CN101578368A - Novel peptides for treating and preventing immune-related disorders, including treating and preventing infection by modulating innate immunity - Google Patents

Abstract

In one aspect, the present invention provides isolated novel peptides that can be used to modulate innate immunity in a subject and/or for the treatment of an immune- related disorder, including treating and preventing infection by modulating innate immunity. Also provided are an agent reactive with the peptide, a pharmaceutical composition that includes the peptide, an isolated nucleic acid molecule encoding the peptide, a recombinant nucleic acid construct that includes the nucleic acid molecule, at least one host cell comprising the recombinant nucleic acid construct, and a method of producing the peptide using the host cell. The present invention further provides a method for treating and/or preventing infection in a subject by administering the peptide of the invention to the subject, thereby modulating innate immunity in the subject. Additionally, the present invention provides a method for predicting whether a subject would be responsive to treatment with a peptide of the invention.

Description

Comprise by regulating congenital immunity treatment and the treatment of preventing infection and the new peptides of epidemic prevention relative disease

Related application before

[0001] the application specifies the U.S., submits to as the part continuation application of the PCT/CA2006/001650 that submitted on October 4th, 2006, and it requires the 60/722nd, 962 of submission on October 4th, 2005; 60/722,958 and 60/722, No. 959, title is all the right of priority of the U.S. Provisional Patent Application of " by regulating the new peptides of congenital immunity treatment and preventing infection ", and it is hereby incorporated by reference.

Invention field

[0002] the present invention relates to be used for prevention and treatment immune correlated disease, comprise by regulating the peptide of congenital immunity treatment and preventing infection.On the one hand, the present invention relates to the purposes of composition and adjusting congenital immunity thereof.On the other hand, the invention provides new peptides and effectively reduce the active purposes of DPP IV (DPPIV).

Background of invention

[0003] various microorganisms comprise virus, bacterium, fungi and parasite, can cause disease.Microorganism cells is different from the animal and plant cell, and they can't be survived alone at nature, can only be as the integral part of multi-cell organism and exist.Microorganism cells can be pathogenic also can be non-virulent, this part depends on microorganism itself and host's situation.For example, in the host of immunologic hypofunction, harmless bacterium can become pathogenic former under the normal circumstances.

[0004] resistance remains an obstacle in the anti-infective at present effort.For example, before bacterium developed immunity to drugs, penicillin can effectively be treated infection of staphylococcus aureus.The whole second half in 20th century, new microbiotic such as vancomycin and methicillinum are developed out, have successfully cured infection of staphylococcus aureus.But differentiation has taken place in the seventies in 20th century in the anti-methicillinum bacterial strain of streptococcus aureus, and is perplexing hospital all over the world so far always.Recently the vancomycin drug-fast strain of streptococcus aureus makes first appearance.

[0005] along with the appearance of the chemical sproof serious day by day and new transmissible disease of antimicrobial drug, exists lasting demand to the novel therapeutic compound.Act on the host and the treatment of non-pathogenic bacteria is an ideal, because it does not impel pathogenic bacterium to produce resistance.Especially the medicine that acts on the host through innate immune system provides a kind of promising treatment source.

[0006] host starts from the epithelium barrier and the innate immune system of health to the defence of microorganism, and finally induces the acquired immune response.Host's innate immune response has comprised the mechanism of a cover high conservative, and identification and combating microorganisms infect.The key element of congenital immunity continues to remain on lower level, is activated rapidly during irriate.Innate immune response starts from and touches the reaction that takes place immediately behind the pathogenic micro-organism.The reaction relevant with acquired immunity as the rearrangement of immunoglobulin receptor gene, is not counted as the part of innate response.

Evidence suggests that [0007] innate response helps to control great majority to be infected, and also Inflammatory response is worked.The inflammatory reaction of infecting initiation is considered to the main mechanism of disease pathogenesis.The importance of Toll sample acceptor (TLRs) in innate immune response is also fully characterized.The identification standpatter of mammiferous TLRs family, wherein many standpatters are found on the surface of pathogenic microorganism or are discharged by pathogenic microorganism.Also have many mechanism that other are not fully characterized, cause and/or help host's congenital defence.

[0008] innate immune system provides a series of protection mechanism, comprises the epithelium barrier function, and the secretion of cytokine and chemokine.Up to the present, according to conservative N end halfcystine motif: the quantity of C, CC, CXC and CX3C (wherein X is non-conservative amino-acid residue), chemokine is divided into four families.Known CXC chemokine comprises monocyte to having the cell of CXCR3 acceptor, and activating T cell (Th1) and NK cell have the chemotactic effect.Former generation human airway epithelium cell and clone 16-HBE constitutive expression CXCR3 acceptor and part thereof, IP-10, I-TAC and MIG (Kelsen et al., The chemokinereceptor CXCR3 and its splice variant are expressed in human airwayepithelial cells (Chemokine Receptors CXCR3 and shearing variant thereof are expressed in the human respiratory epithelial cell), Am.J.Physiol.Lung Cell Mol.Physiol., 287:L584,2004).In addition, the CXCR3 part is induced intracellular chemotactic response of 16-HBE and Actin muscle reorganization (Kelsen et al., The chemokine receptor CXCR3 and its splice variant areexpressed in human airway epithelial cells (Chemokine Receptors CXCR3 and shearing variant thereof are expressed in the human respiratory epithelial cell), Am.J.Physiol.Lung Cell Mol.Physiol., 287:L584,2004).

