CN1668737A - Recombined Bet-V-1 allergen mutants, its preparation method and equipment - Google Patents

Abstract

Novel recombinant allergens with multiple mutations and reduced IgE binding affinity are disclosed. The allergens are mutants of naturally occurring allergens. The overall alpha-carbon backbone tertiary structure is essentially preserved. Also disclosed is a method for preparing such recombinant allergens as well as uses thereof.

Description

Reorganization Bet.v.1 allergen mutants and method and preparation

Invention field

The present invention relates to anaphylactoid diagnosis and treatment.More specifically, the invention provides the method that obtains the sudden change allergen molecule, described molecule can be used for anaphylactoid diagnosis and treatment.The invention still further relates to novel recombinant allergens and application thereof, described anaphylactogen is the mutant of naturally occurring anaphylactogen.In addition, the invention still further relates to the medicinal compositions that contains the recombinant allergens mixture.The pharmaceutical composition and the vaccine that the invention still further relates to the preparation method of this recombination mutation anaphylactogen and contain this recombination mutation anaphylactogen.In some embodiments, the present invention relates in the experimenter, produce immunne response, inoculation or treatment experimenter's method and the method for preparing the present composition.

Background of invention

The individuality of susceptible can be produced by the various environment source that individuality contacted in the heredity antigen or anaphylactogen sensitization (allergy).When before the individuality of sensitization contact identical again or anaphylaxis can take place during the homologous anaphylactogen.Anaphylactoid scope comprises spring fever, nose conduction disease (rhinoconductivitis), rhinitis, asthma and general allergic disease and for example because honeybee or hornet sting or the caused death of insect bite.This reaction is instant and can causes by various atopic allergies are former, for example, from careless class, trees, weeds, insect, food, medicine, the compound of chemical substance and spices etc.

Yet, when this class reaction does not appear in individuality when contact allergy is former first.The adaptability of beginning is replied and is needed the time and do not cause any symptom usually.But when producing can be with the antibody of allergenic response and T cell the time, any contact subsequently all may excite symptom.Therefore, anaphylaxis shows that immunne response itself can cause life-threatening great pathological state.

The antibody that the atopic allergy reaction relates to mainly belongs to IgE type immunoglobulin (Ig).IgE combines with mastocyte and the lip-deep specific receptors of basophilic granulocyte.Specific anaphylactogen be incorporated into after IgE on the mastocyte forms mixture, acceptor is crosslinked at cell surface, causes producing the signal conduction and forming the physiological response of target cell by this receptor.The threshing of mastocyte causes discharging histamine, heparin, and the eosinophilic chemotactic factor, leukotriene C, D4 and E4, they can cause that the bronchial smooth muscle cell continues to shrink.The effect that produces under natural situation can be general or partial.

Antibody-mediated anaphylaxis can be divided into 4 classes, i.e. I type, II type, III type and IV type.Type i allergic reaction is typical instant anaphylaxis, takes place in several seconds or the several minutes behind contact antigen.Its symptom is mediated by allergenic specific IgE.

Usually, observed anaphylaxis is to being present in such as pollen indoor dust mite, animal hair and dandruff, the reaction that the protein allergens in venom and the food takes place.

In order to reduce or to eliminate anaphylaxis, use careful control usually and repeatedly use allergy vaccine.Traditionally through in the parenteral, nose or the hypogloeeis medication carry out allergy vaccination, during considerable time in continuous increase dosage, cause patient to desensitize.Do not know accurate amynologic mechanism as yet, but have the people to think inductive difference particularly important aspect allergen specificity T cell phenotype.

Anaphylactoid vaccine inoculation

The notion of vaccine inoculation is with immune two foundation characteristics, and promptly specificity and memory are the basis.The vaccine inoculation meeting causes the immunity system of acceptor, and when repeating to contact similar protein, intensive can take place the attack such as infected by microbes more immunity system replys.Vaccine is to be used for inoculating to produce the egg white mixture of this protective immunological reaction at acceptor.Protection only comprises composition and the isogeneic that is present in the vaccine.

Compare with the vaccine inoculation of other type, thereby the anaphylaxis inoculation is owing to exist the immune response of just carrying out more complicated in the anaphylaxis patient.There is allergenic specific IgE in this immunoreactive being characterised in that, and the mediation symptoms of allergic occurs when contact allergy is former.Therefore, use anaphylactogen from natural origin to carry out the anaphylaxis inoculation and have inherent danger, the worst consequence of its side effect may threaten patient's life.

Research method around this problem can be divided into 3 classes.Often be used in combination the multiclass method in practice.First kind method is included in the long time and uses some low doses to reach bigger integral dose.Second class methods comprise mixes anaphylactogen such as in the gelatinous mass of aluminium hydroxide anaphylactogen being carried out physical modification.The aluminium hydroxide preparation has adjuvant effect and delays anaphylactogen release to reduce the storage effect of active anaphylactogen composition tissue concentration.The 3rd class methods comprise that the chemically modified anaphylactogen is to reduce allergenicity, the i.e. associativity of IgE.

Also there is dispute in the detailed mechanism of successful anaphylaxis inoculation.Yet, approved that the T cell plays a crucial role in immunoreactive overall adjustment.According to present common recognition, two kinds of extreme phenotypes of T cell, the relation between Th1 and the Th2 has determined individual anaphylaxis state.When stimulating with anaphylactogen, Th1 emiocytosis interleukin-mainly is an interferon-, cause protective immunity, and this individuality is healthy.On the other hand, the main secreting leukocytes mesonium 4 of Th2 cell and 5 cause the synthetic and eosinophilia of IgE, and this individuality is hypersensitive.In vitro study shows, attacks the reaction that then might change allergen specificity T cell with the peptide deutero-anaphylactogen that contains relevant t cell epitope.Therefore at present to the research of new allergy vaccine mainly based at the T cell, purpose is to make T cell (anaphylaxis is induced) silence or makes reaction be converted to the Th1 phenotype from the Th2 phenotype.

The epi-position of binding antibody (B-cell epitope)

F _AbThe understanding to the epi-position of binding antibody has been promoted in the X-radiocrystallography analysis of antigenic compound.According to this alanysis, the epi-position of binding antibody may be defined as the antigenic surface part of the atom that contains 15-25 amino-acid residue, and they remain in certain distance with the antibody atom of energy direct interaction.The affinity of antigen-antibody interaction can not be separately inferred from Van der Waals interaction, hydrogen bond or enthalpy that ionic linkage produced.The size of its contribute energy almost completely breaks away from required enthalpy similar in appearance to water molecules from the interface.This means that the matched between the interactional molecule profile is to carry out the interactional principal element of Ag-Ab high-affinity.

A claimed histone matter molecule in WO 97/30150 (reference 1), this protein molecule is compared the specific sudden change that distributing with parent's protein in aminoacid sequence.As if according to its specification sheets, this invention relates to modified is compared in generation with parent's protein analogue, but they and parent's protein (naturally occurring anaphylactogen) are absorbed in an identical manner, digest and be and passs T cell.Therefore, obtain the t cell responses of change to some extent.The technology preparation that is called PM (mutagenesis of law of parsimony) is used in the library of modifying protein.

Described the dna molecular of reorganization in WO 92/02621 (reference 2), this molecule comprises the DNA that coding has the polypeptide of at least one epi-position of anaphylactogen of setting in the Fagales, and this anaphylactogen is selected from Alng1, Cora1 and Betv1.The recombinant molecule of describing in the literary composition all has aminoacid sequence or the partial amino-acid series corresponding to naturally occurring allergen sequence really.

WO 90/11293 (reference 3) relates to the ragweed pollen peptide of isolating ragweed pollen anaphylactogen polypeptide and modification.Wherein disclosed polypeptide has the aminoacid sequence corresponding to naturally occurring anaphylactogen or its naturally occurring isotype sequence.

The chemically modified of anaphylactogen

Some researchs have been carried out in chemically modified to anaphylactogen.The early stage scientific research of the seventies comprises the anaphylactogen chemical coupling to the polymer and use chemically crosslinked anaphylactogen such as formaldehyde to produce so-called " anaphylactogen analogue (allergoids) ".Thereby the ultimate principle of these researchs is to reduce the IgE combination through adhering to chemical part random disruptions IgE in conjunction with epi-position, keeps its immunogenicity because of the molecular weight that increases mixture simultaneously.What be associated with latent defect that " anaphylactogen analogue " produced is to be difficult to control the chemically crosslinked process and to be difficult to analyze and the high molecular weight component of stdn gained." anaphylactogen analogue " is used for clinical at present, and since random disruptions IgE in conjunction with epi-position, can use higher dosage so compare, but not improve security and efficacy parameter with respect to the use of conventional vaccine with conventional vaccine.

The more recent research of chemically modified anaphylactogen concentrates on the tertiary structure of total failure anaphylactogen, thereby eliminates the IgE combination, supposes that wherein the base therapy target is an allergen specificity T cell.This vaccine contains the synthetic peptide from the minimum t cell epitope of representative of allergen sequence, the longer peptide of the continuous t cell epitope of representative, from longer allergen sequence, represent the synthetic peptide in immundominance t cell epitope zone, or the allergen molecule that cuts in half with recombinant technology.Another program according to this ultimate principle is that the reorganization isotype of " low IgE combination " is used in suggestion.In recent years, clear natural anaphylactogen is a heterology, wherein contains anaphylactogen of the same race (isoallergen) and have to reach the substituted variant of about 25% amino acid.Anaphylactogen of the same race efficient in the IgE combination of having found some reorganization is lower, and this is likely owing to irreversible sex change and has therefore destroyed tertiary structure generally.

Vitro mutagenesis and anaphylaxis inoculation

Use some anaphylactogens to carry out reducing pathergic trial through external site-directed mutagenesis.These anaphylactogens comprise Derf2 (Takai etc., document 4), Derp2 (Smith etc., document 5), 39KDa America man peronium mite allergen (Aki etc., document 6), meltittin venom Phospholipase A2 (F rster etc., document 7), Arah1 (Burks etc., document 8), Arah2 (Stanley etc., document 9), Betv1 (Ferreira etc., document 10 and 11), birch arrestin (Wiedemann etc., and Orys1 (Alvarez etc., document 13) document 12).

Equally, the ultimate principle of these schemes also is at allergen specificity T cell, and process is destroyed the tertiary structure of recombinant mutant anaphylactogen to reduce or eliminate IgE in conjunction with the side effect risk that reduces the IgE mediation simultaneously.

The article of Ferreira etc. (document 11) has been described the use site-directed mutagenesis to reduce the IgE combination.Although in this article, mentioned the three-dimensional structure of Betv1, the amino-acid residue that the author does not use this structure prediction to be exposed to the surface, to can be used for suddenling change, wherein half has lower solvent degree of exposure.They are used for the method for the functional residue of predicted protein matter on the contrary.Although the author has discussed the conservative property of alpha-carbon atom skeleton tertiary structure really, this notion is not the part of therapeutic strategy, and only is used for in-vitro evaluation IgE combination.And the evidence that provides is also insufficient, because the stdn meeting overslaugh of CD-spectrum is to the evaluation of sample sex change part, this is a common problem.Described therapeutic strategy is intended to inducing tolerance in allergen specificity T cell, and the immunne response that starting is new is mentioned at the end.

The article of Wiedemann etc. (document 12) has been described and has been used site-directed mutagenesis and peptide to synthesize the evaluation of carrying out the monoclonal antibody epi-position.This studies show that the amino acid that replaces the surface exposure has the ability of modification monoclonal antibody in conjunction with feature, but this is not wondrous, it is believed that it is a kind of general knowledge.The combination that described experiment is not designed to estimate polyclonal antibody such as anaphylaxis patients serum IgE is regulated.SERUM IgE has been used in an experiment that is wherein comprised really, as if although this experiment is not suitable for quantitative evaluation, IgE is in conjunction with the influence of the sudden change that is not subjected to be carried out.

The article of Smith etc. (document 5) has been described the use site-directed mutagenesis and has been drawn and minimizing IgE combination to realize the monoclonal antibody epi-position.The author does not understand tertiary structure and does not attempt estimating the conservative property of alpha-carbon atom skeleton tertiary structure.Used algorithm can not guarantee that the amino acid of selecting to be used to suddenly change is exposed to molecular surface really.Described mutant has only one to cause IgE in conjunction with obviously reducing.This mutant can not show that its tertiary structure is destroyed in conjunction with whole antibody of being tested.The unqualified therapeutic strategy of author, do not mention the new immunne response of starting yet.

The article of Colombo etc. (document 14) has been described use site-directed mutagenesis and peptide study on the synthesis IgE in conjunction with epi-position.The author has used a three dimensional computer modeling structure to exist with the epi-position of illustrating molecular surface according to the crystalline structure of homologous protein.Use synthetic peptide represent epi-position to confirm also to have an epi-position on the different anaphylactogens of demonstration primary structure homology.On behalf of unit price IgE, therapeutic strategy treat in conjunction with the synthetic peptide of epi-position to use.

The three-dimensional structure and the conservative surperficial expose portion of main birch anaphylactogen described in the article of Spangfort etc. (document 15).This article is not mentioned site-directed mutagenesis, does not set forth treatment yet and uses.

None is to keep the tertiary structure of alpha-carbon skeleton to reduce the IgE combination through replacing the surperficial amino acid that exposes in above-mentioned research.The treatment theory of a new protective immune response of all not mentioned starting of above-mentioned research.

WO01/83559 discloses the method that a kind of screening has improved immunogenic protein variant, utilizes the antibodies peptide sequence to determine epitope sequences in parent's protein three-dimensional structure.Define an epitope regions subsequently, will define one or more amino acid mutations of this epitope regions then.The industrial enzyme that this invention utilization can be used as anaphylactogen is illustrated.

WO99/47680 proposes to introduce artificial amino acid and replaces in specified key position, keep the conservative property of anaphylactogen alpha-carbon skeleton tertiary structure simultaneously.WO 99/47680 also discloses a kind of recombinant allergens especially, it is a kind of non-natural mutant from natural anaphylactogen, wherein at least one is exposed to the conservative amino acid residues on surface by all absent variable another residue replacement on the same position in the aminoacid sequence of any known homologous protein matter in the taxonomy order of described natural anaphylactogen origin in the B cell epitope, described sudden change anaphylactogen has the alpha-carbon skeleton tertiary structure substantially the same with described natural anaphylactogen, and specific IgE is littler with combining of described natural anaphylactogen with the binding ratio of sudden change anaphylactogen.

The disclosed recombinant allergens of WO99/47680 can obtain through following method: a) identify in the natural anaphylactogen to have the conservative amino acid residues that surpasses 70% homogeny in all known homologous proteins in the taxonomy order of this natural anaphylactogen origin; B) to have at least 20% solvent accessibility, determine on allergen molecule three-dimensional structure surface 400A at least ²Scope at least one conservative amino acid residues zone link up connecting, described at least one zone comprises at least one B cell epitope; C) at least one amino-acid residue in the described at least one zone is by at another conservative aminoacid replacement of this specific position, and keeps the overall alpha-carbon skeleton tertiary structure of allergen molecule basically.

Patent application PCT/DK01/00764 relates to the mutant of natural anaphylactogen.Following particular B etv1 mutant is wherein disclosed:

Sudden change A: Asn28Thr, Lys32Gln, Asn78Lys, Lys103Val, Arg145Glu, Asp156His ,+160Asn.

Sudden change B: Tyr5Val, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123Ile, Lys134Glu, Asp156His.

Sudden change 2628: Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu.

Sudden change 2637: Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr, Pro108Gly, Leu152Lys, Ala153Gly, Ser155Pro.

Sudden change 2724: N28T, K32Q, N78K, K103V, P108G, R145E, D156H ,+160N.

Sudden change 2733: Tyr5Val, Lys134Glu, Asn28Thr, Lys32Gln, Glu45Ser, Lys65Asn, Asn78Lys, Lys103Val, Lys97Ser, Pro108Gly, Arg145Glu, Asp156His ,+160Asn.

Sudden change 2744: Tyr5Val, Lys134Glu, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123Ile, Asp156His ,+160Asn.

Sudden change 2753: Asn28Thr, Lys32Gln, Lys65Asn, Glu96Leu, Lys97Ser, Pro108Gly, Asp109Asn, Asp125Tyr, Glu127Ser, Arg145Glu.

Sudden change 2744+2595: Y5V, N28T, K32Q, E42S, E45S, N78K, K103V, P108G, K123I, K134E, D156H ,+160N.

Sudden change 2744+2628: Y5V, E42S, E45S, K65N, N78K, K97S, K103V, K123I, K134E, D156H ,+160N.

Sudden change 2744+2595+2628: Y5V, N28T, K32Q, E42S, E45S, K65N, N78K, K97S, K103V, P108G, K123I, K134E, D156H ,+160N.

Brief description of the drawings

Fig. 1 has shown the theoretical model that passes through the IgE crosslinking reaction between anaphylactogen and the mastocyte.

The location that the Betv1 molecular surface 1-10 that Fig. 2 (left side) shows with black and gray area organizes.(right side) constitutes the amino-acid residue view of 1-10 group.Each group is marked as 1-10.

Fig. 3 has shown the mutant specific oligonucleotide primer that is used for the Betv1 sudden change.The Nucleotide underscore of sudden change.

Fig. 4 has shown for all mutant and has synthesized and 2 blanket primers using (be called " Quan Youyi " (all-sense) and " completely without justice " (all non-sense)).

Fig. 5 has shown DNA and aminoacid sequences natural and some sudden change Betv1 anaphylactogens.

Fig. 6 shows with the Betv1 of abiotic plain mark and the combining of SERUM IgE of suppressing biotin labeled reorganization Betv1 and anaphylaxis patient group with Betv1 Glu45Ser mutant.

Fig. 7 shows with the Betv1 of abiotic plain mark and the combining of SERUM IgE of suppressing biotin labeled reorganization Betv1 and anaphylaxis patient group with Betv1 mutant Asn28Thr+Lys32Gln.

Fig. 8 has shown with the Betv1 of abiotic plain mark and the combining of SERUM IgE of suppressing biotin labeled reorganization Betv1 and anaphylaxis patient group with Betv1 Pro108Gly mutant.

Fig. 9 has shown with the Betv1 of abiotic plain mark and the combining of SERUM IgE of suppressing biotin labeled reorganization Betv1 and anaphylaxis patient group with Betv1 Glu60Ser mutant.

Figure 10 shown reorganization and (Asn28Thr, Lys32Gln, Glu45Ser, the Pro108Gly) CD of mutant spectrum is near record under the isoconcentration.

Figure 11 shown with the Betv1 of abiotic plain mark and with (Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly) mutant suppresses biotin labeled reorganization Betv1 and anaphylaxis patient group's the combining of SERUM IgE.

Figure 12 shows the 2 footwork PCR mutating technology diagrams that are used to form sudden change Betv1 anaphylactogen.

Figure 13 shows formation clone Betv1 (3004), (3005), the PCR sudden change step diagram of (3007) and (3009).Listed the primer that is used to import point mutation.

Figure 14 shows the PCR sudden change step diagram that forms clone Betv1 (3031)-(3045).Listed and be used for 10,20 36,73,87,129 and 149 degenerated primers that import random mutation.Shown the sudden change result that each site may form at the top.

Figure 15 is the synoptic diagram that is used to produce each primer of sudden change.(I) show justice and antisense primer.(II) be presented at the recombinant protein end product of specifying the site to have sudden change.

Figure 16 shows that the structure of Betv1 mutant illustrates and the primer is itemized.This mutant contains 5-9 amino acid.

Figure 17 is presented at the point mutation of Betv1 (2628) and the introducing of Betv1 (2637) surface.In Betv1 (2628) mutant, introduced 5 one-level sudden changes in half zone of Betv1, second half zone maintenance is constant.And in Betv1 (2637) mutant, introduced 5 one-levels sudden changes and 3 secondarys suddenlys change in second half zone, and the first half zone maintenance are constant.

Figure 18 has shown circular dichroism (CD) spectrum of Betv1.2801 (wild-type) and Betv1 (2637) mutant, near record under the isoconcentration.

Figure 19 has shown the Betv1.2801 (wild-type) with abiotic plain mark, with with Betv1 (2628), Betv1 (2637) and combining with 1: 1 blended Betv1 (2628) and Betv1 (2637) inhibition biotin labeled recombinate Betv1 and anaphylaxis patient group's SERUM IgE.

Figure 20 shows Betv1.2801 (wild-type), Betv1 (2628), the histamine release of Betv1 (2637) in people's basophilic granulocyte.

Figure 21 shows Betv1.2801 (wild-type), Betv1 (2628), the histamine release of Betv1 (2637) in people's basophilic granulocyte.

Figure 22 shows the point mutation on Betv1 (2744) surface.

Figure 23 shows the point mutation on Betv1 (2753) surface.

Figure 24 shows the point mutation on Betv1 (2744) and Betv1 (2753) surface.

Figure 25 has shown circular dichroism (CD) spectrum of Betv1.2801 (wild-type) and Betv1 (2744), near record under the isoconcentration.

