CN1146772A

CN1146772A - Synthetic peptide based immunogens for the treatment of allergy

Info

Publication number: CN1146772A
Application number: CN95192778A
Authority: CN
Inventors: C·Y·王
Original assignee: United Biomedical Inc
Current assignee: United Biomedical Inc
Priority date: 1994-03-28
Filing date: 1995-03-24
Publication date: 1997-04-02
Also published as: AU2195395A; JPH09510975A; WO1995026365A1; EP0811016A4; CA2186595A1; EP0811016A1

Abstract

The present invention relates to a method for eliciting the production in healthy mammals, including humans, of high titer antibodies to an effector site in human IgE heavy chain, i.e. a site in the CH4 domain of the ELEMENT -chain, by the use of compositions of synthetic peptide immunogens in either a radially branching multimeric form (such as branching octameric or hexadecameric peptides) or a linearly arranged monomeric form, to inhibit mast cell activation and reduce allergen-induced IgE production. It also relates to the use of such 'optimally' designer, carrier protein free, IgE ELEMENT -chain related immunogens as key components in a synthetic vaccine to provide an immunotherapy for the treatment of allergy. The subject peptides contain immune stimulator sequences, including a built-in helper T cell epitope tandemly linked in a specific orientation, to aid in stimulating the immune response towards the IgE CH4 domain.

Description

Be used for the treatment of anaphylactoid synthetic peptide based immunogens

The cross reference of relevant application

This is the pending application series No.08/218 that submitted on March 28th, 1994,461 part continuation application, No.08/218,461 is the pending application series No.08/060 that submitted on May 10th, 1993,798 continuation application, No.08/060, the 798th, the pending application series No.07/847 that submitted on March 6th, 1 now, 745 part continuation application, No.07/847, the 745th, the application series No.07/637 that submitted on January 4th, 1 now, 364 part continuation application.

The field of the invention

The composition that the present invention relates to the synthetic peptide of line style or radial pattern branch polymer form is induced generation anti human IgE heavy chain ε-chain CH in comprising human healthy mammal as immunogene₄The high titre antibody of district's effector site, and relate to and use said composition to provide immunotherapy as vaccine to the treatment allergic reaction.

Background of the present invention

Since this century is early stage, being used for of known and practice prevents that the anaphylactoid immunotherapy that the IgE such as asthma and hay fever mediates from all carrying out through disallergization effect or desensitization, increase gradually wherein for the amount of patient's anaphylactogen, with the effect (1) that reduces to contact subsequently this anaphylactogen. If this limitation of immunotherapy take anaphylactogen as the basis comprises that identifying that related anaphylactogen has difficulties and identified usually causes adverse reaction (2) by anaphylactogen when using the anaphylactogen of identifying.

Alleviating anaphylactoid other therapies is to use the medicine blocking-up to be responsible for the cascade of anaphylactoid cell event. These medicines comprise antihistamine, decongestant, β₂Antagonist and corticosteroid. Anti--histamine, decongestant and β₂Antagonist is had an effect to IgE downstream event in the allergic reaction cascade, claims that they are that aunt is from medicine, only for allergic symptom. Prophylactic treatment must act on the initial cell event of allergic reaction that more mediates near IgE. These palliatives provide the alleviation of short-term and part. And alleviating of symptom often be accompanied by harmful side effect, and for example, anti--histamine can cause fidgety or sleepy, β₂Antagonist increases relevant with asthmatic patient's incidence of disease sometimes.

Corticosteroid is strong immunodepressant and the treatment symptoms of allergic is had efficient. Yet they stimulate harmful hormonal activity and may cause bad extensive immunosupress.

For fear of the shortcoming of known treatment medicine, more expectation comes the Polyglucan reaction through the selective inhibition for IgE. This can synthesize to realize through suppressing IgE, for example realizes through inconvenient desensitization method, or stimulates mast cell and basocyte to follow the degranulation process that discharges the anaphylaxis chemical mediator to realize through blocking-up IgE-anaphylactogen compound.

On more basic level, Stanworth etc.^(3-7)And other people^(8-13)Use synthetic IgE ε chain peptide and corresponding antibody to study and have a liking for the effect of cell peptide in cell signal mechanism, attempt the molecular basis of explaining that mast cell and basocyte immunology trigger.

In many IgE peptides (table 1) that 20 years study in the past, people ε chain FcCH₄Potential effect thing position (Iys in the district₄₉₇One Phe₅₀₆, in table 2, represent with following stroke of two-wire) and be decapeptide. It is manually synthesized and is used for structure/activity research (3). Find this from IgECH₄The decapeptide in district can activate the dosage dependent histamine in non-molten born of the same parents' mode of the mast cell trigger process that is similar to IgE mediation and discharge (4) from the rat peritoneum mast cell that separates. Infer that by the structure-activity research that a plurality of synthetic analogues that use ε-chain decapeptide carry out this peptide effector site needs accurate structure (3,4,5).

With resisting-IgE CH that relevant " peptide-carrier protein attachment " immunity of ε chain produces₄Peptide antibody also is used for through observing the inhibition activity degranulation of the cell of IgE sensitization being carried out structure function research^{(5，11，12)}。

Stanworth etc.^(14，15)Advised using the possibility that as the vaccine on basis the sensitiveness patient with IgE mediation is provided immunotherapy with peptide. He identifies before using has sequence Lys-Thr-Iys-Gly-Ser-Gly-Phe-Phe-Va1-Phe-NH₂ ⁽³⁾The ε of (SEQ ID No:1)-chain decapeptide is connected with " carrier protein " such as keyhole limpet hemocyanin (KLH) or tuberculin purfied protein derivative (PPD), and finds that " peptide-carrier protein " attachment induces the decapeptide specific antibody. For example, reduce the histamine that the release decapeptide is induced from the rat peritoneum mast cell according to its anti-peptide serum of rabbit that is higher than average anti-peptide titre selection in titre dependence mode. This inhibition is active further to be confirmed in the in vivo studies of models of passive skin irritability of rats (PCA) model system. When carrying out the rat that before anaphylaxis is attacked the anti-peptide serum of this rabbit is given to use in advance multiple anaphylactogen sensitization with anaphylactogen, turns blue area and estimate the anti-peptide serum of rabbit to anaphylactoid effect with the evaluation color depth through measuring.

In identical research, contain the result who obtains in the immunogenic rat of this " decapeptide protein carrier attachment " in use and shown that tentatively they are as the anaphylactoid possibility of vaccine therapy.

Yet this method has run into sizable difficulty. The major defect of this prototype " decapeptide-protein carrier attachment " vaccine comprises less-than-ideal immunostimulation ability and is difficult to limit the production difficulty that the inhomogeneity with coupled reaction causes by the component of carrier protein. Also find usually to be contained than the more anti-protein carrier of the anti-purpose peptide (antibody of the upper epitope of keyhole limpet hemocyanin (KLH) for example by the gained antiserum that this class peptide-protein attachment produces⁽⁵⁾。

Table 1

The IgE heavy chain peptide that is used for structure-activity research

Total number of atnino acid and peptide sequence	The zone	Structure-activity research	List of references
Total number of atnino acid and peptide sequence	The zone	Structure-activity research	List of references	Huε497-506 RTKGSGFFVF	CH ₄	The non-antigen receptor that in mast cell signal mechanism, relates to	Stanworth，Mol.Immunol.，21：1183-1190，1984(4) Stanworth and Burt，Mol.Immunol.，23：1231-1235， 1986(5)
Huε301-376	CH ₂/CH ₃	The passive sensitization of blocking-up people's mast cell and basocyte	He1m et al.，PNAS，86：9465-9469，1989(13)	Huε497-506 RTKGSGFFVF	CH ₄
Huε301-376	CH ₂/CH ₃		He1m et al.，PNAS，86：9465-9469，1989(13)	Huε363-376	CH ₃	Optional in conjunction with FC ε-chain acceptor 1 to peptide	ibid.
Huε367-376	CH ₃	Combination to low-affinity IgE acceptor (CD23)	Chretien et al.，J.Immunol.，141：3128-3134，1988(11)	Huε363-376	CH ₃		ibid.
Huε367-376	CH ₃	Combination to low-affinity IgE acceptor (CD23)	Chretien et al.，J.Immunol.，141：3128-3134，1988(11)	The people FC ε fragment that seven enterobacterias produce	CH ₂/CH ₃/CH ₄	Expression in Escherichia coli and the comparison of activity of being combined with natural people IgE myeloma cell. Restructuring Fc has 20% natural combination activity.	Kenten et al，PNAS 81：2955-2959，1984(12)
The people FC ε fragment that seven enterobacterias produce	CH ₂/CH ₃/CH ₄	The monomeric form non-activity. Fc sample dimeric forms only shows natural IgE cell in conjunction with 1/4 of activity.	Coleman et al.，Eur.J.Immunol.，15：966-969，1985 (13)	The people FC ε fragment that seven enterobacterias produce	CH ₂/CH ₃/CH ₄		Kenten et al，PNAS 81：2955-2959，1984(12)
The people FC ε fragment that seven enterobacterias produce	CH ₂/CH ₃/CH ₄		Coleman et al.，Eur.J.Immunol.，15：966-969，1985 (13)	Rat 414-428 (p129)^* 459-472(p124) 491-503(p128) 542-557(p123) 378-396(p130) 522-535(p122) 560-571(p131)	CH ₃/CH ₄ CH ₃/CH ₄	Concentration is 10^-4-10 ^-5The rat IgE that suppresses 20-50% in the time of between the M is combined with mast cell.^*Even also specific activity rat IgE is active little 1000 times to find maximum activity peptide (p129). The unrestraint effect	Burt and Stanworth，Eur.J.Immunol.，17：437-440， 1987(9)

Total number of atnino acid and peptide sequence	The zone	Structure-activity research	List of references
Total number of atnino acid and peptide sequence	The zone	Structure-activity research	List of references	Rat ε 459-472 (YVFLPPEEEEKDKD) 542-547 (HEAKRELER-TISK)	CH ₄	Be responsible for IgE molecule thermal sensitivity zone that mast cell and basocyte are had cytotropism.	Stanworth and Burt，Mol Immunol，23：1231-1235， 1986(5)
People ε pentapeptide	CH ₂	Non-specific inhibition in the antibody-mediated PK reaction of human IgE.	Hamberger，Science，189：389-390，1975(8)	Rat ε 459-472 (YVFLPPEEEEKDKD) 542-547 (HEAKRELER-TISK)	CH ₄		Stanworth and Burt，Mol Immunol，23：1231-1235， 1986(5)
People ε pentapeptide	CH ₂		Hamberger，Science，189：389-390，1975(8)	Mouse ε peptide 167-180 (p1) 207-218 (p2) 237-251 (p3) 291-305-(p4) 338-352 (p5) 372-385 (p6)	CH ₂/CH ₃/CH ₄ CH ₂ CH ₂/CH ₃ CH ₃ CH ₃ CH ₄ CH ₄	The structure-function relation that is limited by the sequence for antibody is anti--and p1 shows the low adhesion with IgE. Hydrophobic and conformation. Anti--p2 is not in conjunction with IgE. Can not approach. Near IgE acceptor recognition site; Anti--p3 demonstrates minimum IgE in conjunction with vigor. Anti--p4 has stimulated thrombocytin release. Anti--p5 has stimulated thrombocytin release. Anti-p6 and 1gE (free or acceptor combining form) be in conjunction with best, yet invalid in IgE-acceptor compound crosslinked.	Robertson and Liu.，Mol.Immunol，25：103-113，1988 (12)

Little peptide is relatively poor immunogene, and this is known to those skilled in the art. In order to make little peptide have immunogenicity, usually connect or through gene fusion they are connected with larger vector protein through chemistry. Yet these methods generally produce unpredictable conformational change at peptide. And usually mislead immune response for the carrier that immunodominance is arranged. Therefore, expectation further designs immunogene and forms effective vaccine to provide anaphylactoid long-term remission.

In table 2, with rat IgE ε-chain⁽¹⁶⁾With mouse ε-chain⁽¹⁷⁾CH ₂To CH₄Amino acid sequence and people's ε-chain in zone⁽¹⁸⁾The amino acid sequence of (SEQ ID NoS:2-4) has carried out sequence and has contrasted to provide the in the past guidance of the 1gE correlation fragments of peptides of report. It should be noted that at the L near 252 Q on the people IgE ε heavy chain and in original IgE myeloma ND sequence, do not exist. Import breach (representing with billet) to enlarge homology. The homology residue position of coupling is in square frame. Studied position (table 1) underscoring in table 2 on the ε sequence of structure-activity. At people IgECH₄Activated IgE CH structurally in the district₄Draw two-wire (SEQ ID No:1) below the decapeptide sequence. The amino acid code that uses in this table is: A, alanine; R, arginine; N, asparagine; D, aspartic acid; C, cysteine; Q, glutamine; E, glutamic acid; G, glycine; H, histidine; I, isoleucine; L, leucine; K, lysine; M, methionine; F, phenylalanine; P, proline; S, serine; T, threonine; W, tryptophan; Y, tyrosine; V, valine.

Table 2

Table 2 (continuing)

List of references

1.Noon L.″prophylactic inoculation against hay fever.” Lancet，i：1572-1573(1911).

