CN1317013A - Peptide fragments of cholera toxic B or enterotoxin B as vaccine adjuvants - Google Patents

Abstract

The present invention relates to a substance, which comprises one or more amino acid sequences contained in the sequence shown in SEQ ID No2, or its variant, or its homologue, or its fragment, derivative or mimic, and its action form Identical or similar to EtxB and/or CtxB, but does not exhibit GM-1 binding activity.

Description

The peptide fragment of cholera toxin B and enterotoxin B is as vaccine adjuvant

Invention field

The present invention relates to a kind of material.

The very useful material that can show one or more character in a kind of medicine.

For example immunomodulator and/or the adjuvant and/or the inhibitor of this material diarrhoea that can cause as toxin.

Particularly, the present invention relates to immunity modulation material and regulating immunne response, for example with the autoimmune disease associated responses in application.

Again particularly, the present invention relates to together the application during with a kind of relevant or irrelevant antigen of this material as adjuvant.

More specifically, the present invention relates to the application of this material in the diarrhoea that the inhibition toxin causes.

The invention still further relates to analysis to screening with the reagent of this matter interaction.

Background of invention

The Toxins,exo-, cholera (Ctx) that intestinal bacteria (intestinal bacteria) heat labile enterotoxins (Etx) and its homologue derive from vibrio cholerae (Vibrio cholerae) all is the proteotoxin with host cell surface glycolipid receptors bind.Every kind of toxin is made up of six non-covalent bonded polypeptide chains, an A subunit (27kDa) and five B subunits (11.6kDa), B subunit energy and mammalian cell surface GM-1 Sphingolipids,sialo receptors bind (Nashar et al 1996Proc Natl Acad Sci 93:226-230).The A subunit is a toxophore, adenosine diphosphate (ADP) (ADP) ADP-ribosyltransferase activity is arranged, and B subunit (EtxB and CtxB) is nontoxic oligomer, can in conjunction with and the ubiquitous ganglioside fat of crosslinked cell surface GM-1, impel the A subunit to enter cell.

Weak immunogenicity with the A subunit is compared, EtxB and CtxB have very strong immunogenicity and holotoxin separately, when with irrelevant antigen in conjunction with the time, Etx and Ctx (comprising A subunit and B subunit) are strong effectively adjuvant (Ruedl et al 1996 Vaccine 14:792-798; Nashar et al 1993 Vaccine 11:235; Nashar and Hirst 1995Vaccine 13:803; Elson and Ealding 1984 J Immunol133:2892; Lycke and Holmgren 1986 Immunology 59:301).Because their immunogenicity, EtxB and CtxB have been used as other antigenic determinant and antigenic carrier (Nashar et al 1993 ibid), also are used as the vaccine component (Jetborn et al 1992 Vaccine 10:130) of the diarrhea disease of cholera and intestinal bacteria mediation.

In order to develop the vaccine of anti-Toxins,exo-, cholera and heat-labile enterotoxin of E, coli, some carry out about the research of the immunodominant epitope of EtxB and CtxB subunit.Following disclosure has shown the work that has carried out in this field.

UK Patent Application 2415419A discloses the synthetic vaccine of a Toxins,exo-, cholera and heat-labile enterotoxin of E, coli, and it comprises a carrier and one section conjugate that is equivalent to the section of synthesized peptide of CtxB sequence.

WO85/02611 discloses a synthetic peptide that is equivalent to the EtxB particular sequence, is considered to an effective conjugate or produces the activating component of anti-B subunit antibody, can protect host animal not to be subjected to infecting of enterotoxin.

WO89/10967 discloses an aminoacid sequence; represent the 50-64 amino acids of CtxB; energy and an xenogenesis organism in vaccine forms; epi-position combination as flagellum (Flagellum) and salmonella (salmonella); protection is provided; thereby be not subjected to that xenogenesis is organic to be infected, or the condition that causes of organism antigen changes or disorderly.

The bioactive sequence that WO90/03437 relates to a CtxB subunit and heterologous antigen merges the hybrid albumen that forms, and it is meaningful when vaccination that heterologous antigen is considered to, and especially is beneficial to heterologous antigen stablizing in the intestines environment.

WO94/06465 relates to an aminoacid sequence, by or be not connected on the suitable carriers by a connector, like this, the aminoacid sequence that has connected carrier just can produce the immunne response of a conditioning or protectiveness.

WO95/29701 discloses the vaccine of a vibrio cholerae (Vibrio cholera), it is one and comprises the b subunit of cholera toxin (CTB) or the conjugate of its section of synthesized peptide, CTP3 for example, it comprises the amino acid on the 50-64 position that is connected to a B chain on the inert support.

WO96/26282 discloses an expression system, and it expresses the gene prod that comes from recombinant bacterial strain Bordetella (Bordetella), and this gene prod can be the Toxins,exo-, cholera molecule.

WO96/34893 discloses the hybrid molecule of an EtxB and CtxB, and its can stop or cure the disease that individual enterotoxin cause as effective vaccine.

WO98/21344 discloses an EtxB subunit that inserts an antigen peptide, and in host animal, this chimeric antigen-EtxB molecular energy causes the antibody response of this antigen peptide.

These researchs relate to is that following utilization (ⅰ) comprises the peptide section of CtxB/EtxB partial sequence or (ⅱ) comprises the peptide of the CtxB/EtxB sequence that is coupled to second entity (for example antigen), and second entity can cause or amplify the immunne response for this peptide.So these documents relate to immunodominant epitope CtxB/EtxB or its part is causing the effect in the immunne response of these subunits, strengthen the immunity of the diarrhea disease that is caused by cholera or intestinal bacteria.WO91/07979 discloses a chimeric protein that comprises the epi-position of a portion C txB and a target antigen, its objective is as vaccine to cause in the experimenter this antigenic immunne response.In this, the part of CtxB is used as adjuvant rather than immunomodulator.Therefore, do not relate to the utilization of CtxB/EtxB or its part in the above-mentioned document as the immunomodulator of regulating immunne response.

We have pointed out that the EtxB subunit can be as immunomodulator when immunologic derangement.For example, we disclose the EtxB subunit and can be combined on the GM-1 Sphingolipids,sialo acceptor on cells of mamma animals surface in WO97/02045, this combination has different-effect to different lymphocytes, comprises the depletion (depletion) of CD8+T cell and the activation of B cell.EtxB protein mutant (G33D) (lacking GM-1 in conjunction with activity) does not then have above-mentioned effect.Because lacking the mutant (for example EtxB (G33D)) of the binding ability of GM-1 acceptor can not be as adjuvant or immunomodulator, thereby, these experimental results show that all CtxB function relevant with EtxB all is the binding ability of CtxB and EtxB subunit and GM-1 acceptor.Immune modulating action and other effect that CtxB and EtxB can be described like this, so far all are to mediate by the combination with GM-1.Up to now, still but there is not the report of the CtxB/EtxB mutant of immune modulating action about having kept the GM-1 receptor binding capacity.Yet the discovery that we are surprised, not all CtxB and the effect of EtxB all mediate by the combination of GM-1.

Summary of the invention

According to the present invention, we find that the immune modulating action of CtxB and EtxB and some other function are not in conjunction with mediation by GM-1.Each side of the present invention will be addressed in will and discussing in claims and following description.

In one aspect of the invention, the invention provides a kind of material, this material comprise following one or more: one section sequence that includes the aminoacid sequence among the SEQ ID No.2, or its variant, or its homologue, or its fragment, or derivatives thereof, or its stand-in; This material can work in same or similar mode in CtxB or EtxB; But this material does not have the combination of GM-1 active.

Of the present invention one preferred aspect, the invention provides a kind of material, this material comprise following one or more: comprise the aminoacid sequence of sequence shown in the SEQ ID No.2, or its variant, or its homologue, or its fragment, or derivatives thereof, or its stand-in; This material can encircle same or analogous mode with EtxB and/or CtxB and work; But this material discord GM-1 combination.

Aspect highly preferred one of the present invention, the invention provides a kind of material, this material comprise following one or more: the sequence that includes the aminoacid sequence among the SEQ ID No.2, or its variant, or its homologue, or its fragment, or derivatives thereof, or its stand-in; This material can work in the mode of same or similar β 4-α 2 rings on CtxB and/or EtxB; But this material does not have the combination of GM-1 active.

Material among the present invention can be one section aminoacid sequence or its chemical derivative.This material can be the variant of one section synthetic peptide or synthetic peptide, such as retroinverso D peptide.This material even can be an organic compound or other chemical substance.Following example is the stand-in of SEQ ID No.2.

Material among the present invention is with same or similar mode effect in CtxB or EtxB.

" same or similar " is a qualitative rather than quantitative implication.From this point, it can have the enhanced bonding force.