[0009] II type transmembrane serine protease DPP IV (DPPIV) is called CD26 or adenosine deaminase binding protein again in addition, is one and regulates various physiological processs, comprises the important regulon of immunologic function.CD26/DPPIV is the cell surface glycoprotein of a 110kD, mainly at ripe thymocyte, activated T cell, B cell, NK cell, expresses on scavenger cell and the epithelial cell.It has two kinds of functions at least, signal conduction function and proteolysis function (Morimoto C, Schlossman SF.The structure and function of CD26 in the T-cell immune response (26S Proteasome Structure and Function of CD26 in the t cell immune response) .Immunol.Review.1998,161:55-70.).Its cytosis relates to separates dipeptides from chemokine N end check and regulates the chemokine activity.The aminoterminal adjusting of chemokine not only to the combining and following reaction of its acceptor, and all be very important to the receptor-specific that changes treated chemokine.The DPPIV activity is associated with many immune correlated diseases.

Summary of the invention

[0010] inventor finds, has the peptide of the aminoacid sequence (being SEQ ID NO:1-90) of one of listed and described peptide of table 1, or its analogue, derivative, variant, or its obvious chemical equivalent can strengthen host's congenital immunity.On the one hand, immunomodulatory peptides of the present invention shows when improving the infected host survival ability, is found to lack direct anti-microbial activity.On the other hand, the invention provides the active peptide of adjusting DPPIV.The invention provides on the one hand and reduce the active peptide of DPPIV.But then, the invention provides and can be used for diagnosing, treat or prevent peptide as the disease of immune system relevant with DPPIV activity and/or congenital immunity.

[0011] therefore, the invention provides on the one hand the isolated peptides of the aminoacid sequence of arbitrary peptide among a kind of SEQ of comprising ID NO:1-90, or its analogue, derivative, or variant, or obvious chemical equivalent, or comprise the peptide of described peptide.In one embodiment, this peptide is to reach 7,8,9 or 10 amino acid, and it comprises SEQ ID NO:1-43, the described peptide of 45-53 and 55-90, or its analogue, derivative, variant or obvious chemical equivalent.In one embodiment, this peptide is to comprise SEQ ID NO:1-43, nearly 7 amino acid whose peptides of 45-53 and 55-90.In another embodiment, it is to comprise SEQ ID NO:1,3-16,18-43, the peptide of 45-53 and 55-90, or the peptide of its analogue, derivative, variant or obvious chemical equivalent, or comprise same substance up to 7,8,9 or 10 amino acid whose peptides.For instance, this isolated peptides has a C end of modifying (for example amidated C end) and/or a N end of modifying.Isolated peptides of the present invention may further include table 1 aminoacid sequence (SEQ ID NO:1-90) or its analogue, derivative, variant or the obvious chemical equivalent of modifying by at least one D type aminoacid replacement.This isolated peptides can further comprise the skeleton of modification, and for example wherein the N end is modified to the N-methyl by acid amides.On the one hand, these keep the immunocompetent modified peptides of female peptide, and keep described active obvious chemical equivalent and be included in the scope of the present invention.

[0012] on the other hand, the present invention further provides a kind of material, itself and the isolated peptides that comprises table 1 aminoacid sequence, or its analogue, derivative, or variant reaction.In one embodiment, this material is a kind of non-spontaneous antibody (for example a polyclone or monoclonal antibody).In one embodiment, this antibody is to use (TamPJ (1988) .Synthetic peptide vaccine design:Synthesis and properties of ahigh-density multiple antigenic peptide system (the synthetic peptide vaccine design: the synthetic and characteristic of high-density multiple antigenic peptide system) .Proc Natl Acad Sci 85 of MAPS antigen prepd, pp.5409-5413., Briand JP, Barin C, Van Regenmortel MHV, Muller S (1992) .Applicationand limitations of the multiple antigen peptide (MAP) system in theproduction and evaluation of anti-peptide and anti-protein antibodies (multiple antigenic peptide (MAP) system in the production of anti-peptide and anti-protein antibodies and application and limitation in estimating) .JImmunol Meth 156:2, pp.255-265), this antigen is connected on the peptide of the present invention by two glycine residues that the C that is inserted in peptide holds.This construct can be applied to a kind of animal such as rabbit subsequently, can use approach well known to gather antibody.On the one hand, this material can be labeled or be used to mark peptide of the present invention.On the other hand, this material can be used for diagnosing the material that can regulate peptide activity or mensuration peptide quantity with screening method with monitoring.

[0013] more on the one hand, the invention provides a kind of isolated nucleic acid molecule, its coding has or comprises the isolated peptides of table 1 aminoacid sequence, or its analogue, derivative, variant, or obvious chemical equivalent.A kind of recombinant nucleic acid construct also is provided, and it comprises the nucleic acid molecule that is operably connected on the expression vector.

