaction as described in Example 2. and oligonucleotide primers CD-13 and CD. C to 9C9M4N had AGGG-CCC-3', but nucleotide 14 was C or G Will. Add nucleotides 1-8 of CD-13 for cloning purposes. A new XhoI restriction site was created. The remaining nucleotides are the amino acids Ala (I le/Met)-GIyAspLysProG]yPro, and Here amino acid 2 would have been Ice or Met (CyndIa and C Amino acids 1-8 of yndlb (Table 1), CD-15 has the sequence 5'-GCGTACT TCACGAGCAGCGCCAG-GTAATT-3', which is an amino Acid AsnTyrLeuA]aLeuLeuValLysA]a (in Figure 5 Amino acid 1 numbered for clone 2 (C2) and clone 3 (C3) 59-168) complementary to the coding strand sequence encoding This corresponds to the ing sequence. - 5% of the reaction was performed in a secondary PCR as described in Example 2. Oligonucleotide primers CD-13 and CD-16 were added as shown. was used and amplified. CD-16 has the sequence 5'-TTGAATrCGACACGGC GGAACTGCAGCAT-3', where nucleotide 12 is G or It would have been A. Cloning by adding nucleotides 1 to 8 of CD-16 An EcoRI restriction site was created for this purpose. Nucleotides 9-29 are amino Noic acid MetLeuGInPheArgArgVa1 (C2 and and C3 (numbered amino acids 132-138) It corresponds to a non-coding sequence that is complementary to the chain sequence.

PCR amplification was performed as described in Example 2. Collect the amplified product and and ligated into pUC for sequencing as described in Example 2. It matched. KAT-39-1, which has a sequence identified as a Cyndl clone; The indicated clones were isolated. Nucleotide sequence and prediction of KAT-39-1 The determined amino acid sequence is shown in FIG. This clone is a CyndI clone c2 and C3 expansion. Oligonucleotides CD-15 and CD-16 is a single difference with the corresponding in Cyndl clone C18 and its homologues. with their 3' ends. Therefore, only clone c2 or C3, Or close family members will be amplified.

Dane Dandan 1. KAT-39-1 and Cyn displayed as 2/3 (full length) d 1. 2/3 (7) Composite array c, Cyn d 1. CD1 and cyndl , 18 is shown in FIG.

Although the invention has been described with reference to its preferred embodiments, other embodiments may achieve the same results. can do. Variations and modifications to this invention will be apparent to those skilled in the art. and all such modifications and equivalents that fall within the true spirit and scope of the invention. Such embodiments are intended to be encompassed within the scope of the appended claims.

FIG. FI311) FIG 2a Q FIG 2b FIG 3a FIG 3b FIG 5a FIGSb 14a1 PPF FIGB F2O3 I310 FIG 11a T I2 34 5G 78 91011121314 j516171R19 20FIG 12a FIG 15a FIG 1Sb FIG 16a FIG 16b FIG 17a FIG 17b International search report 1-1ie+w1m year N0 international search report! mc+y+++1 6m1+H1ies-N.