Whether a kind of material can be determined by those skilled in the art easily with the mensuration of same or similar mode effect in CtxB or EtxB.For example, this mensuration can be to measure or determine the influence of its pair cell population, as the lymphocyte population.This influence can include but are not limited to that CD8+T is apoptotic to be induced, and the polyclone of the enhancing of CD4+T cell-stimulating and B cell activates.In addition, this mensuration also can be based upon in the decision and based measurement of cell surface marker, and these marks can show the activation (for example measuring the increase that CD25 expresses) of certain incident in the born of the same parents.

Material among the present invention does not have the combination of GM-1 active.

GM-1 can be determined by those skilled in the art by the mensuration of this area easily in conjunction with active shortage.For example, GM-1 can be combined on the solid support, allow this substance flow cross combined GM-1, the elutriant of this material show its can not with the GM-1 combination.One preferred aspect, this is measured described in WO97/02405.

Should be understood that this binding ability also needn't depend on the binding events originally of total length CtxB or EtxB subunit.When having total length CtxB or EtxB subunit, originally be the combination activity of GM-1 in conjunction with activity.From this point, this material can be carried out single binding events.Yet this material can have one or more binding abilities.

Preferably, this material is isolating basically and/or pure basically.

Here, " isolating " and " pure " is meant molecule, no matter is nucleic acid or aminoacid sequence, and they are separated from its physical environment, or originally isolates bonded composition or separate from their at least.A kind of albumen can with carrier or mixing diluents, but do not influence the required characteristic of this material and can also be counted as isolating basically.

This invents based on so surprising discovery: the mutant of some energy and GM-1 receptors bind lacks immunoregulation effect.These mutant have promoted CtxB or EtxB subunit mechanism of action, and especially their immunoregulation effect illustrates.

Others of the present invention are as described below:

Material of the present invention is applied to medicine.

Material of the present invention is as immunomodulator.

The material of this fermentation is as adjuvant.

The inhibitor of the diarrhoea that material of the present invention causes as toxin.

Material of the present invention, wherein this material also comprises antigen or antigenic determinant.

A kind of pharmaceutical composition comprises material of the present invention, depends on the needs to be mixed with one or more pharmaceutically acceptable carrier, thinner or vehicle.

Material of the present invention is used for the treatment of and/or prevents and/or regulate application in relevant disease of the disorder of immunologic derangement and/or toxin mediation and/or the disorderly medicine in preparation.

A kind ofly be used for determining that one or more can and/or influence the measuring method of the reagent of material of the present invention with matter interaction of the present invention; Wherein said mensuration comprises material of the present invention and test agent is contacted; And determine whether that then this reagent can influence this material.

A kind ofly be used for the reagent that measuring method of the present invention identifies.

A kind of methods of treatment comprises to needs treating and/or preventing and/or regulate the relevant disease of immunologic derangement and/or toxin mediated disorders and/or disorderly experimenter uses material of the present invention.

These aspects will present in the title of following each section to some extent.But the content that each paragraph is lectured is not limited to title.

Immunomodulator

Here " immunomodulator " refers to that a kind of material can be by inducing the adjusting immunne response, for example, pair cell such as lymphocytic Different Effects---preferably induce the enhancing of CD8+ apoptosis and/or CD4+ cell-stimulating and/or the polyclone of B cell to activate.

" to leukocytic Different Effects " can comprise but not only be confined to exhaust (for example the passing through apoptosis) of CD8+T cell, the relevant activation of the enhancing of CD4+T cell-stimulating and/or B cell.

Preferably, immunomodulator can be born accent to Th1 and/or the relevant immunne response pathologic reaction of Th2.

Preferably, immunomodulator also can just be transferred the mucomembranous surface production of antibodies.

Adjuvant

Here, adjuvant is meant the material of the non-specific enhancing of energy to antigenic immunne response.

It also comprises can influence an entity, such as arbitrary material of antigen and/or antigenic determinant immunne response degree, by changing antigenic antigenicity, or change specific reversed stress or host's related mechanism nonspecific effect thing, induce the immunne response in the host cell and/or be to guide it to specific direction.More preferred one side, adjuvant can be used as mucosal adjuvants.

Preferably, adjuvant can prolong mammiferous antigen presentation or a lasting immunological memory is provided.

Antigen

Here, " antigen " refers to an entity, when it is introduced among the immunocompetence host, can stimulate to produce specific antibody or energy and this entity bonded antibody.This antigen can be pure material, the mixture of this material or solubility or particulate material (comprising cell or cell debris).From this point, this speech comprises arbitrary suitable antigenic determinant, autoantigen (auto-antigen), autoantigen (self-antigen), the reacting antigen of reporting to the leadship after accomplishing a task, isoantigen, heterologous antigen, toleragen, allergen, haptens and immunogen, or be its part, also can be its composition, these speech can be used instead in the whole text mutually.

" allergen " comprises arbitrary antigen that can stimulate allergic effect reaction and the allergy of I type.

General allergen derives from but not only is confined to artemisiifolia, rye, couchgrass, wild avena sativa, thimothy grass, Bermuda grass (Bermuda), nonirrigated farmland annual bluegrass, mugwortalder, birch, wood-nut, beech, cypress, oak, olive, aspergillus class, branch spore class, chain lattice spore class, sporidium, ascomycetes wheat, rye, oatcat, dog, horse, rabbit, cavy, hamster, parrot, pigeon, duck, chicken, dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus), dust mite (D.farinae), Euroglyphus maynei, cockroach, fly, locust, midge, seafood, leguminous plants, peanut, nut, cereal, daily agricultural-food, egg, fruit, tomato, mushroom, alcoholic beverage, coffee, chocolate, penicillin, sulfonamide and other microbiotic, sulfasalazine, carboxamide benzene , honeybee, wasp, ant and mosquito, serum, vaccine, contrast medium, medicine (comprising anti-asthma medicine and microbiotic).Antigenic determinant

" antigenic determinant " here refers to can be by the site of antibody or TXi Baoshouti identification on the antigen.Preferably, it is little peptide or its part from proteantigen.This term also comprises glycopeptide and carbohydrate epitope.It comprises that also the aminoacid sequence of having been modified maybe can stimulate the carbohydrate of the whole organism reaction of identification.

If this antigenic determinant is the antigenic determinant an of infector that can produce communicable diseases (such as bacterium and virus), that will be more favourable.

For example, if this infector is EBV, that antigenic determinant will be gp340 or a gp350 or a latent protein antigenic determinant (such as EBNAs 1,23A, 3B, 3C and-LP, LMP-1 ,-2A and 2B or EBER).If infectious agent is an influenza virus, that antigenic determinant can be (for example hemagglutinin and neuraminidase) antigenic determinant of a coat protein, or the antigenic determinant of inner albumen (as nucleoprotein).If infectious agent is to select from enteropathogenic, and is enterotoxigenic, the invasion and attack intestinal mucosa, enterorrhagia, in the intestines aggregation intestinal bacteria, that antigenic determinant can be the antigenic determinant of the bacteriotoxin or the adhesion factor.

If antigenic determinant derives from autoantigen, also be favourable.

Reagent

Reagent can be aminoacid sequence or its chemical derivative.This material can be organic compound or other chemical substance.This reagent can be that justice or antisense base sequences are arranged, and also can be antibody.One preferred aspect, this reagent can be can with this material bonded cell receptor.

The inhibitor of the diarrhoea that toxin causes

Term " inhibitor of the diarrhoea that toxin causes " comprises arbitraryly can influence the active material of Etx/Ctx holotoxin, and it can avoid the pathology effect of Etx/Ctx, for example diarrhoea.

Detailed Description Of The Invention

The present invention shows following surprising discovery:

(ⅰ) immunomodulator and/or the adjuvant and/or the inhibitor of the material of the present invention diarrhoea that can cause as toxin, it can influence the diarrhoea of enterotoxin mediation.

(ⅱ) material of the present invention can be with same or similar mode effect in CtxB/EtxB.The activity of the material that the present invention relates to is mediated by 2 rings of the β 4-α on " so-called " CtxB and the EtxB, and this ring is very flexible, contains 45-65 aminoacid sequence.

(ⅲ) undergo mutation (51,56 and 57) in three the different sites of EtxB molecule on β 4-α 2 rings, and it keeps GM-1 in conjunction with activity, but lacks other activity, such as toxicity, induces to the just accent ability of CD25 with to CD8+T is apoptotic.In addition, the Ctx holotoxin that comprises sudden change B subunit also can bring out gives birth to electric muriatic secretion, and this is the initial secretion incident of mediation diarrhoea.These results are beat all, only work when connecting two parts of secondary structure because it has been generally acknowledged that variable ring, and itself seldom has important function.