Figure 26 shows Betv1.2801 (wild-type) and the histamine release of Betv1 (2744) in people's basophilic granulocyte.

Figure 27 shows Betv1.2801 (wild-type) and the histamine release of Betv1 (2744) in people's basophilic granulocyte.

Figure 28 shows the point mutation on Betv1 (2733) surface.

Figure 29 shows the propagation situation of the peripheral blood lymphocyte that is expressed as stimulation index (SI) of different B etv1 preparation.

Figure 30-32 shows the cytokine feature of the T cell that stimulates with different B etv1 preparation.Figure 30 shows the patient with Th0 feature, and feature 31 is the Th1 feature, and Figure 32 is the Th2 feature.

Figure 33 shows rBetv1.2801 () (wild-type) and has circular dichroism (CD) spectrum of rBetv1 3007 mutant [Δ] of 12 sudden changes, near record under the isoconcentration.The coverage diagram that has shown 15 ℃ of circular dichroism (CD) spectrums that obtain down

Figure 34 shows with rBetv1.2801 () (wild-type) and has rBetv1 (3007) mutant [Δ] the biotin labeled rBetv1.2801 of inhibition of 12 sudden changes and combining of birch anaphylaxis patients serum IgE set.

Goal of the invention

The purpose of this invention is to provide improved recombination mutation allergen protein.

Ultimate principle of the present invention

The present invention is based on the principle of uniqueness.According to this ultimate principle, the mechanism of successful anaphylaxis inoculation does not change ongoing Th2 type immunne response, and starting relates to by the tertiary structure epi-position of B cell recognition and a kind of new immunne response that forms antibody simultaneously.Can believe that this novel immune response partly belongs to Th1 type immunne response.When other approach medication by non-respiratory tract of vaccine (or medicinal compositions), supposing that new immunne response is different from health carries out the position that Th2 replys and takes place, thereby making to reply simultaneously for two kinds exists.

In addition, the present invention also based on found allergic symptom be by anaphylactogen with at least two specific IgEs by high affinity IgE acceptor, Fc ε RI and crosslinked initiation the, described IgE and effector cell, the i.e. surface bonding of mastocyte and basophilic granulocyte.For describing, we are referring to Fig. 1, and it has been described a kind of anaphylactogen and has had the theoretical model of 3 kinds of IgE in conjunction with epi-position.Induce amboceptor to discharge from mastocyte, thus the crosslinked initiation allergic symptom of IgE on the mastocyte surface by anaphylactogen mediation, relatively Figure 1A.In the situation shown in Figure 1B, 2 sites of epi-position by sudden change reducing its IgE binding ability, thereby avoided the crosslinked of anaphylactogen mediation.Should be noted that under this connection situation B cell epitope that anaphylactogen contains usually is all more than 3.

For can being produced, the sudden change anaphylactogen comprises that IgG is reflected at interior a kind of novel immunne response, the epi-position that this sudden change anaphylactogen must comprise at least one complete epi-position or only slightly be changed.The slight surfac topography preferred class that changes epi-position can form more IgG antibody like original epi-position.These new IgG antibody have specificity, can compete also some degree ground and replace IgE in conjunction with natural anaphylactogen.In addition, can suppose: by sudden change to eliminate or to reduce the epi-position of its IgE binding ability many more, then crosslinked the and institute of anaphylactogen mediation causes that the risk of allergic symptom is just more little after the allergy vaccine administration.

According to this ultimate principle, anaphylactogen has alpha-carbon skeleton tertiary structure must be basically similar in appearance to natural anaphylactogen.

Having supposed in the past that the site that is suitable for suddenling change only is positioned at is considered to contain the zone of main IgE in conjunction with the conservative amino acid residues of epi-position.But as if according to the present invention, the surface exposed amino acid residue that is suitable for suddenling change comprises high conservative residue and non-conserved residues or slight conservative residue simultaneously.As if this amino-acid residue is distributed in whole molecular surface, tends to form little group, be covered with the specific region of molecular surface.

Therefore, according to the present invention, the surface exposed amino acid that is suitable for suddenling change can be divided into some groups, as shown in Figure 2.Grouping is to carry out according to the proneness of these amino-acid residues formation separated regions, and grouping does not also rely on the conservative degree of amino-acid residue.Every group of amino-acid residue that the many surfaces of representative expose, described amino acid is to find in the limited area on anaphylactogen surface.Each independent group contains a part or at least one complete epi-position of at least one epi-position probably.Each independent group also may contain by sudden change and cause the amino acid sites of the gentle epi-position that changes and the amino acid sites that forms the epi-position of more violent change by sudden change.If original amino-acid residue is by the similar aminoacid replacement of chemical property (as replace another hydrophobic amino acid with hydrophobic amino acid), an independent amino acid can form the gentle epi-position that changes usually so.In a word, can provide a kind of instrument by select sudden change from least four group amino-acid residues of specifying each group, thereby can obtain sudden change anaphylactogen of the present invention, this anaphylactogen is to suddenly change at several B cell epitopes AndHave and the similar alpha-carbon skeleton structure of natural anaphylactogen.

Another importance of the present invention is that the sudden change anaphylactogen is being kept the continuous surface zone, and this region area is approximately 400-800 ², wherein do not contain sudden change or only contain gentle sudden change.It is believed that anaphylactogen contains the calmodulin binding domain CaM of a large amount of potential at specific IgE, wherein each regional area is approximately 800 ²

Invention theory of the present invention is based on the following understanding to the sudden change anaphylactogen: anaphylactogen contains the sudden change that the IgE bonding force can be reduced, and (this sudden change is selected from the group of at least 4 regulations, each group all contains the amino acid that is suitable for suddenling change that the surface exposes), but keep at least one complete or gentle epi-position that changes, this anaphylactogen can reduce the crosslinked of anaphylactogen mediation on the one hand, can produce the IgG reaction of the binding ability of competing with IgE on the other hand.Therefore, described sudden change anaphylactogen has constituted very favorable anaphylactogen, because anaphylactoid risk is greatly reduced.This sudden change anaphylactogen can with suitable high dosage medication with improve its in producing protective immune response effectiveness and do not lose its security.

And; the present invention is based on and recognize that the vaccine anaphylactogen equivalence natural with it on the protective capability of inducing allergic symptom that contains different this sudden change anaphylactogens, wherein many or whole epi-positions do not change ideally or only gentle the change taken place at difference sudden change anaphylactogen.

The invention summary

The present invention relates in anaphylactogen, import aminoacid replacement.This aminoacid replacement is selected from the amino acid group that at least 4 groups are suitable for carrying out aminoacid replacement.Its objective is the specific IgE binding ability that reduces each sudden change epi-position but also on this sudden change anaphylactogen, keep at least one complete or epi-position of gentle change only simultaneously.

The present invention is specifically related to a kind of reorganization Betv1 anaphylactogen, it is characterized in that it is the mutant of natural B etv1 anaphylactogen, wherein:

The alpha-carbon skeleton tertiary structure substantially the same with described natural anaphylactogen kept in sudden change,

Sudden change contains at least 4 one-level sudden changes, compares with the IgE binding ability of natural B etv1 anaphylactogen, and every kind of sudden change can both reduce the specific IgE binding ability of sudden change anaphylactogen,

Every kind of one-level sudden change all is that the amino-acid residue that a kind of surface exposes is replaced by another kind of residue,

The modes of emplacement in this mutational site should meet following: at least one 400-800 ²Area in do not contain sudden change or only contain one or more gentle sudden changes,

This one-level sudden change is selected from least 4 groups in following 10 groups, every group of amino acid sites that all contains the surface exposure that is suitable for aminoacid replacement:

Group 1:A130, E131, K134, A135, K137, E138, E141, T142, R145;

Group 2:V2, F3, N4, Y5, E6, T7, K119;

Group 3:D27, S39, S40, Y41, E42, N43, I44, E45, G46, N47, P50, G51, K55, D72, E73;

Group 4:E8, T10, V12, P14, V105, A106, T107, P108, D109, G110, I113, K115;

Group 5:A16, K20, S149, Y150, L152, A153, H154, S155, D156, Y158, N159 ,+160, wherein+160 represent the increase of-terminal amino acid;

Group 6:L24, D25, N28, K32;

Group 7; H76, T77, N78, F79, K80, E101, K103;

Group 8:K68, R70, I86, E87, E96, K97;

Group 9:G1, G92, D93, T94, K123, G124, D125, H126, E127, K129;

Group 10:P35, Q36, E60, G61, P63, F64, K65, Y66;

Its precondition is: reorganization Betv1 anaphylactogen is not following specified mutant: (Asn28Thr, Lys32Gln, Asn78Lys, Lys103Val, Arg145Glu, Asp156His ,+160Asn); (Tyr5Val, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123Ile, Lys134Glu, Asp156His); (Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu); (Ala16Pro, Asn28Thr, Lys32Gin, Lys103Thr, Pro108Gly, Leu152Lys, Ala153Gly, Ser55Pro); (N28T, K32Q, N78K, K103V, P108G, R145E, D156H ,+160N); (Tyr5Val, Lys134Glu, Asn28Thr, Lys32Gln, Glu45Ser, Lys65Asn, Asn78Lys, Lys103Val, Lys97Ser, Pro108Gly, Arg145Glu, Asp156His ,+160Asn); (Tyr5Val, Lys134Glu, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123Ile, Asp156His ,+160Asn); (Asn28Thr, Lys32Gln, Lys65Asn, Glu96Leu, Lys97Ser, Pro108Gly, Asp109Asn, Asp125Tyr, Glu127Ser, Arg145Glu); (Y5V, N28T, K32Q, E42S, E45S, N78K, K103V, P108G, K123I, K134E, D156H ,+160N); (Y5V, E42S, E45S, K65N, N78K, K97S, K103V, K123I, K134E, D156H ,+160N); (Y5V, N28T, K32Q, E42S, E45S, K65N, N78K, K97S, K103V, P108G, K123I, K134E, D156H ,+160N).

More specifically, the present invention relates to a kind of reorganization Betv1 anaphylactogen, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, every group of amino acid sites that all contains the surface exposure that is suitable for following aminoacid replacement:

Group 1:A130:A130V, A130G, A1301, A130L, A130S, A130H, A130T; E131:E131D, E131H, E131K, E131R, E131S; K134:K134R, K134H, K134S, K134Q, K1341, K134E; A135:A135V, A135G, A1351, A135L, A135S, A135H, A135T; K137:K137R, K137H, K137S, K137Q, K1371, K137E; E138:E138D, E138H, E138K, E138R, E138S, E138N; E141:E141D, E141H, E141K, E141R, E141S; T142:T142A, T142S, T142L, T142V, T142D, T142K, T142N; R145:R145K, R145H, R145T, R145D, R145E;

Group 2:V2:V2A, V21, V2K, V2L, V2R, V2T; F3:F3H, F3W, F3S, F3D; N4:N4H, N4K, N4M, N4Q, N4R; Y5:Y5D, Y5G, Y5H, Y51, Y5K, Y5V; E6:E6D, E6H, E6K, E6R, E6S; T7:T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119:K119R, K119H, K119S, K119Q, K119I, K119E, K119N;

Group 3:D27:D27E, D27H, D27K, D27R, D27S; S39:S39T, S39L, S39V, S39D, S39K; S40:S40T, S40L, S40V, S40D, S40K; Y41:Y41D, Y41G, Y41H, Y41I, Y41K, Y41V; E42:E42S, E42D, E42H, E42K, E42R; N43:N43H, N43K, N43M, N43Q, N43R; I44:I44L, I44K, I44R, I44D; E45:E45S, E45D, E45H, E45K, E45R; G46:G46N, G46H, G46K, G46M, G46Q, G46R; N47:N47H, N47K, N47M, N47Q, N47R; P50:P50G; G51:G51N, G51H, G51K, G51M, G51Q, G51R; K55:K55R, K55H, K55S, K55Q, K551, K55E, K55N; D72:D72E, D72S, D72H, D72R, D72K; E73:E73D, E73S, E73H, E73R, E73K;

Group 4:E8:E8D, E8H, E8K, E8R, E8S; T10:T10P, T10S, T10L, T10V, T10D, T10K, T10N; V12:V12A, V121, V12K, V12L, V12R, V12T; P14:P14G; V105:V105A, V1051, V105K, V105L, V105R, V105T; A106:A106V, A106G, A1061, A106L, A106S, A106H, A106T; T107:T107A, T107S, T107L, T107V, T107D, T107K, T107N; P108:P108G; D109:D109N D109E, D109S, D109H, D109R, D109K; G110:G110N, G110H, G110K, G110M, G110Q, G110R; I113:I113L, I113K, I113R, I113D, K115:K115R, K115H, K115S, K115Q, K151, K115E, K115N;

Group 5:A16:A16V, A16G, A16I, A16L, A16S, A16H, A16T; K20:K20R, K20H, K20S, K20Q, K20I, K20E, K20N; S149:S149T, S149L, S149V, S149D, S149K; Y150:Y150T, Y150L, Y150V, Y150D, Y150K; L152:L152A, L152V, L152G, L1521, L152S, L152H, L152T; A153:A153V, A153G, A153I, A153L, A153S, A153H, A153T; H154:H154W, H154F, H154S, H154D; S155:S155T, S155L, S155V, S155D, S155K; D156:D156H, D156E, D156S, D156R, D156K; Y158:Y158D, Y158G, Y158H, Y158I, Y158K, Y158V; N159:N159H, N159K, N159M, N159Q, N159R, N159G ,+160N;

Group 6:L24:L24A, L24V, L24G, L24I, L24S, L24H, L24T; D25:D25E, D25H, D25K, D25R, D25S; N28:N28H, N28K, N28M, N28Q, N28R, N28T; K32:K32Q, K32R, K32N, K32H, K32S, K321, K32E;

Group 7:H76:H76W, H76F, H76S, H76D; T77:T77A, T77S, T77L, T77V, T77D, T77K, T77N; N78:N78H, N78K, N78M, N78Q, N78R; F79:F79H, F79W, F79S, F79D; K80:K80R, K80H, K80S, K80Q, K801, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S; K103:K103R, K103H, K103S, K103Q, K103I, K103E, K103V;

Group 8:K68:K68R, K68H, K68S, K68Q, K681, K68E, K68N; R70:R70K, R70H, R70T, R70D, R70E, R70N; I86:I86L, I86K, I86R, I86D; E87:E87D, E87H, E87K, E87R, E87S, E87A; E96:E96D, E96H, E96K, E96R, E96S, E96L; K97:K97R, K97H, K97S, K97Q, K971, K97E;

Group 9:G1:G1N, G1H, G1K, G1M, G1Q, G1R; G92:G92N, G92H, G92K, G92M, G92Q, G92R; D93:D93N, D93E, D93S, D93H, D93R, D93K; T94:T94A, T94S, T94L, T94V, T94D, T94K, T94N; K123:K123R, K123H, K123S, K123Q, K123I, K123E; G124:G124N, G124H, G124K, G124M, G124Q, G124R; D125:D125E, D125H, D125K, D125R, D125S, D125Y; H126:H126W, H126F, H126S, H126D; E127:E127D, E127H, E127K, E127R, E127S; K129:K129R, K129H, K129S, K129Q, K129I, K129E, K129N;

Group 10:P35:P35G; Q36:Q36K, Q36R, Q36N, Q36H, Q36S, Q36I, Q36E; E60:E60H, E60K, E60M, E60Q, E60R; G61:G61N, G61H, G61K, G61M, G61Q, G61R; P63:P63G; F64:F64H, F64W, F64S, F64D; K65:K65R, K65H, K65S, K65Q, K651, K65E, K65N; Y66:Y66D, Y66G, Y66H, Y661, Y66K, Y66V.

The invention still further relates to a kind of reorganization Betv1 sudden change anaphylactogen, it contains the replacement that is selected from least 4 groups in following 10 groups:

Group 1:A130V, K134E, E141N,

Group 2:V2L, Y5V, E6S, K119N,

Group 3:E42S, E45S, N47K, K55N, E73S, E73T, E73S,

Group 4:E8S, T10P, P14G, P108G, D19N, K115N,

Group 5:A16G, K20S, S149T L152A A153V, S155T, N159G ,+160N,

Group 6:L24A, D25E, N28T, K32Q,

Group 7:T77A, T77N, N78K, K103V,

Group 8:R70N, E87A, E96S, K97S,

Group 9:D93S, K123I, D125Y, K129N,

Group 10:Q36N, E60S, G61S, P63G.

The invention still further relates to a kind of reorganization Betv1 sudden change anaphylactogen, it contains the replacement that is selected from least 4 groups in following 10 groups:

Group 1:K134E,

Group 2:Y5V, K119N, V2L,

Group 3:E45S, E42S, K55N, N47K, E73S,

Group 4:E96S, K97S, P108G, D109N, T10N, K115N, P14G,

Group 5:N159G ,+160N, S149T, A153V, L152A, A16G, K20S,

Group 6:N28T, K32Q, L24A,

Group 7:K103V, T77N, N78K,

Group 8:E96S, K97S, E87A,

Group 9:K129N, D125Y, K123I, D93S,

Group 10:E60S, Q36N, G61S, P63G.

The invention still further relates to following:

The reorganization Betv1 anaphylactogen variant that can be used as medicine and be used to prevent and/or treat the medicine of birch pollen hypersensitivity reaction

The composition that contains two or more different recombination mutation Betv1 anaphylactogen variants of the present invention, wherein every kind of variant all has at least a one-level sudden change, and this sudden change does not exist in another variant at least.Said composition contains 2-12, preferred 3-10, more preferably 4-9, most preferably 5-8 kind variant.Composition useful as drug of the present invention also can be used for preparing the medicine that prevents and/or treats the reaction of birch pollen hypersensitivity.This pharmaceutical composition preferably contains pharmaceutically acceptable carrier, and/or vehicle, and optional adjuvant.

The pharmaceutical composition of vaccine form, it is at the anaphylaxis that is caused by natural B etv1 anaphylactogen among the birch pollen hypersensitivity patient.

In individual body, produce immunoreactive method, comprise individual administered recombinant anaphylactogen, composition or pharmaceutical composition.

Individual immunization or treatment comprise individual administered recombinant anaphylactogen, composition or pharmaceutical composition.

The method of pharmaceutical compositions comprises recombinant allergens or composition and pharmaceutically acceptable material and/or mixed with excipients.

Treatment prevents or alleviates individual anaphylactoid method, comprises individual administered recombinant Betv1 anaphylactogen, composition or pharmaceutical composition.

Prepare the method for reorganization Betv1 anaphylactogen, be characterised in that aminoacid replacement is by rite-directed mutagenesis, or DNA reorganization (shuffling) (molecular breeding) (Punnonen etc., reference 25) carries out.

The dna sequence dna of coding reorganization Betv1 anaphylactogen, its derivative, its partial sequence, its degenerate sequence or under rigorous condition can with the sequence of its hybridization, wherein said derivative, partial sequence, degenerate sequence or hybridization sequences coding have the peptide of at least a B cell epitope.

Dna sequence dna institute deutero-dna sequence dna by the natural anaphylactogen of coding.The dna sequence dna of this derivative of encoding is to obtain by the dna encoding sequence of natural B etv1 anaphylactogen being carried out rite-directed mutagenesis.

Contain the expression vector of the dna encoding sequence of reorganization Betv1 variant, contain the host cell of this expression vector, and the method for producing reorganization Betv1 mutant, it comprises cultivates this host cell.

Reorganization Betv1 anaphylactogen or by the coded Betv1 anaphylactogen of following dna sequence dna: contain the dna sequence dna of at least one t cell epitope, described epi-position can stimulate T cell clone or the T clone special to natural B etv1 anaphylactogen.

Estimate the dependency of the therapy of using reorganization Betv1 anaphylactogen or combination treatment patient, the diagnostic test of degree of safety or effect, wherein the IgE sample that contains with the patient mixes with described sudden change or composition, estimates the level of reactivity between the IgE and described mutant in the described sample.

Detailed description of the present invention

In the present invention, " Compare specific IgE with the IgE binding ability of natural anaphylactogen Binding ability decreases" be meant that this minimizing is measured to statistical significant level (p＜0.05) at least a immunity test, use serum in the described test to natural anaphylactogen individuality hypersensitive.Preferably, this IgE binding ability is lowered at least 10%, and more preferably at least 30%, more preferably at least 50%, most preferably at least 70%.

" The amino acid that the surface exposes" referring to that amino-acid residue is positioned at the surface of 3 dimension structures, its mode is when anaphylactogen is in solution, at least a portion of at least one atom of this amino-acid residue can be touched by solvent on every side.Preferred this amino-acid residue has at least 20% solvent (water) accessibility in 3 dimension structures, and preferably at least 30%, more preferably at least 40%, most preferably at least 50%.

" The solvent accessibility" be defined as and the suitable come-at-able molecular domains of spheroid of the same solvent molecule of radius (water, r=1.4 )." surface exposes " and " solvent exposure " are used interchangeably.