International Search Report I-Somei 1□Na Continuation of front page (51) Int, C1,' Identification symbol Internal office reference number Cl2P 21108 9161-48GOIN 33153 Q 8310-2J//(C12P 2 1108 C12R1:91) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, SE), AU, CA, JP, KRI

Claims (63)

[Claims] 1. Alleles of Bermuda grass pollen proteins a nucleic acid sequence encoding the gene Cyn d I, or at least one antigenic thereof Fragment or functional equivalent of said nucleic acid sequence. 2. formula: L1NYX In the formula, L1 is a nucleic acid sequence of 0 to 300 nucleotides, and the nucleic acid sequence is Cynd Contains nucleotides encoding the leader sequence of I, where N is up to 600 nucleotides. A nucleic acid sequence consisting of cleotide, and the nucleic acid sequence is an amino acid sequence of mature Cyn dI. Contains nucleotides encoding the terminal portion, Y is clone 2, clone 18, clone 3, clone 22, clone 23 or mature Cyn dI and X is a portion of a nucleic acid sequence in any polymorphic form thereof that encodes It is a nucleic acid sequence of 600 nucleotides, and the nucleic acid sequence consists of the 3' end of Cyn dI. contains the nucleotides of the translated portion, and L1 and X can be 0, or a functional equivalent thereof. 3. formula: L1NYX In the formula, L1 is a nucleic acid sequence of 0 to 300 nucleotides, and the nucleic acid sequence is Cyn d Contains nucleotides encoding the leader sequence of I, where N is up to 600 A nucleic acid sequence consisting of nucleotides, the nucleic acid sequence is a mature Cyn d I Contains nucleotides encoding the mino-terminal portion, Y is clone 2, clone clone 18, clone 3, clone 22, clone 23 or mature Cyn dI. portions of the nucleic acid sequences in any polymorphic form thereof that encode A nucleic acid sequence of ~600 nucleotides, said nucleic acid sequence being the 3′ of Cyn d I. contains an untranslated portion of nucleotides, and the N nucleic acid sequence is the 5' end of the Y nucleic acid sequence. does not overlap the edges, and L1 and X can be 0. or a functional equivalent thereof. 4. L1 is nucleotides 1 to 106 of clone 14a1 shown in FIG. Nucleic acid sequence represented by nucleotides 1 to 103 of clone 14c1 shown in Figure 7 The nucleic acid of claim 3, or a functional equivalent thereof. 5. N is represented by nucleotides 107-244 of clone 14a1 shown in FIG. or nucleotides 104-2 of clone 14c1 shown in FIG. 43, where Y is the nucleonucleotide of clone 18 shown in FIG. 1 to 603, and the nucleic acid sequence of X is shown in Figure 2. The nucleus of claim 4 containing nucleotides 604-775 of clone 18 as shown. acid sequence, or its functional equivalent. 6. N is represented by nucleotides 107-247 of clone 14a1 shown in FIG. or nucleotides 104-2 of clone 14c1 shown in FIG. The nucleic acid sequence represented by 46 is Y or the nucleotide sequence of clone 22 shown in FIG. A nucleic acid sequence represented by octides 1 to 594, and the nucleic acid sequence of X is the 16th Claim 4 containing nucleotides 595-802 of clone 22 as shown in the figure. or a functional equivalent thereof. 7. N is represented by nucleotides 107-246 of clone 14a1 shown in FIG. or nucleotides 104-2 of clone 14c1 shown in FIG. 45, and Y is the nucleotide sequence of clone 23 shown in FIG. A nucleic acid sequence represented by octides 1 to 595, and the nucleic acid sequence of X or the 17th Claim 4 containing nucleotides 596-832 of clone 23 as shown in the figure. or a functional equivalent thereof. 8. L1 is nucleotides 41 to 106 of clone 14a1 shown in FIG. 6, or Nucleic acid represented by nucleotides 28-103 of clone 14c1 shown in FIG. Claim 3, or a functional equivalent thereof, consisting of the sequence. 9. N is nucleotides 107 to 244 of clone 14a1 shown in FIG. Nucleic acid represented by nucleotides 104-243 of clone 14c1 shown in Figure 7 sequence, where Y is represented by nucleotides 1 to 603 of clone 18 shown in Figure 2. and X is 0, or its functional equivalent. 10. N or nucleotides 107 to 247 of clone 14a1 shown in FIG. The nucleus represented by nucleotides 104-246 of clone 14c1 shown in FIG. The acid sequence is Y or nucleotides 1 to 594 of clone 22 shown in Figure 16. the nucleic acid sequence of claim 8, or or its functional equivalent. 