(ⅳ) binding ability of this material also needn't depend on the CtxB of total length and the initial binding events of EtxB.When having the CtxB of total length and EtxB, initial in conjunction with activity be GM-1 in conjunction with activity.

The CtxB/EtxB toxin

Here, " Ctx " is meant Toxins,exo-, cholera, and " CtxB " refers to the B subunit of Toxins,exo-, cholera.In other article, they may be respectively referred to as CT, Ct, CTB or CtB.

Here, " Etx " is meant heat-labile enterotoxin of E, coli, and " EtxB " refers to the B subunit of Etx.In other article, they may be respectively referred to as LT, Lt, LTB or LtB.

β 4-α 2 rings

On the one hand, the present invention relates to the material of a kind of EVPGSQH of comprising (SEQ ID No2) sequence, its mode of action and EtxB or CtxB are same or similar, or its variant, or its homologue, or its fragment, or derivatives thereof, or its stand-in, but lack the GM-1 binding ability.

Be not limited on the basis of original theory, we think that five EtxB/CtxB subunits are a kind of very strong mutual avidity to the bonding force of GM-1, and this makes EtxB/CtxB that the second more weak bonding force relatively be arranged.This combination is to be mediated by 2 rings of the β 4-α on the EtxB/CtxB.The structure of β 4-α 2 rings on the EtxB/CtxB can be referring to (Sixma etal.J.Mol.Biol (1993) 230; Etx molecular structure 890-918), as shown in Figure 1.

In a word, each B subunit of Etx or Ctx all comprises the terminal spiral (α 1) of a N-, and (folding I comprises chain β 2 to two three antiparallel the folding of chain, and β 3, and β 4; Folding II comprises chain β 1, and β 5, and β 6) and a long alpha-helix (α 2).These two βZhe Dies are formed a β bucket.According to two folding ends, the ring that connects these parts of secondary structure in the B subunit is divided into two classes.An end of subunit, " narrow " (or " A ") end, chain rate is shorter, comprises β 1-β 2, and β 3-β 4 is connected and the C-end with α 2-β 5; The other end broad of subunit encircles longlyer, connects secondary structure element α 1-β 1, β 2-β 3, β 4-α 2.Connect the long ring (to call β 4-α 2 rings in the following text) of β 4-α 2, comprise that 51 L-glutamic acid (Glu 51) to 59 aspartic acids (Asp59), extend under the βZhe Die plane always.This ring is very flexible, but according to ((Nature (1992) 355 for Sixma et al; 561-564), after this ring and lactose combination, the handiness meeting obviously reduces.The present invention shows β 4-α 2 ring of EtxB/CtxB, has second in conjunction with active, is promptly lacked first when active so separate from the other parts (for example peptide) of EtxB/CtxB molecule when it, and it can show second in conjunction with activity.The β 4-α 2 that suddenlys change selectively encircles, or the last peptide section of ring, can develop to strengthen second bonding force.Can further get rid of by the interaction that strengthens second bonding force and GM-1.

Here, " β 4-α 2 rings of EtxB/CtxB " are to be responsible for toxin such as Toxins,exo-, cholera and heat-labile enterotoxin of E, coli B subunit second in conjunction with active entity.Separated from the other parts (for example peptide section) of EtxB/CtxB molecule when β 4-α 2 ring and promptly to lack first when active, it can show second in conjunction with active and be called activity hereinafter or in conjunction with active.

Preferably, material of the present invention comprises isolating EtxB/CtxB β 4-α 2 rings.

Preferably, this material comprises the stand-in of isolating EtxB/CtxB β 4-α 2 rings.

Preferably, this material includes highly affine stand-in in conjunction with active isolating EtxB/CtxB β 4-α 2 rings.

Preferably, this material comprises one section 5-40 amino acid whose peptide.

Preferably, this peptide is amino acid contained is less than 25.

If this peptide is a fusion rotein, preferably, this peptide contains 25 with upper amino acid.

Preferably, this material comprises sequence VEVPGSQHIDSQ (SEQ ID No3).

Preferably, this material comprises sequence GATFQVEVPGSQHIDSQKKAI (SEQ IDNo4).

Preferably, this material comprises the EtxB45-65 residue derivative sequence GETFQVEVPGSQHIDSQKKAI (SEQ ID No5) of the pig of escherichia coli expression.

Preferably, this material comprises the people's of escherichia coli expression EtxB45-65 residue derivative sequence.

Aminoacid sequence

The invention provides a kind of material that comprises aminoacid sequence of the present invention, this section aminoacid sequence can be as immunomodulator and/or the adjuvant and/or the inhibitor of the diarrhoea that is caused by toxin, and this material can influence the diarrhea disease of enterotoxin mediation.This material also can be used for to can with mensuration this matter interaction and/or that influence these one or more reagent of material.

Here, " aminoacid sequence " refers to peptide, peptide sequence, protein sequence or its part.

Influence

Term " influence " comprises the active adjusting of material, and for example: handle, prevent, containment improves, and recovers, and raises, and modifies.

Term " modification " includes but are not limited to the active disappearance of material, silence, and sudden change is removed, and strengthens, and increases, and stimulates, and antagonism reduces or retardance.

Variant/homologue/derivative

Invention preferred amino acids sequence is SEQ ID No2, SEQ ID No3, SEQ ID No4, SEQ ID No5 or come from the sequence of material of the present invention, also comprised from other sources and homologue as relevant virus/bacterioprotein, cell homologue and synthetic peptide, with and variant and derivative.

Thereby the variant of aminoacid sequence described here has been contained in the present invention, and homologue or derivative have also comprised variant, homologue or the derivative of the nucleotide sequence of these aminoacid sequences of encoding.

In the context of the present invention, refer to that at least 7 amino acid of the SEQID No 2 on amino acid levels and shown in sequence table have at least 75%, 85% or 90%, preferably at least 95 or 98% conforming aminoacid sequence.Especially, to consider especially, rather than close on the sequence homology of non-important area for the zone (as 51,56,57 amino acid) that plays an important role with the same or similar activity of EtxB/CtxB.Although homology can be considered to similarity (for example: amino-acid residue has similar chemical property/function), among the present invention, the homology that the identical outstanding finger of sequence is expressed.Homology relatively can with the naked eye or be used the existing sequence comparison software.The homology per-cent of these business-like computer softwares between can more two or more sequences.

Homology per-cent also can calculate by continuous sequence, as: a sequence and the contrast of other sequences, its aminoacid sequence directly compares with corresponding amino acid in other sequences, each relatively amino-acid residue, this is called " the non-notch contrast ", clearly, this method is only applicable to less relatively residue number.

Although this method is simple relatively and consistent, but it does not consider, as: in identical paired sequence, just one insert or disappearance can cause amino-acid residue subsequently to place outside the contrast, therefore when adopting the overall sequence contrast, can cause the reduction of percent homology.Correspondingly, most sequence comparative approach has been considered possible insertion and disappearance, has designed the suitableeest contrast, and whole homology score is not had the undue branch that subtracts.This is by inserting " breach " (gap) in sequence contrast, increase local homology and finish.

Yet these more complicated methods have designed " breach compensation " (gap penalty) to each breach in the comparison process, thereby, use dependency between two comparative sequences of sequence contrast reaction of a small amount of breach-more many contrast ratio height of breach as far as possible than having for the same amino acid of similar number." outbreeding breach value " is significantly as desired relative higher value during each amino acid whose subsequently less compensation in the existence of breach and the breach.This is that the breach that is in daily use gets sub-system.High breach compensation can produce the suitable contrast with minority breach certainly.Most contrast allows the breach compensation to be modified.Yet, when using these softwares, preferably use default value.For example: use GCG Wisconsin Bestfit routine package (as follows), the default breach compensation of aminoacid sequence is that each breach is 12, and each extends to 4.

Consider the breach compensation, the calculating of maximum homology at first needs suitable correlated generation.Carry correlated computer program like this GCG Wisconsin Bestfit routine package (University of Wisconsin, U.S.A are arranged; Devereux et al, 1984, nucleic acids research 12:387) other can be used for carrying out the correlated software of sequence include but not limited to the BLAST software package (referring to Ausubel etc., 1999 ibid-chapter 18)), FASTA (Atschulet al, 1990, J.Mol.Bio., 403-410) and GENEWORKS contrast instrument, BLAST and FASTA can be online or online retrieving not.(see Ausubel et al., 1999ibid, pages 7-58 is to 7-60).Yet, preferably use GCG Bestfit program.