[0014] another aspect, the invention provides at least a host cell that comprises recombinant nucleic acid construct of the present invention, also provide a kind of and prepared the peptide that for example has or comprise table 1 aminoacid sequence of the present invention, or the method for its analogue, derivative, variant or obvious chemical equivalent: (a) under the condition that allows peptide to express, cultivate at least a host cell; And (b) recovering peptide from least a host cell or developing medium.

[0015] on the other hand, the invention provides a kind of pharmaceutical composition, it comprises a kind of isolated peptides that has or comprise table 1 aminoacid sequence or be made up of table 1 aminoacid sequence basically, or its analogue, derivative, variant or its obvious chemical equivalent (comprise pharmacy acceptable salt, additive salt, or the ester of above-mentioned any material, and pharmaceutically acceptable carrier, thinner or vehicle or polymorphic).

[0016] on the other hand, the invention provides a kind of peptide by having or comprise table 1 aminoacid sequence or form by table 1 aminoacid sequence basically, or its analogue, derivative, variant or obvious chemical equivalent be applied to the experimenter, treats and/or prevents the method that the experimenter infects (for example infected by microbes).For instance, the experimenter may be infected, or the risk of infection is arranged.In one embodiment, described peptide is regulated experimenter's congenital immunity, infects thereby treat and/or prevent the experimenter.The present invention also provides a kind of method, and it is used to differentiate the infected by microbes of available peptide treatment of the present invention.On the other hand, the invention provides the method for a kind of treatment or prevention DDPIV relative disease.

[0017] the method for the present invention representativeness that can treat and/or prevent infects, and comprises infectation of bacteria (for example Gram-positive or Gram-negative bacteria), fungi infestation, parasitic infection and virus infection.In one embodiment of the invention, this infection is infectation of bacteria (infecting as intestinal bacteria (E.coli), Klebsiella pneumonia (Klebsiella pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Salmonella (Salmonella spp.), streptococcus aureus (Staphylococcus aureus), suis (Streptococcus spp.) or anti-vancocin faecalis).In another embodiment, described infection is fungi infestation (for example mould, yeast or more high-grade fungi infestation).In another embodiment, described infection is parasitiation (for example unicellular or many cells parasitic infection comprises giardia lamblia stiles (Giardia duodenalis), Cryptosporidium parvum (Cryptosporidium parvum), Cayenne ring sporozoite (Cyclospora cayetanensis) and toxoplasma gondii (Toxoplasma gondii)).But in another embodiment, described infection is viral infection (for example AIDS, bird flu, varicella, cold sore, Common Cold, gastroenteritis, gland fever, influenza, measles, parotitis, pharyngitis, pneumonia, rubella, severe acute respiratory syndrome, a following or upper respiratory tract infection correlated virus (for example respiratory syncytial virus)).

[0018] according to method of the present invention, have or comprise the peptide of table 1 aminoacid sequence, or its analogue, derivative, variant or obvious chemical equivalent can directly (promptly be applied to the experimenter to peptide itself) or indirect (for example in the mode that allows this peptide to express the nucleotide sequence of this peptide of coding being applied to the experimenter in the experimenter) is applied to the experimenter.Peptide of the present invention (or nucleic acid of this peptide of encoding) can be oral, parenteral (for example intracutaneous, intramuscular injection, abdominal injection, intravenous injection or subcutaneous injection), part, transdermal, collunarium, lung's (for example tracheae in administration) and/or be applied to the experimenter by osmotic pump.

[0019] but then, the invention provides a kind of by detecting the DPPIV activity of experimenter's diagnostic sample, whether the prediction experimenter is to having the method for replying with comprising the peptide of table 1 aminoacid sequence or treatment that its analogue, derivative, variant or obvious chemical equivalent carry out, wherein said adjusting, active minimizing shows that the experimenter can reply the treatment of carrying out with peptide as DDPIV.On the one hand, the experimenter has or the DDPIV of having relative disease under a cloud.

[0020] in view of follow-up explanation, other aspects of the present invention and advantage will be apparent.But should be appreciated that, because this detailed description makes within the spirit and scope of the present invention many variations and improvement it will be apparent to those skilled in the art that, so when pointing out the preferred embodiment of the invention, detailed description and specific embodiment only provide with illustrational form.

Description of drawings

[0021] relevant accompanying drawing of the present invention is described now, wherein:

[0022] Figure 1A, B and C have described the test-results of embodiment 2.Survival rate %=is with respect to the bacterial growth quantity of solvent control (Tris), for peptide SEQ.ID.NO.1, and 5 and 47, solvent control is configured to 100% bacteria living respectively; Erythr. represent erythromycin.

[0023] Fig. 2 A-G has described the test-results of embodiment 3.Show the every milliliter of colony-forming unit (CFU/ml) of Y-axis and the treatment group (contrast=no peptide of X-axis among the figure; SEQ.ID.NO.1,4,5,6,45 and 47=with the treatment group of peptide treatment with aminoacid sequence separately).The bacterial count of individual mouse as shown in the figure.