2.World Health Organization and International Union of Immunological Societies Working Group Report：Current status of allergen immunotherapy.Lancet，i：259-261 (1989).

3.Stanworth，Kings，Roy，et al.Biochem.J.，180：665- 668(1979).

4.Stanworth.Mol. Immunol.，21：1183-1190(1984).

5.Stanworth，and Burt.Mol-Immunol.，23：1231-1235 (1986).

6.Burt，and Stanworth.Eur.J.Immunol.，17：437-440 (1987).

7.Stanworth. Mol.Immunol.，25：1213-1215(1988).

8.Hamburger. Science，189：389-390(1975).

9.Kenten，Helm，Ishizaka，et al.proc.Natl.Acad Sci.， USA，81：2955-2956(1984).

10.Coleman，Helm，Stanworth，and Gould.Eur.J. Immunol.，15：966-969(1985).

11.Chretien，Helm，Marsh，et al.J.Immunol.，141：3128- 3134(1988).

12.Robertson，and Lin.Mol.Immunol.，25：103-113(1988).

13.Helm，Kebo，Vercelli，et al.Proc.Natl.Acad.Sci.， USA 86：9465-9469(1989).

14.Stanworth，Jones，Lewin，and Nayyar.Allergy treatment with a peptide vaccine.Lancet，336：1279- 1281(1990).

15.Stanworth，Lewin，Nayyar，and Jones.Immunoreactive peptides and antibodies and their use in anti-allergy treatment.EPO 403 312 Al(1990).

16.Kindsroger et al.，DNA 1：335-343(1982).

17.Ishida et al.，EMBO1：1117-1123(1982).

18.Dorrington and Bennich，Immunol.Rev.，41：3-25 (1978).

19.Brett et al.Eur.J.Immunol.，23：1608-1614(1993).

20.Weismuller et al.，Int.J.peptide Res.，40：255-260 (1992).

21.Celis et al.，J.Immunol.，140：1808-1815(1988).

22.Demotz et al.，J.Immunol.，142：394-402(1989).

23.Chong et al.，Infect.Immun.，60：4640-4647(1992).

24.Grant ed.，Synthetic peptides：A User′s Guide， W.H.Freeman&Co.，New York，NY，(1992)pp.382.

25.O′Hagan et al.，Vaccine，9：768-771(1991).

26.Eldridge etal.，Molec.Immunol.，28：287-294(1991).

27.MacGlashan et al.，J.Immunol.，136：2231-2239(1986).

Purpose of the present invention

An object of the present invention is radial branch form or one group of the chemical synthesis peptide immunogene take IgE ε chain as the basis containing line style t helper cell epitope form are used for inducing anti-human ε chain CH when importing comprises human mammal₄The high titre antibody of district's decapeptide effector site.

Another purpose is the best peptide immunogene of design, and it has the people of taking from IgE heavy chain CH₄District (IgECH₄) specific amino acid sequence, be attached on the peptide that contains any people's helper cell epitope with specific orientation, when importing comprises in the human mammal, can stimulate to produce anti human IgE CH₄Effective antibody of the high titre of effector site. These antibody should suppress mast cells activation, reduce the chemical mediator release such as histamine of responsible symptoms of allergic, suppress models of passive skin irritability (PCA) reaction of IgE mediation and the IgE by the bone-marrow-derived lymphocyte generation of inhibition allergen-induced.

Also having a purpose is that formation is effective take the vaccine of IgE ε-chain peptide as the basis, and utilization contains this class branch immunogenic composition polymeric or line style provides immunotherapy for the treatment allergic reaction.

General introduction of the present invention

According to the present invention, with the synthetic preparation of solid phase peptide immunogene. The peptide immunogene comprises ten the amino acid IgE CH that contain of a series of radial branches₄The polymer peptide of peptide (SEQ ID No:1) or its immunogenicity analog; A series of IgE CH that contain₄The peptide of the polymer branch of peptide (SEQ ID No:1) or its immunogenicity analog and a helper cell epitope (Th epitope); With a series of IgE CH that contain₄The line style monomeric peptide of peptide (SEQ ID No:1) or its immunogenicity analog and part helper cell epitope (Th epitope). IgE CH₄Peptide is taken from IgE heavy chain Fc zone, i.e. ε-chain CH₄District (IgE CH₄). In the peptide immunogene of these three series, preferred line style peptide. The composition that contains these peptides is used for immune healthy mammal, and for example: cavy, rat and people produce IgE CH to induce₄Effector site (SEQ ID No:1) has specificity and does not contain the high titre antiserum of irrelevant antibody.

According to the present invention, also the preparation under the condition of suitable adjuvant and/or the existence of transmission carrier of the polymer branch peptide of available effective dose or line style peptide contains synthetic peptide as the immunogenic vaccine of key. Expect that this vaccine composition can bring out than Stanworth etc.⁽¹⁴⁾The anti-IgE reactive polypeptide that normally used those peptides-carrier protein attachment is more concentrated, thus better immunotherapy provided for treating allergic reaction.

Detailed description of the present invention

The present invention relates to use one group of new immunogene take peptide as the basis in comprising human healthy mammal, to produce anti human IgE ε heavy chain CH₄The high titre antibody of district's effector site (SEQ ID No:1) is used for the treatment of the hypersensitivity disorders of IgE-mediation.

Generally accept allergic symptom, namely the hypersensitive direct result of IgE-dependence is to be caused by the chemical mediator that mast cell and basocyte discharge. Discharge when it being had to trigger when specific anaphylactogen is combined with surface conjunction IgE with the mast cell of the IgE sensitization of surface conjunction or basocyte. Carry out combination through the Fab ' part of anaphylactogen and surface conjunction IgE with the interaction of Ag-Ab type and start triggering. Anaphylactogen/antibody is in conjunction with the IgE of crosslinked divalence surface conjunction and the conformational change of inducing IgE far-end Fc district, this IgE district directly with contact at the high-affinity Fc of cell surface acceptor. Through a kind of mechanism of not yet accurately understanding, conformational change active cell-IgE-anaphylactogen compound, cell discharges the medium that comprises histamine as a result. It is believed that the effector site on the IgE participates in trigger event. The existence of the specificity anti-IgE antibodies of anti-this " effector site " can cause suppressing the cell-stimulating process through disturbing the interaction between IgE " effector site " and the cell surface by active or passive immunity in suffering from anaphylactoid host.

By this class of using the specificity anti-IgE antibodies regulate that prophylactic treatment that (that is: a kind of immunotherapy) can be undertaken by specificity " fixed point " antibody with anti-IgE comes passively or more preferably process provide to the host and comprise the immunogenic vaccine of peptide of fixing a point and induce the host to produce self to fix a point anti-IgE antibodies to realize on one's own initiative. It is believed that active immunity will provide more effective and more long-term protection.

In the site in the circulation IgE Fc zone of having studied functional activity, with IgE molecule CH₄The zone (Lys497-Phe506) in district is accredited as the conformational effect thing that relates to mast cell and basocyte triggering^(3-8，14)See Table 1 and table 2 in the zone of underscoring. Discovery has sequence Lys-Thr-Lys-Gly-Ser-G1y-Phe-Phe-Val-Phe-NH₂(SEQ ID No: the conformation from approximate this effector site of the decapeptide at this position 1). This can prove with the ability of bringing out the release of dosage dependent histamine similar in appearance to the mode of immunology trigger process from rat hypertrophy cell through this decapeptide⁽⁴⁾。

Stanworth etc.^(14，15)Confirm by using and carrier protein the IgE CH that keyhole limpet hemocyanin (KLH) is coupled₄Decapeptide (SEQ ID No:1) is made immunogenic experimental vaccine provides immunotherapy to the anaphylactoid patient with IgE mediation possibility. Stanworth etc.^(14，15)The animal immune serum that discovery obtains from this para-immunity relies on the mode moderate with titre and reduces the histamine release of inducing from the decapeptide of rat abdomen abdomen mast cell. Under the condition that a plurality of anaphylactogens are used with body in passive cutaneous anaphylaxis (PCA) test confirmed that further the inhibition that is produced by immune serum is active.

Prototype " IgE CH by foundation such as Stanworth₄Peptide " major defect of vaccine is that its immunogenicity is poor, namely one with problem that almost all autoantigens are inherent relevant.

In the present invention, provide specific immunity former, wherein synthetic immunostimulation composition is with specific orientation and IgE CH₄Decapeptide (SEQ ID No:1) links to each other in order to can produce widely effective antibody of this effector site on the anti-IgE in the different host colony in heredity. Then, these antibody blockings IgE is to the spread effect of mast cell and basocyte, thereby causes the effective treatment to the hypersensitivity disorders of prevention IgE mediation.

Peptide of the present invention immunity proper energy is induced to be had the antibody of serological cross reaction with IgE Fc district target amino acid sequence (SEQ ID No:1) and basically can not mediate the thin amine release of non-molten born of the same parents' property.

Through injection, initial dose is used in preferred intramuscular injection, for example 0.2-2.5mg; The immunogene of preferred 1mg is then used repetition (reinforcement) dosage. Dosage depends on patient's age, body weight and general health situation, and this is that the treatment field is known.

Although the mammal species that is suitable for producing antibody is not specifically limited, general preferred use mouse, rabbit, cavy, pig, goat, rat or sheep etc. are as the host.

For active immunity, term mentioned in this article " immunogene " relates to can induce anti-IgE CH₄The synthetic peptide of the antibody of decapeptide (SEQ ID No:1), wherein antibody causes basocyte and the mast cell degranulation of IgE mediation to be suppressed. Immunogene of the present invention comprises polymer peptide or its analog of the paniculate lysyl core matrix of tool structure.

The polymer peptide of these branches has independently bring out immunoreactive ability in host animal. IgE CH₄Decapeptide (SEQ ID No:1) analog comprises Stanworth etc.^(3，4，5)The synthetic decapeptide analog of describing, this paper quotes for your guidance. For the purpose of suitable, the immunogene molecular weight should be higher than 5,000; The repetition branch unit that preferably is higher than 10,000 peptides should be equal to or higher than 4.

Difunctional amino acid, the lysine that adheres to the amino acid (such as Gly or Ala) with the charged side chain of preferred right and wrong such as the back is useful in the preparation of core matrix structure. Be coupled in the circulation through being inserted into two two Boc-lysines at an amino acid that additionally is coupled in the circulation, this amino acid serves as the introns between the peptide branch, allows the maximum free degree to present necessary conformation to reach the best.

The immunogene that the present invention mentions also comprises the line style peptide that contains any helper cell epitope (Th epitope). These Th epitopes are covalently attached on the decapeptide effector molecules sequence (SEQ ID No:1) in the mode that limits, and have or do not have introns, so as adjacent with decapeptide N end, thus excite effective antibody response. Immunogene also can comprise with such as bacterium Yersinia spp⁽¹⁹⁾The corresponding immunostimulation sequence in invasion protein zone. The invasion functional areas also can be attached on the Th epitope by introns.

" immunogene " of the present invention reduced the generation of irrelevant antibody, is required IgE CH thereby induced more concentrated anti-" target sequence "₄The immune response of cross reactivity (SEQ ID No:1) and not producing can make the complicated bad side effect of the anaphylactoid immunization therapy process for the treatment of.

Yet, as short target sequence, for example 10 amino acid whose IgE CH₄When fragment Lys-Thr-Lys-Gly-Ser-G1y-Phe-Phe-Val-Phe (SEQ ID No:1) is used for design carrier-free protein immunogen, can face serious challenge. Small peptide antigen is T cell dependence antigen normally, that is: the existence of the auxiliary epitope of T is that to cause short " target " peptide to have immunogenicity needed. Short IgE CH₄Decapeptide (SEQ ID No:1) or its immunogenicity analog do not contain the t helper cell epitope. This paper has designed and has comprised short IgE CH₄The polymer of the branch of decapeptide and line style immunogene are to provide artificial built-in function auxiliary merit T-cell epitope.

Peptide immunogene of the present invention is represented by following formula:

(A) _n-(Th) _m-(B) _o-(IgE CH ₄Peptide)_p

Wherein: A is amino acid, α-NH₂, aliphatic acid, aliphatic acid derivative, or invasion functional areas;

B is amino acid;

Th is the helper cell epitope or strengthens immunogenic analog or its fragment;

IgE CH ₄Peptide is Iys-Thr-Lys-G1y-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID No:1) or its immunogenicity analog;

N is from 1 to 10;

M is from 1 to 4;

O is from 0 to 10; P is from 1 to 3.

Peptide immunogene of the present invention comprises about 20 to about 100 amino acid residues; Preferably approximately 20 to about 50 amino acid residues, and more preferably about 20 to about 35 amino acid residues.

When A was amino acid, it can be that any non-natural exists or any naturally occurring amino acid. The amino acid that non-natural exists includes but not limited to Beta-alanine, ornithine, nor-leucine, norvaline, hydroxyproline, thyroxine, GABA, homoserine, citrulling and analog. Naturally occurring amino acid comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. And, when n is when being amino acid greater than 1,2 or a plurality of A group; Each amino acid is identical or different independently.