Although final homology can be measured with homogeny; comparison process itself does not obviously build on the paired comparisons of all or none; in fact, on the basis of chemical similarity and evolutionary distance, the similar value matrix of mass-producing is usually used in the arrangement value of each paired comparisons.The example of this matrix is as the BLOSUM62 matrix of Shi Yonging---the default matrix of blast program usually.The GCGWisconsin program is used public default value, if possible, also can use the user symbol contrast table.(seeing that user manual describes in detail).The public default value of preferred utilization GCG routine package, or the default matrix in other softwares, as: BLOSUM62.

In case Software Production goes out suitable contrast, just may calculate percent homology, especially the sequence same percentage.Software goes this part as the sequence comparison to finish, and draws a numerical result.

With material of the present invention, SEQ ID No 2, SEQ ID No 3, the any of the entity sequence that SEQ ID No 4 " variants " or " derivative " relevant with SEQID No5 have comprised retentive activity substitutes, and variant is modified, replace, or one or (a plurality of) amino acid whose disappearance, increase, preferably to have activity identical and/or similar and CtxB and EtxB at least.

SEQ ID No 2 among the present invention, SEQ ID No 4 or SEQ ID No 5 can be modified.Obviously, modify the activity that is used to strengthen material.Amino acid can be replaced, as: keep active from 1,2 or 3 to 10 or 20 the alternative sequence of modification that makes.

The disappearance of amino-acid residue also can take place in the material among the present invention, inserts or substitutes, and produces the reticent variation material identical with function.Can keep material second in conjunction with active prerequisite under, have similar polarity, electric charge, solvability, hydrophobicity and/or hydrophilic amino-acid residue substitute.For example: electronegative amino acid comprises: L-glutamic acid and aspartic acid; Positively charged amino acid has Methionin and arginine; The amino acid that has identical hydrophobicity non-polar group in addition: leucine, Isoleucine, Xie Ansuan, glycine, L-Ala, l-asparagine, glutamine, Serine, Threonine, phenylalanine and tyrosine.

Conservative substituting can be carried out according to following table.Amino acid in the secondary series in the same square can substitute mutually, and the amino acid in the same square in preferred the 3rd row substitutes.

Aliphatic series	Nonpolar	Glycine, L-Ala, proline(Pro)
			Leucine, Isoleucine, Xie Ansuan
		Polarity does not have electric charge		Halfcystine, Serine, Threonine, methionine(Met)
	L-asparagine, glutamine
			Polarity has electric charge	Aspartic acid, L-glutamic acid
	Methionin, arginine
		Aromatic series			Histidine, phenylalanine, tryptophane, tyrosine

Nucleotide sequence

On the one hand, the invention provides the nucleotide sequence of code book invention material, material of the present invention can be used as template or target thing and is used for analyzing (for example two assorted analyses of yeast) and identifies one or more reagent and/or its derivative that influence this material.

Here, term " nucleotide sequence " refers to nucleotide sequence, oligonucleotide sequence, polymerized nucleoside acid sequence, variant, homologue, fragment and its derivative (for example its part).Nucleotide sequence can be strand or the double-stranded DNA and the RNA of genome or reorganization, or for having a mind to chain or for being not intended to chain or its combination, preferably, nucleotide sequence is prepared (as: recombinant DNA) by recombinant DNA technology.

Preferably, " nucleotide sequence " means DNA.

The material of coding nucleotide sequence can be same as the form of its natural generation.Preferably, the sequence of this material of encoding is non-natural nucleotide sequence, or is its variant, homologue, fragment or derivative.Thereby in the preferred embodiment, the present invention does not comprise under the natural surroundings, by the nucleotide coding sequence corresponding and of the present invention of its natural promoter control.We are called this preferred version " non-natural nucleoside acid sequence ".

Here, " natural generation " refer to have the material of a kind of aminoacid sequence that nature finds.

Here, " biologic activity " refers to have the adjusting of crude substance or the material of biochemical function.

Variant/homologue/derivative

With relevant " variant " of SEQ ID No1 nucleotide sequence of the present invention, " homologue ", " derivative ", substituting of one or more Nucleotide of the synthesizing ribonucleotide sequence that having comprised encodes has active material makes a variation, modify, replace, lose or increase, have active material have at least with sequence table in SEQ ID No 2, SEQ ID No 3, the activity that SEQ ID No 4 and SEQ ID No 5 are identical.

As mentioned above, with the listed sequence of sequence table the sequence of homology is arranged, have 70% homology at least, preferred 80% homology perhaps more preferably has 90% homology.More preferably have 95% homology, further preferably have 98% homology.Nucleotide homology relatively can as above carry out.Aforesaid sequence comparison program is a GCG Wisconsin Bestfit program.The default value matrix is 10 for the matching value of each identical Nucleotide, is 9 to the amino acid number of each non-coupling.For each Nucleotide, default breach produces and is compensated for as 50, and default breach extension is compensated for as 3.

The present invention also comprises can selectivity and the nucleotide sequence of the sequence hybridization here, or any its variant, fragment or derivative, or above-mentioned combination.Nucleotide sequence is at least 15 Nucleotide preferably, more preferably, and at least 20,30,40 or 50 Nucleotide.

Here " disappearance " refers to the variation of one or more Nucleotide in nucleotide sequence or the aminoacid sequence or amino-acid residue, especially disappearance.

Here, " insertion " or " increase " refer to compare the increase of one or more Nucleotide or amino-acid residue in nucleotide sequence or the aminoacid sequence with natural substance.

Here, " substitute " and refer to that one or more Nucleotide or amino acid are that different Nucleotide or amino acid replace.

Hybridization

Here, term " hybridization " comprises " nucleotide chain and another chain base complementrity paired process ", and the amplification procedure that carries out with polymerase chain reaction (PCR) technology.

Nucleotide sequence of the present invention can be optionally with nucleotide sequence disclosed herein or its complementary sequence hybridization.Generally have 75% with Nucleotide disclosed herein at least, preferably at least 85% or 90%, 95% or 98% homology is more preferably arranged.Comprise at least 20, preferred 25 or 30, more preferably at least 40,60 or 100 or more continuous nucleotide.The preferred nucleotide sequence of the present invention comprises the zone with SEQ ID No1 nucleotide homology, preferably has 80% or 90% with SEQ ID No1 at least, and the zone of 95% homology is more preferably arranged.

" selective cross " refers to template nucleotide sequence of the present invention under being significantly higher than the background situation during with probe hybridization, and nucleotide sequence is as probe.The generation of background be since other nucleotide sequence for example: cDNA or genome dna library are detected.In this incident, background includes the signal that probe interacts and produces with non-specific DNA in the library, and this signal hangs down 10 times than the specific reaction of target DNA, preferably, and low 100 times.The intensity of reaction can detect, as; By radio-labeling as: ³²P.

Say as Berger and kimmol, hybridization conditions is based on the melting temperature(Tm) (Tm) (1987 of Nucleotide in conjunction with complex body, Guide to Molecular CloningTechniques, Methods in Enzymology, Vol 152, Academic Press, SanDiego CA), resemble as described belowly, be called as " stringency ".

Maximum stringency approximately occurs in Tm-5 ℃ the Tm value of probe (be lower than 5 ℃); High stringency is less than about Tm5 ℃ to 10 ℃ greatly; Medium stringency is less than about Tm10 ℃ to 20 ℃ greatly; Low stringency is less than about Tm20 ℃ to 25 ℃ greatly.As those skilled in the art known, the hybridization of maximum stringency is used for identifying or detecting identical nucleotide sequence, and medium stringency hybridization (or low stringency) is used for identifying or detecting similar or related nucleotide sequences.

Preferably, present invention includes under tight situation the nucleotide sequence of (as: 65 ℃, 0.1 * SSC{1 * SSC=0.15NaCl, 0.015 M Trisodium Citrate pH7.0 }) and nucleotide sequence hybridization of the present invention.No matter nucleotides sequence of the present invention is classified two strands as, and double-helical two strands, or both mixtures all are contained among the present invention.When nucleotides sequence is classified strand as, can think that the complementary sequence of nucleotide sequence also is contained among the present invention.

Expression vector

Nucleotide sequence of the present invention can be participated in the recombinant replication carrier.Carrier can be used for duplicating and/or express and fit nucleotide sequence host cell or that come from it mutually.Use comprises promotor/enhanser and other expression conditioning signals are controlled expression.Promotor can play the promotor of function for prokaryotic promoter or in eukaryotic cell.Also can using-system specificity or stimulation specificity promoter.The chimeric promoters that comes from above-mentioned two or more different promotor also can use.

Depend on used sequence and/or carrier, the material that host's reconstitution cell produces can be secreted or is contained in the born of the same parents.The material encoding sequence can be designed as and contains the signal sequence that directly secretion of material encoding sequence is passed eucaryon or prokaryotic cell prokaryocyte cytolemma.