11. N or nucleotides 107 to 246 of clone 14a1 shown in FIG. The nucleus represented by nucleotides 104-245 of clone 14c1 shown in FIG. Acid sequence, where Y is nucleotides 1 to 595 of clone 23 shown in Figure 17. the nucleic acid sequence of claim 8, or or its functional equivalent. 12. L1 is 0 and N is nucleotide 10 of clone 14a1 shown in FIG. 7-244 or nucleotides 104-243 of clone 14c1 shown in FIG. The nucleic acid sequence represented by Y is the nucleotide of clone 18 shown in FIG. 1 to 603, and X is 0. or a functional equivalent thereof. 13. Ll is 0 and N is nucleotide 10 of clone 14a1 shown in FIG. 7-247 or nucleotides 104-246 of clone 14c1 shown in FIG. 16, where Y is the nucleotide sequence of clone 22 shown in FIG. 1 to 594, and X is 0. 3, or a functional equivalent thereof. 14. L1 is 0 and N is nucleotide 10 of clone 14a1 shown in FIG. 7-246 or nucleotides 104-245 of clone 14c1 shown in FIG. 17, where Y is the nucleotide sequence of clone 23 shown in FIG. 1 to 595, and X is 0. 3, or a functional equivalent thereof. 15. Nuclei where Y is represented by nucleotides 1 to 438 of clone 2 shown in FIG. Claim 3, or a functional equivalent thereof, which is an acid sequence. 16. Y is represented by nucleotides 1-417 of clone 3 shown in FIG. Nucleic acid sequence, and X is nucleotide 418 to nucleotide 418 of clone 3 shown in FIG. 594, or a functional equivalent thereof. . 17. L1 is 0 and Y is nucleotides 1 to 438 of clone 2 shown in FIG. and wherein X is 0, or the nucleic acid sequence represented by functional equivalent of. 18. L1 is 0 and Y is nucleotides 1 to 41 of clone 3 shown in FIG. 7, or a functional equivalent thereof. 19. Protein array of Bermuda grass pollen The nucleic acid sequence shown in FIG. 18 encoding Cyn d I, or clone C A functional equivalent of said nucleic acid sequence consisting of the nucleic acid sequence of D1. 20. Mature Cyn dI. shown in FIG. Encodes the amino acid sequence of CD1 nucleic acid sequence. 21. Protein array of Bermuda grass pollen Nucleic acid sequence encoding Cyn d I or Cyn d shown in FIG. I. Said nucleus consisting of a nucleic acid sequence encoding a 2/3 (full length) amino acid sequence Functional equivalents of acid sequences. 22. An expression vector comprising the nucleic acid sequence of claim 1, 3, 12, 19 or 20 ctor. 23. Encoded by the nucleic acid sequence of claim 1, 3, 12, 19 or 20 A host cell that has been transformed to express a protein or peptide. 24. Isolated Bermuda grass pollen protein Cyn d I, or claims 1, 3, 12, 19, or at least one of the isolated antigenic molecules encoded by 20 nucleic acid sequences; piece. 25. Cyn dI. shown in FIG. represented by 18 amino acids 1-246 An isolated Bermuda gr. ass) pollen protein allergen Cyn d I. 26. Bermuda grass pollen according to claim 25. Isolated antigenic fragments of protein allergens. 27. Cyn dI. shown in FIG. represented by amino acids 1-246 of CD1 An isolated Bermuda g. Cyn d I, a pollen allergen of Cyn d I. 28. Bermuda grass pollen according to claim 27 Isolated antigenic fragments of protein allergens. 29. Cyn dI. shown in FIG. 2/3 (full length) amino acids 1 to 244 An isolated Bermud grass comprising the amino acid sequence represented by a grass) pollen protein allergen Cyn d I. 30. Bermuda grass pollen according to claim 29 Isolated antigenic fragments of protein allergens. 31. The isolation of claim 24, 26, 28 or 30, which has T cell stimulating activity. antigenic fragments. 32. Amino acids 5-246, amino acids 10-246, amino acids 20-246, and amino acids 25-246; Cyn d I. as shown in FIG. All 18? An isolated fragment of Cyn d I having an amino acid sequence selected from the group consisting of peptide or an isolated antigenic fragment thereof. 33. Amino acids 5-246, amino acids 10-246, amino acids 20-246, and amino acids 25-246; CyndI. Everything about CD1 An isolated peptide of Cyn d I having an amino acid sequence selected from the group consisting of: or an isolated antigenic fragment thereof. 