" Amino acid whose group" be interpreted as the amino acid whose set that is suitable for suddenling change that the surface exposes.Every group of amino-acid residue that the many surfaces of representative expose, described amino acid is to find in the limited area on anaphylactogen surface.Each independent group contains many amino acid, and these amino acid are parts of at least one epi-position.Each independent group also may contain covers a zone that comprises a complete epi-position.One or more sudden changes in a group are defined as a kind of one-level sudden change.The sudden change anaphylactogen that has at least 4 one-level sudden changes can guarantee that several epi-positions decrease to the IgE binding affinity.Can further guarantee also that from the amino acid whose sudden change of at least 4 groups sudden change approximately is evenly distributed at molecular surface, thereby and guarantee several epi-positions produce sudden change cause several epi-positions decrease with the IgE binding affinity (with have the mutant that is less than 4 sudden changes that suddenlys change compare).

" The taxonomy kind in natural anaphylactogen source" be meant that the taxonomy in described natural anaphylactogen origin belongs to, preferred subfamily, more preferably section, more preferably superfamily, more preferably group's (legion), more preferably suborder, most preferably kind in the order.

" The gentle epi-position that changes" refer to keep the epi-position of learning with essentially identical tertiary structure of the corresponding epi-position of not suddenling change and surface topology.Gentle change is normally used with this original amino acid of aminoacid replacement like the original chemistry of amino acids property class and is obtained.A kind of preparation method is the amino acid that expose with can detectable aminoacid replacement in the taxonomy order at natural anaphylactogen place one or more surfaces.The gentle epi-position that changes also may contain such aminoacid replacement: the amino acid of wherein one or more replacements do not find in the taxonomy order at natural anaphylactogen place, as long as this replacement can influence the tertiary structure of epi-position and/or IgE slightly in conjunction with affinity.This sudden change anaphylactogen can be estimated in conjunction with the affinity angle from structure and IgE subsequently.Change the opposite epi-position that is acutely changed of epi-position with gentleness, as can significantly reducing the sudden change of IgE binding affinity.Usually, the violent change of epi-position comprises with the amino acid of different chemical character and replaces one or more amino acid whose replacements.

In addition, " Has sudden change with the essentially identical alpha-carbon skeleton of natural anaphylactogen tertiary structure Anaphylactogen" refer to when relatively suddenling change and during the structure of natural anaphylactogen, the average root-mean-square difference of atomic coordinate is preferably less than 2 .By before sudden change and after the sudden change, carrying out x ray crystallography or NMR obtains the conservative property that same structure is accurately measured alpha-carbon skeleton tertiary structure.If compare with the data that the determined molecule of analytical structure is obtained, when not describing the structured data of sudden change, indiscriminate CD spectrum or immunochemistry data (as antibody response) can be thought guarding of alpha-carbon skeleton tertiary structure.

Among the present invention " Sudden change" refer to compare with the aminoacid sequence of natural anaphylactogen, there is amino acid whose disappearance, replace or increase.This term " sudden change " and " replacement " are used interchangeably.Recombination mutation Betv1 of the present invention also can further contain aminoacid insertion or aminoacid deletion, particularly at the surperficial exposed region of this molecule, as " ring zone ". The ring zoneGetting in touch the secondary structure element, as βZhe Die, α spiral and winding arrangement at random.Ala16 is arrived for Val12 in ring zone among the Betv1, and val33 is to ser40, and glu45 is to Thr52, and pro54 is to tyr66, and his76 is to asn78, and gly89 is to glu96, and val105 is to gly111, and thr122 is to glu131.Mutant can contain 1-5 in the ring zone, preferred 1-3, most preferably 1-2 replacement.

The one-level sudden changeBe defined as the one or more sudden changes in amino acid whose single group that exposes, is suitable for suddenling change on the surface.Each group of at least one mutating acid can reduce IgE binding affinity (with comparing on the same group mutually with sudden change not).Preferred recombinant allergens of the present invention contains 5-10, preferred 6-10, more preferably 7-10, most preferably 8-10 one-level sudden change.

The secondary sudden changeExtra sudden change in being defined as single group.This recombinant allergens preferably contains a plurality of secondary sudden changes, compares the binding ability of each secondary sudden change all can the reduction this sudden change anaphylactogen and IgE with the binding ability of natural anaphylactogen.Therefore, its IgE binding affinity may be lower than the one-level sudden change that only has a kind of sudden change in many cases to contain the one-level sudden change of several secondarys sudden change.Each one-level sudden change of recombinant allergens of the present invention contains 1-15, preferred 1-10, most preferably 1-5 secondary sudden change.

Conserved residues: all known homologous proteins have and are higher than 70% in the conserved residues in the natural anaphylactogen and this anaphylactogen origin species, and are preferred 80%, most preferably 90% homology.The height solvent exposes and conservative amino-acid residue has constituted the target position that replaces.

It is the ability that sudden change suppresses histamine release (HR) that IgE bonding force due to the sudden change reduces and mediate another evaluation method that crosslinked ability reduces.Can be by the release of several histamine release test determination histamine.The histamine release reduction of mutant comes from the minimizing with the special avidity of cell surface bonded IgE, with and to the reduction of crosslinked promoter action ability.Compare with natural anaphylactogen, the histamine of preferred mutant of the present invention is lowered 5-100%, more preferably 25-100%, more preferably 50-100%, most preferably 75-100%.

In a preferred embodiment of the invention, the surface area that does not contain sudden change or only contain gentle sudden change is 800 ², preferred 600 ², more preferably 500 ², 400 most preferably ² Common 800 that do not contain sudden change or only contain gentle sudden change ²The area surf zone contains the atom of 15-25 amino-acid residue.

Among another embodiment, at least one amino-acid residue taxonomy under this naturally occurring anaphylactogen that will be incorporated into allergen mutants belongs to, preferred subfamily, more preferably section, more preferably superfamily, more preferably group (legion), more preferably suborder and most preferably anyly in the order do not occur on the same position as homologous protein matter aminoacid sequence.

According to the present invention, the amino-acid residue that the surface is exposed is according to the ordering of solvent accessibility, and at least 4 amino acid in the bigger amino acid of solvent accessibility are substituted.

Among another embodiment, recombinant allergens is characterised in that the amino-acid residue that the surface is exposed sorts by the conservative degree in its all known homologous proteins in the taxonomy kind of this naturally occurring anaphylactogen, and will be in the one or more replacements in the amino acid of higher conservative degree.

Principle disclosed in this invention comprises and will be selected from the amino acid whose surface exposed amino acid residue sudden change of at least 4 groups, wherein every group of separated region of representing this molecular surface.This principle also is applicable to the anaphylactogen outside the Betv1.Recombinant allergens of the present invention can suitably be the inhalant allergens mutant, derives from tree, grass, herbaceous plant, fungi, dermatophagoides pteronyssinus, cockroach and animal hair and scurf in theory.From tree, careless and herbal important pollen allergens is to be derived from beech (Fagales), sweet-scented osmanthus order (Oleales) and pinales (Pinales) classification purpose these, comprise birch (Betula (Betula)) in theory, alder (Alder (Alnus)), hazel (Corylus (Corylus)), hornbeam (Carpinus (Carpinus)) and olive (olea belongs to (OIe)), standing grain order (Poales) order comprises lolium (Lolium) in theory, Kittentails (Phleum), annual bluegrass belongs to (Poa), Cynodon (Cynodon), the grass that orchardgrass (Dactylis) and Secale (Secale) etc. belong to, chrysanthemum order (Asterales) and Urticales (Urticales) comprise the herbaceous plant of Ambrosia (Ambrosia) and artemisia (Artemisia) in theory.From the important inhalant allergens of fungi in theory for being derived from these of Alternaria (Alternaria) and Blastocladia (Cladosporium).Other important inhalant allergens are from the dermatophagoides pteronyssinus of dermatophagoides, from cockroach with from Mammals such as cat, dog and horse.Further, recombinant allergens of the present invention can be the mutant of venom allergens, comprise from thorn the people's or gallinipper, for example from Hymenoptera, comprise honeybee (Apoidea (Apidae)), wasp (Vespoidea (Vespidae)) and ant (Formicoidea (Formicoidae)).

Special anaphylactogen composition comprises the (white birch (B.verrucosa) as beech (Fagales) purpose Betv1, birch), Alng1 (glue alder (Alnus glutinosa) alder), Cora1 (Corylus avelana, fibert) and Carb1 (birch leaf carpinus turczaninowii (Carpinus betulus) hornbeam).Other be Cryj1 (pinales), Amba1 and 2, Artv1 (chrysanthemum order), Parj1 (Urticales), Olee1 (sweet-scented osmanthus order), Avee1, Cynd1, Dacg1, Fesp1, Hol11, Lolp1 and 5, Pasn1, Phlp1 and 5, Poap1,2 and 5, Secc1 and 5, and Sorh1 (pollen of various grass), Alta1 and Clah1 (fungi), Derf1 and 2, Derp1 and 2 (dermatophagoides pteronyssinuses, be respectively dust mite (D.farinae) and dermatophagoides pteronyssinus (D.pteronyssinus)), Eurm1 (mite, Euroglyphus maynei), (Lepd1 and 2 (Lepidoglyphus destructor; Storage mite (storage mite), Blag1 and 2, Pera1 (cockroach, be respectively Groton bug (Blatella germanica) and periplaneta americana (Periplaneta americana)), Feld1 (cat), Canf1 (dog), Equc1,2 and 3 (horses), Apism1 and 2 (honeybee), Vesv1,2 and 5, Pola1,2 and 5 (being wasp) and Soli1,2,3 and 4 (fiery ants).

In specifying mutant, import extra some examples that replace

The sudden change anaphylactogen has further been imported replacement in an example of the present invention, and can guarantee finally the to suddenly change replacement of anaphylactogen of this replacement mode can be evenly distributed in molecular surface, and the number of the importing sudden change that contains of each group is basic identical.This available following embodiment explanation, the mutant that wherein contains specific replacement has preferably further been imported the replacement that is selected from tabulation, and amino acid whose consecutive reaction goes out to import more polysubstituted preferred sequence in this table.How these embodiment representatives design a kind of application of sudden change, and also unrestricted the present invention, those skilled in the art can change to guarantee that sudden change is evenly distributed it apparently.Sudden change can be designed to contain one or more replacements of following appointment tabulation.

Betv1 mutant (" 3004A ") anaphylactogen: the Y5V that contains following replacement, E45S, N78K, K97S, K103V, K134E ,+160N.Also can further contain one or more following replacements: E8 or K115, D125 or H126, E138 or K137 or E141, D25 or N28, E87 or K55, S155 or H154 or N159, N47 or P50 or H76 or N43 or I44 or R70, E73 or P50 or D72, A130, N28 or D25, P108, V2 or K119 or N4 or E6 or E96.

Betv1 mutant (" 3004B ") anaphylactogen: the Y5V that contains following replacement, E45S, L62F, N78K, K97S, K103V, K134E ,+160N.Also can further contain one or more following replacements: T10P, K65N, N28 or D25 or K32Q or E141X or K137X or E138X, D125X or K123I or H126, P108X or D109N, E42S or K55X or I44X or N43X, E73X or D72X, E87X, E96X or K119, A130X, V2X or E6X, E8X or K115, N47X or P50X or R70X or H76X or T77A, S155X or D156H or N159X, E6X or V2X.

Betv1 mutant (" 3005A ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, K134E ,+160N.Also can further contain one or more following replacements: E8X or K115X, D125 or H126, E138X or K137X or E141X, E87X or K55X, S155X or H154X or N159X, N47X or P50X or H76X or N43X or I44X or R70X, E73X or P50X or D72X, A130X, D25X, P108X, V2X or K119X or N4X or E6X or E96X.

Betv1 mutant (" 3005B ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, K134E ,+160N.Also can further contain one or more following replacements: T10P, K65N, E141X or K137X or E138X, D125X or K123I or H126X, P108X or D109N, E42S or K55X or I44X or N43X, E73X or D72X, E87X, E96X or K119X, A130X, V2X or E6X, E8X or K115X, N47X or P50X or R70X or H76X or T77A, S155X or D156H or N159X, E6X or V2X.

Betv1 mutant (" 3006A ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, N78K, E87S, K97S, K103V, K134E, N159G ,+160N.Also can further contain one or more following replacements: K55, A138 or K137 or E141, D125 or H126, P108, V2 or N4 or K119 or E6, S155 or H154, N47 or P50 or H76, E73, R70, A130, E8 or K115, E96.

Betv1 mutant (" 3006B ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, N78K, E87S, K97S, K103V, K134E, N159G ,+160N.Also can further contain one or more following replacements: K65N, T10P, D125, K123I, P108, D109N, N47 or P50 or H76, E138 or K137 or E141, E42S or K55 or I44 or N43, S155 or D156H, E73 or D72, E6 or V2, E96.

Betv1 mutant (" 3007A ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, P108G, D125Y, K134E ,+160N.Also can further contain one or more following replacements: E87, E141, E138, K55, N47 or N43X or I44 or H76, S155 or H154, A130, E8, E73, V2 or K119, D25.

Betv1 mutant (" 3007B ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, P108G, D125Y, K134E ,+160N.Also can further contain one or more following replacements: K65N, T10P or E8, E87, S155 or D156H, E138, E141, E42S, A130, E8 or T10P, N47, H76, R70, E96.

Betv1 mutant (" 3008A ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, E73S, E96S, P108G, D125Y, N159G ,+160N.Also can further contain one or more following replacements: E134, N78, E87, K119, E8, K55, E138, E141, S155, N47, E6, K103, D25, A130, V2, R70.

Betv1 mutant (" 3008B ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, E73S, E96S, P108G, D125Y, N159G ,+160N.Also can further contain one or more following replacements: K65N or K55, T10P or E8 or E141, E138 or K134, E87, E42S or K55 or I44, S155 or D156H, N78, K119 or V2 or N4, N47 or P50, H76 or T77A, A130, D25, E6 or K115 or K103.

Betv1 mutant (" 3009A ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, E96S, P108G ,+160N.Also can further contain one or more following replacements: E134, N78, E87, K119, E8, K55, E138, E141, S155, N47, E6, K103, D25, A130, V2, R70.

Betv1 mutant (" 3009B ") anaphylactogen: the Y5V that contains following replacement, N28T, K32Q, E45S, L62F, E96S, P108G ,+160N.Also can further contain one or more following replacements: N78 or T77A, K103, E134 or E138, K65N or K55, T10P, D125 or H126, S155 or D156H or HIS154, K119 or V2, E87, N47 or P50 or H76, E42S or K55, I44 or N43, A130.

The ring sudden change

In the present invention suddenlys change another embodiment of anaphylactogen, also further contain aminoacid insertion or aminoacid deletion,, as encircle the zone particularly at the surperficial exposed region of molecule.The ring regional connection the secondary structure element, as βZhe Die, and α spiral and winding arrangement at random.Ring zone among the Betv1 is: Val12 is to ala16, and val33 is to ser40, and glu45 is to Thr52, and pro54 is to tyr66, and his76 is to asn78, and gly89 is to g1u96, and Val105 is to gly111, and thr122 is to glu131.Mutant of the present invention can contain 1-5 in the ring zone, preferred 1-3, most preferably 1-2 replacement.In a preferred embodiment, the sudden change anaphylactogen contains and is selected from least 4 sudden changes of 10 groups, and some extra " ring sudden changes ".The example of this " ring sudden change " (wherein amino-acid residue of x representative adding) is:

The Betv1 (3007-L1) that between residue B 60 and residue G61, has aminoacid insertion:

GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTI

KKISFPExGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVA

TGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYLLAHSDA

YNN

The Betv1 (3007-L2) that between residue D93 and residue T94, has aminoacid insertion:

GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTI

KKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDxTLESISNEIVIVA

TGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYLLAHSDA

YNN

The Betv1 (3007-L3) that between residue V12 and residue 113, has aminoacid insertion:

GVFNVETETTSVxIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGT

IKKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVA

TGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYLLAHSDA

YNN

The Betv1 (3007-L4) that between residue 156 and residue S57 and residue K65 and residue T66, has aminoacid insertion:

GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTI

KKIxSFPExGFPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLESISNEIVIV

ATGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYLLAHSD

AYNN

The Betv1 (3007-L5) of residue G111 site disappearance:

GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTI

KKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVAT

GDGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYLLAHSDAYN

N

The method of recombinant allergens produced according to the present invention

The amino acid that the surface that is suitable for replacing according to the present invention exposes can its solvent (water) accessibility information be that the basis is determined, this information representation the degree that exposes of its surface.A preferred embodiment of the inventive method is characterised in that with the solvent accessibility amino-acid residue of wherein determining is sorted, and replaces one or more in the amino acid with big solvent accessibility.

In addition, a particularly preferred embodiment of the inventive method is characterised in that about coming the amino-acid residue of wherein determining is sorted with the conservative degree in all known homologous proteins in this naturally occurring anaphylactogen institute species, and replaces one or more in the amino acid with big conservative property.

The inventive method further embodiment preferred comprises the definite amino acid of selection to form the mutant anaphylactogen, and it has the α carbon backbone chain tertiary structure substantially the same with naturally occurring anaphylactogen.

Another preferred embodiment of the inventive method is characterised in that the replacement to amino-acid residue realizes by rite-directed mutagenesis.

Another preferred embodiment of the inventive method is characterised in that the replacement of amino-acid residue reorganized by DNA and realizes, or contains suitable site and the preferred library that replaces is realized by foundation.

The standard that replaces

The mutant that has substituted amino acid for the definite molecule (for example by X-radiocrystallography or NMR electron microscope method) of tertiary structure preferably should satisfy following standard:

1. the overall alpha-carbon skeleton tertiary structure of molecule is preferably conservative.Conservative being defined as when relatively suddenling change and during the structure of natural anaphylactogen, the average root-mean-square difference of atomic coordinate is less than 2 .Its importance has 2 reasons: can expect the overlapping continuum of the potential antibodies epi-position of whole surface composition of this natural anaphylactogen.The influence that the major part of this molecular surface is not replaced, thus its antibodies characteristic kept, and this is important for producing at the new protection antibody specificity that also is present in the epi-position on the natural anaphylactogen.B) stability relates to preservation period and the stability that is injected into body fluid two aspects.

By before sudden change and after the sudden change, carrying out x ray crystallography or NMR obtains the conservative property that same structure is accurately measured alpha-carbon skeleton tertiary structure.If compare with the data that the determined molecule of analytical structure is obtained, under the situation of the structured data of not describing sudden change, indifference CD spectrum or immunochemistry data (as antibody response) can be thought guarding of alpha-carbon skeleton tertiary structure.

2. amino acid to be replaced is preferably placed at the surface, and is come-at-able for antibodies therefore.The amino acid that is positioned at the surface has at least 20% solvent (water) accessibility usually, and that suitable is 20-80%, more suitably is the solvent accessibility of 30-80%.The solvent accessibility is defined as with radius and the suitable come-at-able molecular domains of spheroid of solvent molecule (water, r=1.4 ).

3. the amino acid of Qu Daiing is selected from least 4 groups.Every group of amino-acid residue that the many surfaces of representative expose, described amino acid is to find in the limited area on anaphylactogen surface.One or more sudden changes in a group are defined as an one-level sudden change.Each independent group contains many amino acid, and these amino acid are parts of at least one epi-position.Each independent group also may comprise a complete epi-position.The sudden change anaphylactogen that has at least 4 one-level sudden changes can guarantee that several epi-positions decrease to the IgE binding affinity.Can further guarantee also that from the amino acid whose sudden change of at least 4 groups sudden change approximately is evenly distributed at molecular surface, thereby and guarantee that several epi-positions produce sudden change and cause several epi-positions and IgE binding affinity to decrease.

For keeping 3 dimension structures of anaphylactogen substantially, can on the basis that compares with this anaphylactogen homologous structure albumen, select the amino acid that imports, as belonging to the same category target protein with anaphylactogen, and there are not any crosslinking reaction in this albumen and this anaphylactogen.

Vaccine

Vaccine to be prepared as this area well-known.Vaccine generally prepares with the form of injectable liquor or suspension.Such vaccine also can emulsification or be mixed with can intranasal and peroral administration form, comprises oral cavity and sublingual administration.Immunogenic components in question (as the recombinant allergens that is limited herein) can suitably with on the pharmacology can be accepted and further compatible with activeconstituents mixed with excipients.The example of appropriate excipients is water, salt, dextrose, glycerine, ethanol etc. and their combination.Vaccine can contain other material in addition, as the adjuvant of wetting agent, emulsifying agent, buffer reagent or enhancing vaccine potency.

Vaccine the most common by subcutaneous or intramuscularly with the administration of parenteral mode.The preparation that is adapted to pass through other administrations comprises oral preparations and suppository.Peroral administration vaccine can be suitably be allocated with the vehicle that is generally used for this preparation, as the N.F,USP MANNITOL of pharmaceutical grade, lactose, starch, Magnesium Stearate, soluble saccharin (sodium saccharine), Mierocrystalline cellulose, magnesiumcarbonate etc.Composition can be prepared to solution, suspend tuck in, emulsion, tablet, pill, capsule, sustained release preparation, aerosol, powder or particle.