When A is aliphatic acid, such as stearic acid or palmitic acid or aliphatic acid derivative, such as three palmitin acyl cysteine (Pam₃Cys) during group, its is through the immunostimulation characteristic that strengthens Th epitope and serve as a kind of adjuvant⁽²⁰⁾ When A was the aliphatic acid or derivatives thereof, it was usually located at the amino terminal of peptide. And, when an A is aliphatic acid, 2 or 3 additional amino acid A parts are arranged. Useful aliphatic acid has the hydrocarbon chain of 8 to 24 carbon atoms among the present invention, and it can be saturated or unsaturated.

When A was the invasion functional areas, it was the immunostimulation epitope from the invasion protein of Yersinia bacterial classification. This immunostimulation feature is from this invasion functional areas and T cell, the immunity that particularly activates or the β on the memory T cell₁The ability of integrin interaction of molecules. Find and β₁The concrete sequence of the interactional invasion of integrin functional areas is by Brett etc.⁽¹⁹⁾Describe. In preferred embodiments, the invasion functional areas (Inv) that are connected on any Th epitope have sequence: Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe (SEQ ID No:25) or from its immunostimulation analog in another corresponding zone of Yersinia bacterial classification invasion protein. This analog can contain replacement, disappearance or insertion, and so that the variation between bacterial strain to be provided, prerequisite is that analog has kept the immunostimulation characteristic.

In one embodiment, n is that 4, A is α-NH₂, lysine, lysine and lysine are also arranged in this order. In another embodiment; N is that 1, A is α-NH₂ Also have in the embodiment, n is that 4, A is α-NH₂, invasion functional areas (Inv), glycine and glycine, and arranged sequentially with this.

B comprises the amino acid that aforesaid naturally occurring or non-natural exists. Each B can be identical or different independently. When B is lysine, can form the polymer of branch. For example, if 0 be 7 and all 7 B groups be lysine, so when not protecting lysyl side chain ε amino group to carry out the K core (K that peptide forms branch when synthetic₄K ₂K). Peptide with K core has 8 branch arms, and each branch arm is identical and with " (A)_n-(Th) _m-" or " (the IgE CH with inherent Th₄Peptide)-" representative. In addition, B amino acid can form the elasticity twisting, or spacer region is to strengthen Th epitope and IgE CH₄The immune response of decapeptide or its analog. The example of coding elasticity twisting sequence is found in heavy chain immunoglobulin twisting district. The normal Pro-rich of elasticity twisting sequence. A useful especially elasticity twisting that provides is sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID No:24), and wherein Xaa is arbitrary amino acid, preferred aspartic acid. Also can pass through at any Th epitope and IgE CH₄Add intervening sequence residue (for example Gly-Gly) between decapeptide or its analog and improve immunogenicity. Except Th epitope and B cell antigen being determined base (that is: IgE CH₄Decapeptide site or its analog) outside physics separated, glycine residue can destroy by Th epitope and IgE CH₄Decapeptide (SEQ ID No:1) or its analog connect any artificial secondary structure that produces, thereby eliminate the interference between T and/or B cell effect. Therefore, the separation of the conformation between auxiliary cell and the antibody induction district allows to produce more effective interaction in the immunogene that provides and suitable Th and B iuntercellular.

Th is the Th epitope that comprises natural or alpha-non-natural amino acid. The Th epitope can be comprised of continuous or discrete epitope; Not that each amino acid of Th must be the part of epitope. Be applicable to Th epitope of the present invention, comprise that its analog and fragment can strengthen or stimulate to IgE CH₄The immune response of decapeptide (SEQ ID No:1) or its analog. Have immunodominance and in animal and human's class colony of large-scale various different MHC types, have height and reactive widely with Th epitope arbitrarily^(21-23) Be suitable for Th of the present invention district and have about 10 to about 50 amino acid, preferably approximately 10 to about 30 amino acid. When having a plurality of Th epitope (m 〉=2), each Th epitope can be independently identical or different.

Th epitope analog is included on the Th epitope and replaces, and adds, and lacks and insert one to about 10 amino acid residues. The Th fragment is to be enough to strengthen or stimulate IgE CH₄The continuous part of the Th epitope of decapeptide (SEQ ID No:1) or its analog.

Epitope of the present invention comprises hepatitis B surface and cAg helper cell epitope (HB_sTh and HB_cTh), there is day to cough toxin helper cell epitope (PT Th), tetanus toxin helper cell epitope (TT Th), measles virus F protein helper cell epitope (MV_FTh), the main outer membrane protein helper cell of Chlamy dia trachomatis epitope (CT Th), diphtheria toxin helper cell epitope (DT Th), Ploasmodium Falciparum ring spore is in auxiliary T cell epitope (PF Th), the different poplar enzyme of Schistosoma mansoni triose phosphate helper cell epitope (SM Th), Escherichia coli TraT helper cell epitope (SM Th), Escherichia coli TraT helper cell epitope (TraT Th) and immunity strengthen any fragment of analog and these Th epitopes. The example of Th epitope sequence provides as follows: HB_aTh：Phe-Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr-Ile-Pro-Gln-

Ser-Leu-Asp (SEQ ID NO：5) PT ₁Th：Lys-Lys-Leu-Arg-Arg-Leu-Leu-Tyr-Met-Ile-Tyr-Met-

Ser-Gly-Leu-Ala-Val-Arg-Val-His-Val-Ser-Lys-Glu-

Glu-Gln-Tyr-Tyr-Asp-Tyr (SEQ ID NO：6) TT ₁Th：Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-

Gly-Ile-Thr-Glu-Leu (SEQ ID NO：7) TT ₂Th：Lys-Lys-Phe-Asn-Asn-Phe-Thr-Val-Ser-Phe-Trp-Leu-

Arg-Val-Pro-Lys-Val-Ser-Ala-Ser-His-Leu

(SEQ ID NO：8) PT _1ATh：Tyr-Met-Ser-Gly-Leu-Ala-Val-Arg-Val-His-Val-Ser-

Lys-Glu-Glu (SEQ ID NO：9) TT ₃Th： Tyr-Asp-Pro-Asn-Tyr-Leu-Arg-Thr-Asp-Ser-Asp-Lys-

Asp-Arg-Phe-Leu-Gln-Thr-Met-Val-Lys-Leu-Phe-Asn-

Arg-Ile-Lys (SEQ ID NO：10) PT ₂Th： Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-Gly-Thr-Arg-Ala-

Leu-Thr-Val-Ala-Glu-Leu-Arg-Gly-Asn-Ala-Glu-Leu

(SEQ ID NO：11) MVF ₁Th：Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-Glu-

Gly (SEQ ID NO：12)

and

Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-

Glu-Gly-Val (SEQ ID NO：61) HB _CTh： Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala-

Tyr-Arg-Pro-Pro-Asn-Ala-Pro-Ile-Leu

(SEQ ID NO：14) MV _F2Th：Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-Lys-Ala-Arg-Ile-

Thr-His-Val-Asp-Thr-Glu-Ser-Tyr (SEQ ID NO：26) TT ₄Th： Trp-Val-Arg-Asp-Ile-Ile-Asp-Asp-Phe-Thr-Asn-Glu-

Ser-Ser-Gln-Lys-Thr (SEQ ID NO：27) TT ₅Th： Asp-Val-Ser-Thr-Ile-Val-Pro-Tyr-Ile-Gly-Pro-Ala-

Leu-Asn-His-Val (SEQ ID NO：28) CT Th： Ala-Leu-Asn-Ile-Trp-Asp-Arg-Phe-Asp-Val-Phe-Cys-

Thr-Leu-Gly-Ala-Thr-Thr-Gly-Tyr-Leu-Lys-Gly-Asn-

Ser (SEQ ID NO：29) DT ₁Th： Asp-Ser-Glu-Thr-Ala-Asp-Asn-Leu-Glu-Lys-Thr-Val-

Ala-Ala-Leu-Ser-Ile-Leu-Pro-Gly-Ile-Gly-Cys

(SEQ ID NO：30) DT ₂Th： Glu-Glu-Ile-Val-Ala-Gln-Ser-Ile-Ala-Leu-Ser-Ser-

Leu-Met-Val-Ala-Gln-Ala-Ile-Pro-Leu-Val-Gly-Glu-

Leu-Val-Asp-Ile-Gly-Phe-Ala-Ala-Thr-Asn-Phe-Val-

Glu-Ser-Cys (SEQ ID NO：31) PF Th： Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Glu-Lys-Ala-

Ser-Ser-Val-Phe-Asn-Val-Val-Asn-Ser

(SEQ ID NO：32) SM Th： Lys-Trp-Phe-Lys-Thr-Asn-Ala-Pro-Asn-Gly-Val-Asp-

Glu-Lys-Ile-Arg-Ile (SEQ ID NO：33) TraT ₁Th：Gly-Leu-Gln-Gly-Lys-Ile-Ala-Asp-Ala-Val-Lys-Ala-

Lys-Gly (SEQ ID NO：34) TraT ₂Th：Gly-Leu-Ala-Ala-Gly-Leu-Val-Gly-Met-Ala-Ala-Asp-

Ala-Met-Val-Glu-Asp-Val-Asn (SEQ ID NO：35) TraT ₃Th：Ser-Thr-Glu-Thr-Gly-Asn-Gln-His-His-Tyr-Gln-Thr-

Arg-Val-Val-Ser-Asn-Ala-Asn-Lys (SEQ ID NO：36)

Preferably, the Th epitope is BH_sTh，PT ₁ Th，PT ₂ Th，TT ₁ Th，TT ₃Th, or MV_F1Th。

In monomer line style peptide of the present invention, suc as formula (A)_n-(Th) _m-(B)。-(IgE CH ₄Peptide) described, the Th epitope is covalently attached to IgE CH by spacer region B₄On the N end of decapeptide (SEQ ID No:1). IgE CH₄Peptide is Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID No:1) decapeptide. IgE CH₄Peptide can substitute with an immunogenicity analog. Its immunogenicity analog can contain a replacement to about 4 amino acid residues, adds, and disappearance or insertion, prerequisite is that this analog can be induced the CH with IgE₄The immune response of decapeptide (SEQ ID No:1) cross reaction. Can the natural or alpha-non-natural amino acid that this paper limits be replaced, increase and insertion. Stanworth etc.^(3，4，5)Define IgE CH₄The immunogenicity analog of peptide (SEQ ID No:1), this paper quotes for your guidance.

Therefore, the preferred peptide immunogene of the present invention is to contain IgE CH₄The monomeric peptide of decapeptide (SEQ ID No:1) or its immunogenicity analog and Th. More particularly, preferred peptide immunogene is that those contain IgE CH₄(SEQ ID NO:1) or its immunogenicity analog; Introns (for example Gly-Gly); Be selected from BH_sTh，PT ₁ Th，PT ₂ Th，TT ₁ Th，TT ₃Th and MV_F1The Th epitope of Th (being respectively SEQ ID Nos:5,6,11,7,10,61); The line style construct in optional InV district (SEQ ID No:25). Preferred peptide immunogene composition comprises: for example, and peptide Nos.19-23 and 28 (table 5 and 6, SEQ ID Nos:51-55,62).

Peptide immunogene of the present invention can prepare with chemical synthesis well-known to those having ordinary skill in the art. For example, see Grant ed.Synthetic Peptides⁽²⁴⁾ Therefore, the immunogenic synthetic automatic Merrifield technology that can use solid phase to synthesize of peptide is with t-Boc or F.moc chemical method protection α-NH₂, the amino acid that uses side chain protected is for example being used biosystem peptide synthesizer 430A or 431 types carry out. So that synthetic peptide branch thereon uses Di-α, ε (t-Boc) lysine residue replaces the bad hydracid of t-Boc with 2,4-dichloro benzyl protection epsilon-amino for synthetic K core.

When A was aliphatic acid, the available carbodiimides method of knowing was added to the N end of resin binding peptide at an easy rate. In order to add Pam₃Cys, and chemical synthesis lipoamino acid S-(two (palmitin acyl-oxygen)-(the 2R)-propyl group of 2,3--N-palmitin acyl-(R)-cysteine (Pam₃Cys)). Then available solid-phase synthesis is with Pam₃Cys is coupled to peptide N end, in the end is coupled and uses Pam in the step₃Cys-OH is connected to lipoamino acid on the peptide chain of resin combination.

In order to improve the solubility of the lipopeptid product that finally is coupled, can prolong solid-phase peptide with another serine and lysine residue at the N end.

After finishing the immunogenic assembling of required peptide, according to the standard method process resin, cracking peptide and the functional groups on the amino acid side chain gone protection from the resin. The purifying of free peptide uses HPLC and carries out biochemistry and identify, for example, carries out propylhomoserin analysis or order-checking. The Purification and Characterization method of peptide is that those of ordinary skill in the art is known.

Produce line style Th-IgE CH of the present invention₄Other chemical method of decapeptide construct comprises by forming the connection halo acetylation of " thioether " key and cysteinyl peptide. For example, it is terminal that cysteine can be added to the peptide C that contains Th, the sulfydryl of cysteine be used for such as N^αChloracetyl lysine residue α that modify or that maleimide is derived-or ε-NH₂The electrophilic group of base forms covalent bond, and this lysine residue is attached to IgE CH₄The N-of decapeptide (SEQ ID No:1) or its immunogenicity analog is terminal.