Fusion rotein

For the ease of extracting and purifying, material of the present invention also can be with the form production of fusion rotein, the pairing body (partners) of fusion rotein can be glutathione S-transferase (GST), 6 * Histidine, GAL4 (DNA combination and/or transcriptional activation zone) and beta-galactosidase enzymes.Can match at fusion rotein and add a proteolysis site between body (partners) and the institute's protein of interest so that the removal of fusion rotein sequence.Preferably, fusion rotein should not hinder the activity of the material that comprises aminoacid sequence of the present invention.

In one embodiment of the invention, fusion rotein has comprised the antigen or the antigenic determinant of merging with material of the present invention.In this embodiment, fusion rotein is that comprise can be as the non-spontaneous fusion rotein of the material of the adjuvant of extensive stimulating immune system.Antigen or antigenic determinant adhere to the amino or the C-terminal of this material.

In another embodiment of the present invention, material of the present invention can be connected on the heterologous sequence of an encoding fusion protein, for example: influence the peptide of the active reagent of material storehouse in order to screen, the utmost point is usefully encoded one and can be expressed chimeric material by the allos epi-position of industrialization antibody recognition.

In another embodiment, using can Analysis and Identification such as reagent such as target acceptor in conjunction with fusion rotein, or can regulate the reagent of CD25 transcriptional activity.

Antibody

In one embodiment of the invention, material of the present invention can be an antibody.This antibody can be used as stand-in of the present invention.

Antibody can pass through standard techniques, as: utilize material of the present invention to carry out immunity or utilization phage display library and producing.

" antibody " unless specifically refer to a certain opposite meaning, includes but are not limited to polyclonal antibody, monoclonal antibody, embedding and thing, strand, Fab fragment and the fragment that is produced by the Fab expression library.These fragments comprise with the target material having the fragment that combines active complete antibody, Fv, F (ab ') and F (ab ') ₂, and the antibody of strand (scFv), the fusion rotein of the antigen binding site of formation antibody and other synthetic proteins.In addition, antibody and its fragment can be humanized antibody, for example: described in the substance A-239400.Neutralizing antibody, as: the antibody of inhibitory substance polypeptide biologic activity is particularly useful for diagnosis and treatment.

In one embodiment, the present invention also provides monoclonal antibody or polyclonal antibody for material of the present invention such as polypeptide and its fragment.Thereby, the present invention be the monoclonal antibody of material or polyclonal antibody as: polypeptide of the present invention provides a production approach.

Polyclonal antibody

If preparation polyclonal antibody, select Mammals (as: mouse, rabbit, goat, horse etc.), carry out immunity with the immunogenicity polypeptide that obtains from appraisable reagent and/or material of the present invention,, adds different adjuvant raising immune responses according to used host's kind attribute with epi-position.These adjuvants include but are not limited to:, formula adjuvant not, mineral glue be as aluminium hydroxide, and surfactant is as lysolecithin and pluronic polyvalent alcohol, polyanion, peptide, oil-emulsion, keyhole  hemocyanin, dinitrophenol(DNP), BCG (bacille Calmette-Guerin vaccine) and diphtheria toxin (Corynebacterium).If the peptide material of purifying applies to the individuality of immunologic injury, stimulate its system's defence, above-mentioned substance will be potential human adjuvant.

Immune serum is collected the back and is handled with known program, if contain in the serum of polyclonal antibody, except containing the epi-position that comes from a certain appraisable reagent and/or material of the present invention, also comprise other antigenic antibody, can carry out purifying to it through immunoaffinity chromatography.Production and the sero-fast technology of purifying polyclonal antibody are well known in the art.In order to prepare this antibody-like, the present invention also provides polypeptide of the present invention and the fragment thereof as animal and immunogenic other polypeptide haptenizations of people.

Monoclonal antibody

Directly be incorporated into come from can certified reagent and/or the monoclonal antibody of the epi-position of material of the present invention also can be with technology production well known to those skilled in the art.Preparing monoclonal antibody method by hybridoma is widely known by the people, permanent antibody producing cells is to pass through cytogamy, also can by other technologies as: bone-marrow-derived lymphocyte is with the generation that directly is converted of carcinogenic DNA or EB-virus, a series of monoclonal antibodies in conjunction with the orbit epi-position that produced can screenedly be used for different property testings: as isotype and epi-position affinity.

The MONOCLONAL ANTIBODIES SPECIFIC FOR of material and/or reagent of the present invention can obtain by the production technology of cultivating antibody molecule in the continuous cell line.This comprises but not only comprises Koehler, the described hybridoma technology of Milstein (1975 Nature 256:495-497), human B cell hybridoma technology (Kosbor etc., (1983) Immunol Today 4:72, Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and EBV-hybridoma technology (Cote et al (1985) Monoclonal AntibodiesTherapy, Alan R Liss Inc, pp77-96), in addition, also can use " chimeric antibody " production technology, technology (Morrison et al (1984) the Nature 312:604-608 that the montage acquisition of mouse antibody genes and human immunoglobulin gene has suitable antigen-specific and biologic activity; Takeda et al (1985) Nature 314:452-454).The technology (U.S. Patent number 4,946,779) that single-chain antibody is produced is applicable to produces the material specific single-chain antibody.

But directly, comprise monoclonal antibody and polyclonal antibody, especially in diagnosis, be carried out utilization in conjunction with the antibody that comes from the epi-position of indentifying substance and/or material of the present invention.Those neutralizing antibodies then have utilization in passive immunotherapy.Monoclonal antibody is particularly useful for the plantation (raising) of antiidiotypic antibody.Antiidiotypic antibody is a kind of immunoglobulin (Ig) with the protected material of design and/or reagent " inner video " (" internal image ").The technology of plantation antiidiotypic antibody is widely known by the people these antiidiotypic antibodys thereby highly significant in the art.

Antibody induce or screen in also can body by lymphocyte populations the recombination immunoglobulin library or as: Orlandi et al (1989, Proc Natl Acad Sci 86:3833-3837) and one group of experiment of being showed of Winter G and Milstein et al (1991, Nature 349:293-299) with high degree of specificity binding reagents obtain.

The antibody fragment that comprises the material specific binding site also can produce.For example: this fragment including, but not limited to through the pepsin hydrolysis antibody molecule and (Fab ') ₂Fragment, the Fab fragment can be by reducing (Fab ') ₂Disulfide linkage and obtain.The Fab expression library is beneficial to and makes that to have a segmental evaluation of required specific polyclone Fab simple, easily row (Huse WD et al (1989) Science 256:1275-1281).

Immunity modulation species analysis

Immunoreactive immune modulation can be measured by the situation of transcribing, as: the transcriptional activity of the signal analysis CD25 cell surface marker thing of the reporter gene that is connected by mensuration.

Reporter gene

The signal surveyed that preferred reporter gene can be provided convenience can be used in the analytical procedure of the present invention, (for example: spectroscopy).For example: but the enzyme of a kind of catalysis change of reporter gene codified optical absorption characteristics.

The example of reporter gene includes but are not limited to beta-galactosidase enzymes, saccharase, green fluorescent protein, luciferase, paraxin, Transacetylase, β-glucuronidase, exoglucanase and glucoamylase.Radio-labeling or fluorescently-labeled Nucleotide can participate in new life's the transcript, are identified out when being attached to oligonucleotide probe.

In an embodiment preferred, the generation of reporter gene molecule by the enzymic activity of reporter gene product as: beta-galactosidase enzymes is measured.

The analysis of the inhibitor of toxin-induced diarrhoea

Material or derivatives thereof of the present invention or homologue and/or the clone of expressing material or derivatives thereof of the present invention or homologue can be used for influencing the screening (as antibody, peptide, organic molecule or non-organic molecule) of the active reagent of material.For example: any inhibitor that can the active reagent of inhibitory substance can be used as the diarrhoea of toxin-induced screens, thereby identifies the diarrhea disease that can influence cholera and/or enterotoxin mediation.

In one embodiment, screening of the present invention can be identified the antagonist of material of the present invention, as: can be as the antibody of the diarrhoea inhibitor of toxin-induced, peptide, or little organic molecule

Reagent analysis

Phage display can be used for the evaluation of reagent, for example: by can with the cell surface receptor that combines.The positive identification of this acceptor is beneficial to the utilization combinatorial library and identifies the stand-in that have with the same or similar function of material of the present invention.

Phage display is the program of the molecular screening of a utilization recombinant bacteria phage, present technique comprised can same desired substance (or derivatives thereof or homologue) or the encoding gene of the suitable aglucon (being candidate agent in this example) of nucleotide sequence (or derivatives thereof or the homologue) reaction of coding identical sequence be transformed in the bacteriophage.The bacteriophage (preferably adhering to a solid support) that transforms is expressed suitable part (for example candidate agent), and is showed in its phage ghost.The separated and amplification of entity (as cell) with desired substance molecule of identification candidate agent.The candidate agent that obtains carries out characterized.Phage display has superiority with respect to the aglucon avidity triage techniques of standard.Phage surface more approaches the form of its natural structure phase and shows candidate agent with three-dimensional structure phase, and this makes that just the purpose of screening has more the binding affinity of specificity and Geng Gao.