The route of administration of vaccine should be compatible with dosage formulation, and its amount has treatment validity and tool immunogenicity.The amount of the activeconstituents that comprises in vaccine depends on treatment target, the subject is in response to the immunity system of treatment responsibility, route of administration and subject's age and the body weight to treatment in theory.Suitable dosage range is at 0.0001 μ g～1000 μ g.

As mentioned above, by in prepared product, adding the effect that adjuvant can obtain to increase.The example of such adjuvant is the aluminium hydroxide and the phosphoric acid salt (alum) that exist with 0.05%～0.1% solution in phosphate buffered saline(PBS), lactide (polylactid glycolid) has been (PLG) for synthetic carbohydrate polymer that the solution with 0.25% uses or polylactide.Also can adopt and bacterial cell such as the intracellular toxin of spillikin bacillus (C.parvum), gram negative bacterium or the mixture of lipopolysaccharides composition, emulsion on such as mannitan (mannide monoaleate) physiology (AracelA) in the acceptable oils carrier, or (for example, the emulsion of solution Fluosol-DA) is as the sealing substituent to contain 20% perchloro-carbon.Oil-emulsion, also available such as MF-59.Also can use other adjuvant, complete and Freund and QuilA as Fu Shi, Qs-21 and ISCOM, RIBI.

The most frequently, the multiple administration of vaccine is to guaranteeing that its effect is essential.Vaccine is often to follow the mode of follow-up inoculation or other administrations to come administration after the first administration.The quantity of vaccine inoculation is generally 1～50 time, is no more than 35 vaccine inoculation usually.3 months to 5 years time is carried out in vaccine inoculation twoly usually thoughtful month.Expect that this will reach the aspiration level of prevention or result of treatment.

Recombinant allergens can be used as pharmaceutical preparations, symptom occur place year during be suitable for providing some to resist anaphylactoid protection (prevention).Usually, must annual repetitive therapy to keep provide protection.Be formulated as the prepared product of using in nose, mouth and hypogloeeis and be particularly suitable for this purpose.

DNA of the present invention

Dna sequence dna of the present invention is the mutant of natural B etv1 anaphylactogen coding DNA.The example of natural B etv1 molecule is SEQ ID NO 1 (database login Z80104) and SEQID NO 2 (database login P15494).Other Betv1 variant comprises the Betv1 sequence with following database login number: P15494=X15877=Z80106, Z80101, AJ002107, Z72429, AJ002108, Z80105, Z80100, Z80103, AJ001555, Z80102, AJ002110, Z72436, P43183=X77271, Z72430, AJ002106, P43178=X77267, P43179=X77268, P43177=X77266, Z72438, P43180=X77269, AJ001551, P43185=X77273, AJ001557, Z72434, AJ001556, Z72433=P43186, AJ001554, X81972, Z72431, P45431=X77200, P43184=X77272, P43176=X77265, S47250, S47251, Z72435, Z72439, Z72437, and S47249.

Preferably, this DNA derivative be by natural anaphylactogen coding DNA is fixed a point at random or half random mutation produce.

" sudden change library " is the library of allergen mutants.This library is to make up by the DNA Oligonucleolide primers with degeneracy to form, and this primer can import 0,1 or several different amino-acid residue in each site.Such library method can make amino-acid residue by conservative or non-conservative replacement.Because conservative replacement can import this " slightly " or gentle sudden change at specific position, can not influence the globality of structure too much, therefore can increase and form the chance of stablizing mutant.Making up this mutant library can overcome by the caused sudden change allergenic protein stability of the sudden change of single or particular combination defective." half random library " refers to that site to be suddenlyd change is limited among those amino-acid residues of surface exposure.This method also can improve the possibility that obtains stable sudden change anaphylactogen." partly at random " means that also the primer of design can allow to replace at the amino acid of select location with selected number.But this two and half random devices independent utility, or applied in any combination.In theory, many rBetv1 sudden change anaphylactogens are contained in library of the present invention, compare with the Betv1 of not sudden change, and each sudden change anaphylactogen all has at least 4 aminoacid replacement.

In one embodiment, made up one based on rBetv1 (2744) (mutational site Y5, E42, E45, N78, K103, K123, K134, D156 ,+160) and rBetv1 (2628) (site, site Y5, E45, K65, K97, half random library K134), and outside anaphylactogen surface targets quota 7 site: T10, K20, Q36, E73, E87, K129 and S149.These 7 sites are selected from the surf zone that is arranged in the coherent surf zone in the total outside of Fagales anaphylactogen.This library is based on the application of degeneracy DNA Oligonucleolide primers, and this primer can import the different aminoacids residue in each site.In addition, the several mutating acid residues site in rBetv1 (2744) and rBetv1 (2628) can continue to keep, or the residue of finding among the WT rBet v1.2801 that suddenlys change back.

Having made up based on rBetv1 (2744) and rBetv1 (2628) and rBetv1 (2595) among another embodiment is N28, K32, E45, half random library of P108, wherein extra 7 target site: T10, K20, Q36 have been decided at the anaphylactogen surface targets, E73, E87, K129 and S149.

Mutant

Below listed the example of particular B etv1 allergen mutants of the present invention.The amino acid sites of sudden change shows with the black matrix small letters:

Betv1(“3004”)(SEQ?ID?NO?3)：

GVFNvETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVsNIEGNGGPGTIK

KISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPD

GGSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn

Betv1(“3005”)(SEQ?ID?NO?4)：

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDG

GSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn

Betv1(“3007”)(SEQ?ID?NO?5)：

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATgDG

GSILKISNKYHTKGyHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn

Betv1(“3009”)(SEQ?ID?NO?6)：

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLsKISNEIKIVATgD

GGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYNn

Betv1(“3006”)(SEQ?ID?NO?7)：

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDG

GSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYgn

Betv1(“3008”)(SEQ?ID?NO?8)：

GVFNvETETTSVIPAARLFKAFILDGDtLFPkVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDsVDHTNFKYNYSVIEGGPIGDTLsKISNEIKIVATgDG

GSILKISNKYHTKGyHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYgn

The present invention also comprises following specified mutant:

Betv1(“3005-7”)(SEQ?ID?NO?9)：

Y5V，N28T，K32Q，E45S，N78K，K97S，K103V，K134E，+160N，E8S，D125Y，E141S，D25T，E87A，S155T，N47K，K55N.

GVFNvETsTTSVIPAARLFKAFILtGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS

FPEGLPFKYVKDRVDEVDHTkFKYNYSVIaGGPIGDTLEsISNEIvIVATPDGG

SILKISNKYHTKGyHEVKAEQVeASKEMGsTLLRAVESYLLAHtDAYNn

Betv1(“3005-12”)(SEQ?ID?NO?10)：

Y5V，N28T，K32Q，E45S，N78K，K97S，K103V，K134E，+160N，E8S，D125Y，E141S，D25T，E87A，S155T，N47K，K55N，E73T，A130V，P108G，V2L，

GIFNvETsTTSVIPAARLFKAFILtGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS

FPEGLPFKYVKDRVDtVDHTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDGGS

ILKISNKYHTKGyHEVKvEQVeASKEMGsTLLRAVESYLLAHtDAYNn

Betv1(“3005-22”)(SEQ?ID?NO?11)：

Y5V，N28T，K32Q，E45S，N78K，K97S，K103V，K134E，+160N，T10K，K65N，E141N，K123I，D109N，E42S，E73T，E87A，V2L，N47K.

GIFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKI

SFPEGLPFnYVKDRVDtVDHTtFKYNYSVIaGGPIGDTLEsISNEIvIVATPnGG

SILKISNKYHTiGDHEVKAEQVeASKEMGnTLLRAVESYLLAHSDAYNn

Betv1(“3005-27”)(SEQ?ID?NO?12)：

Y5V，N28T，K32Q，E45S，N78K，K97S，K103V，K134E，+160N，T10K，K65N，E141N，K123I，D109N，E42S，E73T，E87A，K119N，A130V，V2L，E8S，N47K，D156H，E6S.

GIFNvsTsTpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKIS

FPEGLPFnYVKDRVDtVDHTtFKYNYSVIaGGPIGDTLEsISNEIvIVATPnGGSI

LKISNKYHTiGDHEVKAEQVeASKEMGnTLLRAVESYLLAHShAYNn

Betv1(“3007-6”)(SEQ?ID?NO?13)：

Y5V，N28T，K32S，E45S，N78K，K97S?K103V，P108G，D125Y，K134E，+160N，E87A，E141N，K55N，N47K，S155T，

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnI

SFPEGLPFKYVKDRVDEVDHTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDG

GSILKISNKYHTKGyHEVKAEQVeASKEMGnTLLRAVESYLLAHtDAYNn

Betv1(“3007-10”)(SEQ?ID?NO?14)：

Y5V，N28T，K32S，E45S，N78K，K97S?K103V，P108G，D125Y，K134E，+160N，E87A，E141N，K55N，N47K，S155T，A130V，E8S，E73T，V2L.

GIFNvETsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS

FPEGLPFKYVKDRVDtVDHTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDGGS

ILKISNKYHTKGyHEVKvEQVeASKEMGnTLLRAVESYLLAHtDAYNn

Betv1(“3007-17”)(SEQ?ID?NO?15)：

Y5V，N28T，K32Q，E45S，N78K，K97S，K103V，P108G，D125Y，K134E，+160N，K65N，T10P?E87A，D156H，E141N，E42S.

GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK

ISFPEGLPFnYVKDRVDEVDHTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDG

GSILKISNKYHTKGyHEVKAEQVeASKEMGnTLLRAVESYLLAHShAYNn

Betv1(“3007-22”)(SEQ?ID?NO?16)：

Y5V，N28T，K32Q，E45S，N78K，K97S，K103V，P108G，D125Y，K134E，+160N，K65N，T10P?E87A，D156H，E141N，E42S，A130V，E8S，N47K，H76T，V2L.

GIFNvETsTpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKIS

FPEGLPFnYVKDRVDEVDtTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDGGS

ILKISNKYHTKGyHEVKvEQVeASKEMGnTLLRAVESYLLAHShAYNn

Betv1(“3008-8”)(SEQ?ID?NO?17)：

Y5V，N28T，K32Q，E45S，E73S，E96S，P108G，D125Y，N159G，+160N，K134E，N78K，E87A，K119N，E8S，K55N，E141N，N47K.

GVFNvETsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnI

SFPEGLPFKYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIKIVATgDG

GSILKISNnYHTKGyHEVKAEQVeASKEMGnTLLRAVESYLLAHSDAYgn

Betv1(“3008-13”)(SEQ?ID?NO?18)：

Y5V，N28T，K32Q，E45S，E73S，E96S，P108G，D125Y，N159G，+160N，K134E，N78K，E87A，K119N，E8S，K55N，E141N，N47K，S155T，E6S，K103V，A130V，V2L.

GIFNvsTsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS

FPEGLPFKYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIvIVATgDGG

SILKISNnYHTKGyHEVKvEQVeASKEMGnTLLRAVESYLLAHtDAYgn

Betv1(“3008-20”)(SEQ?ID?NO?19)：

Y5V，N28T，K32Q，E45S，E73S，E96S，P108G，D125Y，N159G，+160N，K65N，T10P，E138N，E87A，E42S，D156H，N78K.

GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK

ISFPEGLPFnYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIKIVATgDG

GSILKISNKYHTKGyHEVKAEQVKASKnMGETLLRAVESYLLAHShAYgn

Betv1“3008-25”)(SEQ?ID?NO?20)：

Y5V，N28T，K32Q，E45S，E73S，E96S，P108G，D125Y，N159G，+160N，K65N，T10P，E138N，E87A，E42S，D156H，N78K，K119N，N47K，T77A，E130V，K115N.

GIFNvsTETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKKI

SFPEGLPFnYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIvIVATgDG

GSILKISNKYHTKGyHEVKvEQVKASKnMGETLLRAVESYLLAHthAYgn

Betv1(“3009-9”)(SEQ?ID?NO?21)：

Y5V，N28T，K32Q，E45S，E96S，P108G，+160N，K134E，N78K，E87A，K119N，E8S，K55N，E138N，E141N，S155T，N47K，E6S，K103V，A130V，V2L，R70N，D125Y.

GVFNvETsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnI

SFPEGLPFKYVKDRVDEVDHTkFKYNYSVIaGGPIGDTLsKISNEIKIVATgDG

GSILKISNnYHTKGDHEVKAEQVeASKnMGnTLLRAVESYLLAHtDAYNn

Betv1(“3009-15”)(SEQ?ID?NO?22)：

Y5V，N28T，K32Q，E45S，E96S，P108G，+160N，K134E，N78K，E87A，K119N，E8S，K55N，E138N，E141N，S155T，N47K，E6S，K103V，A130V，V2L，R70N，D125Y.

GIFNvsTsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS

FPEGLPFKYVKDnVDEVDHTkFKYNYSVIaGGPIGDTLsKISNEIvIVATgDGG

SILKISNnYHTKGyHEVKvEQVeASKnMGnTLLRAVESYLLAHtDAYNn

Betv1(“3009-22”)(SEQ?ID?NO?23)：

Y5V，N28T，K32Q，E45S，E96S，P108G，+160N，T77A，K103V，E138N，K65N，T10P，D125Y，E42S.

GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK

ISFPEGLPFnYVKDRVDEVDHaNFKYNYSVIEGGPIGDTLsKISNEIvIVATgD

GGSILKISNKYHTKGyHEVKAEQVKASKnMGETLLRAVESYLLAHSDAYNn

Betv1(“3009-28”)(SEQ?ID?NO?24)：

Y5V，N28T，K32Q，E45S，E96S，P108G，+160N，T77A，K103V，E138N，K65N，T10P，D125Y，D156H，K119N?E87A，E42S，A130V.

GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK

ISFPEGLPFnYVKDRVDEVDHaNFKYNYSVIaGGPIGDTLsKISNEIvIVATgDG

GSILKISNnYHTKGyHEVKvEQVKASKnMGETLLRAVESYLLAHShAYNn

Betv1 clones (" 3031 ") (SEQ ID NO 25):

GVFNVETETASVIPAARLFNAFILDGDTLFPQVAPQAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIEGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN

N

Betv1 clones (" 3032 ") (SEQ ID NO 26):

GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDPVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN

N

Betv1 clones (" 3033 ") (SEQ ID NO 27):

GVFNVETETPSVIPAARLFHAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN

N

Grand (" 3034 ") (the SEQ ID NO 28) of Betv1 gram I:

GVFNVETETTSVIPAARLFHAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3035 ") (SEQ ID NO 29):

GVFNVETETPSVIPAARLFMAFILDGDTLFPQVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAYLLAHSHAYN

N

Betv1 clones (" 3036 ") (SEQ ID NO 30):

GVFNVETETPSVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDTVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3037 ") (SEQ ID NO 31):

GVFNVETETPSVIPAARLFQAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN

N

Betv1 clones (" 3038 ") (SEQ ID NO 32):

GVFNVETETASVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3039 ") (SEQ ID NO 33):

GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPEAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVA

TPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAYLLAHSHAY

NN

Betv1 clones (" 3040 ") (SEQ ID NO 34):

GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETYLLAHSHAYN

N

Betv1 clones " 3041 ") (SEQ ID NO 35):

GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3042 ") (SEQ ID NO 36):

GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3043 ") (SEQ ID NO 37):

GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN

N

Betv1 clones (" 3044 ") (SEQ ID NO 38):

GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVA

TPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETYLLAHSHAY

NN

Betv1 clones (" 3045 ") (SEQ ID NO 39):

GVFNVETETPSVIPAARLFMAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN

N

Betv1(3010)(SEQ?ID?NO?40)

Y5V，N28T，K32Q，E45S，K97S，P108G，+160N，E60S，T10N，K103V，K65N，

K129N，D125Y，E42S，S149T.

GVFNvETETnSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK

ISFPsGLPFnYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEsISNEIvIVATgDG

GSILKISNKYHTKGyHEVnAEQVKASKEMGETLLRAVEtYLLAHSDAYNn

Betv1(3011)(SEQ?ID?NO?41)

Y5V，N28T，K32Q，E45S，K97S，P108G，+160N，E60S，T10N，K103V，K65N，

K129N，D125Y，E42S，S149T，K134E，N47K，T77N，V2L.

GIFNvETETnSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKI

SFPsGLPFnYVKDRVDEVDHnNFKYNYSVIEGGPIGDTLEsISNEIvIVATgDG

GSILKISNKYHTKGyHEVnAEQVeASKEMGETLLRAVEtYLLAHSDAYNn

Betv1(3012)(SEQ?ID?NO?42)

Y5V，N28T，K32Q，E45S，K97S，P108G，+160N，E60S，T10N，K103V，K65N，

K129N，D125Y，E42S，S149T，K134E，N47K，T77N，V2L，E87A，A16G，

Q36N，E73S，D93S.

GIFNvETETnSVIPAgRLFKAFILDGDtLFPqVAPnAISSVsNIsGkGGPGTIIKKIS

FPsGLPFnYVKDRVDsVDHnNFKYNYSVIaGGPIGsTLEsISNEIvIVATgDGGS

ILKISNKYHTKGyHEVnAEQVeASKEMGETLLRAVEtYLLAHSDAYNn

Diagnostic test

In addition, recombinant allergens mutant according to the present invention has diagnosis possibility and advantage.The extract that allergy vaccine in the current field is originated based on naturally occurring anaphylactogen, thereby represented multiple isotype.Anaphylaxis is individual irritated and produce IgE at the isotype of one or more existence at first.Because reaching subsequently, homology with making individuality the cross reactivity of anaphylactoid isotype takes place, some isotypes are correlated with about the anaphylaxis of anaphylaxis individuality, yet other isotypes are uncorrelated owing to do not have that any anaphylaxis individuality has the IgE conjugated antigen decision epi-position of specific IgE to it.Since the heterogeneity of IgE population characteristic, some isotypes thereby administration safely, promptly they do not cause the anaphylaxis via IgE, however other isotype may cause bad side effect nocuously.

Thereby the mutant of the present invention of plan therapeutic administration and the present composition also can be used as in the body or in-vitro diagnosis is measured appropriateness, security or the result who uses this mutant or composition to treat with monitoring.The diagnosis sample of using comprises the human sample, as blood or serum.

Thereby, the present invention also relates to estimate diagnostic assay that wherein the IgE sample that contains with individuality mixes with this mutant or said composition and assesses in this sample or the reactive level between the IgE in this mutant to appropriateness, security or the result of the treatment of individuality with recombinant allergens mutant of the present invention or composition of the present invention.The available any known immunoassay of the assessment of the reactive level between the IgE in this sample or in this mutant are implemented.

The present invention illustrates further by following non-limiting example.

Embodiment

Embodiment 1

This embodiment has described the feature of the reorganization Betv1 allergen mutants of IgE binding affinity reduction.PCT/DK 01/00764 also discloses this specific sudden change anaphylactogen.Below how representative prepares the illustrative embodiment of mutant of the present invention.

The evaluation of common epitope in beech (Fagales) pollen allergens

Main birch pollen allergens Betv1 and main anaphylactogen from the relevant trees pollen of taxonomy have about 90% aminoacid sequence homogeny, that is, Fagales (for example fibert and hornbeam) and birch pollen hypersensitivity patient often demonstrate the clinical symptom of irritated cross reaction to these Betv1 homologous proteins.

Betv1 also show with some fruit (for example apple and cherry) and vegetables (as celery and Radix Dauci Sativae) in the anaphylaxis protein that exists demonstrate the sequence homogeny of about 50-60%, and have clinical evidence to show between Betv1 and these food associated protein to have the anaphylaxis cross reactivity.

In addition, Betv1 and one group of vegetable-protein that is called as pathology related protein (PR-10) have obvious sequence homogeny (20-40%), yet these PR-10 albumen are not also had the report of irritated cross reactivity.

Molecular model shows that Fagales (Fagales) is identical with the structure proximate of Betv1 with food allergens and the proteinic structure of PR-10.

The architecture basics of pathergy Betv1 cross reactivity is at (Gajhede etc., 1996, document 17) middle report, therefore, any IgE that identification Betv1 goes up these sections can combine with the main pollen allergens generation cross reaction of other Fagales and with it, produces symptoms of allergic.

Select amino-acid residue to be used for site-directed mutagenesis

The amino-acid residue that is used for rite-directed mutagenesis is selected from the surperficial exposed residue on the Betv1.The relative positioning and the solvent that calculate each amino-acid residue according to their atomic coordinate expose percentage ratio.Residue with low solvent degree of exposure (＜20%) is regarded as with mutagenesis irrelevant owing to destroying structure or lacking the antibody interaction.Remaining residue sorts according to its solvent degree of exposure.