These peptides also can get up in polymerization. Can be through using the conventional method NH of glutaraldehyde and lysine residue₂Reaction between the group realizes polymerization. Line style " A-Th-introns-IgE CH₄" peptide construct (for example, peptide Nos, 19-23 and 28, SEQ ID Nos:51-55 and 62) also can be added to line style " A-Th-introns-IgE CH through using an extra cysteine₄" the N end of construct carries out polymerization or copolymerization. N-end cysteine sulfydryl can be used for and the α that the halo chloracetyl is modified or maleimide is derived of lysine residue-or ε-NH₂Base forms " thioether " key, and this lysine residue is attached to poly lysyl core element (for example, the K of branch₂K，K ₄K ₂K，K ₈K ₄K ₂K) N end.

As selection, the available recombinant DNA technology of knowing is synthesized longer line style peptide immunogene. The detailed scheme of production peptide of the present invention all can be provided about any manual of standards of dna technique. In order to make up the gene of code book invention peptide, amino acid sequence can reversely be translated into nucleotide sequence, and the preferred the suitableeest codon of the biology of expressing this gene that uses uses. Then, if necessary, can prepare synthetic gene, typically through composite coding peptide and any overlapping oligonucleotides of regulating composition. Synthetic gene is inserted in the suitable cloning vector, obtain and identify recon. Then select to express under expression system and host's the condition this peptide in suitable being suitable for. With standard method Purification and Characterization peptide.

The immunogenic effectiveness of peptide of the present invention can through immunogene is administered to animal, then be monitored the CH to IgE₄Decapeptide (SEQ ID No:1) or the HI of its immunogenicity analog are set up, such as the detailed description of carrying out among the embodiment. Suitable animal comprises mouse, rat, rabbit, cavy, pig, goat, sheep or similar animal.

Another aspect of the present invention provides one or more peptide immunogenes that comprise effective dose of the present invention vaccine composition in the acceptable transmission system pharmaceutically. These vaccine compositions are used for prevention and comprise allergic rhinitis, food hypersenstivity reaction, asthma, allergic reaction and other anaphylactoid atopic hypersensitivity reaction of inducing such as other IgE of the asthma of virus induction.

Therefore, this peptide immunogene can be used adjuvant, and medicinal upper acceptable carrier or other conventional other composition that provides in the vaccine composition is mixed with the vaccine composition. This class prescription those of ordinary skill in the art is easy to determine and comprises for discharging immediately and/or lasting release, with be used for the inducible system immunity and/or induce the prescription of the mucous membrane immunity of location, it can be through for example realizing with the immunity of particulate embedding is original. Prescription also can comprise adjuvant or emulsifying agent, such as alum, and incomplete FreundShi adjuvant, Liposyn, saponarin, squalene, L₁₂₁, emulsifying agent and ISA720 and analog.

Vaccine of the present invention can be subcutaneous, oral through comprising, in the muscle or any conventional route medication of other parenteral or intestines approach. Can be used as single dosage or a plurality of dosage medication. Those of ordinary skill in the art is easy to determine immune method.

Vaccine composition of the present invention comprise effective dose one or more contain IgE CH₄The synthetic peptide based immunogens of decapeptide or its immunogenicity analog and medicinal upper acceptable carrier. The every kilogram of body weight that can be mixed with dosage unit form contains about 0.5 μ g to every kind of peptide of about 1mg. When discharging with multiple dose, can easily effective dose be divided into the per unit dosage form and contain suitable amount.

Vaccine composition of the present invention can be made into and contain the immunogenic mixture of two or more these peptides strengthening the immune efficacy in large group more, thereby better anti-IgE CH is provided₄The immune response of decapeptide. For example, peptide Nos.19,20,21,23 and 4 mixture is useful. Said composition also can be made into and contain lipopeptid so that inherent adjuvant to be provided. To containing immunogenic synthetic IgE CH₄The immune response of decapeptide also can be by being embedded in O ' Hagan etc.⁽²⁵⁾Transmit in the biodegradable particulate of described type or on it and improve. Immunogene can be made into capsule, uses or does not use adjuvant, comprises the Pam of covalent attachment₃Cys, this microparticle portability immunostimulation adjuvant is such as FreundShi Freunds incomplete adjuvant or alum. Microparticle performance function stimulates the immune response that immunogene is comprised the mucous membrane immunity of location. The immunity of this location needs especially to for example allergic reaction of mucous membrane location. The vaccine composition of microparticle form also can be made into provides the time control that continues or periodically react to discharge, and is used for oral medication, and surperficial medication^(25-26)。

The below provides the immunogenic embodiment of concrete peptide with explanation the present invention and is used for limiting the scope of the invention.

Embodiment 1

Eight mer peptides are immunogenic synthetic

Eight mer peptides: peptide No.1 (LysThrLysGlySerGlyPhePheValPheGlyProGlyLysThrLysGlySer GlyPhePheValPheGlyLysMet) below synthetic₈Lys ₄Lys ₂Lys, (SEQ ID No:23) peptide No.2 (LysThrLysGlySerGlyPhePheValPheGlyProGlyLysThrLysGlySer GlyPhePheValPheGlyProGlyLysThrLysGlySerGlyPhePheValPhe GlyLysMet)₈Lys ₄Lys ₂Lys， (SEQ ID No：13)

Limited order propagation through three functional aminos is carried out the synthetic of polymer peptide, and three functional aminos are as low-molecular-weight matrix core, and it is the basis that forms branch polymer peptide antigen systems. Three functional amino Boc-Lys (Boc) or two-(Boc)-Lys is optimum, because N^α-and N^ε-amino acid group can be used as reactive terminal and obtains. Therefore, two-(Boc)-order of Lys propagation can produce 2ⁿReactive end.

For example, two-(Boc)-Lys is coupled to two reactive amino ends that can produce on the solid-phase resin in conjunction with two peptide chains for the first time. Therefore for two-(Boc)-Lys sequentially produced for the second, the third and fourth step and produce respectively tetravalence, octavalence and ten sexavalence ends are used in conjunction with polymer peptide chain antigen. This class polymer peptide is useful as immunogene. Eight mer peptides Nos.1 of described branch and 2 are as immunogene above synthetic. Branch antigen contains seven little lysyl cores, and this core is surrounded around the highdensity even peptide antigen of one deck of core matrix. This design is different from and contains such as the large-scale protein carrier of PPD or KLH and be randomly distributed in the conventional peptide of the little peptide antigen on protein carrier surface-carrier attachment antigen with many not finite forms.

Phenylhydroxylamine and the tBoc-chemical bond of the combination of the immunogenic synthetic employing Boc amino-acid resin of eight mer peptides. For example, prepare at Biosearch 9500 instruments on 0.14nmol/g MBHA (the 4-aminomethyl phenyl azanol) resin of seven lysyl core resins of 8 branches through two-t-Boc Lys being coupled to utmost point underload. Two-(Boc)-two couplings circulation of Lys after, then use the 0.3M acetyl imidazole that is dissolved in the dimethyl formamide to carry out capping twice in every circulation.

Two other, two-(Boc)-Lys conjugate is added to first pair (NH₂) on the Lys resin. Then measure the substitution level of synthetic eight polyimide precursor resins through ninhydrin test, find the freedom-NH with proper level according to theoretical coupling productive rate₂Base, subsequently according to standard t-Boc program for the synthesis of eight mer peptides immunogenes.

Acid unstability tertbutyloxycarbonyl (t-Boc) is for the protection of the N-alpha amino acid. Functional side blocking group below using: the O-benzyl is used for Thr, Ser, Glu and Tyr; N^δ-tosyl is used for Arg; BOM is BOC-N^im-benzyloxy methyl is used for His, N^ε-dichloro benzyloxycarbonyl group is used for Lys; The S-4-methyl-benzyl is used for Cys; The O-cyclohexyl is used for Asp, and CHO is used for Trp.

Press the described continuous amino acid that adds peptide No.1 and No.2 of from C to N, holding of sequence of peptide Nos.1 and 2 (SEQ ID Nos:23,13). The gained eight mer peptides resins of peptide No.1 and peptide No.2 under the condition that the 10%v/v anisole exists at 0 ℃ with anhydrous HF cracking 1 hour. The polymer antigen that discharges extracts with acetic acid, and also freeze-drying is extremely dry to wash twice with ether. The polymer peptide of freeze-drying is as immunogene.

Embodiment 2

Use CFA and IFA to make adjuvant and carry out active immunity with eight mer peptides immunogenes of branch

(a) immune method:

According to following method, with two IgE CH₄-relevant polymer peptide immunogene (peptide Nos.1 with are connected) with the peptide No.3 that is connected with KLH (SEQ ID No:1) immunity respectively organize cavy (every group of N=3): the immunogenic hypodermic injection of peptide that mixes with incomplete FreundShi adjuvant (IFA) to each animal via hypodermic injection peptide immunogene mixture (200 μ L) or with the attachment (100 μ g/ml) of isopyknic complete FreundShi adjuvant (CFA) emulsification is the 21st, 42, and repeated in 63 days.

(b) through measuring its anti--IgE CH₄Relevant reactive polypeptide test cavy immune serum

It is active that the flat little titer plate in 96 holes that use covers with corresponding immunogene is measured anti-peptide antibody through ELISA (EUSA). Concentration be 5 μ g/ml the peptide immunogen solution equal portions sample (100 μ L) 37 ℃ the insulation 1 hour. It is dull and stereotyped to be incubated sealing in 1 hour with 3% gelatin/PBS solution at 37 ℃ again. Then dry sealing is dull and stereotyped also for test. Beginning subsequently with 1: 10 dilution factor in the Sample Dilution buffer solution, the sample aliquot of the test GPS of 10 times of serial dilutions (100 μ L) joins on the flat board of peptide covering. Dull and stereotyped 37 ℃ of insulations 1 hour. Normal GPS is with comparing.

Use 0.05%PBS/Tween^_Dull and stereotyped 6 times of buffer solution washing. Be added in and connect in the dilution buffer liquid (contain 0.5MNaCl, and the phosphate buffer of normal lowlenthal serum) 1: 1, the anti-cavy antibody of goat of 100 μ L horseradish peroxidase marks of 000 dilution. Be incubated 1 hour by dull and stereotyped before the above-mentioned washing at 37 ℃. Then the sample aliquot (100 μ L) that adds the o-phenylenediamine substrate solution. Develop the color and add 50 μ L2N H after 5-15 minute₂SO ₄Stop the enzymatic color reaction. Read the A492nm of each hole content at dull and stereotyped reader.

By the immunogene of ELISA inhibition test discovery branch polymer form, peptide No.1 and its closely-related peptide No.2 that derives are inducing the CH to IgE₄Target sequence (SEQ ID No: 1) effective in the specific antibody. These results and contrast immunogene, monomeric peptide No.3 (IgE CH₄Decapeptide SEQ ID No:1) KLH attachment is compared demonstration, and these two polymer peptide antigens produce than the higher levels of antibody titer of KLH attachment.

The successful result of these immunization experiments shows owing to insert Cly-Lys-Met and produced Th epitope (see SEQ ID Nos: 23 and 13, peptide Nos.1 and 2) and show that to pass immune system be important to some orientation to effectively being in the polymer system at peptide sequence C end. Other experiment shows: only with other orientation produce 8 or even the 16-branch contain IgE CH₄The IgE peptide immunogene of decapeptide (SEQ ID No:1) or its a plurality of repetitions is being induced anti-IgE CH₄Invalid in the reaction. In fact, in the polymer construct of 19 branches altogether, peptide Nos.1 and 2 is that only table reveals the peptide that immunogenicity strengthens. About this point, need experiment with polymer peptide Nos.1 and 2 high immunogenicities of observing, and be that those skilled in the art is unpredictable.

In addition, acquired results shows the short IgE CH of insertion₄Intervening sequence between the fragment, namely freely to present for the epitope that allows the subunit sequence to provide be essential to Gly-Pro-Gly. Find that also inserting introns at the C end before being connected to the lysine core resin of branch is that Gly-Lys-Met is to the IgE CH of polymer branch₄The immunogenicity of the synthetic construct of decapeptide (SEQ ID No:1) is essential.

Embodiment 3

With line style immunogene (SEQ ID Nos:15-22) immune rat

A. immunogene preparation: through solid-phase synthesis synthetic peptide based immunogens A-H (table 3), use the F-moc chemical method to carry out at application biosystem peptide synthesizer 430A or 431 types according to producer's explanation. After finishing the assembling of peptide, according to the standard method process resin, cracking peptide and make the functional groups on the amino acid side chain go protection from the resin. Peptide immunogene structure from amino terminal to the carbonyl end is as follows: peptide immunogene A is the line style peptide that 3 functional areas are arranged: 3 lysine residues (3K), hepatitis B surface antigen helper cell epitope (HB_sThe Th epitope) and IgE CH₄Peptide. Therefore, peptide immunogene A is with 3K-HB_sTh-IgE CH ₄Peptide represents. The actual sequence of peptide immunogene A and peptide immunogene B-H represents (SEQ ID Nos:15-22) in table 5.

For in the immunity of the 0th, 2 and 5 weeks, dissolve each peptide immunogene and merge the final concentration that produces 0.5mg/ml with assist agent solution (complete FreundShi adjuvant, not exclusively FreundShi adjuvant, or 0.2% alum). Solution be stored in 4 ℃ for subsequent use, the injection before the rotation 3 to 5 minutes. 100 μ g are accepted in every rat per injection.