[0024] Fig. 3 A and B have described the test-results of embodiment 4.Show the every milliliter of colony-forming unit (CFU/ml) of Y-axis and the treatment group (contrast=no peptide of X-axis among the figure; SEQ.ID.NO.1 and 5=are with having the treatment group of the peptide treatment of aminoacid sequence separately).The bacterial count of individual mouse as shown in the figure.

[0025] Fig. 4 has described the result of the human blood infection research of description among the embodiment 5.Show the every milliliter of colony-forming unit (CFU/ml) of Y-axis and the treatment group (contrast=no peptide of X-axis among the figure; SEQ.ID.NO.5=is with having the treatment group of the peptide treatment of aminoacid sequence separately).

[0026] Fig. 5 has described to treat the effect that LPS stimulating cytokine in the body of earlier external back is replied as the peptide of describing among the embodiment 6.

[0027] Fig. 6 has described as SEQ ID No.5 in the human blood of describing among the embodiment 8,51 and 83 plasma D PPIV dose response curve.

[0028] Fig. 7 has described the dose response curve to SEQ.ID.NO.5 of the present invention as the SEQ.ID.NO.7 (KSRIVPAIPVSLL) of the PCT/CA02/01830 that submitted in 2002 12 years 2 days that describes among the embodiment 9.

[0029] Fig. 8 A and B have described to unite as the antibiotic therapy of describing among the embodiment 10 the enhanced effect of SEQ.ID.NO.1 and 5.

Detailed Description Of The Invention

Definition

[0030] " DDPIV relevant disease " used herein refers to active relevant any doctor with DDPIV Learn disease, the adjusting of wherein said activity can be used for treating and/or preventing or diagnoses described disease. The example of these diseases includes but not limited to: AIDS virus/AIDS, autoimmune disease as Rheumatic arthritis, multiple sclerosis, cancer (for example colon cancer and lung cancer), diabetes With the Graves disease.

[0031] " immune correlated disease " is a kind of passing through or activation or Immunosuppression system, thereby with The disease that experimenter's immune system is relevant, or a kind of by target experimenter immune response as The a certain special component of innate immune response, thus the disease of prevention or diagnosis can be treated Sick.

[0032] " immunocompetence " used herein refers to that the congenital immunity activity (for example can regulate the experimenter Or the activity of regulating DDPIV the activity of innate immune response or its component).

[0033] " adjusting " used herein for example regulates the active or a certain specific reaction of DDPIV, bag Draw together under the specified conditions, with the contrast of active or reaction normal or baseline values is relevant active or Increase or the minimizing of reaction. It also can comprise, is usually increasing or reducing the active or anti-of peptide Answer under the condition of level the keeping of activity or reaction level.

[0034] " pharmaceutically acceptable salt " refers to be usually used in non-toxic bases metal, the alkaline earth of pharmaceutical industry Metal, ammonium salt comprise sodium, potassium, lithium, calcium, magnesium, barium, ammonium, protamine zinc salt, are to use Method preparation well known in the art. This term also comprises non-toxicity acid-addition salts, generally be with Compound of the present invention and suitable organic or inorganic acid reaction make. Representational salt bag Draw together chloride, bromide, sulfate, disulfate, acetate, oxalates, valerate, Oleate, laruate, borate, benzoate, lactate, phosphate, toluenesulfonic acid Salt, citrate, maleate, fumarate, succinate, tartrate, benzene sulfonic acid Salt, trifluoroacetate etc.

[0035] " pharmaceutically acceptable acid-addition salts " refers to biopotency and the property that those have kept free alkali Matter, and do not have the salt of biology or other undesirable properties, with inorganic acid example hydrochloric acid, hydrogen bromine Acid, sulfuric acid, nitric acid, phosphoric acid etc., organic acid such as trifluoroacetic acid, acetic acid, propionic acid, ethanol Acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, wine Stone acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, pyrovinic acid, ethylsulfonic acid, right Toluenesulfonic acid, salicylic acid etc. make. Relevant pharmaceutically acceptable acid-addition salts is as prodrug Illustrate, referring to Bundgaard, H., ed., (1985) Design of Prodrugs, Elsevier Science Publishers, Amsterdam.

[0036] " pharmaceutically acceptable ester " refers to keep behind those ester linkage hydrolyzings the biology effect of Carboxylic acid and alcohol Can and character, and do not have the ester of biology or other undesirable properties. Relevant pharmaceutically acceptable Ester as the explanation of prodrug, referring to Bundgaard, H., supra. These esters are by corresponding carboxylic Sour and pure typical reaction forms. In general, the formation of ester can be real by traditional synthetic technology Existing. (referring to for example March, Advanced Organic Chemistry, 3rd Ed., John Wiley ﹠ Sons, New York (1985) p.1157 and the document of wherein quoting and Mark et al., Encyclopedia of Chemical Technology, John Wiley ﹠ Sons, New York (1980) .). Alkoxide component in the ester generally comprises (i) and can maybe cannot comprise one or more Two keys, and the C. that can maybe cannot contain the branch branched chain₂-C. ₁₂Fatty alcohol or (ii) C.₇- C. ₁₂Fragrance or assorted fragrant and mellow. It is ester as herein described that the present invention also considers to use those, simultaneously again It is the component of its pharmaceutically acceptable acid-addition salts.