The sequence contrast

Derive from GenBank and EMBL sequence library (Altschul etc., document 18) with described sequence (Betv1 No.2801, WHO IUIS anaphylactogen name sub-committee) homologous sequence by the BLAST retrieval.Consider BLAST report probability less than 0.1 all sequences, the homologous sequence that will contain irredundant sequence is made tabulation.Carry out sequence contrast by CLUSTAL W (Higgins etc., document 19), at full sequence or the same percentage of each position in the akin kind sequence of calculation on the taxonomy only.Always have 122 sequences and Betv1No.2801 homology, wherein 57 sequences are from the relevant kind of taxonomy.

The gene of clones coding Betv1

Precipitate from white birch (Betula verrucosa) pollen (Allergon, Sweden) preparation RNA by phenol extraction and LiCl.In Eppendorf tube, carry out oligomerization (dT)-Mierocrystalline cellulose affinity chromatography in batches, use commercial reagent box (Amersham) synthetic double chain cDNA.The DNA of coding Betv1 is through pcr amplification and clone.Briefly, use cDNA to make template and be designed to and carry out PCR corresponding to the primer of the cDNA sequences match of Betv1 aminoterminal and 3 '-non-translational region position respectively.Primer advances pKK233-2 in 5 ' end extension to provide restriction site (Ncol and HindIII) to be used for directed cloning.

Subclone advances pMAL-c

The gene of coding Betv1 is advanced among the maltose binding protein fusion vector pMAL-c (New England Biolabs) by subclone subsequently.The pcr amplification gene also meets frame ground subclone with malE and merges operon to produce maltose binding protein (MBP)-Betv1 albumen, wherein MBP and Betv1 are by factor Xa protease cracking site separately, this cracking site present position makes can recover the real amino terminal sequence of Betv1 after cracking, as described in document 15.Briefly, use the pKK233-3 that has inserted Betv1 to make template, the primer corresponding to this protein amino and carboxyl terminal carries out PCR respectively.Promotor near-end primer extends to comprise 4 codons of the in-frame factor Xa protease cracking site of encoding at 5 ' end.Two primers all are used for the clone further the extension to comprise restriction site (KPn1) of 5 ' end.The gene that uses 20 round-robin PCR subclone coding Betv1 is to reduce the frequency of PCR illusion.

Vitro mutagenesis

Use has Betv1 and inserts segmental reorganization pMAL-c and make template and carry out vitro mutagenesis through PCR.The Betv1 gene of each sudden change uses 4 primers to produce through 3 PCR reactions.Following sudden change example is according to prior art PCT/DK 01/00764.Mutant of the present invention can prepare and test in a similar manner.

The individual sudden change specific oligonucleotide primer that comprises each sudden change of Synthetic 2, every primer is seen Fig. 3 and 4 at a DNA chain.Nucleotide with sudden change is made starting point, and two primers all extend 7 Nucleotide at 5 '-end, extend 15 Nucleotide at 3 '-end.The Nucleotide that extends is identical with Betv1 gene actual area on sequence.

Also synthesize two universal primers (in Fig. 4, being called " Quan Youyi " and " completely without justice ") and be used for all mutant.Long 15 Nucleotide of these primers, on sequence with the pMAL-c carrier in about 1 kilobase of Betv1 upstream and downstream corresponding to the sequence of locating the zone.The sequence of upstream primer is from sense strand, and the sequence of downstream primer is seen Fig. 4 from nonsense strand.

Except only carrying out 20 temperature cycle, carry out 2 independently PCR reactions according to standard method (Saiki etc., 1988, document 20) basically with the frequency that reduces the PCR illusion.Each PCR reaction uses the pMAL-c that has inserted Betv1 to make template, and a sudden change special primer and the formed meaningful combination of universal primer.

According to progressively method recited above in mutant, import 4 aminoacid replacement (Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly).Use respectively and inserted Betv1 the 2801st, Betv1 (Glu45Ser), (Glu45Ser, pMAL-c Pro108Gly) makes template to Betv1, at first imports the Glu45Ser sudden change, imports the Pro108Gly sudden change then, imports Asn28Thr at last, the Lys32Gln sudden change.

Through agarose gel electrophoresis and electroelution, ethanol sedimentation purified pcr product subsequently.Use makes template from the mixing PCR product of preceding twice PCR reaction and 2 universal primers carry out the 3rd PCR reaction.Equally, use 20 round-robin Standard PC R.Through agarose gel electrophoresis and electroelution, carry out the ethanol sedimentation purified pcr product subsequently, with Restriction Enzyme (Bs/WI/EcoRI) cutting and directedly connected to advance with the insertion of same restrictions enzymic digestion among the pMAL-C of Betv1.

Fig. 5 has shown the general introduction of all 9 Betv1 sudden changes, and these 9 sudden changes are as follows: Thr10Pro, Asp25Gly, Asn28Thr+Lys32Gln, Glu45Ser, Asn47Ser, Lys55Asn, Glu60Ser, Thr77Ala and Pro108Gly.Also prepared another one mutant with 4 places sudden changes (Asn28Thr, Lys32Gln, Glu45Ser, Pro108my).Wherein, select 5 mutant to be used for further test: Asn28Thr+Lys32Gln, Glu45Ser, Glu60Ser, Pro108Gly and Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly mutant.

Nucleotide sequencing

Before the subclone, afterwards with vitro mutagenesis after respectively the nucleotide sequence of Betv1 encoding gene is measured.

With 10ml come leisure replenished incubated overnight in the LB substratum of 0.1g/l acillin to the plasmid DNA of saturated bacterial cultures with Qiagen-tip 20 column purification, and use Sequenase version 2.0 dna sequencing kits (USB) order-checking according to the suggestion of manufacturer.

The expression and purification of reorganization Betv1 and mutant

The reorganization Betv1 (Betv1 the 2801st and mutant) that is fused on the maltose binding protein crosses expression and presses document 15 described purifying in escherichia coli DH5a.Briefly, optical density(OD) is 1.0 to recombinant Bacillus coli cells under the 436nm growing under 37 ℃, and add IPTG and induce the Betv1 expressing fusion protein this moment.Induce back 3 hours centrifugal cell harvestings, be resuspended in the lysis buffer and through the supersound process fragmentation.Behind supersound process and the recentrifuge, separate recombination fusion protein through the starch affinity chromatography and also pass through subsequently and factor Xa incubation cracking (document 15).After the factor Xa cracking, separate reorganization Betv1, if necessary, carry out another and take turns the starch affinity chromatography to remove the maltose binding protein of trace through gel-filtration.

Reorganization Betv1 is purified to about 5mg/ml and is stored in 4 ℃ through ultrafiltration and concentration.The ultimate yield of the reorganization Betv1 goods of purifying is every liter of Bacillus coli cells culture 2-5mg.

The single band of 17.5kDa apparent molecular weight appears having in the reorganization Betv1 goods of purifying after SDS-polyacrylamide gel electrophoresis silver dyes, the order-checking of N-end shows the expected sequence from the cDNA nucleotide sequence, and quantitatively amino acid analysis demonstrates the amino acid composition of expection.

We showed in the past that (document 15) reorganization Betv1 the 2801st can not distinguish on immunochemistry with naturally occurring Betv1.

Use the immunoelectrophoresis of rabbit polyclonal antibody

Produced 7 kinds of mutant Betv1 and by purifying mentioned above with reorganization Betv1 proteic form, tested itself and reactivity at the multi-clone rabbit antibody that produces from the isolating Betv1 of birch pollen.When immunoelectrophoresis (rocket immunoelectrophoresis) under non-sex change condition was analyzed, the rabbit antibody capable precipitated all mutant, showed that these mutant have conservative alpha-carbon atom tertiary structure.

In order to analyze influence to people's polyclone IgE-reaction, select mutant Glu45Ser, Pro108Gly, Asn28Thr+Lys32Gln and Glu60Ser further analyze.

Betv1 Glu45Ser mutant

Show height solvent exposure property (40%) at 45 L-glutamic acid.Find that in some Betv1 homologous PR-10 protein serine residue plants oneself 45, show that L-glutamic acid can be replaced by Serine and can not destroy the tertiary structure of alpha-carbon skeleton.In addition, because the known Fagales allergen sequence of none 45 has Serine in the position, therefore replace L-glutamic acid and produce the Betv1 molecule that non-natural exists with Serine.

Use reorganization Glu45Ser Betv1 mutant to carry out the T cell proliferation test

Press Spangfort etc., 1996a is described to be analyzed.Find that reorganization Betv1Glu45Ser sudden change physical efficiency induces the T cell line proliferation from 3 kinds of different birch pollen hypersensitivity patients, its stimulation index is similar in appearance to reorganization and naturally occurring anaphylactogen.

Crystallization and the structure determination of reorganization Glu45Ser Betv1

Basically form reorganization Glu45Ser Betv1 crystal at 25 ℃ through vapor diffusion by 1996b such as (, document 21) Spangfort is described.The Glu45Ser Betv1 of 5mg/ml concentration and isopyknic 2.0M ammonium sulfate, 0.1M Trisodium Citrate, the dioxane of 1% (V/V), pH6.0 mixes, and with the 2.0M ammonium sulfate of 100 times of volumes, 0.1M Trisodium Citrate, the dioxane of 1% (V/V), pH6.0 balance.After the balance 24 hours, use reorganization wild-type Betv1 crystal to do the crystal seed source, use document 21 described seeding techniques and induce crystal growth.

After about 2 months, the results crystal also rotates the anodal X-ray that produces by document 21 described use Rigaku and analyzes, and uses molecule to replace analytic structure.

The structure of Betv1 Gln45Ser mutant

Resolve the influence of sudden change by the 3 long Betv1 Glu45Ser albumin crystals of supporting one's family to structure, this crystal is when going out to distinguish that with the anodal x-ray analysis time-division diffraction that produces of rotation rate is 3.0 , confirm that with Bet vIGlu45Ser structure electron density map the L-glutamic acid at 45 is replaced to Serine, this electron density map shows that also overall α carbon skeleton tertiary structure guards.

The IgE binding characteristic of Betv1 Gln45Ser mutant

Use is carried out liquid phase IgE-inhibition test from birch anaphylaxis patients serum IgE storehouse, relatively the IgE binding characteristic of Betv1 Glu45Ser mutant and reorganization Betv1.

(Betv1 the 2801st: vitamin H) with biotin labeling reorganization Betv1 the 2801st with 1: 5 mol ratio.Inhibition test is carried out as follows: the anti-IgE7 insulation of serum sample (25 μ l) and solid phase, and washing, resuspended and further be incubated with the mixture of biotin labeled Betv1 the 2801st (3.4nM) and given mutant (0-2.6nM).Be incubated the RLU that measures the back according to streptavidin and estimate the amount that is attached to the biotin labeled Betv1 the 2801st on the solid phase with the acridinium ester mark.Calculate the degree that suppresses with the ratio that uses damping fluid and mutant to do between the RLU of inhibitor acquisition.

Fig. 6 has shown by the Betv1 of the plain mark of lifeless matter with by the bonded of Betv1 Glu45Ser mutant to biotin labeled reorganization Betv1 and anaphylaxis patients serum IgE and has suppressed.

The combination of the SERUM IgE that exists in the Serum Bank is reached 50% amount that suppresses required each recombinant protein tangible difference is arranged.Reorganization Betv1 reaches 50% inhibition at about 6.5ng, and the respective concentration of Betv1 Glu45Ser mutant is about 12ng.The point mutation that this explanation imports in the Betv1Glu45Ser mutant will approximately reduce by 2 times to the affinity of specific serum IgE.

The maximum inhibition level that is reached with Betv1 Glu45Ser mutant is obviously lower than reorganization Betv1.After this showed that Glu45Ser replaces, some specific IgEs that are present in the Serum Bank can not be discerned Betv1 Glu45Ser mutant.

Betv1 mutant Asn28Thr+Lvs32Gln

The solvent that 28 and 32 s' aspartic acid and Methionin demonstrate height respectively exposes property (being respectively 35% and 50%).Structurally, aspartic acid 28 and Methionin 32 are adjacent to each other on molecular surface, and most possibly pass through interaction of hydrogen bond.Find that in some Betv1 homology PR-10 protein Threonine and glutaminic acid residue plant oneself 28 and 32 respectively, show that aspartic acid and Methionin can be replaced by Threonine and L-glutamic acid respectively and do not destroy alpha-carbon skeleton tertiary structure.In addition, because the naturally occurring allergen sequence of the same race of none has Threonine and L-glutamic acid at 28 and 32 respectively, therefore this replacement has produced natural non-existent Betv1 molecule.

The IgE binding characteristic of Betv1 mutant Asn28Thr+Lvs32Gln

Use the IgE binding characteristic that in liquid phase IgE inhibition test, compares mutant Asn28Thr+Lys32Gln and reorganization Betv1 from birch anaphylaxis patients serum IgE storehouse mentioned above.

Fig. 7 has shown by the Betv1 of abiotic plain mark with by the bonded restraining effect of Betv1 mutant Asn28Thr+Lys32Gln to biotin labeled reorganization Betv1 and anaphylaxis patients serum IgE.

The amount that the combination of the SERUM IgE that exists in the Serum Bank is reached each required recombinant protein of 50% restraining effect has tangible difference.Reorganization Betv1 reaches 50% inhibition when about 6.5ng, and the respective concentration of Betv1 mutant Asn28Thr+Lys32Gln is about 12ng.This shows that the point mutation that imports among the Betv1 mutant Asn28Thr+Lys32Gln will reduce about 2 times to the avidity of specific serum IgE.

Compare obviously lower with the maximum inhibition level that Betv1 mutant Ash28Thr+Lys32Gln mutant reaches with reorganization Betv1.After this may show that Asn28Thr+Lyr32Gln replaces, some specific IgEs that are present in the Serum Bank can not be discerned Betv1 mutant Asn28Thr+Lys32Gln.

Betv1 mutant Pro108Gly

The solvent that the proline(Pro) of position 108 demonstrates height exposes surname (60%).Find that in some Betv1 homologous PR-10 albumen glycine residue occupies 108, this shows that proline(Pro) can be replaced by glycine and do not destroy the alpha-carbon atom tertiary structure.In addition, because the naturally occurring allergen sequence of the same race of none has glycine at 108, therefore produced the Betv1 molecule that non-natural exists with the glycine substituted prolines.

The IgE binding characteristic of Betv1 mutant Pro108Gly

Use the above-mentioned IgE-binding characteristic that in liquid phase IgE-inhibition test, compares Betv1 Pro108GIy mutant and reorganization Betv1 from birch anaphylaxis patient's SERUM IgE storehouse.

Fig. 8 has shown by the Betv1 of abiotic plain mark with by the bonded restraining effect of Betv1 Pro108Gly mutant to biotin labeled reorganization Betv1 and anaphylaxis patients serum IgE.

The combination of the SERUM IgE that exists in the Serum Bank is reached 50% amount that suppresses required each recombinant protein tangible difference is arranged.Reorganization Betv1 reaches 50% inhibition when about 6.5ng, and the respective concentration of Betv1 Pro108Gly is 15ng.This shows that a single point sudden change that imports has reduced to the affinity of specific serum IgE about 2 times in Betv1 Pro108Gly.

Betv1 compares the horizontal decrease to some degree of maximum inhibition that Betv1 Pro108GIy mutant reaches with reorganization.This may show that after Pro108Gly replaced, some specific IgEs that exist in the Serum Bank can not be discerned B6t VI Pro108Gly mutant.

Betv1 mutant Glu60Ser

The L-glutamic acid of position 60 demonstrates the solvent exposure property (60%) of height.Find that in some Betv1 homologous PR-10 albumen serine residue occupies 60, this shows that L-glutamic acid can be replaced by Serine and do not destroy the alpha-carbon atom tertiary structure.In addition, because the naturally occurring allergen sequence of the same race of none has Serine at 60, therefore replace L-glutamic acid and can produce the Betv1 molecule that non-natural exists with Serine.

The IgE binding characteristic of Betv1 mutant Glu60Ser

Use the above-mentioned IgE-binding characteristic that in liquid phase IgE-inhibition test, compares Betv1 Glu60Ser mutant and reorganization Betv1 from birch anaphylaxis patient's SERUM IgE storehouse.

Fig. 9 has shown by the Betv1 of abiotic plain mark with by the bonded restraining effect of Betv1 Glu60Ser mutant to biotin labeled reorganization Betv1 and anaphylaxis patients serum IgE.With Glu45Ser, Pro108Gly is opposite with the Asn28Thr+Lys32Gln mutant, and L-glutamic acid 60 is replaced to Serine its IgE-binding characteristic is not shown any tangible influence.

Betv1 Glu45Ser, Asn28Thr+Lvs32Gln and Pro108Glv mutant Structural analysis

Analyze the proteic structural integrity of purifying with circular dichroism (CD) spectroscopy.Figure 10 has shown recombinant mutant and the proteic CD spectrum of recombinant natural that writes down near isoconcentration the time.Two recombinant protein c D spectrographic peak amplitudes and location overlap show that two goods contain the secondary structure of equivalent, and this effectively shows the influence of the aminoacid replacement that the alpha-carbon atom tertiary structure is not imported.

Betv1 Glu45Ser, Asn28Thr+Lvs32Gln and Pro108Gly mutant The IgE binding characteristic

Use the IgB binding characteristic that in liquid phase IgE inhibition test, compares mutant and reorganization Betv1 from birch anaphylaxis patient's SERUM IgE storehouse mentioned above.

Figure 11 has shown by the Betv1 of abiotic plain mark with by the bonded restraining effect of Betv1 mutant to biotin labeled reorganization Betv1 and anaphylaxis patients serum IgE.Opposite with single mutant as herein described, the inhibition curve of mutant is no longer similar to recombinant chou.This shows, compares with recombinant chou, and the replacement that imports in the mutant has changed IgE binding characteristic and epi-position situation.The shortage similarity makes and is difficult to the reduction of qualitative assessment mutant and specific serum IgE affinity.

Reorganization Betv1 reaches 50% at about 6ng to be suppressed, and the Betv1 mutant (Asn28Thr, Lys32Gln, Glu45Ser, respective concentration Pro108Gly) is 30ng, promptly affinity reduces by 5 times.Yet, suppressing in order to reach 80%, corresponding value is respectively 20ng and 400ng, promptly reduces by 20 times.

Use reorganization Betv1 Glu45Ser, Asn28Thr+Lys32Gln and Pro108Gly are prominent The T cell proliferation test of variant

By document 15 described analyses.Find that reorganization Betv1 sudden change physical efficiency is to induce T cell line proliferation from three kinds of different birch pollen hypersensitivities reaction patients with the reorganization stimulation index similar with the natural Betv1 of existence.This shows that mutant can start antibody and produce necessary cellullar immunologic response.

Embodiment 2

The vitro mutagenesis of mutant of the present invention

The reorganization pMAL-c that Betv1 has been inserted in use carries out PCR as template, realizes vitro mutagenesis.The preparation of recombination mutation anaphylactogen comprises two PCR step: step I and II.At first, use comprises the justice and the antisense sudden change specific oligonucleotide primer of each sudden change, with the justice and the antisense oligonucleotide primer that comprise the contiguous sudden change in upstream or downstream or Betv1 N-terminal/C-terminal, each is suddenlyd change separately (if the location is very tight in dna sequence dna, also can be several sudden changes) import in the continuous DNA sequence of Betv1.2801 derivative, be Betv1 (2595) or Betv1 (2628) or Betv1 (2733), respectively shown in Figure 12 (I).Secondly, the PCR product of purifying PCR reaction I mixes, and as template, carries out another PCR reaction (II) with the Oligonucleolide primers that contains Betv1 N-terminal/C-terminal, shown in Figure 13 (II).Agarose gel electrophoresis, PCR gel-purified (Life Techhnologies), this PCR product of ethanol sedimentation purifying cuts among the also directed pMAL-c that connects to advance with the same restrictions enzymic digestion with Restriction Enzyme (Sacl/EcoRI) or (Sacl/Xbal) then.

Figure 13 has shown the structure synoptic diagram of synthetic Oligonucleolide primers and Betv1 mutant.Following Betv1 mutant cloned and check order (order-checking of nucleic acid molecule is described in embodiment 1):

Betv1(3004)

GVFNvETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVsNIEGNGGPGTIK

KISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPD

GGSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn

Betv1(3005)

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDG

GSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn

Betv1(3007)

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATgDG

GSILKISNKYHTKGyHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn

Betv1(3009)

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLsKISNEIKIVATgD

GGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYNn

Betv1(3006)

GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDG

GSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYgn

Betv1(3008)

GVFNvETETTSVIPAARLFKAFILDGDtLFPkVAPQAISSVENIsGNGGPGTIKK

ISFPEGfPFKYVKDRVDsVDHTNFKYNYSVIEGGPIGDTLsKISNEIKIVATgDG

GSILKISNKYHTKGyHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYgn

Other mutant prepared in accordance with the present invention

In the Betv1 mutant, import a plurality of point mutation folding pattern of α carbon skeleton of saboteur potentially.Importing random amino acid replaces and can raising form the chance of stablizing the Betv1 mutant molecule.Therefore we have made up the Betv1 mutant library that contains the Betv1 mutant, and described mutant has 17-20 point mutation, and it is at random aminoacid replacement that 7 sites are wherein arranged.Up to a hundred different clones are contained in this library.15 Betv1 mutant that are called as Betv1 (3031)-(3045) are available from this Betv1 mutant library that uses degeneracy oligonucleotide primer to generate.These primers are at the T10 of Betv1, K20, and Q36, E73, E87, K129 and S149 site are contained the random amino acid residue and are replaced (Figure 14 and 15).These sites can't cover the point mutation that has imported among Betv1 (3002) and the Betv1 (2595), and described Betv1 (3002) and Betv1 (2595) are used to carry out rite-directed mutagenesis PCR reaction as dna profiling, as shown in figure 15.