B. immune programme for children and serum are collected:

Subcutaneous (S.C.) immune Sprague-Dawley rat (n=5). The 2nd the week and the 5th week gave the S.C booster shots. The 3rd, 6,7 and 11 weeks were collected blood.

Through intraperitoneal injection 1mL, in 0.9%NaCl, dilute 1 to 10 times yellow Jackets (64.8mg/mL; Anthony Products Co., Accadia (A) behind the anesthetized rat, carries out blood from central arteria caudalis and collects. Tail left standstill in 48 ℃ ± 0.5 ℃ water 2 minutes and massaged (i.e. extruding) with paper handkerchief rapidly. Blood is collected immediately in the 5ml syringe that No. 23 syringe needles are housed. Typically, obtain 2 to 2.5ml blood. Collected serum in centrifugal 25 minutes with 3000rpm. Serum is divided into the equal portions of 300 μ L volumes and refrigerated storage until be used for the ELISA test.

Table 3

Peptide immunogene A-H sequence

Peptide immunogene amino acid sequence A 3K-HB_sTh-IgECH ₄ Lys-Lys-Lys-Phe-Phe-Leu-Leu-Thr-Arg-

Ile-Leu-Thr-Ile-Pro-Gln-Ser-Leu-Asp-

Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-

Phe

(SEQ ID No：15) B PT ₁Th-IgECH ₄ Lys-Lys-Leu Arg-Arg-Leu-Leu-Tyr-Mel-

Ile-Tye-Met-Ser-Gly-Leu-Ala-Val-Arg-

Val-His-Val-Ser-Lys-Glu-Glu-Gln-Tyr-

Tyr-Asp-Tyr-Lys-Thr-Lys-Gly-Ser-Gly-

Phe-Phe-Val-Phe

(SEQ ID No：16) C PT _1ATh-IgECH ₄ Tyr-Met-Ser-Gly-Leu-Ala-Val-Arg-Val-

His-Val-Ser-Lys-Glu-Glu-Lys-Thr-Lys-

Gly-Ser-Gly-Phe-Phe-Val-Phe

(SEQ ID No：17) D TT ₁Th-IgECH ₄ Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-

Lys-Phe-Ile-Gly-Ile-Thr-Glu-Leu-Lys-

Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe

(SEQ ID No：18) E TT ₂Th-IgECH ₄ Lys-Lys-Phe-Asn Asn-Phe-Thr Val-Ser-

Phe-Trp-Leu-Arg-Val-Pro-Lys Val-Ser-

Ala-Ser-His-Leu-Lys-Thr-Lys GLy-Ser-

Gly-Phe-Phe-Val-Phe

(SEQ ID No：19) F TT ₃Th-IgECH ₄ Tyr-Asp-Pro-Asn-Tyr-Leu-Arg-Thr-Asp-

Ser-Asp-Lys-Asp-Arg-Phe-Leu-Gln-Thr-

Met-Val-Lys-Leu-Phe-Asn-Arg-Ile-Lys-

Lys-Thr-Lys Gly-Ser-Gly-Phe-Phe-Val-

Phe

(SEQ ID No：20) G PT ₂Th-IgECH ₄ Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-Gly-

Thr-Arg-Ala-Leu-Thr-Val-Ala-Glu-Leu-

Arg-Gly-Asn-Ala-Glu-Leu-Lys-Thr-Lys-

Gly-Ser-Gly-Phe-Phe-Val-Phe

(SEQ ID No：21) H MV _F1Th-IgECH ₄ Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-

Arg-Leu-Glu-Gly-Val-Leu-Lys-Thr-Lys

Gly Ser-Gly-Phe-Phe-Val-Phe

(SEQ ID No：22)

Embodiment 4

With line style immunogene (SEQ ID Nos:37-50) immune rat line style peptide immunogene with A-Th-GG-IgE CH₄Expression, wherein A can be NH₂-, Lys-Lys (2K), Lys-Lys-Lys (3K) or invasion functional areas (Inv) (SEQ ID No:25), Th are the auxiliary peptides of T, GG is the Gly-G1y introns, IgE CH₄Be target decapeptide (SEQ ID No:1), press embodiment 3 described synthetic A-Th-GG-IgE CH₄ This this peptide immunogene represents with peptide immunogene Nos.4-17 (SEQ ID Nos:37-50) in table 4. These peptides synthetic and cracking are used for immune rat with the test drug effect.

With the experiment immunization evaluate alternatives listed the below drug effect to 5 rats in each group.

Experimental design:

Immunogene: peptide Nos.4-17 (at every turn testing 1)

Dosage: each immune 100 μ g

Approach: in the muscle.

Adjuvant: FreundShi fully/Freunds incomplete adjuvant

Dosage: the 0th (FCA), 3 and 6 (IFA) weeks

The blood drawing scheme: the 0th, 3,6,8,10 weeks

Kind: Sprague-Dawley rat

Group size: 5

Test: the ELISA of antipeptide activity, Solid phase Immunoadsorbent is IgE CH₄The decapeptide order

The monomeric peptide No.3 of row (SEQ ID No:1).

Collect blood, add 2 one-tenths serum, store to carrying out titration by embodiment 2 is described through ELISA, except the goat of using the horseradish peroxidase mark anti--the anti-cavy IgG of rat IgG antibody replacement goat makes tracer.

Table 4

Peptide immunogene Nos.4-17 sequence

Peptide immunogene amino acid sequence 4 TT₁Th-GG-IgECH ₄ Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-

Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-

Leu-Gly-Gly-Lys-Thr-Lys-Gly-Ser-

Gly-Phe-Phe-Val-Phe

(SEQ ID No：37) 5 TT ₂Th-GG-IgECH ₄ Lys-Lys-Phe-Asn-Asn-Phe-Thr Val-

Ser-Phe-Trp-Leu-Arg-Val-Pro-Lys-

Val-Ser-Ala-Ser-His-Leu-Gly-Gly-

Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-

Val Phe

(SEQ ID No：38) 6 PT _1ATh-GG-IgECH ₄ Tyr-Met-Ser-Gly-Leu-Ala-Val-Arg-

Val-His-Val-Ser-Lys-Glu-Glu-Gly-

Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-

Phe-Val-Phe

(SEQ ID No：39) 7 MV _F2Th-GG-IgECH ₄ Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-

Lys-Ala-Arg-Ile-Thr-His-Val-Asp-

Thr-Glu-Ser-Tyr-Gly-Gly-Lys-Thr-

Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe

(SEQ ID No：40) 8 TT ₄Th-GG-IgECH ₄ Trp-Val-Arg-Asp-Ile-Ile-Asp-Asp-

Phe-Thr-Asn-Glu-Ser-Ser-Gln-Lys-

Thr-Gly-Gly-Lys-Thr-Lys-Gly-Ser-

Gly-Phe-Phe-Val-Phe

(SEQ ID No：41) 9 TT ₅Th-GG-IgECH ₄ Asp-Val-Ser-Thr-Ile-Val-Pro-Tyr-

Ile-Gly-Pro-Ala-Leu-Asn-His-Val-

Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-

Phe-Phe-Val-Phe

(SEQ ID No：42) 10 CTTh-GG-lgECH ₄ Ala-Leu-Asn-Ile-Trp-Asp-Arg-Phe-

Asp-Val-Phe-Cys-Thr-Leu-Gly-Ala-

Thr-Thr-Gly-Tyr-Leu-Lys-Gly-Asn-

Ser-Gly-Gly-Lys-Thr-Lys-Gly-Ser

Gly Phe-Phe-Val-Phe

(SEQ ID No：43) 11 DT ₁Th-GG-IgECH ₄ Asp-Ser-Glu-Thr-Ala-Asp-Asn-Leu-

Glu-Lys-Thr-Val-Ala-Ala-Leu-Ser-

Ile-Leu-Pro-Gly-Ile-Gly-Cys-Gly-

Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-

Phe-Val-Phe

(SEQ ID No：44) 12 DT ₂Th-Gg-IgECH ₄ Glu-Glu-Ile-Val-Ala-Gln-Ser-Ile-

Ala-Leu-Ser-Ser-Leu-Met-Val-Ala-

Gln-Ala-Ile-Pro-Leu-Val-Gly-Glu-

Leu-Val-Asp-Ile-Gly-Phe-Ala-Ala-

Thr-Asn-Phe-Val-Glu-Ser-Cys Gly-

Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-

Phe-Val-

(SEQ ID No：45) 13 PFTh-GG-IgECH ₄ Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-

Met-Glu-Lys-Ala-Ser-Ser-Val-Phe-

Asn-Val Val-Asn-Ser-Gly-Gly-Lys

Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-

Phe

(SEQ ID No：46) 14 SMTh-GG-IgECH ₄ Lys-Trp-Phe-Lys-Thr-Asn-Ala-Pro-

Asn-Gly-Val-Asp-Glu-Lys-Ile-Arg-

lle-Gly-Gly-Lys-Thr-Lys-Gly-Ser-

Gly-Phe-Phe-Val-Phe

(SEQ ID No：47) 15 TraT ₁Th-GG-IgECH ₄ Gly-Leu-Gln-Gly-Lys-Ile-Ala-Asp-

Ala-Val-Lys-Ala-Lys-Gly-Gly-Gly-

Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-

Val-Phe

(SEQ ID No：48) 16 TraT ₂Th-GG-IgECH ₄ Gly-Leu-Ala-Ala-Gly-Leu-Val-Gly-

Met-Ala-Ala-Asp-Ala-Met-Val-Glu-

Asp-Val-Asn-Gly-Gly-Lys-Thr-Lys-

Gly-Ser-Gly-Phe-Phe-Val-Phe

(SEQ ID No：49) 17 TraT ₃Th-GG-IgECH ₄ Ser Thr-Glu-Thr-Gly-Asn-Gln-His-

His-Tyr-Gln-Thr-Arg-Val-Val-Ser-

Asn-Ala-Asn-Lys-Gly-Gly-Lys-Thr-

Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe-

(SEQ ID No：50)

Embodiment 5

With line style immunogene (SEQ ID Nos:51-56,62) and reversed polarity

Line style immunogene (SEQ ID Nos:57-60) immune rat

Press the peptide immunogene Nos:18-23 (SEQ ID Nos:51-56) shown in the embodiment 3 described synthetic tables 5. The general formula of peptide immunogene NoS18-23 can be used A-Th-GG-IgE CH₄Representative, wherein A is N end Lys-Lys (2K), Lys-Lys-Lys (3K) or be spaced apart the invasion functional areas (Inv) (SEQ ID No:25) that district GG and Th sequence are separated; Th is selected from HBsTh, PT₁Th，PT ₂Th，MV _F1Th, or TT₃Th; GG is the Gly-Gly spacer region; IgECH₄IgE CH₄Decapeptide (SEQ ID No:1).

Also pressing the mode identical with peptide 18-23 as the peptide immunogene with SEQ ID Nos:57-60 shown in the peptide 24-27 in table 5 synthesizes. These peptides can be used IgECH₄-GG-Th represents. These peptides are with regard to IgECH₄Decapeptide, spacer region and Th sequence are equivalent to peptide number 19,20,21,23 (table 5), except decapeptide/Th polarity opposite, i.e. IgE CH₄Decapeptide (SEQ ID No:1) is at the N end, and Th is positioned at the C end.

These peptides exempt to hold former described for immune rat by following experimental program, are used for comparing and confirming its drug effect.

Experimental design:

Immunogene: peptide number: 18-28 (1 every group) (SEQ ID No:51-60 and 62)

Dosage: each immune 100 μ g

Approach: in the muscle

Adjuvant: FreundShi fully/Freunds incomplete adjuvant is used for peptide 18-27,0.4% alum

Be used for peptide numbers 28

Dosage: at the 0th (FCA), 3 and 6 weeks (IFA) were used peptide 8-27, the 0th,

3, and 6 weeks alum was used for peptide numbers 28.

The blood drawing scheme: the 0th, 3,6,8,10 weeks.

Kind: Sprague-Dawley rat

Group size: 5 are used for peptide 27-28, and 4 are used for peptide numbers 28

Test: the ELISA of antipeptide activity, solid phase substrate are peptide number 3 (SEQ ID No:1).

Collect blood, be processed into serum, storage is described with the ELISA titration to pressing embodiment 2, except the goat Chinese People's Anti-Japanese Military and Political College mouse IgG antibody with the horseradish peroxidase mark replaces anti-cavy IgG as tracer. Test whole serum with anti-peptide ELISA, those samples of the A492nm value that produces during with dilution in 1: 100 〉=0.2 are registered as seropositivity.

With anti-peptide ELISA estimate the immune efficacy of peptide immunogene Nos.1 8-28 (SEQ ID Nos:51-60, and 62) and in table 6 with in the reaction to experiment immunization, every group of 4 or 5 rats are transformed into the CH to IgE in the 6th and 8 weeks₄The number that peptide No.3 produces seropositivity reaction (that is A492mm 〉=0.2 when, diluting with 1: 100) represents.