[0037] " pharmaceutically acceptable acid amides " refers to have kept after the hydrolysis of those amido links carboxylic acid or amine Biopotency and character, and do not have the acid amides of biology or other undesirable properties. Relevant pharmacy Go up acceptable acid amides as the explanation of prodrug, referring to Bundgaard, H., ed., supra. These Acid amides is formed by corresponding carboxylic acid and amine typical reaction. In general, the formation of amine can be led to Crossing traditional synthetic technology realizes. (referring to for example March, Advanced Organic Chemistry, 3rd Ed., John Wiley ﹠ Sons, New York (1985) p.1152 and Mark et al., Encyclopedia of Chemical Technology, John Wiley ﹠ Sons, New York (1980) .). It is acid amides as herein described that the present invention also considers to use those, is again its medicine simultaneously The component of acceptable acid-addition salts on.

[0038] " pharmaceutically or treatment upper acceptable carrier " refer to that neither the biology of interferon activity composition is lived Property usefulness is again to host or the avirulent mounting medium of patient.

[0039] " stereoisomer " refers to have the same molecular amount with another compound, chemical composition and Structure, but the different compound of former subgroup. Just some identical chemical group is in not Spatial orientation together, so sterling can make the polarized light flat rotation. But some pure optical siomerisms The body optically-active is very little, so that can't detect with present instrument. Compound of the present invention can One or more asymmetric carbon atoms can be arranged, therefore multiple stereoisomer is arranged. All have immunity Active stereoisomer all comprises within the scope of the invention.

[0040] " treatment or pharmacy effective dose " of the present composition be meant the amount of the composition that is enough to cause required biology effect.This effect can be that sign minimizing, sx or the cause of disease of disease alleviated, or any other required change of biosystem.For example in the present invention, this effect is usually directed to the enhancing of innate immune response, the active reduction of DPPIV, and/or to infecting or the adjusting (as inhibition or minimizing or non-stimulation) of the inflammatory reaction of tissue injury.

[0041] the amino-acid residue abbreviation is as follows in the peptide: phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine(Met) is Met or M; Xie Ansuan is Val or V; Serine is Ser or S; Proline(Pro) is Pro or P; Threonine is Thr or T; L-Ala is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; L-asparagine is Asn or N; Methionin is Lys or K; Aspartic acid is Asp or D; L-glutamic acid is Glu or E; Halfcystine is Cys or C; Tryptophane is Trp or W; Arginine is Arg or R; Glycine is Gly or G.In addition, abbreviation Nal is meant the 1-naphthylalanine; Ornithine is Orn or O, and Cit is a citrulline; Hci is a citrulline of being with a methylene group more, Vx or Valine x, and wherein " X " refers to the variation of amino acid backbone, and wherein to connect no longer be amido linkage but the amine that methylates to amino acid, and this is equally applicable to other the amino acid of " X " sign.In addition, 2,4-diamino-butanoic is Dab; 2, the 3-diaminopropionic acid is Dpr or Dapa; (the amino butyl of 4-)-glycine is Nlys to N-; HSer is a homoserine; Hyp is an oxyproline; Val (betaOH) is a hydroxyvaline; D-Pro is 3, the 4-dehydroproline; Pyr is Pyrrolidonecarboxylic acid (on the ring of proline(Pro) C=O being arranged); There is fluorine to replace on the ring of proline(Pro); 1,3-thiazoles alkane 4-carboxylic acid (having sulphur to replace on the proline(Pro) ring); Thi is β-(2-thienyl)-L-Ala; Abu is the 2-aminobutyric acid; Nva is a norvaline; Nle is a nor-leucine; Hol is a homoleucine; With Aib be α-An Jiyidingsuan.Pip used herein refers to (S)-(-)-piperidines-2-formic acid (L-(-)-pipecolinic acid); Dhpr is 3,4-dehydrogenation-L-proline(Pro); Fpro is 2S, 4S-4-fluoro-tetramethyleneimine-2-formic acid (cis-4-fluoro-l-proline); And R-thiazolidine-4-formic acid (L-Thioproline).