Clone's process is same as shown in Figure 12, just in first round PCR used primer at specific site by degeneracy, G is used in these sites in Figure 15, C, the letter representation outside T or the A.G, C, this primer of the letter representation outside T or the A contain several different Nucleotide in these sites.Prepared 8 kinds of PCR products that cover the Betv1 genes, and in the first round PCR purifying, use terminal primer (3076s and 3067a) to take turns among the PCR then and assemble second, wherein use 8 kinds of products from first round PCR as template.

As to Betv1 3004,3005,3007 and 3007 mutant are described, and 3031-3045 carries out dna sequencing to the Betv1 mutant, to identify number and the character that imports point mutation:

Betv1 clones (" 3031 ") (SEQ ID NO 25):

GVFNVETETASVIPAARLFNAFILDGDTLFPQVAPQAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIEGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN

N

Betv1 clones (" 3032 ") (SEQ ID NO 26):

GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDPVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN

N

Betv1 clones (" 3033 ") (SEQ ID NO 27):

GVFNVETETPSVIPAARLFHAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN

N

Betv1 clones (" 3034 ") (SEQ ID NO 28):

GVFNVETETTSVIPAARLFHAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3035 ") (SEQ ID NO 29):

GVFNVETETPSVIPAARLFMAFILDGDTLFPQVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAYLLAHSHAYN

N

Betv1 clones (" 3036 ") (SEQ ID NO 30):

GVFNVETETPSVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDTVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3037 ") (SEQ ID NO 31):

GVFNVETETPSVIPAARLFQAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN

N

Betv1 clones (" 3038 ") (SEQ ID NO 32):

GVFNVETETASVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3039 ") (SEQ ID NO 33):

GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPEAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVA

TPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAYLLAHSHAY

NN

Betv1 clones (" 3040 ") (SEQ ID NO 34):

GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETYLLAHSHAYN

N

Betv1 clones " 3041 ") (SEQ ID NO 35):

GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3042 ") (SEQ ID NO 36):

GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN

N

Betv1 clones (" 3043 ") (SEQ ID NO 37):

GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN

N

Betv1 clones (" 3044 ") (SEQ ID NO 38):

GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVA

TPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETYLLAHSHAY

NN

Betv1 clones (" 3045 ") (SEQ ID NO 39):

GVFNVETETPSVIPAARLFMAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI

KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT

PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN

N

Embodiment 3

Identify and screen the amino acid that is used to replace

With the amino acid that solvent accessibility and conservative extent index are determined and the surface of selecting to be suitable for anaphylactogen Betv1, DerP2 and Vesv5 are replaced exposes.

The solvent accessibility

The solvent accessibility is calculated with the radius of investigation (Connolly Surface) of software I nsightII version 97.0 (MSI) and 1.4 .

From analyze, get rid of inner chamber by using software PASS (the spheroid avtive spot of inferring) to be full of with probe.Manually remove the probe on surface subsequently.

Conservative property

Betv1

The 3-D structure is based on accession number Z80104 (1bv1.pdb).

Be included in the conserved residues analysis 38 other Betv1 sequences and comprise accession number:

P15494＝X15877＝Z80106，Z80101，AJ002107，Z72429，AJ002108，Z80105，Z80100，Z801O3，AJ001555，Z80102，AJ002110，Z72436，P43183＝X77271，Z72430，AJ002106，P43178＝X77267，P43179＝X77268，P43177＝X77266，Z72438，P43180＝X77269，AJ001551，P43185＝X77273，AJ001557，Z72434，AJ001556，Z72433＝P43186，AJ001554，X81972，Z72431，P45431＝X77200，P43184＝X77272，P43176＝X77265，S47250，S47251，Z72435，Z72439，Z72437，S47249.

Betv1

59 amino acid that the height solvent exposes:

K-129，E-60，N-47，K-65，P-108，N-159，D-93，K-123，K-32，D-125，R-145，D-109，T-77，E-127，Q-36，E-131，L-152，E-6，E-96，D-156，P-63，H-76，E-8，K-134，E-45，T-10，V-12，K-20，L-62，S-155，H-126，P-50，N-78，K-119，V-2，L-24，E-42，N-4，A-153，I-44，E-138，G-61，A-130，R-70，N-28，P-35，S-149，K-103，Y-150，H-154，N-43，A-106，K-115，P-14，Y-5，K-137，E-141，E-87，E-73.

57 height solvents expose and guard the amino acid of (＞70%):

K-129，E-60，N-47，K-65，P-108，N-159，D-93，K-123，K-32，D-125，R-145，D-109，E-127，Q-36，E-131，L-152，E-6，E-96，D-156，P-63，H-76，E-8，K-134，E-45，T-10，V-12，K-20，S-155，H-126，P-50，N-78，K-119，V-2，L-24，E-42，N-4，A-153，1-44，E-138，G-61，A-130，R-70，N-28，P-35，S-149，K-103，Y-150，H-154，N-43，A-106，K-115，P-14，Y-5，K-137，E-141，E-87，E-73.

Table 1 is the tabulation that shows Betv1 amino acid solvent degree of exposure with descending.The 1st has listed and has originated in aminoterminal amino acid number, and the 2nd row have been listed amino acid with the single-letter abbreviation, and the 3rd lists standardized solvent exposure index, and the 4th lists known array has conserved amino acid in this position per-cent number.

Table 1:Betv1

NO AA Solv_ex?Cons％

p

129 K 1,000 90

60 E 0,986 97

47 N 0,979 100

65 K 0,978 100.

108 P 0,929 100

159 N 0,869 100

93 D 0,866 100

123 K 0,855 100

32 K 0,855 100

125 D 0,821 74

145 R 0,801 90

109 D 0,778 82

77 T 0,775 56

127 E 0,760 100

36 Q 0,749 95

131 E 0,725 100

152 L 0,718 97

6 E 0,712 100

96 E 0,696 100

156 D 0,693 97

63 P 0,692 97

76 H 0,683 90

8 E 0,638 97

134 K 0,630 100

45 E 0,623 100

10 T 0,613 97

12 V 0,592 100

20 K 0,584 100

62 L 0,575 5

155 S 0,568 97

126 H 0,551 95

50 P 0,541 100

78 N 0,538 100

119 K 0,529 100

2 V 0,528 100

24 L 0,528 100

42 E 0,519 100

4 N 0,517 95

153 A 0,513 100

44 1 0,508 97

138 E 0,496 100

61 G 0,488 100

130 A 0,479 97

70 R 0,474 100

28 N 0,469 90

35 P 0,467 100

149 S 0,455 92

103 K 0,447 100

150 Y 0,438 100

154 H 0,436 100

43 N 0,412 100

106 A 0,411 95

115 K 0,411 100

14 P 0,410 97

5 Y 0,410 100

137 K 0,396 100

141 E 0,387 95

87 E 0,385 100

73 E 0,384 100

16 A 0,367 100

79 F 0,362 100

3 F 0,355 100

158 Y 0,346 100

105 V 0,336 100

101 E 0,326 100

64 F 0,325 100

86 I 0,322 100

39 S 0,314 100

124 G 0,310 100

72 D 0,308 97

142 T 0,293 67

66 Y 0,289 100

55 K 0,288 100

7 T 0,279 67

40 S 0,274 95

25 D 0,271 87

135 A 0,267 92

68 K 0,262 100

97 K 0,247 100

46 G 0,235 100

27 D 0,232 97

1 G 0,227 100

113 I 0,225 77

51 G 0,220 100

92 G 0,218 100

80 K 0,212 100

110 G 0,211 100

107 T 0,203 85

94 T 0,202 92

41 V 0,201 97

48 G 0,198 100

91 I 0,192 18

31 P 0,188 100

75 D 0,188 97

33 V 0,183 100

49 G 0,176 100

17 R 0,172 100

99 S 0,158 64

89 G 0,154 100

53 I 0,154 100

121 H 0,153 100

9 T 0,150 72

74 V 0,148 97

132 Q 0,146 72

57 S 0,137 49

148 E 0,135 100

82 N 0,133 41

128 V 0,125 64

117 S 0,124 87

90 P 0,117 67

116 I 0,112 100

122 T 0,107 100

139 M 0,104 62

95 L 0,104 97

54 K 0,096 100

146 A 0,095 100

59 P 0,088 97

157 A 0,088 100

133 V 0,077 44

88 G 0,068 100

140 G 0,053 85

37 A 0,042 95

81 Y 0,041 100

23 I 0,036 95

104 I 0,036 92

15 A 0,036 97

58 F 0,029 100

29 L 0,028 100

19 F 0,027 100

100 N 0,022 97

22 F 0,021 97

71 V 0,014 100

111 G 0,014 100

13 I 0,014 100

18 L 0,014 97

114 L 0,014 100

11 S 0,007 100

151 L 0,007 97

144 L 0,007 90

52 T 0,007 100

84 S 0,007 97

118 N 0,007 97

102 I 0,007 100

21 A 0,000 97

26 G 0,000 97

30 F 0,000 44

34 A 0,000 100

38 I 0,000 87

56 I 0,000 100

67 V 0,000 97

69 D 0,000 62

83 Y 0,000 95

85 V 0,000 72

98 I 0,000 95

112 S 0,000 77

120 Y 0,000 95

136 S 0,000 67

143 L 0,000 100

147 V 0,000 100

Embodiment 4

This embodiment describes the preparation and the feature of reorganization Betv1 allergen mutants, and these mutant have more than 4 sudden changes, and have the IgE binding affinity of comparing reduction with prior art PCT/DK 01/00764.Mutant of the present invention is by corresponding preparation and test.

Be used for the selection of the amino-acid residue of Betv1 rite-directed mutagenesis

Press embodiment 1 described selection amino-acid residue.

Vitro mutagenesis

Vitro mutagenesis is undertaken by PCR as template with the reorganization pMAL-c that has inserted Betv1.The preparation that comprises the recombinant allergens mutant of 5～9 one-level sudden changes comprises two PCR step: step I and II.At first, use comprises the justice and the antisense sudden change specific oligonucleotide primer of each sudden change, with the justice and the antisense oligonucleotide primer that comprise the contiguous sudden change in upstream or downstream or Betv1 N-terminal/C-terminal, each is suddenlyd change separately (if the location is very tight in dna sequence dna, also can be several sudden changes) import in the continuous DNA sequence of Betv1.2801 or Betv1.2801 derivative, respectively shown in Figure 15 (I).Secondly, the PCR product of purifying PCR reaction I mixes, and as template, uses the Oligonucleolide primers of the N-terminal/C-terminal that contains Betv1 to carry out another PCR reaction (II) together, shown in Figure 15 (II).Agarose gel electrophoresis, PCR gel-purified (Life Techhnologies), this PCR product of ethanol sedimentation purifying cuts among the also directed pMAL-c that connects to advance with the same restrictions enzymic digestion with Restriction Enzyme (Sac1/EcoRI) or (Sac1/Xba1) then.

Figure 16 represents the synthetic Oligonucleolide primers and has the structure synoptic diagram of the Betv1 mutant that suddenlys change greater than 4 one-levels.The amino acid of sudden change preferably is selected from and is characterised in that the height solvent exposes and the amino acid of conservative property as having as described in the embodiment 3.The Betv1 mutant is as follows:

Mutant Betv1 (2628): Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys1 34GIu.

Mutant Betv1 (2637): Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr, Pro108Gly, Leu152Lys, Ala153Gly, Ser155Pro.

Mutant Betv1 (2733): Tyr5Val, Lys134Glu, Asn28Thr.Lys32Gln，Glu45Ser，Lys65Asn，Asn78Lys，Lys103Val，Lys97Ser，Pro108Gly，Arg145Glu，Asp156His，+160Asn。

Mutant Betv1 (2744): Tyr5Val, Lys134Glu, Glu42Ser, G1u45Ser, Asn78Lys, Lys103Val, Lys123Ile, Asp156His ,+160Asn.

Mutant Betv1 (2753): Asn28Thr, Lys32Gln, Lys65Asn, Glu96Leu, Lys97Ser, Pro108Gly, Asp109Asn, Asp125Tyr, Glu127Ser, Arg145Glu.

Nucleotide sequencing and the expression and the purifying of reorganization Betv1 and mutant

The order-checking of recombinant protein and expression are carried out as described in embodiment 1.

Betv1 (2628) and Betv1 (2637) mutant

Figure 17 has shown the point mutation in Betv1 (2628) and the importing of Betv1 (2637) molecular surface.

The crystallization and the structure of reorganization Betv1 (2628) mutain are determined.

Structure is determined to carry out as described in embodiment 1.

The structure of Betv1 (2628) mutant

The structure function of sudden change is resolved by growing three-dimensional Betv1 (2628) protein crystal, and this crystalline diffraction is the resolving power of 2.0 when the X-ray that generates with rotating anode is analyzed.The structure electron density map that Tyr5Val, GIn45Ser, Lys65Asn, Lys97Ser, Lys134Glu replace by Betv1 (2628) confirms, this figure shows that also total alpha-carbon skeleton tertiary structure guards.

The structural analysis of Betv1 (2637) mutant

The structural integrity of the Betv1 of purifying (2637) mutant circular dichroism (CD) spectrum analysis.Figure 18 shows the CD spectrum with reorganization Betv1.2801 (wild-type) that is close to identical concentration records and Betv1 (2637) mutant.Crossover from acrometron and position in the CD spectrum of 2 recombinant proteins shows that these 2 kinds of prepared products contain the secondary structure of same amount, and this hints that forcefully alpha-carbon skeleton tertiary structure is not subjected to amino acid and replaces the influence of introducing.

The IgE binding characteristic of Betv1 (2628) and Betv1 (2637) mutant

Relatively 1: 1 mixture of Betv1 (2628) and Betv1 (2637) and Betv1 (2628) and Betv1 (2637) combines character with the IgE of reorganization wild-type Betv1.2801 in liquid phase IgE suppress to measure, and this mensuration use is derived from birch anaphylaxis patient's SERUM IgE storehouse.

As described in the embodiment 1, reorganization Betv1.2801 is to carry out biotinylation at 1: 5 (Betv1 the 2801st: vitamin H) with mol ratio.Suppress to measure following carrying out: with serum sample (25 μ l) and the anti-IgE incubation of solid phase, washing, resuspended and further with biotinylated Betv1.2801 and given mutant or 1: 1 mixture incubation of two kinds of mutant.Be incubated the RLU that measures the back according to streptavidin and estimate the amount that is attached to the biotin labeled Betv1.2801 on the solid phase with the acridinium ester mark.Calculate the degree that suppresses with the ratio that uses damping fluid and mutant to do between the RLU of inhibitor acquisition.

Figure 19 represent by the Betv1.2801 of abiotic elementization and by 1: 1 mixture of Betv1 (2628), Betv1 (2637) and Betv1 (2628) and Betv1 (2637) to biotinylated reorganization Betv1.2801 and bonded restraining effect from the SERUM IgE in anaphylaxis patient storehouse.

The combination of the SERUM IgE that exists in the Serum Bank is reached 50% amount that suppresses required each recombinant protein tangible difference is arranged.Reorganization Betv1.2801 reaches 50% inhibition at about 5ng, and the respective concentration of Betv1 (2628) mutant is about 15-20ng.The point mutation that this explanation imports in Betv1 (2628) mutant will approximately reduce 3-4 to the affinity of specific serum IgE doubly.

The maximum inhibition level that is reached with Betv1 (2628) mutant is obviously lower than reorganization Betv1.2801.After this showed the importing point mutation, some specific IgEs that are present in the Serum Bank can not be discerned Betv1 (2628) mutant.

Betv1 (2637) reaches 50% inhibition at about 400-500ng, is presented at the point mutation of introducing in Betv1 (2637) mutant and it is compared with Betv1.2801 the avidity to special SERUM IgE has been reduced 80-100 doubly.The remarkable inclination angle difference of gained was supported after greatest differences in the IgB combination was further compared by Betv1.2801 inhibition curve and Betv1 (2637) mutein inhibition curve.Different inclination angles produces evidence to show that the IgE bonded reduces owing to the distinct epi-position pattern of this mutant (comparing with Betv1.2801).

Except that suppressing to measure with the anaphylactogen of modifying separately, to testing with the Betv1 (2628) of the Betv1 of volumetric molar concentrations such as the sample of Betv1 (2628) or Betv1 (2637) has and 1: 1 mixture of Bet11 (2637) respectively, their show can thoroughly suppress the combination (100%) of rBetv1.2801 to IgE.Thoroughly suppress IgE bonded ability and clearly show that all are present in the anaphylactogen mixture that active epi-position on the Betv1.2801 also is present in 1: 1.Further proof is from Betv1.2801 and two kinds of suitable inclination angles that suppress curve of anaphylactogen mixture.The IgE reactivity that blended anaphylactogen sample reduces is by the anaphylactogen mixture of high 4 times of concentration proves need be than Betv1.2801 the time in conjunction with suppressing in order to obtain 50% IgE.

Reorganization Betv1 anaphylactogen with sudden change carries out the T cell proliferating determining

This analysis is carried out as method as described in the reference 15.Both all can induce propagation from the T clone among the birch pollen hypersensitivity patient Betv1 (2628) and Betv1 (2637), and its stimulation index is similar with naturally occurring situation to recombinant chou.This shows that Betv1 (2628) and Betv1 (2637) mutein all can produce necessary cell immune response by initial antagonist.

The histamine release of people's basophilic granulocyte is measured

The basophil histamine release is following carries out.Blood (20ml) from each birch pollen hypersensitivity patient taking heparinization is stored in room temperature, and uses in 24 hours.With the whole blood of 25 microlitre heparinizations place microtiter well (ReferenceLaboratory, Copenhagen, Denmark) that glass fibre covers and with the anaphylactogen of 25 microlitres or anti-IgE in 37 ℃ of incubations 1 hour.Rinsing is dull and stereotyped and remove interfering substance then.At last, the histamine that is attached on the primitive fiber carries out the fluorescence spectrophotometry measurement.

The histamine release of Betv1 (2628) and Betv1 (2637) mutein Matter

The data of histamine release show in Figure 20 and Figure 21.Measured Betv1 (2628) and Betv1 (2637) mutein and in people's basophilic granulocyte, induced the potential of histamine release from two birch pollen hypersensitivity patients.Compare with the release profiles of Betv1.2801, the release profiles of two kinds of sudden change anaphylactogens all moves to right significantly.Should move and show that the potential of Betv1 (2628) and Betv1 (2637) had reduced 3～10 times.

Mutant Betv1 (2744) and mutant Betv1 (2753)

Betv1 (2744) and Betv1 (2753) mutant have been made up equally as mixing the anaphylactogen vaccine.Shown in Figure 22 and 23, in these sudden change anaphylactogens, point mutation is adjusted and is distributed in whole surface, and also is designed to influence different surfaces respectively in two molecules, as shown in figure 24.Yet the anaphylactogen of these modifications also can be used alone as single anaphylactogen vaccine.

The structural analysis of Betv1 (2744) mutant protein

The structural integrity of the Betv1 of purifying (2744) mutant circular dichroism (CD) spectrum analysis.Figure 25 shows the CD spectrum with reorganization Betv1.2801 (wild-type) that is close to identical concentration records and Betv1 (2744) mutant.Crossover from acrometron and position in the CD spectrum of 2 recombinant proteins shows that 2 kinds of prepared products contain the secondary structure of same amount, and this shows the influence of the aminoacid replacement that alpha-carbon skeleton tertiary structure is not imported forcefully.

The histamine release character of Betv1 (2744)

The histamine release data that get from 5 experiments from 5 kinds of different pollen hypersensitivity patients' basophil show in Figure 26 and Figure 27 A-D.Measure Betv1 (2744) mutein and in people's basophilic granulocyte, induced the potential of histamine release.Compare with the release profiles of Betv1.2801, the release profiles of sudden change anaphylactogen moves to right significantly.Should move and show that the potential of Betv1 (2744) had reduced 3～5 times.

Mutant Betv1 (2733)

Made up also recombinant expressed mutant Betv1 (2733).The distribution of point mutation has kept several＞400 in Betv1 (2733) mutant ²Surf zone be not changed.Figure 28 is illustrated in the point mutation that Betv1 (2733) molecular surface is introduced.