Model analysis

In one embodiment, screening of the present invention can be identified the stand-in of material of the present invention, as: antibody or other have the compound of immunity modulation and/or adjuvant effect.

This analoglike thing can be used for treatment of diseases of the present invention separately or with the other treatment method jointly.

Screening

Material of the present invention can be used to identify immunomodulator in any class drug screening technology, adjuvant, and analogue is or/and the diarrhoea inhibitor that toxin causes.The material that applies to screen can be the solution shape, is bonded to the upholder of solid phase, is carried on cell surface, or is positioned at cell.The formation that combines complex body between active forfeiture of material or material and test agent can be detected.

Another technology that is used for high flux screening has the suitable bonding force of commaterial, is dependent on the method that describes in detail in WO84/03564.

Analytical procedure of the present invention will be suitable for a small amount of of testing compound or screening and quantitative analysis in a large number.

Pharmaceutical composition

The present invention also provides a kind of pharmaceutical composition, comprises the material of the present invention of administering therapeutic significant quantity and acceptable carrier pharmaceutically, thinner or vehicle (comprising its combination).

Pharmaceutical composition comprises people's medication or the veterinary medicine that is applied to the human or animal, generally also comprises one or more pharmaceutically acceptable carrier, thinner or vehicle.Acceptable carrier pharmaceutically, thinner or vehicle widely pharmaceutical field personnel are known, and are documented in the Sciences such as Remington ' s Pharmceutical, among the Mack PublishingCo. (A.R.Gennaro version in 1985).Pharmaceutically acceptable carrier, the selection of thinner or vehicle depend on the pharmacy practice of desiring administering mode and standard.Pharmaceutical composition also may comprise (or additionally comprising) carrier, vehicle or any suitable binder, lubricant, suspension agent, embedding medium, solubilizing agent.

Sanitas, stablizer, dyestuff even odorant all can be applied in the pharmaceutical composition.Examples of preservatives comprises Sodium Benzoate, Sorbic Acid and p-Hydroxybenzoate.Antioxidant and suspension agent also can be used among the pharmaceutical composition.

According to different drug delivery systems, has different compositions/prescription requirement.For example, pharmaceutical composition of the present invention can be made into little pump or makes the spray nose because of the mucous membrane approach, or makes aerosol and suck, or makes the solution that can take in, or makes injectable reagent, for example vein, muscle or subcutaneous injection because of parenteral absorption.Perhaps, pharmaceutical composition also can be designed to two kinds of administering modes.

When medicament during via the gastrointestinal mucosa administration, it should keep stable in being transported to GI process.For example, it must resist proteolysis, and acid pH is stable and resist biliary degraded.

The words that compatibility is suitable, this pharmaceutical composition can be used in the following manner: suck, bolt medicine or medicated vaginal suppository generally are washing lotions, solution, creme, ointment or epipasxtic, skin patch, oral with the form that adds the tablet that vehicle such as starch or lactose make, add or do not add the capsule that vehicle is made, vaginal suppository adds the elixir or the suspension of toner or odorant; But also parenteral injection, for example, vein, muscle or subcutaneous injection.For administered parenterally, pharmaceutical composition is preferably made the form of aseptic aqueous solution, and this aqueous solution also may contain other materials, oozes to keep solution and blood etc. as the salt and the monose of capacity.For oral or sublingual administration, composition can be made tablet or lozenge administration according to ordinary method.

Vaccine

In one of embodiment of the present invention, this material is incorporated into as adjuvant and is used for resisting in the vaccine composition of autoimmune disease, HTL, organ-graft refection and supersensitivity or communicable disease.

In other embodiments of the present invention, vaccine composition also may be extra comprises antigen or antigenic determinant.The antigen or the antigenic determinant that are fit to are disclosed in WO99/34817.

Preferably, vaccine composition includes antigen or antigenic determinant.

Preferably, antigen is autoantigen or its homologue.

Preferably, one or more materials of the present invention are used to the preparation of therapeutic or preventative vaccine.

" preventative vaccine " is meant and is used to the vaccine that normal individual stops disease progression.

" therapeutic vaccine " is meant that being used to infected individuals dispels or reduce infection, perhaps is used to cancel the vaccine of the immunopathology process of disease.

Comprising one or more materials is widely known to these those skilled in the art as the preparation method of the vaccine of activeconstituents.Typically, this vaccine is used to injection, and it should be liquor or suspension; And the solid form before liquor that is used to inject or suspension also should be prepared.Vaccine also can be prepared into emulsification or by the proteic form of liposome bag quilt.Active ingredient usually with that pharmaceutically accept and the mixed with excipients compatible with activeconstituents.For example, the vehicle of Shi Heing has water, salts solution, glucose, glycerine, ethanol or the like, or its combination.

In addition, if be ready, vaccine also can comprise the wetting agent or the auxiliary substances such as emulsifying agent and pH buffer reagent of trace.

The vaccine compound also may comprise the adjuvant that is used for strengthening vaccine effect.Effectively adjuvant has following severally, but is not limited to that these are several: aluminium hydroxide, aluminum phosphate, potassium aluminium sulfate, beryllium sulfate, silicon-dioxide, kaolin, carbon, water-in-oil emulsifier, oil-water emulsifiers, Muramyl dipeptide, bacterial endotoxin, lipid X, Propionibacterium (Propionbacterium acnes), bordetella pertussis, polyribonucleotide, sodiun alginate, lanolin, Ultrapole L, vitamin A, saponin(e, liposome, LEVAMISOLE HCL, DEAE-dextran, block interpolymers or other synthetic adjuvants.These adjuvants can obtain from multiple commercial sources.For example, Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.) or Freund ' s IncompleteAdjuant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan)

Typically, generally use Amphigen (oil-in-water), Alhydrogel (aluminium hydroxide), or both mixtures.Have only aluminium hydroxide to be permitted for the mankind.

Administration

Usually, be fit to individual actual dose and determine, and change with age, body weight, concrete patient by the doctor.Following dosage is the typical amounts of average example.Certainly, in the example above-mentioned dosage may appear also being more or less than individually.

The compounds of this invention can be used to direct injection.This composition can be via parenteral, mucous membrane, muscle, vein, subcutaneous or percutaneous dosing.Typically, every kind of proteinic dosage is a per kilogram of body weight 0.01-30 milligram, is preferably the 0.1-10 milligram, and per kilogram of body weight 0.1-1 milligram is the most preferred.

" administration " comprises through virus and non-viral approach.The virus delivery mechanism is including but not limited to following several carriers: adenovirus carrier, gland relevant viral vector, herpesvirus vector, retrovirus vector, lentiviral vectors and baculovirus vector.Non-viral delivery mechanism comprises the transfection of fat mediation, liposome, immunoliposome, lipofectin reagent, surperficial cationic hydrophilic fat molecule and combination thereof.These delivery mechanism paths include but not limited to from mucous membrane, nose, mouthful, parenteral, stomach and intestine, send in part or hypogloeeis.

" administration " includes but not limited to utilize the approach of the per mucous membrane that sprays nose or aerosol suction; Parenteral route by vein, muscle or subcutaneous injection form.

" altogether administration " is meant to obtaining necessary immunity system adjustment and additionally adds antigen and/or antigenic determinant, they and any material of the present invention at one time, same site is applied to individuality.Yet, though this material can with antigen at one time, the altogether administration of same site, this material and antigen are at different time, the different loci administration also has certain advantage.This material and antigen even can in same vehicle, be sent.This material and antigen can be coupled and/or non-be coupled and/or gene level on be coupled and/or non-being coupled.

Antigenic determinant and peptide or homologue or stand-in can with single dose or multiple doses be individually dosed or administration altogether.

Vaccine composition among the present invention can pass through different administrations, as: injection (comprising administered parenterally, subcutaneous and intramuscular injection), intranasal, mucous membrane, organ, intravaginal, urethra or dosing eyes.

The vaccine that comprises material among the present invention is generally by parenteral admin, for example by subcutaneous or intramuscular injection.The prescription that is fit to other administering mode comprises suppository and the oral preparation used etc. in certain a few example.For suppository, traditional tackiness agent and carrier comprise polyalkylene glycol or triglyceride level; These suppositorys are mostly made active ingredient and are accounted for 0.5%-10%, general mixture at 1%-2%.For oral preparation, the vehicle of normal use comprises the N.F,USP MANNITOL of pharmaceutical grade, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate or the like.The form of these mixtures can be solution, suspension, tablet, pill, capsule, slowly-releasing or pulvis.These preparations contain 10% to 95% activeconstituents, and preferred 25% to 70%.When vaccine composition was made lyophilized powder, lyophilized powder should be reconstructed into the form such as suspension before administration.The preferred process of reconstruction in damping fluid.