[0042] except the peptide that the amino acid that is only generated by nature is formed, the present invention also provides simulating peptide or peptide analogs.Peptide analogs generally uses in pharmaceutical industry as non-peptide medicament, and its character and template peptide are similar.The non-peptide compound of these types is called as " simulating peptide " (Fauchere, J., Adv.Drug Res.15:29 (1986); Veber and Freidinger, TINSp.392 (1985); And Evans et al., J Med.Chem.30:1229 (1987), at this that it is incorporated by reference).The simulating peptide that goes up useful peptide similar with treatment can be used for producing equal or enhanced treatment or prophylactic effect.In general, the simulating peptide similar is in typical peptide (promptly having pharmacy or bioactive peptide), as the receptor-binding peptides that generates naturally, but one or more peptide connecting keys are arranged with the method that further describes in this area or the following reference, optional being selected from--CH ₂NH--,--CH ₂.S--,--CH ₂=CH ₂--,--CH=CH--(cis and trans),--COCH ₂--,--CH (OH) CH ₂--and--CH ₂The connecting key of SO--replaces.Spatola, A.F.in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B.Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, A.F., Vega Data (March 1983), Vol.1, Issue 3, PEPTIDE BACKBONEMODIFICATIONS (general summary); Morley, Trends Pharm Sci (1980) pp.463 468 (general summary); Hudson, D.et al., Int J Pept Prot Res 14:177 185 (1979): (--CH ₂NH--, CH ₂CH ₂--); Spatola et al., Life Sci 38:1243 1249 (1986): (--CH ₂--S); Hann J.Chem.Soc.Perkin Trans.I 307 314 (1982): (--CH--CH--, cis and trans); Almquist et al., J Med Chem 23:1392 1398 (1980): (--COCH ₂--); Jennings-White et al., Tetrahedron Lett 23:2533 (1982): (--COCH ₂--); Szelke et al., European Application.EP 45665 CA:97:39405 (1982) (--CH (OH) CH ₂.); Holladay et al., Tetrahedron Lett24:4401 4404 (1983): (--C (OH) CH ₂--); And Hruby Life Sci 31:189 199 (1982): (--CH ₂--S--); Wherein each piece of writing all is hereby incorporated by reference.On the one hand, non-peptide connecting key is--CH ₂NH--, the polypeptide in the embodiment, this simulating peptide has significant advantage, it comprise as: produce more economical, chemical stability is better, and (transformation period absorbs, and tires in the pharmacological property enhancing, curative effects etc.), specificity changes (for example broad-spectrum biological activity), and antigenicity reduces or the like.The mark of simulating peptide generally includes one or more labels directly or by connecting arm (for example amide group), is covalently attached to by quantitative structure to imitate on the noiseless site of simulating peptide of data and/or molecule modeling and forecasting.This noiseless site be generally not with the direct position contacting of macromole (for example immunoglobulin superfamily molecule), simulating peptide is bonded on this macromole and produces result of treatment.The derivatize of simulating peptide (for example mark) should not produce materially affect to required biology of simulating peptide or pharmaceutical active.In general, the simulating peptide of receptor-binding peptides is attached on the acceptor with high-affinity, and has detectable biological activity (promptly the one or more receptor-mediated phenotypes of competition or antagonism change).

[0043] the whole one or more amino acid (for example replacing L-Methionin with D-Methionin) that replace consensus sequence of homotype D-amino acid can be used for generating more stable peptide.Should be appreciated that, those D-aminoacid replacement, what wherein the immunocompetence of peptide was kept is desirable.

Explanation

[0044] as herein described, the inventor has confirmed to have and/or comprise the new peptides of aminoacid sequence shown in the table 1, or the analogue of aminoacid sequence disclosed in this invention, derivative, variant or obvious chemical equivalent.The inventor has also confirmed a kind ofly have or that comprise one of described aminoacid sequence of table 1 and peptide monoamide C end, and it is strengthening aspect the congenital immunity treatment effectiveness being arranged.Especially the inventor illustrates, and the peptide that comprises table 1 aminoacid sequence lacks the curative effect of anti-streptococcus aureus, but provides endogenous protective to the mouse that infects streptococcus aureus.This peptide has strengthened the host to replying of infecting, thereby has improved bacteria clearance and host's survival rate.Therefore, described new peptides can be used for treating communicable disease.In another embodiment, peptide of the present invention has been proved and can have reduced the DDPIV activity, and it is relevant with the panimmunity relative disease that the DDPIV activity has been proved, for example acquired immune deficiency syndrome (AIDS) and virus of AIDS progression of disease (Blazquezet al.1992; Vanham et al.1993; Schols et al.1998 Oravecz et al.1995), Graves disease (Eguchi et al.1989; Nishikawa et al.1995) and cancer (Stecca etal.1997), as lung cancer and colorectal carcinoma, and diabetes (Hinke et al.2000; Marguet etal.2000).Further, DDPIV has been proved to be and several autoimmune diseases as a kind of indicator of T-cell-stimulating, as rheumatoid arthritis (Nakao et al., 1989) and autoimmune thyroiditis ((Eguchi et al., 1989) make parallel undulations.DPPIV has been described to and the fine relevant mark of the activity level of these diseases., carried out further research (Constantinescu et al., 1995) to the indicator of DPPIV as chronic progressive multiple sclerosis.Peptide of the present invention can be used for treating these diseases.

Peptide of the present invention

[0045] therefore, the invention provides the isolated peptides that has or comprise table 1 aminoacid sequence, or its immunocompetence analogue, derivative, variant or obvious chemical equivalent, pharmacy acceptable salt, acid salt and the ester of these peptides also are provided, analogue of the present invention, derivative and variant comprise that those are as herein described, replace as conservative, N end and C are terminal modified, and backbone modification.Peptide of the present invention can also be a cyclic." separation " of the present invention used herein peptide is or does not have the counterpart of natural generation, or separated or peptide that purifying comes out from the composition of natural coexistence.Isolated peptides of the present invention can from, for example the expression or the chemosynthesis of the recombinant nucleic acid by encoded peptide obtain.Because its essence of the peptide of chemosynthesis is to separate from the composition of natural coexistence, so synthetic peptide is " separated ".Similarly, the present invention also comprises fusion rotein and the peptide that comprises the peptide different with natural generation environment.Such peptide can comprise that metabolism is the peptide of peptide of the present invention.