Embodiment 5

This embodiment describes the feature of reorganization Betv1 allergen mutants, and these mutant have more than 4 sudden changes, and have the IgE binding affinity of comparing reduction with prior art PCT/DK 01/00764.Mutant of the present invention is by corresponding preparation and test.

The t cell responses of reorganization and sudden change Betv1

Purpose

External t cell responses by propagation and cytokine production research sudden change anaphylactogen.

Method:

In following research, use PBL (peripheral blood lymphocyte) from the allergy patient.

In order to keep the kind of the betv1 isotype that the T-cell presented, 8 betv1 specificity T cell line/lineages have been set up with the betv1 of natural purifying from PBL, as (26) as described in the previously disclosed method.

Betv1 (nBetv1), reorganization Betv1 (rBetv1 or wt with birch extract (Betv), natural purifying; 27) and 4 kinds of different mutants (described elsewhere) of rBetv1: 2595,2628,2637,2744,2773 stimulate 10 PBL and 8 T-clones.Find that subsequently 2637 mutant partly separate foldingly, will not discuss.

Simply say: every hole adds 2 * 10 on one 96 hole circle base plate ⁵PBL.Add different birch samples and make its growth 6 days so that 3 kinds of different concns are quadruplicate.At the 6th day, results had half volume (the 100 μ l) cell in each hole of maximum concentration birch, to be used for the production of cytokine.Radiolabeled thymidine is joined in the hole.Second day (the 7th day) harvested cell in filter.Scintillation solution is joined in the filter, and its radioactivity is measured with scintillometer.

Similarly, on one 96 hole circle base plate, every hole adds 3 * 10 ⁴The T-cell, and with self radiating PBL (1 * 10 ⁵Cells/well) stimulates with the different birch samples of 3 kinds of different concns.After 1 day, gather in the crops from each porocyte to be used for the production of cytokine with maximum concentration birch.Radiolabeled thymidine is joined in the hole.At the 2nd day harvested cell and as described in to PBL, counting in filter.

Collect from four parts supernatant, and use CBA (cytokine pearl array (cytokine bead array)) test kit to measure cytokine from Becton Dickinson.

The result:

10 PBL cultures demonstrate the differential stimulus to birch.Usually, more similar to the PBL breeding ratio of different birch samples, although can see variation.In 3 PBL, nBetv1 can better stimulate proliferation than rBetv1 and mutant.Sudden change birch sample and rBetv1 stimulate PBL (Figure 29) about the samely.Figure 29 represents the stimulation index of above-mentioned Betv1 prepared product.Stimulation index (SI) is by the propagation of stimulated samples (cpm: the reading of per minute) calculate divided by the propagation (cpm) of substratum (medium) contrast.PPD refers to that it serves as positive control from the protein purification derivative of mycobacterium tuberculosis (mucobacterium tuberculosis).

The output of cytokine is subjected to the control of IFN-γ and increases in proportion with the propagation of PBL.The signal not obvious (Figure 30-32) that Th1/Th2 replaces.Figure 30 represents to have the patient of Th0 feature, and Figure 31 represents the Th1 feature and Figure 32 represents the Th2 feature.Cytokine production is measured (shown in bar graph) with pg/ml, and the ratio between the IL-5/IFN-γ is the dotted line (Y-axis on right side) by the below.Measure propagation with cpm, shown on the Y-axis of right side, be expressed as solid line.Contrast as a setting with substratum (medium) and MBP (maltose binding protein matter).

On nBetv1, set up 8 T-clones, bar one, remaining all has the propagation of equal good degree to all birch samples.4 T clone is based on the ratio of IL-5 and IFN-γ (Th2＞5,5＞Th0＞0.2,0.2＞Th1) and the secretion Th0 like cell factor.3 T clone secretion Th1 cytokines, 1 T clone secretion Th2 cytokine.The ratio of IL-5/IFN-γ is not subjected to the influence of different birch samples.

Conclusion:

All PBL cultures and 7/8 T clone of nBetv1 being shown differential stimulus also react to rBetv1 and mutant.These data representations are for the stimulation of T cell, the mixture of the isotype of single Betv1 or the replaceable independent isotype of finding in natural anaphylactogen prepared product of these 4 mutant.Thereby, will be based on the vaccine of recombinant allergens or these 4 mutant at existing Betv1 specific T-cells colony.

Embodiment 6

This embodiment describes the feature of reorganization Betv1 allergen mutants, and these mutant have more than 4 sudden changes, and have the IgE binding affinity of comparing reduction with prior art PCT/DK 01/00764.Mutant of the present invention is by corresponding preparation and test.

After with reorganization and mutant Betv1 protein immunization, induce Betv1 special Property IgG antibody and blocking antibody

In this part, term " blocking antibody " is defined as and the different antibody of people IgE antibody, and it can conjugated antigen and stops people IgE antibody to this antigenic combination.

Check reorganization Betv1 2227 wild-type proteins (rBetv1) and Betv1 2595,2628,2744 and 2773 muteins are induced the ability of Betv1 specific IgG antibodies and blocking antibody in the mouse immune inoculation experiments.

Carry out immunization with reorganization Betv1 2227 wild-type proteins or 4 mutant protein confrontation BALB/cA mouse (8 every group) peritoneal injection.This mouse is with 14 days spacing of doses immunization 4 times.The Alhydrogel that different protein is connected to 1.25mg/ml go up (aluminum hydroxide gel, 1,3%pH 8.0-8.4, SuperfosBiosector).This mouse is with 1 μ g protein/dose or 10 μ g protein/dose immunizations.Blood sample is the 0th, 14, obtains from the eye socket blood sampling in 35,21,49 and 63 days.

Antibody is surveyed in microtiter plate and the anti-mouse lgG of biotinylation rabbit antibody (Jackson) conduct with rBetv1 bag quilt, by direct elisa assay specific IgG antibodies level.The immunization of reorganization Betv1 2227 wild-type proteins or 4 kinds of muteins has been induced very strong rBetv1 specific IgG reaction.The protein of 4 kinds of sudden changes of this discovery proof can be induced the antibody with Betv1 2227 wild-type protein height cross reactions.

In order to estimate inducing of blocking antibody, will be from birch allergy patient's serum sample and bag by the paramagnetic beads incubation of mono-clonal mouse anti human IgE antibody.Behind the incubation, with pearl washing and be resuspended in damping fluid or dilute sample (1: 100) from the mice serum of immunized mice (contrast) or above-mentioned immunized mice not in.Biotinylated rBetv1 is joined in the mixture of this pearl and mice serum antibody then.After the incubation, the washing pearl is surveyed its bonded biotinylation rBetv1 with the strepto-microbiotic of acridine (acridinium) mark.Pearl and do not change combining of rBetv1 and pearl from the incubation of immunization mice serum not.Opposite, pearl and the incubation of the mice serum that comes personal reorganization Betv1 1227 wild-type proteins or 4 kinds of mutein immunizations have reduced combining of Betv1 and pearl significantly, and this has proved and have Betv1 specific inhibition antibody in serum sample.As a result, at the 63rd day, can reduce 80% or more with combining of pearl Betv1 from one or more serum samples of all high dosages (10 μ g/ dosage) immunization group.These protein of finding 4 kinds of sudden changes of proof can be induced the antibody that can serve as Betv1 specific inhibition antibody.

Embodiment 7

This embodiment describes the constitutional features and the IgE binding characteristic of the mutant of the present invention that heavily has 12 sudden changes.The sudden change that imports in mutant 3007 is described in embodiment 2.

The structural analysis of Betv1 (3007) mutant protein

As described in embodiment 1, the structural integrity of the Betv1 of purifying (3007) mutant circular dichroism (CD) spectrum analysis.Figure 33 shows with the reorganization Betv1.2801 (wild-type) that is close to identical concentration records and the CD spectrum of Betv1 (3007) mutant.Crossover from acrometron and position in the CD spectrum of 2 recombinant proteins shows that these 2 kinds of prepared products contain the secondary structure of same amount, and this shows that forcefully alpha-carbon skeleton tertiary structure is not subjected to amino acid and replaces the influence of introducing.

The IgE binding characteristic of Betv1 (3007) mutain

Figure 34 represents by the Betv1.2801 of abiotic elementization (wild-type) and Betv1 (3007) according to embodiment 4 described methods biotinylation reorganization Betv1.2801 and bonded restraining effect from the SERUM IgE in anaphylaxis patient storehouse.The combination of the SERUM IgE that exists in the Serum Bank is reached 50% amount that suppresses required each recombinant protein tangible difference is arranged.Reorganization Betv1.2801 reaches 50% inhibition at about 5ng, and the respective concentration of Betv1 (3007) mutant is about 200ng.The inhibition level that Betv1 (3007) mutant reaches is starkly lower than reorganization Betv1.2801.12 point mutation that this explanation imports in Betv1 (3007) mutant have reduced the affinity to specific serum IgE.

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Claims (61)

1. reorganization Betv1 anaphylactogen is characterized in that it is the mutant of natural B etv1 anaphylactogen, wherein:

A. this mutant keeps and the essentially identical alpha-carbon skeleton structure of described natural anaphylactogen,

B. this sudden change contains at least 4 one-level sudden changes, compares with the IgE binding ability of natural B etv1 anaphylactogen, and every kind of sudden change can both reduce the IgE specific binding capacity of sudden change anaphylactogen,

C. every kind of one-level sudden change all is that the amino-acid residue that a kind of surface exposes is replaced by another kind,

D. the modes of emplacement in described mutational site makes: at least one 400-800 ²Area in do not contain sudden change or only contain one or more gentle sudden changes,

E. described one-level sudden change is selected from least 4 groups in following 10 groups, every group of amino acid sites that all contains the surface exposure that is suitable for aminoacid replacement:

Group 1:A130, E131, K134, A135, K137, E138, E141, T142, R145;

Group 2:V2, F3, N4, Y5, E6, T7, K119;

Group 3:D27, S39, S40, Y41, E42, N43, I44, E45, G46, N47, P50, G51, K55, D72, E73;

Group 4:E8, T10, V12, P14, V105, A106, T107, P108, D109, G110, I113, K115;

Group 5:A16, K20, S149, Y150, L152, A153, H154, S155, D156, Y158, N159 ,+160, wherein+160 represent the increase of-terminal amino acid;

Group 6:L24, D25, N28, K32;

Thin 7:H76, T77, N78, F79, K80, E101, K103;

Group 8:K68, R70, I86, E87, E96, K97;

Group 9:G1, G92, D93, T94, K123, G124, D125, H126, E127, K129;

Group 10:P35, Q36, E60, G61, P63, F64, K65, Y66;

Its precondition is: reorganization Betv1 anaphylactogen is not following specified mutant: (Asn28Thr, Lys32Gln, Asn78Lys, Lys103Val, Arg145Glu, Asp156His ,+160Asn); (Tyr5Val, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123Ile, Lys134Glu, Asp156His); (Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu); (Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr, Pro108Gly, Leu152Lys, Ala153Gly, Ser55Pro); (N28T, K32Q, N78K, K103V, P108G, R145E, D156H ,+160N); (Tyr5Val, Lys134Glu, Asn28Thr, Lys32Gln, Glu45Ser, Lys65Asn, Asn78Lys, Lys103Val, Lys97Ser, Pro108Gly, Arg145Glu, Asp156His ,+160Asn); (Tyr5Val, Lys134Glu, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123Ile, Asp156His ,+160Asn); (Asn28Thr, Lys32Gln, Lys65Asn, Glu96Leu, Lys97Ser, Pro108Gly, Asp109Asn, Asp125Tyr, Glu127Ser, Arg145Glu); (Y5V, N28T, K32Q, E42S, E45S, N78K, K103V, P108G, K123I, K134E, D156H ,+160N); (Y5V, E42S, E45S, K65N, N78K, K97S, K103V, K123I, K134E, D156H ,+160N); (Y5V, N28T, K32Q, E42S, E45S, K65N, N78K, K97S, K103V, P108G, K123I, K134E, D156H ,+160N).

2. the reorganization Betv1 anaphylactogen of claim 1, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, and every group all contains the amino acid sites that the surface that is suitable for aminoacid replacement exposes:

Group 1:A130, K134, A135, K137, E138, E141, T142, R145;

Group 2:V2, F3, N4, Y5, E6, T7, K119;

Group 3:D27, Y41, E42, N43, I44, E45, G46, N47, P50, G51, K55, D72, E73;

Group 4:E8, T10, P108, D109, I113, K115;

Group 5:H154, S155, D156, N159 ,+160;

Group 6:D25, N28, K32;

Group 7:H76, T77, N78, K80, E101, K103;

Group 8:K68, R70, I86, E87, E96, K97;

Group 9:G1, G92, T94, K123, G124, D125, H126;

Group 10:K65, Y66.

3. claim 1 or 2 reorganization Betv1 anaphylactogen, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, and every group all contains the amino acid sites that the surface that is suitable for aminoacid replacement exposes:

Group 1:A130, K134, A135, K137, E138, E141, T142;

Group 2:V2, F3, N4, Y5, E6, T7, K119;

Group 3:D27, Y41, N43, I44, E45, G46, N47, P50, G51, K55, D72, E73;

Group 4:E8, P108, I113, K115;

Group 5:H154, S155, N159 ,+160;

Group 6:D25, N28;

Group 7:H76, N78, K80, E101, K103;

Group 8:K68, R70, I86, E87, E96, K97;

Group 9:G1, G92, T94, G124, D125, H126;

Group 10:Y66.

4. each reorganization Betv1 anaphylactogen of claim 1-3, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, and every group all contains the amino acid sites that the surface that is suitable for aminoacid replacement exposes:

Group 1:A130, A135, K137, E138, E141, T142;

Group 2:F3, N4, E6, T7, K119;

Group 3:D27, Y41, N43, I44, G46, P50, G51, D72, E73;

Group 4:E8, I113, K115;

Group 5:H154, S155, N159;

Group 7:H76, N78, K80, E101;

Group 8:K68, R70, I86, E87;

Group 9:G1, G92, D93, G124, H126;

Group 10:Y66.

5. the reorganization Betv1 anaphylactogen of claim 1, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, and every group all contains and is suitable for the amino acid sites that surface that following specific amino acids replaces exposes:

Group 1:A130:A130V, A130G, A130I, A130L, A130S, A130H, A130T; E131:E131D, E131H, E131K, E131R, E131S; K134:K134R, K134H, K134S, K134Q, K134I, K134E; A135:A135V, A135G, A135I, A135L, A135S, A135H, A135T; K137:K137R, K137H, K137S, K137Q, K137I, K137E; E138:E138D, E138H, E138K, E138R, E138S, E138N; E141:E141D, E141H, E141K, E141R, E141S; T142:T142A, T142S, T142L, T142V, T142D, T142K, T142N; R145:R145K, R145H, R145T, R145D, R145E;

Group 2:V2:V2A, V2I, V2K, V2L, V2R, V2T; F3:F3H, F3W, F3S, F3D; N4:N4H, N4K, N4M, N4Q, N4R; Y5:Y5D, Y5G, Y5H, Y5I, Y5K, Y5V; E6:E6D, E6H, E6K, E6R, E6S; T7:T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119:K119R, K119H, K119S, K119Q, K119I, K119E, K119N;

Group 3:D27:D27E, D27H, D27K, D27R, D27S; S39:S39T, S39L, S39V, S39D, S39K; S40:S40T, S40L, S40V, S40D, S40K; Y41:Y41D, Y41G, Y41H, Y41I, Y41K, Y41V; E42:E42S, E42D, E42H, E42K, E42R; N43:N43H, N43K, N43M, N43Q, N43R; I44:I44L, I44K, I44R, I44D; E45:E45S, E45D, E45H, E45K, E45R; G46:G46N, G46H, G46K, G46M, G46Q, G46R; N47:N47H, N47K, N47M, N47Q, N47R; P50:P50G; G51:G51N, G51H, G51K, G51M, G51Q, G51R; K55:K55R, K55H, K55S, K55Q, K55I, K55E, K55N; D72:D72E, D72S, D72H, D72R, D72K; E73:E73D, E73S, E73H, E73R, E73K;

Group 4:E8:E8D, E8H, E8K, E8R, E8S; T10:T10P, T10S, T10L, T10V, T10D, T10K, T10N; V12:V12A, V12I, V12K, V12L, V12R, V12T; P14:P14G; V105:V105A, V105I, V105K, V105L, V105R, V105T; A106:A106V, A106G, A106I, A106L, A106S, A106H, A106T; T107:T107A, T107S, T107L, T107V, T107D, T107K, T107N; P108:P108G; D109:D109N D109E, D109S, D109H, D109R, D109K; G110:G110N, G110H, G110K, G110M, G110Q, G11OR; I113:I113L, I113K, I113R, I113D, K115:K115R, K115H, K115S, K115Q, K115I, K115E, K115N;

Group 5:A16:A16V, A16G, A16I, A16L, A16S, A16H, A16T; K20:K20R, K20H, K20S, K20Q, K20I, K20E, K20N; S149:S149T, S149L, S149V, S149D, S149K; Y150:Y150T, Y150L, Y150V, Y150D, Y150K; L152:L152A, L152V, L152G, L152I, L152S, L152H, L152T; A153:A153V, A153G, A153I, A153L, A153S, A153H, A153T; H154:H154W, H154F, H154S, H154D; S155:S155T, S155L, S155V, S155D, S155K; D156:D156H, D156E, D156S, D156R, D156K; Y158:Y158D, Y158G, Y158H, Y158I, Y158K, Y158V; N159:N159H, N159K, N159M, N159Q, N159R, N159G ,+160N;

Group 6:L24:L24A, L24V, L24G, L24I, L24S, L24H, L24T; D25:D25E, D25H, D25K, D25R, D25S; N28:N28H, N28K, N28M, N28Q, N28R, N28T; K32:K32Q, K32R, K32N, K32H, K32S, K32I, K32E;

Group 7:H76:H76W, H76F, H76S, H76D; T77:T77A, T77S, T77L, T77V, T77D, T77K, T77N; N78:N78H, N78K, N78M, N78Q, N78R; F79:F79H, F79W, F79S, F79D; K80:K80R, K80H, K80S, K80Q, K80I, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S; K103:K103R, K103H, K103S, K103Q, K103I, K103E, K103V;

Group 8:K68:K68R, K68H, K68S, K68Q, K68I, K68E, K68N; R70:R70K, R70H, R70T, R70D, R70E, R70N; I86:I86L, I86K, I86R, I86D; E87:E87D, E87H, E87K, E87R, E87S, E87A; E96:E96D, E96H, E96K, E96R, E96S, E96L; K97:K97R, K97H, K97S, K97Q, K97I, K97E;

Group 9:G1:G1N, G1H, G1K, G1M, G1Q, G1R; G92:G92N, G92H, G92K, G92M, G92Q, G92R; D93:D93N, D93E, D93S, D93H, D93R, D93K; T94:T94A, T94S, T94L, T94V, T94D, T94K, T94N; K123:K123R, K123H, K123S, K123Q, K123I, K123E; G124:G124N, G124H, G124K, G124M, G124Q, G124R; D125:D125E, D125H, D125K, D125R, D125S, D125Y; H126:H126W, H126F, H126S, H126D; E127:E127D, E127H, E127K, E127R, E127S; K129:K129R, K129H, K129S, K129Q, K129I, K129E, K129N;

Group 10:P35:P35G; Q36:Q36K, Q36R, Q36N, Q36H, Q36S, Q36I, Q36E; E60:E60H, E60K, E60M, E60Q, E60R; G61:G61N, G61H, G61K, G61M, G61Q, G61R; P63:P63G; F64:F64H, F64W, F64S, F64D; K65:K65R, K65H, K65S, K65Q, K65I, K65E, K65N; Y66:Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.