The polarity Th-GG-IgE CH of present embodiment₄Peptide immunogene (peptide 18-23 and 28SEQ ID Nos:51-56 and 62) to inducing anti-IgE CH₄The antibody of decapeptide (peptide No.3, SEQ ID No:1) demonstrates obvious effectiveness. Demonstrate compared with the control with all 6 groups of rats of this polarity peptide immunogene (peptide 18-23,28) immunity and obviously to change seropositivity into. Till the 6th week, each organizes seroconversion prevalence rate scope is 1/5 to 5/5, between the 6th week and 8 weeks to the immunogenic reaction of dosage for the third time in the seroconversion prevalence rate continue to rise. Contain the peptide immunogene No.18 of HBsTh peptide sequence, have MV_F1The peptide immunogene No.19 of Th peptide and contain PT₁The peptide of Th peptide sequence numbers 28 is the most effective, and till the 8th week, its seroconversion prevalence rate is respectively 4/5,5/5 and 4/4. Relatively the immunogenicity of peptide immunogene Nos.21 and 22 (SEQ ID Nos:54,55) confirms till Inv district's 8 weeks of peptide to the containing PT₂The immunostimulation ability of Th peptide (table 6) obviously improves.

In contrast, the similar peptide immunogene (peptide immunogene number: 24-27, SEQ ID Nos:57-60) that has opposite Th polarity changes rat blood serum and does not demonstrate obvious immune efficacy. This weak immune efficacy shows Th-GG-IgE CH₄Amino is crucial to c-terminus polarity for the immunogenic immunogenicity of line style peptide of the present invention. A kind of orientation of target peptide and Th is better than the determining that those skilled in the art pushes away and do not measure of effectiveness of another kind of orientation, and is unpredictable.

Table 5

The sequence of peptide immunogene Nos.18-28

Peptide immunogene amino acid sequence 18 3K-HB_sTh-GG-IgECH ₄ Phe-Phe-Leu-Leu-Thr-Arg-Ile-Leu-

Thr-Ile-Pro-Gln-Ser-Leu-Asp-Gly-

Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-

Phe-Val-Phe

(SEQ ID No：51) 19 MV _F1Th-GG-IgECH ₄ Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-

Val-His-Arg-Leu-Glu-Gly-Val-Gly-

Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-

Phe-Val-Phe

(SEQ ID No：52) 20 PT ₁Th-GG-IgECH ₄ Lys-Lys-Leu-Arg-Arg-Leu-Leu-Tyr-

MeL-Ile-Tyr-Met-Ser-Gly-Leu-Ala-

Val Arg-Val-His-Val-Ser-Lys-Glu-

Glu-Gln-Tyr-Tyr-Asp-Tyr-Gly-Gly-

Lys-Thr Lys-Gly-Ser-Gly-Phe-Phe-

Val-Phe

(SEQ ID No：53) 21 PT ₂Th-GG-IgECH ₄ Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-

Gly-Thr-Arg-Ala-Leu-Thr-Val-Ala-

Glu-Leu-Arg-Gly-Asn-Ala-Glu-Leu-

Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-

Phe-Phe-Val-Phe

(SEQ ID No：54) 22 Tnv-GG-PT ₂Th-GG-IgECH ₄ Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-

Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe-

Gly-Gly-Gly-Ala-Tyr-Ala-Arg-Cys-

Pro-Asn-Gly-Thr-Arg-Ala-Leu-Thr-

Val-Ala-Glu-Leu-Arg-Gly-Asn-Ala-

Glu-Leu-Gly-Gly-Lys-Thr-Lys-Gly-

Ser Gly-Phe-Phe-Val-Phe

(SEQ ID No：55) 23 TT ₃Th-GG-IgECH ₄ Tyr-Asp-Pro-Asn-Tyr-Leu-Arg-Thr-

Asp-Ser-Asp-Lys-Asp-Arg-Phe-Leu-

Gln-Thr-Met-Val-Lys-Leu-Phe-Asn-

Asp-Arg-Phe-Leu-Gln-Thr-Met-Val-

Lys-Leu-Phe-Asn-Arg-Ile-Lys-Gly

Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-

Phe-Val-Phe

(SEQ ID No：56) 24 IgECH ₄-GG-MV _F1Th Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-

Val-Phe-Gly-Gly-Leu-Ser-Glu-Ile-

Lys-Gly-Val-Ile-Val-His-Arg-Leu-

Glu-Gly-Val

(SEQ ID No：57) 25 IgECH ₄-GG-PT ₁Th Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-

Val-Phe-Gly-Gly-Lys-Lys-Leu-Arg-

Arg-Leu-Leu-Tyr-Met-Ile-Tyr-Met-

Ser-Gly-Leu-Ala-Val-Arg-Val-His-

Val-His-Lys-Glu-Glu-Gln-Tyr-Tyr-

Asp-Tyr

(SEQ ID No：58) 26 IgECH ₄-GG-PT ₂Th Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-

Val-Phe-Gly-Gly-Gly-Ala-Tyr-Ala-

Arg-Cys-Pro-Asn-Glu-Thr-Arg-Ala-

Leu-Thr-Val-Ala-Glu-Leu-Arg-Gly-

Asn-Ala-Glu-Leu

(SEQ ID No：59) 27 IgECH ₄-GG-TT ₃Th Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-

Val-Phe-Gly-Gly-Tyr-Asp-Pro-Asn-

Tyr-Leu-Arg-Thr-Asp-Ser-Asp-Lys-

Asp-Arg-Phe-Leu-Gln-Thr-Mel-Val-

Lys-Leu-Phe-Asn-Asp-Arg-Phe-Leu-

Gln-Thr-Met-Val-Lys-Leu-Phe-Asn-

Arg-Ile-Lys

(SEQ ID No：60) 28 PT ₁Th-IgECH ₄ Lys-Lys-Leu-Arg-Arg-Leu-Leu-Tyr-

Met-Ile-Tyr-Met-Ser-Gly-Leu-Ala-

Val-Arg-Val-His-Val-Ser-Lys-Glu-

Glu-Gln-Tyr-Tyr-Asp-Tyr-Lys-Thr-

Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe-

(SEQ ID No：62)

Table 6

The animal of seroconversion/group^*

The peptide immunogene

The 6th week the 8th all 18 3K-HB_STh-GG-IgECH ₄ 4 4 (SEQ ID No：51) 19 MV _F1Th-GG-IgECH ₄ 5 5 (SEQ ID No：52) 20 PT ₁Th-GG-IgECH ₄ 2 3 (SEQ ID No：53) 21 PT ₂Th-GG-IgECH ₄ 1 1 (SEQ ID No：54) 22 Inv-GG-PT ₂-GG-IgECH ₄ 1 3 (SEQ ID No：55) 23 TT ₃ Th-GG-IgECH ₄ 3 3 (SEQ ID No：56) 24 IgECH ₄-GG-MV _F1Th 0 0 (SEQ ID No：57) 25 IgECH ₄-GG-PT ₁Th 1 1 (SEQ ID No：58) 26 IgECH ₄-GG-PT ₂Th 0 0 (SEQ ID No：59) 27 IgECH ₄-GG-TT ₃Th 0 0 (SEQ ID No：60) 28 PT ₁Th-IgECH ₄Every group of 5 animals of 44 (SEQ ID No:62) Control Immunization 00 (No peptide) * are used for peptide Nos.18-27, and 4 animals are used for peptide No.28

Embodiment 6

Further enlarge the line style immunogene mixture of reaction colony

Many different IgE CH that contain₄The foundation of the relative effectivenes of the line style construct of decapeptide and Th (embodiment 3-5) allows to select useful peptide immunogene preparation polyvalent vaccine composition. Carry the protein from measles virus F, each Th-GG-IgECH of any Th peptide of the immunodominance of tetanus toxin and pertussis toxin₄Construct (peptide 19-23) is being induced IgECH through studies confirm that of embodiment 5₄Decapeptide SEQ ID No:1) effective in the antibody response. The composition that contains these line style peptide mixtures can provide required maximum immunogenicity in genetic diversity colony.

In rat, estimate to be made into to comprise with embodiment 5 described methods and have preferred " A-Th-GG-IgECH₄" synthetic peptide of polarity, the immune efficacy of these compositions of the mixture of peptide immunogene number 19,20,21,23 (table 5) and peptide immunogene number 4 (tables 4, embodiment 4). Every animal in one group of 5 rat is waited a mole preparation, i.e. every kind of peptide 20 μ g immunity with 5 kinds of peptides of 100 μ g dosage. Change the reactive rat number of seropositivity till the 5th week and the 8th week in two time intervals into and be 5 merely hit 5 (namely 100%).

This result shows that the vaccine that contains the immunogenic mixture of peptide of the present invention provides improved immunogenicity. Shown that also this mixture and the cocktail analog that is comprised of different effective peptides induce the potentiality of immunization therapy antibody response in the genetic diversity human colony.

Embodiment 7

With effective line style immunogene mixture immunity

Contain IgE CH₄The foundation of the relative effectivenes of the construct (embodiment 3-5) of the many different line style of decapeptide and Th allows to select useful peptide to be used for the immunogene mixture. In the immunogene mixture carry the Gly-Gly spacer region and from measles virus F protein, HBsAg, each construct of any Th peptide of tetanus toxin and pertussis toxin confirm it is effectively (table 6). Therefore these confirm that the immunogenic mixture of line style peptide that effectively has specific polarity can provide maximum immunogenicity in genetic diversity colony. Scheme confirmation below the design is mixed with and contains preferred " A-Th-GG-IgECH₄" effectiveness of composition of the present invention of synthetic peptide based immunogens mixture of construct. Experimental design:

Immunogene: (1) mixture 1: peptide numbers 18,19,20

(2) mixture 2: peptide numbers 18,19,22

(3) the KIH attachment of positive control-peptide 3 (every group of a kind of immunity of rat

Former)

Dosage: each immunity equals equimolar each synthetic peptide of 100 μ g or IgE CH₄Of equal value

Thing, or 33.3 each peptide of μ g.

Approach: in the muscle

Adjuvant: (1) FreundShi fully/Freunds incomplete adjuvant

(2) 0.4% alum (aluminium hydroxide) (every group of a kind of adjuvant of each immunogene)

Dosage: (CFA/IFA organized for the 0th week and accepted CFA, the 2nd He the 0th, 2 and 4 weeks

4 weeks were accepted IFA. All 3 kinds of dosage of alum group are accepted the alum preparation

Product).

Blood drawing scheme: the 0th, 3,6 and 8 weeks

Kind: Sprague-Dawley rat/group

Group size: 5,6 groups

Test: antipeptide activity ELISA, Solid phase Immunoadsorbent amount peptide No.3 (SEQ ID No

：1)

Collect blood, be processed into serum, and store to pressing embodiment 5 described through the ELISA titration.

Design the known immunogene of this experiment confirm immunogene of the present invention and prior art^(14，15)Compare the improvement of performance. This result is useful to estimating the immunogenic two kinds of mixtures of effective peptide that respectively contain three kinds of Th peptides, confirmed that (mixture 2 contains Inv for the serviceability of immunostimulation Inv functional areas, mixture 1 does not contain), and adjuvant, the effectiveness of alum in vaccine composition of the present invention.

Embodiment 8

Use the clinical testing of immunogene mixture

Contain IgE CH₄The foundation of the relative effectivenes of the many different constructs of decapeptide and Th (embodiment 3-5) allows to select representative peptide to be used for the immunogene mixture. In the immunogene mixture, carry the Gly-Gly introns and from measles virus F, hepatitis B surface antigen, each construct of tetanus toxin and pertussis toxin Th peptide sequence turns out to be effectively (table 6) and is random to various human HLA DR antigen, in order to provide maximum immunogenicity in genetic diversity people colony. And, be effective source of Th memory because these Th peptides from children's vaccine, are seeded among the immune human colony childhood. Therefore, children's vaccine has potentiality to the higher immune efficacy of antianaphylaxis vaccine of giving by this class Th peptide compositions of mixtures. Clinical scheme below the design confirms to be mixed with this class line style " A-Th-introns-IgE CH in extensive acceptable adjuvant (alum)₄Decapeptide " effectiveness of composition of the present invention of mixture of peptide immunogene. Experimental design:

Experimenter: hay fever patient

Season and during: hay fever season, 8 weeks

Group: 4 groups, 1 group/immunogene/dosage,

Every group of N=15 accepts immunogene for 12, accepts placebo for 3.

Immunogene: mixture 1: peptide numbers 18,19,20,23

Adjuvant: 0.2% alum

Dosage: equimolar each synthetic peptide of every dosage equals 500 μ g, or every kind of peptide 125 μ g.

Approach: in the muscle

The dosage time: the 0th and 4 weeks

The estimation time: the 0th, 4 and 0,6 week

Estimate effectiveness and the security of vaccine composition " cocktail 1 " with serum, test by dermoreaction, patient uses the utilization rate of hay fever medication, patient's allergic symptom and the physical examination of adverse reaction, and the access patient realizes the subjective assessment with this product effect with acquisition. Serological evaluation comprises the ELISA of aforementioned antagonism peptide titre, and the automatic light splitting fluorescent test of standard and the definite product of measuring the minimizing of histamine level do not trigger histamine release. Skin-test is intracutaneous test, wherein the anaphylactogen standard liquid is injected into the skin upper strata. The typical case who produces in the reaction to anaphylactogen after measured " bubble and speck " area quantitative is to the reaction of anaphylactogen. Allergic symptom obviously improved when the result of expectation was included in the research termination, did not have vaccine composition of the present invention to trigger the sign that histamine discharges.