[0046] on the one hand, isolated peptides of the present invention comprises and has structural formula " X ₁X ₂P " (SEQ.ID.NO.55) aminoacid sequence, wherein P can be a proline analogs, as Pip, Thz, Fpro, Dhp, wherein: X ₁Be selected from K, R, S, O, or N the end compound based on glycine (for example Nlys), hSer, the Val (betaOH) that are replaced by basic functionality, or be selected from K, R, S and O in another embodiment, or be R in another embodiment; X wherein ₂Be selected from V, I, R and W, or be selected from V, I and R in one embodiment.In one embodiment, work as X ₂When being V, X ₁Can be G (for example SEQ.ID.NO.88).In another embodiment, work as X ₂When being H, X ₁Can be K (for example SEQ.ID.NO.89).In one embodiment, isolated peptides of the present invention is SEQ.ID.NO.55.On the other hand, it is up to 6,7,8 or 9 amino acid whose peptides, or in another embodiment, is nearly ten the amino acid whose peptides that comprise aminoacid sequence SEQ.ID.NO.55.In one embodiment, the isolated peptides of SEQ.ID.NO.55 is SEQ.ID.NO 83-87, SEQ.ID.NO 8,9,26,39,40,41,45,46 and 48-53, or comprises nearly ten amino acid whose isolated peptides of described sequence.In one embodiment, described peptide is to comprise described sequence, as SE Q.ID.NO 62,8,9,13,26,39,40,41,45,46,48,49,50,52 and 53 up to 7 amino acid.In another embodiment, the isolated peptides that comprises SEQ.ID.NO.55 is 13 amino acid whose SEQ.ID.NO.44 nearly.

[0047] in another embodiment, the invention provides a kind of structural formula " X that comprises ₁X ₂X ₃P " (SEQ.ID.NO.56) isolated peptides, wherein P can be a proline analogs, for example Pip, Thz, Fpro, Dhp, wherein X ₁Be selected from K, H, R, S, T, O, or N the end compound based on glycine (for example Nlys), hSer, the Val (betaOH) that are replaced by basic functionality, or in another embodiment, be selected from K, H, R and O, or in another embodiment, be selected from K, H, R, S and T, or in another embodiment, be selected from K, H, R, S and O, or in another embodiment, be selected from R, H, K and S, or in another embodiment, be selected from R, H and K; X wherein ₂Be selected from A, I, L, V, K, P, G, H, R, S, O, Dab, Dpr, Cit, Hci, Abu, Nva, Nle, X ₂Can be that nitrogen is methylated, or in another embodiment, be selected from A, I, L, V, K, P, G, H and R, wherein X ₂Can be that nitrogen is methylated; X wherein ₃Be selected from I, V, P, G, H, W, E, wherein in one embodiment, X ₃Be that non-nitrogen is methylated.In one embodiment, isolated peptides can be nearly 5,6,7,8,9 or 10 the amino acid whose aminoacid sequences that comprise SEQ.ID.NO.56, or comprise SE Q.ID.NO.1,3-7,10-16,18,21-25,27,28,31-39,42,47,61,77,72,79,81 or 90 up to 5 or 7 amino acid whose aminoacid sequences, or comprise the nearly isolated peptides of 5,6,7,8,9,10 or 11 aminoacid sequences of SEQ.ID.NO.54.But in one embodiment, when SEQ.ID.NO.56 was six aggressiveness, it was selected from SEQ.ID.NO.1,3,61,64 or 90, but was not SEQ.ID.NO.2, or in one embodiment, when it was pentamer, it was not SEQ.ID.NO.17.In one embodiment, isolated peptides of the present invention does not comprise the peptide that comprises SEQ.ID.NO.2 or 17.In one embodiment, when described peptide was heptamer, it was selected from SEQ.ID.NO.18,32 and 79.

[0048] in another embodiment, the invention provides a kind of isolated peptides that comprises peptide, this involved peptide comprises the formula SEQ.ID.NO.56 in pentamer or six aggressiveness.In one embodiment, described peptide has immunocompetence.

[0049] in one embodiment, isolated peptides of the present invention comprises a kind of structural formula " aX ₁X ₂X ₃P " (SEQ.ID.NO.57) peptide, wherein P can be a proline analogs, for example Pip, Thz, Fpro, Dhp, wherein X ₁, X ₂And X ₃Identical with its definition in SEQ.ID.NO.56, wherein " a " is selected from S, P, I, R, C, T, L, V, A, G, K, H, R, O, C, M and F, or in another embodiment, be selected from S, P, I, R, C, T, L, V, A, G, K, H, R, O, C and M, or in another embodiment, be selected from S, P, I, R and C, or be S in another embodiment.In one embodiment, isolated peptides comprises SEQ.ID.NO.57, or a kind of nearly 6,7,8,9 or 10 amino acid whose peptides that comprise described sequence.In another embodiment, isolated peptides is SEQ.ID.NO.4,12,32,39 or 47, or a kind of nearly 7,8,9 or 10 amino acid whose isolated peptides that comprise described sequence.