6. claim 1 or 2 reorganization Betv1 anaphylactogen, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, and every group all contains and is suitable for the amino acid sites that surface that following specific amino acids replaces exposes:

Group 1:A130:A130V, A130G, A130I, A130L, A130S, A130H, A130T; K134:K134R, K134H, K134S, K134Q, K134I, K134E; A135:A135V, A135G, A135I, A135L, A135S, A135H, A135T; K137:K137R, K137H, K137S, K137Q, K137I, K137E; E138:E138D, E138H, E138K, E138R, E138S, E138N; E141:E141D, E141H, E141K, E141R, E141S; T142:T142A, T142S, T142L, T142V, T142D, T142K, T142N; R145:R145K, R145H, R145T, R145D, R145E;

Group 2:V2:V2A, V2I, V2K, V2L, V2R, V2T; F3:F3H, F3W, F3S, F3D; N4:N4H, N4K, N4M, N4Q, N4R; Y5:Y5D, Y5G, Y5H, Y5I, Y5K, Y5V; E6:E6D, E6H, E6K, E6R, E6S; T7:T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119:K119R, K119H, K119S, K119Q, K119I, K119E, K119N;

Group 3:D27:D27E, D27H, D27K, D27R, D27S; Y41:Y41D, Y41G, Y41H, Y411, Y41K, Y41V; E42:E42S, E42D, E42H, E42K, E42R; N43:N43H, N43K, N43M, N43Q, N43R; I44:I44L, I44K, I44R, I44D; E45:E45S, E45D, E45H, E45K, E45R; G46:G46N, G46H, G46K, G46M, G46Q, G46R; N47:N47H, N47K, N47M, N47Q, N47R; P50:P50G; G51:G51N, G51H, G51K, G51M, G51Q, G51R; K55:K55R, K55H, K55S, K55Q, K55I, K55E, K55N; D72:D72E, D72S, D72H, D72R, D72K; E73:E73D, E73S, E73H, E73R, E73K;

Group 4:E8:E8D, E8H, E8K, E8R, E8S; T10:T10P, T10S, T10L, T10V, T10D, T10K, T10N; P108:P108G; D109:D109ND109E, D109S, D109H, D109R, D109K; I113:I113L, I113K, I113R, I113D, K115:K115R, K115H, K115S, K115Q, K115I, K115E, K115N;

Group 5:H154:H154W, H154F, H154S, H154D; S155:S155T, S155L, S155V, S155D, S155K; D156:D156H, D156E, D156S, D156R, D156K; N159:N159H, N159K, N159M, N159Q, N159R, N159G ,+160N;

Group 6:D25:D25E, D25H, D25K, D25R, D25S; N28:N28H, N28K, N28M, N28Q, N28R, N28T; K32:K32Q, K32R, K32N, K32H, K32S, K32I, K32E;

Group 7:H76:H76W, H76F, H76S, H76D; T77:T77A, T77S, T77L, T77V, T77D, T77K, T77N; N78:N78H, N78K, N78M, N78Q, N78R; K80:K80R, K80H, K80S, K80Q, K80I, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S; K103:K103R, K103H, K103S, K103Q, K103I, K103E, K103V;

Group 8:K68:K68R, K68H, K68S, K68Q, K68I, K68E, K68N; R70:R70K, R70H, R70T, R70D, R70E, R70N; I86:I86L, I86K, I86R, I86D; E87:E87D, E87H, E87K, E87R, E87S, E87A; E96:E96D, E96H, E96K, E96R, E96S, E96L; K97:K97R, K97H, K97S, K97Q, K97I, K97E;

Group 9:G1:G1N, G1H, G1K, G1M, G1Q, G1R; G92:G92N, G92H, G92K, G9 2M, G92Q, G92R; T94:T94A, T94S, T94L, T94V, T94D, T94K, T9 4N; K123:K123R, K123H, K123S, K123Q, K123I, K123E; G124:G124N, G124H, G124K, G124M, G124Q, G124R; D125:D125E, D125H, D125K, D125R, D125S, D125Y; H126:H126W, H126F, H126S, H126D;

Group 10:K65:K65R, K65H, K65S, K65Q, K65I, K65E, K65N; Y66:Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.

7. the reorganization Betv1 anaphylactogen of each of claim 1-3, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, and every group all contains and is suitable for the amino acid sites that surface that following specific amino acids replaces exposes:

Group 1:A130:A130V, A130G, A130I, A130L, A130S, A130H, A130T; K134:K134R, K134H, K134S, K134Q, K134I, K134E; A135:A135V, A135G, A135I, A135L, A135S, A135H, A135T; K137:K137R, K137H, K137S, K137Q, K137I, K137E; E138:E138D, E138H, E138K, E138R, E138S, E138N; E141:E141D, E141H, E141K, E141R, E141S; T142:T142A, T142S, T142L, T142V, T142D, T142K, T142N;

Group 2:V2:V2A, V2I, V2K, V2L, V2R, V2T; F3:F3H, F3W, F3S, F3D; N4:N4H, N4K, N4M, N4Q, N4R; Y5:Y5D, Y5G, Y5H, Y5I, Y5K, Y5V; E6:E6D, E6H, E6K, E6R, E6S; T7:T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119:K119R, K119H, K119S, K119Q, K119I, K119E, K119N;

Group 3:D27:D27E, D27H, D27K, D27R, D27S; Y41:Y41D, Y41G, Y41H, Y411, Y41K, Y41V; N43:N43H, N43K, N43M, N43Q, N43R; I44:I44L, I44K, I44R, I44D; E45:E45S, E45D, E45H, E45K, E45R; G46:G46N, G46H, G46K, G46M, G46Q, G46R; N47:N47H, N47K, N47M, N47Q, N47R; P50:P50G; G51:G51N, G51H, G51K, G51M, G51Q, G51R; K55:K55R, K55H, K55S, K55Q, K55I, K55E, K55N; D72:D72E, D72S, D72H, D72R, D72K; E73:E73D, E73S, E73H, E73R, E73K;

Group 4:E8:E8D, E8H, E8K, E8R, E8S; P108:P108G; I113:I113L, I113K, I113R, I113D, K115:K115R, K115H, K115S, K115Q, K115I, K115E, K115N;

Group 5:H154:H154W, H154F, H154S, H154D; S155:S155T, S155L, S155V, S155D, S155K; N159:N159H, N159K, N159M, N159Q, N159R, N159G ,+160N;

Group 6:D25:D25E, D25H, D25K, D25R, D25S; N28:N28H, N28K, N28M, N28Q, N28R, N28T;

Group 7:H76:H76W, H76F, H76S, H76D; N78:N78H, N78K, N78M, N78Q, N78R; K80:K80R, K80H, K80S, K80Q, K80I, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S; K103:K103R, K103H, K103S, K103Q, K103I, K103E, K103V;

Group 8:K68:K68R, K68H, K68S, K68Q, K68I, K68E, K68N; R70:R70K, R70H, R70T, R70D, R70E, R70N; I86:I86L, I86K, I86R, I86D; E87:E87D, E87H, E87K, E87R, E87S, E87A; E96:E96D, E96H, E96K, E96R, E96S, E96L; K97:K97R, K97H, K97S, K97Q, K97I, K97E;

Group 9:G1:G1N, G1H, G1K, G1M, G1Q, G1R; G92:G92N, G92H, G92K, G92M, G92Q, G92R; T94:T94A, T94S, T94L, T94V, T94D, T94K, T94N; G124:G124N, G124H, G124K, G124M, G124Q, G124R; D125:D125E, D125H, D125K, D125R, D125S, D125Y; H126:H126W, H126F, H126S, H126D;

Group 10:Y66:Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.

8. the reorganization Betv1 anaphylactogen of each of claim 1-4, wherein the one-level sudden change is selected from least 4 groups in following 10 groups, and every group all contains and is suitable for the amino acid sites that surface that following specific amino acids replaces exposes:

Group 1:A130:A130V, A130G, A130I, A130L, A130S, A130H, A130T; A135:A135V, A135G, A135I, A135L, A135S, A135H, A135T; K137:K137R, K137H, K137S, K137Q, K137I, K137E; E138:E138D, E138H, E138K, E138R, E138S, E138N; E141:E141D, E141H, E141K, E141R, E141S; T142:T142A, T142S, T142L, T142V, T142D, T142K, T142N;

Group 2:F3:F3H, F3W, F3S, F3D; N4:N4H, N4K, N4M, N4Q, N4R; E6:E6D, E6H, E6K, E6R, E6S; T7:T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119:K119R, K119H, K119S, K119Q, K119I, K119E, K119N;

Group 3:D27:D27E, D27H, D27K, D27R, D27S; Y41:Y41D, Y41G, Y41H, Y41I, Y41K, Y41V; N43:N43H, N43K, N43M, N43Q, N43R; I44:I44L, I44K, I44R, I44D; G46:G46N, G46H, G46K, G46M, G46Q, G46R; N47:N47H, N47K, N47M, N47Q, N47R; P50:P50G; G51:G51N, G51H, G51K, G51M, G51Q, G51R; D72:D72E, D72S, D72H, D72R, D72K; E73:E73D, E73S, E73H, E73R, E73K;

Group 4:E8:E8D, E8H, E8K, E8R, E8S; I113:I113L, I113K, I113R, I113D, K115:K115R, K115H, K115S, K115Q, K115I, K115E, K115N;

Group 5:H154:H154W, H154F, H154S, H154D; S155:S155T, S155L, S155V, S155D, S155K; N159:N159H, N159K, N159M, N159Q, N159R, N159G ,+160N;

Group 7:H76:H76W, H76F, H76S, H76D; N78:N78H, N78K, N78M, N78Q, N78R; K80:K80R, K80H, K80S, K80Q, K80I, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S;

Group 8:K68:K68R, K68H, K68S, K68Q, K68I, K68E, K68N; R70:R70K, R70H, R70T, R70D, R70E, R70N; 186:186L, 186K, 186R, 186D; E87:E87D, E87H, E87K, E87R, E87S, E87A;

Group 9:G1:G1N, G1H, G1K, G1M, G1Q, G1R; G92:G92N, G92H, G92K, G92M, G92Q, G92R; D93:D93N, D93E, D93S, D93H, D93R, D93K; G124:G124N, G124H, G124K, G124M, G124Q, G124R; H126:H126W, H126F, H126S, H126D;

Group 10:Y66:Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.

9. the reorganization Betv1 anaphylactogen of claim 1, it contains following sudden change: Y5V, E45S, N78K, K97S, K103V, K134E ,+160N.

10. the reorganization Betv1 anaphylactogen of claim 9, it further contains at least a following replacement, and wherein the replacement carried out of expectation is listed at first: E8/K115, D125/H126, E138/K137/E141, D25/N28, E87/K55, S155/H154/N159, N47/P50/H76/N43/I44/R70, E87/K55, E73/P50/D72, A130, N28/D25, P108, V2/K119/N4/E6/E96.

11. the reorganization Betv1 anaphylactogen of claim 9 or 10, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: T10P most, K65N, N28/D25/K32Q/E141/K137/E138, D125/K123I/H126, P108/D109N, E42S/K55/I44/N43, E73/D72, E87, E96/K119, A130, V2/E6, E8/K115, N47/P50/R70/H76/T77A, S155/D156H/N159, E6/V2.

12. the reorganization Betv1 anaphylactogen of claim 1, it contains following sudden change: Y5V, N28T, K32Q, E45S, N78K, K97S, K103V, K134E ,+160N.

13. the reorganization Betv1 anaphylactogen of claim 12, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: E8/K115, D125/H126, E138/K137/E141, E87/K55, S155/H154/N159 most.N47/P50/H76/N43/I44/R70，K55，E73/P50/D72，A130，D25，P108，V2/K119/N4/E6/E96。

14. the reorganization Betv1 anaphylactogen of claim 12 or 13, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: T10P, K65N most, E141/K137/E138, D125/K123I/H126, P108/D109N, E42S/K55/I44/N43, E73/D72, E87, V2/E6, N47/P50/R70/H76/T77A.E96/K119，A130，E8/K115，S155/D156H/H154/N159，E6/V2。

15. the reorganization Betv1 anaphylactogen of claim 1, it contains following sudden change: Y5V, N28T, K32Q, E45S, N78K, E87S, K97S, K103V, K134E, N159G ,+160N.

16. the recombinant allergens of claim 15, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: K55 most, A138/K137/E141, D125/H126, P108, V2/N4/K119/E6, S155/H154, N47/P50/H76, E73, R70, A130, E8/K115, E96.

17. the recombinant allergens of claim 15 or 16, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: K65N most, T10P, D125, K123I, P108, D109N, N47/P50/H76, E138/K137/E141, E42S/K55/I44/N43, S155/D156H, E73/D72, E6/V2, E96.

18. the reorganization Betv1 anaphylactogen of claim 1, it contains following sudden change: Y5V, N28T, K32Q, E45S, N78K, K97S, K103V, P108G, D125Y, K134E ,+160N.

19. the reorganization Betv1 anaphylactogen of claim 18, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: E87, E141, K55, N47/N43/I44/H76, S155/HIS154, A130, E8, E73, V2/K119 most.

20. the reorganization Betv1 anaphylactogen of claim 18 or 19, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: K65N, T10P/E8, E87 most, S155/D156H, E141, E42S, A130, E8/T10P, N47, H76T, V2.

21. the reorganization Betv1 anaphylactogen of claim 1, it contains following sudden change: Y5V, N28T, K32Q, E45S, E73S, E96S, P108G, D125Y, N159G ,+160N.

22. the reorganization Betv1 anaphylactogen of claim 21, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: K134, N78, E87, K119, E8, K55X, E141, N47, S155, E6, K103, A130, V2 most.

23. the reorganization Betv1 anaphylactogen of claim 21 or 22, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: K65N/K55 most, T10P/E8/E141, E138/K134, E87, E42S/K55/I44, S155/D156H, N78, K119/V2/N4, N47/P50, H76/T77A, A130, E6/K115/K103.

24. the reorganization Betv1 anaphylactogen of claim 1, it contains following sudden change: Y5V, N28T, K32Q, E45S, E96S, P108G ,+160N.

The reorganization Betv1 anaphylactogen of 25 claims 24, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: K134, N78, E87, K119, E8, K55X, E141, S155, N47, E6, K103, A130, V2, R70, D125 most.

26. the reorganization Betv1 anaphylactogen of claim 24 or 25, it further contains at least a following replacement, expects that wherein the replacement of carrying out is listed at first: N78/T77A most, K103X, K134/E138, K65N/K55, T10P, D125/H126, E42S/K55, S155/D156H/HIS154, K119/V2, E87, N47/P50/H76, A130.

27. the reorganization Betv1 anaphylactogen of aforementioned arbitrary claim, it contains at least a following replacement: Y5, N28, K32, E45, E96/K97, P108/D109, N159/+160, E60, T10, K103/K115, K65, K129, K134, E42/K55, S149/A153/L152, D125/K123, N47/L24, T77/N78, K119, E87, A16/K20/P14, Q36/G61/P63, E73, D93, V2.

28. the reorganization Betv1 anaphylactogen of aforementioned arbitrary claim, it contains at least a following replacement: Y5V, N28T, K32Q, E45S, E96S/K97S, P108G/D109N, N159G/+160N, E60S, T10N, K103V/K115N, K129N, K134E, E42S/K55N, S149T/A153V/L152A, D125Y/K123I, N47K/L24A, T77N/N78K, K119N, E87A, A16G/K20S/P14G, Q36N/G61S/P63G, E73S, D93S, V2L.

29. the reorganization Betv1 anaphylactogen of claim 1, it contains replacement, and described replacement is selected from least 4 groups in following 10 groups:

Group 1:A130V, K134E, E141N,

Group 2:V2L, Y5V, E6S, K119N,

Group 3:E42S, E45S, N47K, K55N, E73S, E73T, E73S,

Group 4:E8S, T10P, P14G, P108G, D109N, K115N,

Group 5:A16G, K20S, S149TL152AA153V, S155T, N159G ,+160N,

Group 6:L24A, D25E, N28T, K32Q,

Group 7:T77A, T77N, N78K, K103V,

Group 8:R70N, E87A, E96S, K97S,

Group 9:D93S, K123I, D125Y, K129N,

Group 10:Q36N, E60S, G61S, P63G.

30. the invention further relates to the reorganization Betv1 anaphylactogen according to claim 1, it contains the replacement that is selected from least 4 groups in following 10 groups:

Group 1:K134E,

Group 2:Y5V, K119N, V2L,

Group 3:E45S, E42S, K55N, N47K, E73S,

Group 4:E96S, K97S, P108G, D109N, T10N, K115N, P14G,

Group 5:N159G ,+160N, S149T, A153V, L152A, A16G, K20S,

Group 6:N28T, K32Q, L24A,

Group 7:K103V, T77N, N78K,

Group 8:E96S, K97S, E87A,

Group 9:K129N, D125Y, K123I, D93S,

Group 10:E60S, Q36N, G61S, P63G.

31. the reorganization Betv1 anaphylactogen of each of claim 1-30, it contains 5 at least, preferred 6, and more preferably 7, most preferably 8-10 one-level sudden change.

32. the reorganization Betv1 anaphylactogen of each of claim 1-31, it further contains at least one secondary sudden change.

33. the reorganization Betv1 anaphylactogen of each of claim 1-32, it further contains claim 1, at least one secondary sudden change of listed each group of claim 2 or claim 3.

34. the reorganization Betv1 anaphylactogen of each of claim 1-33, it further contains at least one additional mutations, and wherein this sudden change is increase or the disappearance that the surface exposes the cyclic amino acids residue.

35. the reorganization Betv1 anaphylactogen of arbitrary aforementioned claim is as the application of medicine.

36. arbitrary recombinant allergens of claim 1-34 prevents and/or treats application in the medicine of beech (Fagales) pollen hypersensitivity in preparation.

37. arbitrary recombinant allergens of claim 1-34 prevents and/or treats application in the medicine of birch pollen hypersensitivity in preparation.

38. composition, it comprises arbitrary reorganization Betv1 anaphylactogen variant of two or more claims 1-34, and wherein each variant is defined as and has at least a one-level sudden change, and this sudden change does not exist in another variant at least.

39. the composition of claim 38, it contains 2-12, preferred 3-10, more preferably 4-8, most preferably 5-7 kind variant.

The composition of 40 claim 38-39 is as the application of medicine.

41. the composition of claim 38-40 prevents and/or treats application in the medicine of beech (Fagales) pollen hypersensitivity in preparation.

42. the composition of claim 38-40 prevents and/or treats application in the medicine of birch pollen hypersensitivity in preparation.

43. pharmaceutical composition is characterized in that it contains the arbitrary recombinant allergens of claim 1-34 or the composition of claim 38-40, also randomly contains pharmaceutically acceptable carrier and/or vehicle, and optional assistant agent.

44. the pharmaceutical composition of claim 43 is characterized in that it is the anaphylactoid vaccine form of being brought out by naturally occurring Betv1 anaphylactogen in suffering from the anaphylactoid patient of birch.

45. in individuality, produce the method for immunne response, comprise to individuality and use arbitrary recombinant allergens or the composition of claim 38-40 or the pharmaceutical composition of claim 42-43 among at least a claim 1-34.

46., comprise individuality used arbitrary recombinant allergens or the composition of claim 38-40 or the pharmaceutical composition of claim 42-43 among at least a claim 1-34 to the individual vaccination or the method for the treatment of.

47. the method for the medicinal compositions of preparation claim 42-43 comprises each composition and pharmaceutically acceptable material and/or mixed with excipients among arbitrary recombinant allergens among the claim 1-34 or the claim 37-39.

48. the pharmaceutical composition that method obtained of claim 47.

49. individuality is treated, prevent or alleviate anaphylactoid method, comprise to individuality use the arbitrary reorganization Betv1 anaphylactogen of claim 1-34 or claim 38-40 each composition or arbitrary pharmaceutical composition of claim 43-44 and 48.

50. the method for arbitrary reorganization Betv1 anaphylactogen of preparation claim 1-34, wherein amino acid whose replacement is to be undertaken by rite-directed mutagenesis.

51. the method for arbitrary reorganization Betv1 anaphylactogen of preparation claim 1-34, wherein anaphylactogen is by DNA reorganization (molecular breeding) preparation.

52. the method in arbitrary reorganization Betv1 anaphylactogen library of preparation claim 1-34, wherein this anaphylactogen is to use and contains the Oligonucleolide primers preparation that at least 4 seed amino acid residues replace at random.

53. the method for claim 52, wherein amino-acid residue is selected from: Y5, T10, K20, N28, K32, Q36, E42, E45, E73, K65, N78, E87, K97, K103, P108, K123, K129, K134, S149, D156 and+160.

54. the dna sequence dna of the reorganization Betv1 anaphylactogen of coding claim 1-34, its derivative, its part fragment, its degenerate sequence or under rigorous condition can with the sequence of its hybridization, wherein said derivative, the part fragment, degenerate sequence or hybridization sequences coding have the peptide of at least a B cell epitope.

55. the dna sequence dna of claim 54, it is the derivative of natural anaphylactogen dna encoding sequence.

56. the dna sequence dna of claim 55, wherein this derivative is to obtain by natural B etv1 anaphylactogen dna encoding sequence being carried out rite-directed mutagenesis.

57. contain each the expression vector of DNA of claim 54-56.

58. contain the host cell of the expression vector of claim 57.

59. prepare the method for reorganization Betv1 allergen mutants, comprise the host cell of cultivating claim 58.

60. each reorganization Betv1 anaphylactogen or each the coded reorganization Betv1 anaphylactogen of dna sequence dna of claim 54-56 of claim 1-34, comprise at least one t cell epitope, described epi-position can stimulate T cell clone or the T clone special to natural B etv1 anaphylactogen.

61. estimate to use claim 1-34 each reorganization Betv1 allergen mutants or the dependency of the therapy of the combination treatment individuality of claim 38-40, the diagnostic test of degree of safety or effect, the individual sample that wherein will contain IgE is mixed with described mutant or described composition, and estimates the level of reactivity between the IgE and described mutant in the described sample.

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