Design this experiment confirm clinical effectiveness of the present invention. These results use acceptable adjuvant (" the A-Th-introns-IgE CH that contain 4 kinds of Th peptide sequences of alum preparation to medication₄Decapeptide " immunogenic mixture estimates.

Embodiment 9

In vitro test confirms IgE CH₄The effectiveness of decapeptide specific antibody

People's basocyte that the histamine that is used for knowing discharges the passive sensitization of test is used for external evaluation through using IgECH₄The antibody that the immunity of decapeptide immunogene is induced. Prepare human basocyte from volunteer's venous blood, then use benzylpenicillin-special IgE passive sensitization of human serum albumins attachment (BPO-HSA), it prepares from the donor blood that BPO-HSA specificity IgE is had the hyperimmune globulin associativity. The histamine of sensitization basocyte discharges the impact that is added BPO-HSA or IgE CH4 peptide No.3 (SEQ ID No:1). Before the reagent of histamine release is induced in adding, the anti-IgE CH of basocyte and serial dilution₄Control serum merges before the antiserum of decapeptide (SEQ ID No:1) or the immunity. Histamine with automatic fluorescent technique analytic sample discharges. From both, deduct after the spontaneous release and to calculate histamine from the ratio of sample and total basocyte histamine content and discharge percentage.⁽²⁷⁾External effectiveness has confirmed that experimental antiserum suppresses the ability that histamine discharges.

This test has confirmed IgE CH₄The ability that peptide No. (SEQ ID No:1) induces histidine to discharge in the concentration dependent mode. The result provides in table 7, has shown IgE CH₄Peptide number 3 (SEQ ID No:1) induce the histamine of people's basocyte to discharge and are used for confirming SEQ ID No:1 and corresponding antibody and the anaphylactoid correlation of people.

Table 7

The clean histamine of inducer % discharges^*

Peptide No.3

1 50μg/mL(1.3×10 ^-4M) 30％

60μg/mL(7×10 ^-5M) 13

6μg/mL(7×10 ^-6M) 2 BPO-HSA

0.1μg/mL 63％

^*Proofread and correct with deducting spontaneous histamine release 9%

Sequence table physical data: (i) applicant: United Biomedical, Inc.﹠WANG, Chang Yi (ii) invention exercise question: be used for the treatment of anaphylactoid synthetic peptide based immunogens (iii) sequence number: 62 (iv) contact address: (A) contact A:Maria C.H.Lin (B) street: 345 Park Avenue (C) cities: New York (D) state: NY (E) country: USA (F) postcode: 10154 (V) computer-reader form: (A) medium type: Floppy disk (B) computer: IBM PC compatible (C) operating system: PC-DOS/MS-DOS (D) software: WordPerfect 5.1 (vi) are application materials at present: (A) application number: (B) applying date: classification on March 24 nineteen ninety-five (C): the application materials (vii): (A) application number: US08/328,912 (B) applyings date: the application materials before on October 25th, 1994 (vii): (A) application number: US08/218,461 (B) applyings date: the application materials before on March 28th, 1994 (vii): (A) application number: US08/060,798 (B) applyings date: the application materials before on May 10th, 1993 (vii): (A) application number: US07/847,745 (B) applyings date: the application materials before on March 6th, 1992 (vii): (A) application number: US07/637,364 (B) applyings date: January 4 (viii) in 1991: lawyer/agent's data: (A) name: Maria C.H.Lin (B) registration number: 29,323 (C) are with reference to/summary number: 1151-4061US4 (ix) telecommunication data:: (A) phone: 212-758-4800 (B) fax: 212-751-6849 (2) SEQ ID NO:1 data:: (i) sequence signature:: (A) length: 10 (B) types: amino acid (C) chain: improper (D) topology: line style (ii) molecule type: peptide (x) documents and materials:: (A) author: Stanworth et al. (B) exercise question: The Role of Non-antigen Receptors

In Mast Cell Signalling Processes ( C ) ：Molecular Immunology ( D ) ：21 ( E ) ：12 ( F ) ：1183-1190 ( G ) ：1984 ( J ) ： ( K ) ：497 to 506 ( xi ) ：SEQ ID NO：1： Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 1 5 10 ( 3 ) SEQ ID NO：2：： ( i ) ：： ( A ) ：10 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：Polypeptideε-chain of human IgE ( x ) ：： ( A ) ：Dorrington and Bennich ( B ) ： ( C ) ：Immunology Review ( D ) ：41 ( E ) ：12 ( F ) ：3-25 ( G ) ：1978 ( xi ) ：SEQ ID NO：2： Val Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln

5 10 15 Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu

20 25 30 Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr

35 40 45 Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala

50 55 60 Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Gln Leu

65 70 75 Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys

80 85 90 Gln Val Thr Tyr Gln Gly His Thr Phe Gln Asp Ser Thr Lys Lys

95 100 105 Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg

110 115 120 Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr

125 130 135 Cys Leu Val Leu Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu

140 145 150 Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn Asn Ser Thr Arg

155 160 165 Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr

170 175 180 Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln

185 190 195 Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser

200 205 210 Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala

215 220 225 Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu

230 235 240 Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln

245 250 255 Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr

260 265 270 Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser

275 280 285 Arg Leu Glu Val Thr Arg Ala Glu Trp Gln Glu Lys Asp Glu Phe

290 295 300 Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln Thr Val

305 310 315 Gln Arg Ala Val Ser Val Asn Pro Gly Lys

320 325 (4) SEQ ID NO:3 data:: (i) sequence signature:: (A) length: 175 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: Polypeptide ε-chain of rat IgE (x) documents and materials:: (A) author: Kindsrogel et al. (B) exercise question: (C) periodical: DNA (D) volume: 1 (E) issue: (F) page or leaf: 335-343 (G) time: 1982 (xi): SEQ ID NO:3:Asn Leu Asn Ile Thr Gln Gln Gln Trp Met Ser Glu Ser Thr Phe

5 10 15 Thr Cys Lys Val Thr Ser Gln Gly Glu Asn Tyr Trp Ala His Thr

20 25 30 Arg Arg Cys Ser Asp Asp Glu Pro Arg Gly Val Ile Thr Tyr Leu

35 40 45 Ile Pro Pro Ser Pro Leu Asp Leu Tyr Glu Asn Gly Thr Pro Lys

50 55 60 Leu Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Glu Asn Ile Thr

65 70 75 Val Thr Trp Val Arg Glu Arg Lys Lys Ser Ile Gly Ser Ala Ser

80 85 90 Gln Arg Ser Thr Lys His His Asn Ala Thr Thr Ser Ile Thr Ser

95 100 105 Ile Leu Pro Val Asp Ala Lys Asp Trp Ile Glu Gly Glu Gly Tyr

110 115 120 Gln Cys Arg Val Asp His Pro His Phe Pro Lys Pro Ile Val Arg

125 130 135 Ser Ile Thr Lys Ala Leu Gly Leu Arg Ser Ala Pro Glu Val Tyr

140 145 150 Val Phe Leu Pro Pro Glu Glu Glu Glu Lys Asn Lys Arg Thr Leu

155 160 165 Thr Cys Leu Ile Gln Asn Phe Phe Pro Glu

170 175 (5) SEQ ID NO:4 data: (i) sequence signature:: (A) length: 315 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: mouse IgE polypeptide ε chain (x) documents and materials:: (A) author: Ishida et al. (B) exercise question: (C) periodical: EMBO (D) volume: 1 (E) issue: (F) page or leaf: 1117-1123 (G) time: 1982 (xi) sequence description: SEQ ID NO:4:Val Arg Pro Val Thr His Ser Leu Ser Pro Pro Trp Ser Tyr Ser

5 10 15 Ile His Arg Cys Asp Pro Asn Ala Phe His Ser Thr Ile Gln Leu

20 25 30 Tyr Cys Phe Ile Tyr Gly His Ile Leu Asn Asp Val Ser Val Ser

35 40 45 Trp Leu Met Asp Asp Arg Glu Ile Thr Asp Thr Leu Ala Gln Thr

50 55 60 Val Leu Ile Lys Glu Glu Gly Lys Leu Ala Ser Thr Cys Ser Lys

65 70 75 Leu Asn Ile Thr Glu Gln Gln Trp Met Ser Glu Ser Thr Phe Thr

80 85 90 Cys Arg Val Thr Ser Gln Gly Cys Asp Tyr Leu Ala His Thr Arg

95 100 105 Arg Cys Pro Asp His Glu Pro Arg Gly Ala Ile Thr Tyr Leu Ile

110 115 120 Pro Pro Ser Pro Leu Asp Leu Tyr Gln Asn Gly Ala Pro Lys Leu

125 130 135 Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Lys Asn Val Asn Val

140 145 150 Thr Trp Asn Gln Glu Lys Lys Thr Ser Val Ser Ala Ser Gln Trp

155 160 165 Tyr Thr Lys His His Asn Asn Ala Thr Thr Ser Ile Thr Ser Ile

170 175 180 Leu Pro Val Val Ala Lys Asp Trp Ile Glu Gly Tyr Gly Tyr Gln

185 190 195 Cys Ile Val Asp Arg Pro Asp Phe Pro Lys Pro Ile Val Arg Ser

200 205 210 Ile Thr Leu Pro Gln Val Ser Gln Arg Ser Ala Pro Glu Val Tyr

215 220 225 Val Phe Pro Pro Pro Glu Glu Glu Ser Glu Asp Lys Arg Thr Leu

230 235 240 Thr Cys Leu Ile Gln Asn Phe Phe Pro Glu Asp Ile Ser Val Gln

245 250 255 Trp Leu Gly Asp Gly Lys Leu Ile Ser Asn Ser Gln His Ser Thr

260 265 270 Thr Thr Pro Leu Lys Ser Asn Gly Ser Asn Gln Gly Phe Phe Ile

275 280 285 Phe Ser Arg Leu Glu Val Ala Lys Thr Leu Trp Thr Gln Arg Lys

290 295 300 Gln Phe Thr Cys Gln Val Ile His Glu Ala Leu Gln Lys Pro Arg

305 310 315 （ 6 ） SEQ ID NO：5：（ i ）：（ A ）：15 （ B ）：（ C ）：（ D ）：（ ii ）：（ xi ）：SEQ ID NO：5： Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp 1 5 10 15 （ 7 ） SEQ ID NO：6：（ i ）：（ A ）：28 （ B ）：（ C ）：（ D ）：（ ii ）：（ xi ）： SEQ ID NO：6： Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu 1 5 10 15 Ala Val Arg Val His Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr。

20 25 30 （ 8 ） SEQ ID N0：7：（ i ）：（ A ）：17 （ B ）：（ C ）：（ D ）：（ ii ）：（ xi ）： SEQ ID NO：7： Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr 1 5 10 15 Glu Leu （ 9 ） SEQ ID NO：8：（ i ）：（ A ）：22 （ B ）：（ C ）：（ D ）：（ ii ）：（ xi ）：SEQ ID NO：8： Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro 1 5 10 15 Lys Val Ser Ala Ser His Leu。

20 （ 10 ） SEQ ID NO：9：（ i ）：（ A ）：15 （ B ）：（ C ）：（ D ）：（ ii ）：（ xi ）：SEQ ID NO：9： Tyr Met Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu Glu 1 5 10 15 （ 11 ） SEQ ID NO：10：（ i ）：（ A ）：27 （ B ）：（ C ）：（ D ）：（ ii ）：（ xi ）：SEQ ID NO：10： Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp Arg Phe 1 5 10 15 Leu Gln Thr Met Val Lys Leu Phe Asn Arg Ile Lys。

20 25 (12) SEQ ID NO:11 data: (i) sequence signature: (A) length: 24 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:11:Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val 15 10 15 Ala Glu Leu Arg Gly Asn Ala Glu Leu

20 (13) SEQ ID NO:12 data: (i) sequence signature: (A) length: 15 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:12:Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Leu 15 10 15 (14) SEQ ID NO:13 data: (i) sequence signature: (A) length: 39 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:13:Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Pro Gly Lys Thr

5 10 15 Lys Gly Ser Gly Phe Phe Val Phe Gly Pro Gly Lys Thr Lys Gly

20 25 30 Ser Gly Phe Phe Val Phe Gly Lys Met

35 (15) SEQ ID NO:14 data: (i) sequence signature: (A) length: 21 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:14:Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro 15 10 15 Pro Asn Ala Pro Ile Leu

20 (16) SEQ ID NO:15 data: (i) sequence signature: (A) length: 28 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:15:Lys Lys Lys Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln 15 10 15 Ser Leu Asp Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (17) SEQ ID NO:16 data: (i) sequence signature: (A) length: 38 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:16:Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu 15 10 15 Ala Val Arg Val His Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr

20 25 30 Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

35 40 (18) SEQ ID NO:17 data: (i) sequence signature: (A) length: 25 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:17:Tyr Met Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu Glu 15 10 15 Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (19) SEQ ID NO:18 data: (i) sequence signature: (A) length: 27 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:18:Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr 15 10 15 Glu Leu Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (20) SEQ ID NO:19 data: (i) sequence signature: (A) length: 32 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:19:Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro 15 10 15 Lys Val Ser Ala Ser His Leu Lys Thr Lys Gly Ser Gly Phe Phe

20 25 30 Val Phe (21) SEQ ID NO:20 data: (i) sequence signature: (A) length: 37 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:20:Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp Arg Phe 15 10 15 Leu Gln Thr Met Val Lys Leu Phe Asn Arg Ile Lys Lys Thr Lys