Diseases related

The material that relates among the present invention often is used to treatment or stops autoimmune disease, HTL, organ-graft refection, allos or xenotransplantation, graft versus host disease (GVH disease) (GVHD), allergy or communicable disease.Communicable disease is meant that in course of infection infectious factor is attached to colon as other immunopathology mechanism diseases, especially the disease of mucous membrane.

The present invention relates to communicable disease including but not limited to HSV-1, HSV-2, EBV, VZV, CMV, HHV-6, HHV-7 and HHV-8, hepatitis A, B, C, D and E, neisserial meningitis, hemophilus influenzae B and streptococcus pneumoniae, legionella pneumophilia and mycobacterium tuberculosis, neisserial gonorrhoea, HIV-1, HIV-2 and chlamydia trachomatis, intestinal bacteria, rotavirus, Salmonella enteritidis, Corynebacterium diphtheriae, helicobacter pylori, Helicobacter pylori, campylobacter jejuni and vibrio cholerae, staphylococcus aureus, Streptococcus pyrogenes, mutan suis, malaria, trypanosomiasis, Taxoplasma gondii, Leishmaniadonovani and parcel parasitosis.

The example of the supersensitivity that the present invention relates to imbalance is including but not limited to following disease: asthma, allergic cough, allergic rhinitis and conjunctivitis, specificity eczema and dermatitis, urticaria, insect sting allergy, diet and drug allergy.

The example of autoimmune disease is including but not limited to following several, as rheumatoid arthritis, and multiple sclerosis and diabetes.

Test kit

The present invention further provides the diagnosis test kit that comprises material of the present invention.These test kits are applied to stoping and/or treat and/or adjust disease of the present invention.

In one embodiment of the present invention, test kit also includes antigen and/or antigenic determinant and/or independent adjuvant, for the common administration of treatment and prevention.

Additionally, test kit also may provide stand-in of the present invention with the form of the antibody that the present invention relates to, and it can be attached to solid support and/or with suitable reagent, and contrast illustrates etc. to be packaged in the test kit together.

Brief summary

In brief, the present invention relates to a kind of material, it comprise following one or more: a kind of aminoacid sequence wherein comprises the sequence that is disclosed among the SEQ ID NO.2, or its variant, homologue, fragment, derivative, stand-in, these materials can work in the mode identical with EtxB and CtxB; But these materials do not have GM-1 in conjunction with activity simultaneously.

The invention still further relates to a kind of measuring method, whether this method is used to measure one or more reagent can or influence material of the present invention with matter interaction of the present invention, wherein said mensuration comprises that described material contacts with test agent, determines then whether these reagent influence described material.

The present invention relates to the other aspect is listed below:

1. a peptide comprises the EVPGSQH sequence, or its homologue or stand-in.

2.1 in the peptide section, comprise the EVPGSQHIDSQ sequence.

3.2 in peptide, comprise GATFQEVPGSQHIDSQKKAI or GETFQEVPGSQHIDSQKKAI sequence.

4. one kind is prevented or therapeutic composition, comprises peptide or their homologue or stand-in among the above-mentioned 1-3.

5. prevention described in a kind 4 or therapeutic composition also comprise antigen or antigenic determinant.

6. prevention described in a kind 4 or 5 or therapeutic composition, application of treatment or preventative reagent are as adjuvant and immunomodulator simultaneously.

7. prevention described in a kind 4 or 5 or therapeutic composition, application of treatment or preventative reagent come the output to the antibody of adjusted mucomembranous surface simultaneously.

8. prevention described in a kind 4 or 5 or therapeutic composition, application of treatment or preventative reagent prolong antigen presentation and keep the immunological memory that Mammals is tried body simultaneously.

9. prevention described in a kind 4 or 5 or therapeutic composition, application of treatment or preventative reagent are reduced the immunoreactive pathology composition that links to each other with Th2 with Th1 simultaneously.

10. one kind 4 to prevention described in 9 or therapeutic composition, is used to treatment or stops autoimmune disease, HTL, organ transplantation, GVHD or communicable disease.

11. prevention or therapeutic composition comprise the reagent component on β 4-α 2 rings that can specificity be attached to EtxB or CtxB.

12. prevention described in a kind 11 or therapeutic composition, reagent component wherein are antibody.

13. prevention or therapeutic composition in one kind 11 or 12 are used to treatment diarrhoea.

14. a vaccine composition that is used to disease treatment comprises the described peptide section of 1-3 kind or its homologue or stand-in.

15. the vaccine composition described in a kind 14 also comprises antigenic determinant.

16. the vaccine composition described in a kind 14 or 15 is used for the treatment of or stops communicable disease, autoimmune disease, HTL, organ transplantation, GVHD etc.

17. a test kit comprises described treatment of any 4-13 or prophylactic compositions.

The present invention will be specifically described by the following example, and with reference to the accompanying drawings.

Fig. 1 has shown the three-dimensional band chart of the B subunit of EtxB/CtxB.(draw from Sixma etc., J.Mol.Biol. (1993) 230:890-918, figure index person adds).

Fig. 2 has shown the ring-type residue with the apoptosis-related CtxB of CD8+T.

Fig. 3 has shown that CD8+T apoptosis defective B subunit mutant still remains with the ability in conjunction with cell surface receptor.

When Fig. 4 has shown the intranasal immunity of 10ugB subunit, the whole immunoglobulin level of the EtxB of mice serum and CtxB (H57S).

Fig. 5 has shown the necessary zone of His-57 decision adjuvanticity of EtxB and CtxB.

Fig. 6 has shown that E51-158B subunit peptides section has the ability of inducing CD8+T cellular immunization to regulate.

Embodiment

Embodiment 1

Residue triggers the evaluation of leukocytic immunity regulating effect on the Glu-51-Ile-58 ring

The NIH male mice is put to death, and gets the mesenteric lymph nodes tissue and puts into Hanks balanced salt solution (no calcium ion of HBSS and magnesium ion, and adding 20mM Hepes).Push gently from fibrous tissue by nylon mesh and to divide the lymphocyte that sheds, HBSS to give a baby a bath on the third day after its birth time, lymph-node cell is suspended with Eagle improved culture medium (Gibco), this substratum contains 20mM Hepes, 4mM L-glutaminate, 100IU/ml penicillin, 100 μ g/ml Streptomycin sulphates, 5 * 10 ^-5The M2-mercaptoethanol, cell concn is controlled at 2*10 ⁶Cell/ml.Add or do not add wild-type EtxB or the CtxB of 3.45 μ M (40 μ g/ml) then, or various B subunit mutant, as EtxB (G33D), CtxB (E51A), CtxB (V52A), CtxB (P53A), CtxB (G54A), CtxB (S55A), CtxB (Q56A), CtxB (H57A) or 37 ℃ of incubations of CtxB (I58A) 96 hours.Then, with 0.4ml HBSS/20mM Hepes/0.1%NaN ₃/ 10% mice serum is washed and suspension cell, by 1/400 add that anti--CD8 that phycoerythrobilin (PE) is coupled and FITC-be coupled resist-CD4 ice bath 30 minutes.Behind the antibody incubation, cell suspension is washed once with ISOTON, and resuspended with ISOTON 0.4ml.Every group of sample chosen 10,000 examples and done facs analysis, result's WinMDI software processes.Anti--CD4 that the upper left FITC-of showing of Fig. 2 is coupled; The bottom right shows anti--CD8 that phycoerythrobilin is coupled.Every figure expresses percentage ratio.

As a result 1

As shown in Figure 2, wild-type EtxB or CtxB make CD8 ⁺The T cell reduces; And next this phenomenon that do not take place of situation that exists at PBS (contrast) or EtxB mutant (EtxB (G33D) not can be incorporated on the Sphingolipids,sialo of cell surface).CtxB (E51A) and CtxB (H57A) can not trigger CD8 ⁺The T cell reduces; In addition, CtxB (V52A) and CtxB (I58A) cause triggering CD8 ⁺The hypocellular part defective of T.These results show that E51 and H57 play an important role on triggering lymphocytic regulating effect, V52 and I58 then play booster action.