34. Amino acids 5-244, amino acids 10-244, amino acids 20-244, and amino acids 25-244; Cyn d I. as shown in FIG. 2/3 (total length) all of which have an amino acid sequence selected from the group consisting of: isolated peptide or isolated antigenic fragment thereof. 35. 29. The isolated immunoglobulin E-stimulating activity of claim 28 has minimal immunoglobulin E stimulating activity. Antigenic fragment. 36. Cynodon dactylon species protein A substantial percentage of individuals susceptible to protein allergens does not bind to immunoglobulin E specific for the quality allergen, or If binding of the fragment to immunoglobulin E occurs, such binding is essential. Claim 24, which does not result in the release of histamine from cells or basophil cells; 26, 28 or 30 isolated antigenic fragments. 37. The allergen of the protein that induced the fragment binds to immunoglobulin E. Claims 28, 32, which bind to immunoglobulin E to a substantially lesser extent. 33 or 34 isolated antigenic fragments. 38. Individuals susceptible to Bermuda grass pollen When administered to the body, the pollen of Bermuda graSS Altering the allergic response of said individual to a protein allergen. Allergen or isolated antigenic fragment of the isolated protein of Box 24. 39. Individuals susceptible to Bermuda grass pollen When administered to the body, the pollen of Bermuda graSS Altering the allergic response of said individual to a protein allergen. Isolated protein counterfeit allergen of Box 27. 40. Individuals susceptible to Bermuda grass pollen When administered to the body, the pollen of Bermuda graSS Altering the allergic response of said individual to a protein allergen. Isolated antigenic fragment of box 28. 41. Protein array of Bermuda grass pollen Response of an individual's B cells to rugen, Bermuda gras s) an individual's T cell response to pollen protein allergens, or Against the protein allergen of Bermuda grass pollen Claim 24: altering both the B cell response and the T cell response of an individual who An isolated protein allergen or an isolated antigenic fragment of. 42. Protein array of Bermuda grass pollen Response of an individual's B cells to rugen, Bermuda gras s) an individual's T cell response to pollen protein allergens, or Against the protein allergen of Bermuda grass pollen Claim 27: altering both the B cell response and the T cell response of an individual who isolated protein allergens. 43. Protein array of Bermuda grass pollen Response of an individual's B cells to rugen, Bermuda gras s) an individual's T cell response to pollen protein allergens, or Against the protein allergen of Bermuda grass pollen Claim 33: altering both the B cell response and the T cell response of an individual who An isolated antigenic fragment of. 44. The pollen of Bermuda grass is administered to susceptible individuals. When given, the protein of Bermuda grass pollen modified Cy that reduces the allergic response of said individual to the allergen of ndI protein allergen. 45. The pollen of Bermuda grass is administered to susceptible individuals. the allergic response of said individual to said protein allergen when given; at least one modification of a Cyn d I protein allergen that reduces fragment. 46. Greater than 73% phase with Cyn d I or its isolated antigenic fragment Isolated protein allergens with the same sex. 47. has greater than 73% homology with Cyn d I and Cyn d I or immunologically cross-reactive with antibodies specific for the isolated antigenic fragment thereof. Isolated protein allergen that is. 48. Cyn cells specific for Cyn d I or an isolated antigenic fragment thereof Isolated protein allergens that can irritate. 49. Cyn d of isolated Bermuda grass an allergen of a protein of I, or at least one isolated antigenic fragment thereof; A therapeutic composition comprising a pharmaceutically acceptable carrier or diluent and a pharmaceutically acceptable carrier or diluent. 50. The allergen of the protein is CyndI. CD1 Ami The therapeutic of claim 49 comprising the amino acid sequence represented by amino acids 1-246. therapeutic composition. 51. The protein allergen is Cyn dI. 18 a The treatment of claim 49 having an amino acid sequence represented by amino acids 1-246. Composition for use. 52. The allergen of the protein is Cyn d I. as shown in FIG. 2/3( the claimed amino acid sequence represented by amino acids 1 to 244 of Range 49 therapeutic composition. 