20 25 30 Gly Ser Gly Phe Phe Val Phe

35 (22) SEQ ID NO:21 data: (i) sequence signature: (A) length: 34 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:21:Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val 15 10 15 Ala Glu Leu Arg Gly Asn Ala Glu Leu Lys Thr Lys Gly Ser Gly

20 25 30 Phe Phe Val Phe (23) SEQ ID NO:22 data: (i) sequence signature: (A) length: 25 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:22:Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Leu 15 10 15 Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (24) SEQ ID NO:23 data: (i) sequence signature: (A) length: 26 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:23:Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Pro Gly Lys Thr

5 10 15 Lys Gly Ser Gly Phe Phe Val Phe Gly Lys Met

20 25 (25) SEQ ID NO:24 data: (i) sequence signature: (A) length: 6 (B) types: amino acid (C) chain: improper (D) topology: unknown (ii) molecule type: linking group (xi) sequence description: SEQ ID NO:24:Pro Pro Xaa Pro Xaa Pro

5 (2) SEQ ID NO:25 data: (i) sequence signature: (A) length: 16 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala Thr Tyr Gln Phe 15 10 15 (2) SEQ ID NO:26 data: (i) sequence signature: (A) length: 20 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:26:Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val Asp 15 10 15 Thr Glu Ser Tyr

20 ( 2 ) SEQ ID NO：27： ( i ) ： ( A ) ：17 ( B ) ： ( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：27： Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys 1 5 10 15 Thr ( 2 ) SEQ ID NO：28： ( i ) ： ( A ) ：16 ( B ) ： ( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：28： Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn His Val 1 5 10 15 ( 2 ) SEQ ID NO：29： ( i ) ： ( A ) ：25 ( B ) ： ( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：29： Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala 1 5 10 15 Thr Thr Gly Tyr Leu Lys Gly Asn Ser

20 25 (2) SEQ ID NO:30 data: (i) sequence signature: (A) length: 23 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:30:Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val Ala Ala Leu Ser 15 10 15 Ile Leu Pro Gly Ile Gly Cys

20 (2) SEQ ID NO:31 data: (i) sequence signature: (A) length: 39 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:31:Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala 15 10 15 Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala

20 25 30 Thr Asn Phe Val Glu Ser Cys

35 (2) SEQ ID NO:32 data: (i) sequence signature: (A) length: 21 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:32:Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe 15 10 15 Asn Val Val Asn Ser

20 ( 2 ) SEQ ID NO：33： ( i ) ： ( A ) ：17 ( B ) ： ( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：33： Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp Glu Lys Ile Arg 1 5 10 15 Ile ( 2 ) SEQ ID NO：34： ( i ) ： ( A ) ：14 ( B ) ： ( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：34： Gly Leu Gln Gly Lys Ile Ala Asp Ala Val Lys Ala Lys Gly 1 5 10 ( 2 ) SEQ ID NO：35： ( i ) ： ( A ) ：19 ( B ) ： ( C ) ： ( D ) ： ( ii ) ： ( xi ) ： SEQ ID NO：35： Gly Leu Ala Ala Gly Leu Val Gly Met Ala Ala Asp Ala Met Val Glu 1 5 10 15 Asp Val Asn ( 2 ) SEQ ID NO：36： ( i ) ： ( A ) ：20 ( B ) ： ( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：36： Ser Thr Glu Thr Gly Asn Gln His His Tyr Gln Thr Arg Val Val Ser 1 5 10 15 Asn Ala Asn Lys

20 (2) SEQ ID NO:37 data: (i) sequence signature: (A) length: 29 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:37:Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu 15 10 15 Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:38 data: (i) sequence signature: (A) length: 34 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:38:Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 15 10 15 Val Ser Ala Ser His Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe

20 25 30 Val Phe (2) SEQ ID NO:39 data: (i) sequence signature: (A) length: 27 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:39:Tyr Met Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu Glu Gly 15 10 15 Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:40 data: (i) sequence signature: (A) length: 32 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:40:Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val Asp 15 10 15 Thr Glu Ser Tyr Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 30 (2) SEQ ID NO:41 data: (i) sequence signature: (A) length: 29 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:41:Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys 15 10 15 Thr Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:42 data: (i) sequence signature: (A) length: 28 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:42:Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn His Val 15 10 15 Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:43 data: (i) sequence signature: (A) length: 37 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:43:Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala 15 10 15 Thr Thr Gly Tyr Leu Lys Gly Asn Ser Gly Gly Lys Thr Lys Gly Ser

20 25 30 Gly Phe Phe Val Phe

35 (2) SEQ ID NO:44 data: (i) sequence signature: (A) length: 35 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:44:Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val Ala Ala Leu Ser 15 10 15 Ile Leu Pro Gly Ile Gly Cys Gly Gly Lys Thr Lys Gly Ser Gly Phe

20 25 30 Phe Val Phe

35 (2) SEQ ID NO:45 data: (i) sequence signature: (A) length: 51 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:45:Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala 15 10 15 Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala

20 25 30 Thr Asn Phe Val Glu Ser Cys Gly Gly Lys Thr Lys Gly Ser Gly Phe

35 40 45 Phe Val Phe

50 (2) SEQ ID NO:46 data: (i) sequence signature: (A) length: 33 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:46:Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe 15 10 15 Asn Val Val Asn Ser Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val

20 25 30 Phe (2) SEQ ID NO:47 data: (i) sequence signature: (A) length: 29 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:47:Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp Glu Lys Ile Arg 15 10 15 Ile Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:48 data: (i) sequence signature: (A) length: 26 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:48:Gly Leu Gln Gly Lys Ile Ala Asp Ala Val Lys Ala Lys Gly Gly Gly 15 10 15 Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:49 data: (i) sequence signature: (A) length: 31 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:49:Gly Leu Ala Ala Gly Leu Val Gly Met Ala Ala Asp Ala Met Val Glu 15 10 15 Asp Val Asn Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 30 (2) SEQ ID NO:50 data: (i) sequence signature: (A) length: 32 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:50:Ser Thr Glu Thr Gly Asn Gln His His Tyr Gln Thr Arg Val Val Ser 15 10 15 Asn Ala Asn Lys Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 30 (2) SEQ ID NO:51 data: (i) sequence signature: (A) length: 27 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:51:Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Gly 15 10 15 Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:52 data: (i) sequence signature: (A) length: 25 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:52:Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Gly 15 10 15 Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

20 25 (2) SEQ ID NO:53 data: (i) sequence signature: (A) length: 42 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:53:Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu Ala 15 10 15 Val Arg Val His Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr Gly Gly

20 25 30 Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

35 40 (2) SEQ ID NO:54 data: (i) sequence signature: (A) length: 36 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:54:Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val Ala 15 10 15 Glu Leu Arg Gly Asn Ala Glu Leu Gly Gly Lys Thr Lys Gly Ser Gly

20 25 30 Phe Phe Val Phe

35 (2) SEQ ID NO:55 data: (i) sequence signature: (A) length: 54 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:55:Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala Thr Tyr Gln Phe 15 10 15 Gly Gly Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr

20 25 30 Val Ala Glu Leu Arg Gly Asn Ala Glu Leu Gly Gly Lys Thr Lys Gly

35 40 45 Ser Gly Phe Phe Val Phe

50 (2) SEQ ID NO:56: data: (i) sequence signature: (A) length: 51 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:56:Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp Arg Phe Leu 15 10 15 Gln Thr Met Val Lys Leu Phe Asn Asp Arg Phe Leu Gln Thr Met Val

20 25 30 Lys Leu Phe Asn Arg Ile Lys Gly Gly Lys Thr Lys Gly Ser Gly Phe

35 40 45 Phe Val Phe

50 (2) SEQ ID NO:57: data: (i) sequence signature: (A) length: 27 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:57:Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Leu Ser Glu Ile 15 10 15 Lys Gly Val Ile Val His Arg Leu Glu Gly Val

20 25 (2) SEQ ID NO:58 data: (i) sequence signature: (A) length: 42 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:58:Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Lys Lys Leu Arg 15 10 15 Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu Ala Val Arg Val His

20 25 30 Val His Lys Glu Glu Gln Tyr Tyr Asp Tyr

35 40 (2) SEQ ID NO:59 data: (i) sequence signature: (A) length: 36 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:59:Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Gly Ala Tyr Ala 15 10 15 Arg Cys Pro Asn Glu Thr Arg Ala Leu Thr Val Ala Glu Leu Arg Gly

20 25 30 Asn Ala Glu Leu

35 (2) SEQ ID NO:60 data: (i) sequence signature: (A) length: 52 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:60:Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Tyr Asp Pro 15 10 15 Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met

20 25 30 Val Lys Leu Phe Asn Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe

35 40 45 Asn Arg Ile Lys

50 (2) SEQ ID NO:61 data: (i) sequence signature: (A) length: 16 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:61:Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val 15 10 15 (2) SEQ ID NO:62 data: (i) sequence signature: (A) length: 40 (B) types: amino acid (D) topology: line style (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:62:Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu Ala 15 10 15 Val Arg Val His Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr Lys Thr

20 25 30 Lys Gly Ser Gly Phe Phe Val Phe

35 40

Claims

1. with general formula (A) n-(Th) m-(B) o-(IgECH₄Peptide) the peptide immunogene that represents of p, wherein: A is amino acid, α-NH₂, aliphatic acid or derivatives thereof, or invasion; B is amino acid; Th is the helper cell epitope, its analog or fragment; IgE CH₄Peptide is SEQ ID No:1 or its immunogenicity analog; N is from 1 to 10; M is from 1 to 4; O is from 0 to 10; P is from 1 to 3.

2. the peptide immunogene of claim 1; Wherein p is 1.

3. the peptide immunogene of claim 1; Wherein Th is selected from SEQ ID Nos:5-12,14,26-36,61 and its immunogenicity analog or its fragment.

4. the peptide immunogene of claim 2, wherein Th is selected from SEQ ID Nos:5-12,14,26-36,61 and its immunogenicity analog or fragment.

5. the peptide immunogene of claim 1 is selected from SEQ ID Nos:13,15-23, and 37-50,51-56 and 62.

6. the peptide immunogene of claim 3 is selected from SEQ ID Nos:51-56 and 62.

7. the peptide immunogene of claim 1, wherein A is aliphatic acid.

8. the peptide immunogene of claim 2, wherein A is aliphatic acid.

9. the peptide immunogene of claim 1, wherein A is the aliphatic acid derivative.

10. the peptide immunogene of claim 2, wherein A is the aliphatic acid derivative.

11. the peptide immunogene of claim 9, wherein the aliphatic acid derivative is Pam3 Cys.

12. the peptide immunogene of claim 10; Wherein the aliphatic acid derivative is Pam3 Cys.

13. a vaccine composition comprises the peptide immunogene of the claim 1 of the effective dose in the acceptable transmission system pharmaceutically.

14. a vaccine composition comprises the peptide immunogene of the claim 2 of the effective dose in the acceptable transmission system pharmaceutically.

15. a vaccine composition comprises the peptide immunogene of the claim 3 of the effective dose in the acceptable transmission system pharmaceutically.

16. a vaccine composition comprises the peptide immunogene of the claim 4 of the effective dose in the acceptable transmission system pharmaceutically.

17. a vaccine composition comprises the peptide immunogene of the claim 5 of the effective dose in the acceptable transmission system pharmaceutically.

18. a vaccine composition comprises the peptide immunogene of the claim 6 of the effective dose in the acceptable transmission system pharmaceutically.

19. a vaccine composition comprises the peptide immunogene of the claim 7 of the effective dose in the acceptable transmission system pharmaceutically.

20. a vaccine composition comprises the peptide immunogene of the claim 8 of the effective dose in the acceptable transmission system pharmaceutically.

21. a vaccine composition comprises the peptide immunogene of the claim 9 of the effective dose in the acceptable transmission system pharmaceutically.

22. a vaccine composition comprises the peptide immunogene of the claim 10 of the effective dose in the acceptable transmission system pharmaceutically.

23. a vaccine composition comprises the peptide immunogene of the claim 11 of the effective dose in the acceptable transmission system pharmaceutically.

24. a vaccine composition comprises the peptide immunogene of the claim 12 of the effective dose in the acceptable transmission system pharmaceutically.

25. a vaccine composition comprises the immunogenic mixture of peptide of the claim 9 of the effective dose in the acceptable transmission system pharmaceutically.

26. a vaccine composition comprises the immunogenic mixture of peptide of the claim 10 of the effective dose in the acceptable transmission system pharmaceutically.

27. a vaccine composition comprises the immunogenic mixture of peptide of the claim 11 of the effective dose in the acceptable transmission system pharmaceutically.

28. a vaccine composition comprises the immunogenic mixture of peptide of the claim 12 of the effective dose in the acceptable transmission system pharmaceutically.

29. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 13 of effective dose.

30. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 14 of effective dose.

31. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 15 of effective dose.

32. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 16 of effective dose.

33. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 17 of effective dose.

34. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 18 of effective dose.

35. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 19 of effective dose.

36. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 20 of effective dose.

37. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 21 of effective dose.

38. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 22 of effective dose.

39. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 23 of effective dose.

40. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 24 of effective dose.

41. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 25 of effective dose.

42. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 26 of effective dose.

43. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 27 of effective dose.

44. the anaphylactoid method for the treatment of is through using the vaccine composition according to claim 28 of effective dose.