Embodiment 2

B subunit defective mutant is at CD8 ⁺Keep ability in the t cell proliferation in conjunction with cell surface receptor

The NIH male mice is put to death, and gets the mesenteric lymph nodes tissue and puts into Hanks balanced salt solution (no calcium ion of HBSS and magnesium ion, and adding 20mM Hepes).Push gently from fibrous tissue by nylon mesh and to divide the lymphocyte that sheds, HBSS to give a baby a bath on the third day after its birth time, lymphocyte be resuspended in the 300ml precooling and the degassing the MACS damping fluid (PBS, 5mM EDTA, 0.5%BSA, pH7.2) in.In cell, add anti-CD4 of 50ml and anti-B220MACS antibody, select to be purified into CD8 by magnetic MACS negativity ⁺The T cell.With CD8 ⁺The T cell suspends with Eagle improved culture medium (Gibco), and this substratum contains 20mM Hepes, 4mM L-glutaminate, 100IU/ml penicillin, 100 μ g/ml Streptomycin sulphates, 5 * 10 ^-5The M2-mercaptoethanol, cell concn is controlled at 2 * 10 ⁶Cell/ml.Add or do not add wild-type EtxB or the CtxB of 3.45 μ M (40 μ g/ml) then, or various B subunit mutant, as EtxB (G33D), CtxB (E51A), CtxB (V52A), CtxB (P53A), CtxB (G54A), CtxB (S55A), CtxB (Q56A), CtxB (H57A), EtxB (H57S) or 37 ℃ of ice of CtxB (I58A) were educated 20 minutes.Then, use the 0.4ml HBSS/20mM Hepes/0.1%NaN of precooling on ice ₃/ 10% mice serum is washed and suspension cell, by 1/500 anti-EtxB monoclonal antibody 118-8 of adding and EtxB, and EtxB (G33D), EtxB (H57S) is hatched; Adding anti-CtxB monoclonal antibody LT-39 and CtxB and mutant thereof by 1/800 hatches.After 30 minutes, use 0.4mlHBSS/20mM Hepes/0.1%NaN ₃/ 10% mice serum is washed and suspension cell, and adds the anti-mouse IgG antibody of FITC mark, two anti-hatching 30 minutes.Cell suspension is washed once with ISOTON (Becton-Dickinson), and resuspended with ISOTON 0.4ml.The FITC fluorescence intensity that facs analysis shows is represented EtxB, and CtxB and mutant thereof are in conjunction with the ability of CD8+T cell.10,000 examples of every group of sample show the CD8+T cells at the fluorescence that does not have B subunit to being combined in the fluorescence intensity of B subunit.

As a result 2

As shown in Figure 2, all B subunits, identical in conjunction with the CD8+T cell ability except that no binding ability mutant EtxB (G33D).The a little higher than wild-type B of the detectable fluorescence intensity subunit that combines CtxB (H57A) and EtxB (H57S) illustrates that these two kinds of mutant have higher cell surface avidity.This result and CtxB (H57A), EtxB (H57S) have higher affinity with the microtiter plate of GM-1 embedding, and the resonance of cytoplasmic mass surface is measured it has the result of higher Kd value consistent with GM-1.(data not shown)

Embodiment 3

The His-57 residue of EtxB is by inducing the anti-EtxB reaction of intensive necessary

Get 8 groups of female mouse intranasal immunity 10ugEtxB of NIH or EtxB (H57S) 20 μ l, in a week, divide three immunity.After the immunity 14 days are put to death careful puncture and are got blood for the third time.Wrap the level of the microtiter plate of quilt with 1 μ g/mlEtxB through the anti-EtxB IgG of the methods analyst of GM-1-ELISA.Endpoint titration is determined.(equaling the dilution that absorption value is higher than background 0.1)

As a result 3

High anti-EtxB IgG antigen titration serum (titre is 5757+/-785) appears in intranasal immunization EtxB as shown in Figure 4; Immunization EtxB (H57S) then induces the reaction that significantly (p=0.001) is low (titre is 1205+/-222).

Embodiment 4

The His-57 residue of EtxB and CtxB is necessary as the mucous membrane adjuvant by B subunit

Get 8 groups of female mouse intranasal immunity 10ug ovalbumins of NIH or be mixed with EtxB, CtxB, immune 10 μ g ovalbumins under the situation of EtxB (H57S) or CtxB (H57A), 20 μ l divide three immunity in a week.In addition, two groups of only intranasal immunity EtxB or CtxB are as negative control.After the immunity 14 days are put to death careful puncture and are got blood for the third time.With the microtiter plate of 5 μ g/ml ovalbumin bag quilts level through the methods analyst antiovalbumin IgG of ELISA.Endpoint titration is determined.(equaling the dilution that absorption value is higher than background 0.1)

As a result 4

As shown in Figure 5, and only compare with the ovalbumin immune mouse, wild-type EtxB and CtxB have increased antiovalbumin reaction (relatively swimming lane 4,5 and swimming lane 1) widely as the mucous membrane adjuvant.On the contrary, when ovalbumin is mixed with EtxB (H57S) (swimming lane 6) or CtxB (H57A) (swimming lane 7), the reaction that the antiovalbumin response intensity triggers less than wild-type B subunit.The no adjuvanticity of data presentation CtxB (H57A).This result more proves the importance and the vital role of H57 residue on the immunoloregulation function of reporter molecule of E51 one I58 of B subunit ring.

Embodiment 5

Encircle corresponding one section section of synthesized peptide EVPGSQHI with EtxB and CtxBE51-I58 and have immunoloregulation function.

For encircling corresponding one section section of synthesized peptide, research and EtxB and CtxBE51-I58 whether cause that the CD8+T cell reduces, the method of pressing in the embodiment 1 is separated the mesentery lymphocyte, the control peptide section IRNETTTTKGDYC incubation that adds different concns (0.1 μ M-20 μ M) section of synthesized peptide EVPGSQHI or select at random.37 ℃, use 0.4ml HBSS/20mMHepes/0.1%NaN after 96 hours ₃/ 10% mice serum is washed and suspension cell, draws CD4 through facs analysis then ⁺And CD8 ⁺The relative proportion of cell, concrete grammar are seen example 1.The per-cent of CD8+ cell is figure (Fig. 6) to different concns after the control peptide section (solid mass colour square curve) of section of synthesized peptide EVPGSQHI (black circle red curve) or selection is at random handled.

As a result 5

The section of synthesized peptide of presentation of results shown in Fig. 6 EVPGSQHI causes that the CD8+T cell reduces, and control treatment does not then have.This explanation and EtxB and CtxBE51-I58 encircle corresponding one section section of synthesized peptide EVPGSQHI lymphocyte are produced immunoloregulation function.

All publications that this specification sheets is mentioned are hereby incorporated by document.Under the prerequisite of the scope of the invention and spirit, obviously be suitable by the numerous modifications and variations that those skilled in the art did.Although the present invention in conjunction with specific embodiment, should not think in view of the above that the present invention only is confined to above-mentioned specific embodiment yet when describing.In fact, concerning molecular biology or association area skilled person, above-mentioned embodiment of the present invention is obvious.Diversified modification will be comprised in subsequently claims.

Sequence table SEQ ID No 1GAA GTA CCA GGT AGT CAA CAT ATA GATSEQ ID No 2EVPGSQHSEQ ID No 3VEVPGSQHIDSQSEQ ID No 4GATFQVEVPGSQHIDSQKKAISEQ ID No 5GETFQVEVPGSQHIDSQKKAI

Claims (11)

1. one kind comprises following one or more material: one section aminoacid sequence that comprises sequence shown in the SEQ ID No.2, or its variant, or its homologue, or its fragment, or derivatives thereof, or its stand-in; This material can work with EtxB and/or the same or analogous mode of CtxB; But this material does not have GM-1 in conjunction with activity.

2. a kind of material of definition is used for medicine in the claim 1.

3. a kind of material of definition is used for immunomodulator in the claim 1.

4. a kind of material of definition is used as adjuvant in the claim 1.

5. a kind of material of definition is used for the inhibitor of the diarrhoea that toxin causes in the claim 1.

6. the defined material of above-mentioned each claim, wherein this material also contains antigen or antigenic determinant.

7. pharmaceutical composition comprises the material of claim 1-6 described in each, depends on the needs, and also can mix with one or more pharmaceutically acceptable carrier, thinner or inert matter.

8. each defined material of claim 1-7 is used for the treatment of and/or prevents and/or regulate application in relevant disease of the diarrhoea of immunologic derangement and/or toxin-induced and/or the disorderly medicine in preparation.

9. measuring method, be used for determining can with claim 1-7 each matter interaction and/or can influence one or more reagent of each described material of claim 1-7; Wherein said mensuration comprises described material and test agent is contacted, and determines whether that then this reagent can influence described material.

10. the determined material of the measuring method described in the claim 9.

11. a methods of treatment comprises that relevant eqpidemic disease and/or the disorderly experimenter of disorder who treats and/or prevents and/or regulate the mediation of immunologic derangement and/or toxin to needs uses each defined material of claim 1-7.

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