53. administering to an individual a therapeutically effective amount of the therapeutic composition of claim 49. Bermuda grass pollen protein comprising or Bermuda graSS pollen allergens. susceptibility in said individual to an allergen that is immunologically cross-reactive with the allergen; How to treat susceptibility. 54. The therapeutic composition of claim 50, 51 or 52 in an amount therapeutically effective for an individual. of Bermuda graSS, comprising administering Pollen protein allergens or Bermuda gra ss) against allergens that are immunologically cross-reactive with pollen allergens. Methods of treating susceptibility in individuals. 55. Blood samples obtained from individuals were collected using Bermudagra SS. or the nucleic acid sequence of claim 1. produced in a host cell that has been transformed into a host cell, or chemically synthesized. antigenic fragments of and the association of blood components with said protein allergens or fragments thereof. combination, and the extent to which such combination occurs. comprising measuring Cyn d I protein allergen. A method of detecting susceptibility in said individual. 56. T cell function, antibodies present in the blood and the above proteins or fragments thereof the degree to which combination occurs by evaluating the combination with or combinations thereof; The square of claim 55 which determines. 57. Sufficient amount of Astragalus to produce an allergic response in an individual (Bermuda grass) pollen allergen Cynd I, or produced in a host cell transformed with the nucleic acid sequence of the desired scope 1 or chemically administering to said individual at least one antigenic fragment thereof, synthesized by The allergen of Bermuda grass pollen or determining the occurrence of an allergic response in said individual to said antigenic fragment; An allele of Bermuda grass pollen containing A method of determining an individual's susceptibility to gen. 58. specifically reactive with Cyn d I or at least one antigenic fragment thereof monoclonal antibody. 59. a) Encodes Cyn d I or at least one antigenic fragment thereof A host cell transformed with the DNA sequence described above is cultured in a suitable medium. Japanese cedar pollen allergen Cyn d I or producing a mixture of cells and medium containing at least one antigenic fragment thereof; and b) purifying said mixture to obtain substantially pure Cyn d I, or at least Cyn d I or less comprising a step of producing one of its fragments. How to produce both pieces. 60. a) recombinantly or synthetically producing a fragment of Cyn d I; b) producing a Cyn d I fragment; In individuals susceptible to Bermuda grass pollen, B cells and testing said fragments for the ability to influence C and/or T cell responses; ) selecting suitable fragments containing the epitope recognized by the cell; Bermuda gr. When administered to individuals susceptible to the pollen allergen of B. changing the symptoms of an individual's allergy to the pollen of Ermuda grass A method for designing antigenic fragments of Cyn d I. 61. An isolated peptide of Cyn d I or an isolated fragment thereof, , said peptide or fragment thereof is an epitope of at least one CyndI cell. The peptide consists of Cyn dI. 18 amino acids An isolated peptide of Cyn d I having an amino acid sequence consisting of 25-246 or isolated fragments thereof. 62. An isolated peptide of Cyn d I or an isolated fragment thereof, , said peptide or fragment thereof is an epitope of at least one CyndI cell. The peptide consists of Cyn dI. CD1 amino An isolated version of Cyn d I having an amino acid sequence comprising acids 25-246. isolated peptide or isolated fragment thereof. 63. An isolated peptide of Cyn d I or an isolated fragment thereof, , the peptide or a fragment thereof is present in at least one cell of Cyn dI. containing a pitope, and the peptide is Cyn d I2/3 ( Cyn d, which has an amino acid sequence comprising amino acids 25 to 244 of An isolated peptide of I or an isolated fragment thereof.