[0050] in another embodiment, isolated peptides of the present invention comprises a kind of structural formula " X that has ₁X ₂X ₃Pb " (SEQ.ID.NO.58) peptide, wherein P can be a proline analogs, for example Pip, Thz, Fpro, Dhp, wherein X ₁X ₂X ₃Identical with its definition in SEQ.ID.NO.56, " b " is any aliphatics, aromatic series, negative charge or positive charge amino acid.Or be selected from A, A in one embodiment ^*, E, G, S, L, F, K, C, I, V, T, D, Y, R, H, O, Q, N, P and M, but be not P in one embodiment, or be selected from A, A in another embodiment ^*, E, H, W, G, S, L, F and K, or be selected from A, A in another embodiment ^*, G, S, L, K and C, or be selected from A, A in one embodiment ^*, G, S, L and K, wherein A ^*The D type amino acid that refers to L-Ala.In one embodiment, work as X ₁Be R, X ₂When being A for I and b, X ₃Can be W (for example SEQ.ID.NO.71).In one embodiment, isolated peptides is nearly 7,8,9 or 10 the amino acid whose amino acid that comprise SEQ.ID.NO.58..In one embodiment, isolated peptides is or comprises SEQ.ID.NO.5-7,10,11,13-16,21-25,27,28,31,33-38 and 42-43.In another embodiment, the peptide of SEQ.ID.NO.58, wherein " b " is not P or is not RIVPP (SEQ.ID.NO.17); Or X wherein ₃Not Vx or be not RIVxPA.

[0051] in one embodiment, isolated peptides of the present invention is or comprises the peptide of a kind of SEQ.ID.NO.58 of being similar to, but X wherein ₁Be selected from G, GG or Cit, or wherein " b " is A, X ₂Be I, X ₃Be V, X ₁Be G, GG or Cit, or this peptide is SEQ.ID.NO.19,20 and 36.In one embodiment, this isolated peptides comprises SEQ.ID.NO.31.In another embodiment, isolated peptides comprises a kind of reverse sequence of SEQ.ID.NO.58, or comprises SEQ.ID.NO.30.

[0052] in one embodiment, isolated peptides of the present invention is or comprises the peptide (reverse sequence of SEQ.ID.NO.29) of the aminoacid sequence of a kind of SEQ.ID.NO.29 of having.

[0053] the present invention also provides a kind of structural formula " a that comprises ₁a ₂X ₁X ₂X ₃P " (SEQ.ID.NO.59) isolated peptides, wherein P can be a proline analogs, for example Pip, Thz, Fpro, Dhp, wherein X ₁, X ₂And X ₃Identical with its definition in SEQ.ID.NO.56, a ₁Be selected from K, I, R, H, O, L, V, A and G, or be selected from K and I in one embodiment, or be K in one embodiment, a ₂Be selected from S, P, R, T, H, K, O, L, V, A, G, S and I, or be selected from S, P and R in one embodiment, or be selected from S and P in another embodiment, or be P in another embodiment.In one embodiment, a ₁Not acetylizad, or a wherein ₁Be K, K is not acetylizad or is not SEQ.ID.NO.2.In one embodiment, isolated peptides is or comprises SEQ.ID.NO.1 and 47 or comprise nearly ten amino acid whose peptides of SEQ.ID.NO.59.

[0054] in another embodiment, isolated peptides of the present invention is or comprises structural formula " aX ₁X ₂X ₃Pb " (SEQ.ID.NO.60) peptide, wherein P can be a proline analogs, for example Pip, Thz, Fpro, Dhp, wherein X ₁, X ₂And X ₃Identical with its definition in SEQ.ID.NO.56, wherein " a " is selected from S, R, K, H, O, T, I, L, V, A, G, or be selected from S, R and I in another embodiment, or be selected from S and R in another embodiment, wherein " b " is selected from A, V, I, L, G, K, H, R, O, S, T, F, or is A in another embodiment.In another embodiment, the peptide of SEQ.ID.NO.60 is SEQ.ID.NO.3,12 and 39, or a kind of SEQ.ID.NO.60 or SE Q.ID.NO.3, nearly ten amino acid whose peptides of 12 or 39 of comprising.

[0055] a kind of " peptide that comprises table 1 aminoacid sequence " used herein comprises peptide itself, its obvious chemical equivalent, isomer (isomers for example, stereoisomers, trans-isomer(ide), trans-the counter-rotating isomer, all D type isomer, all L type isomer, or its blended L type and D type isomer), its conservative substituent, precursor forms, the form that the proteolysis inscribe is handled (for example rupture from the N end of peptide of the present invention or C end or its immunocompetence metabolite to single amino acids), its pharmacy acceptable salt and ester, and other form of posttranslational modification formation.Also comprising nearly and comprise any auxiliary sequence (ring-like, wire, or the parent nucleus sequence has ramose) of 10,9,8,7,6,5 and 4 amino acid lengths, is a subsequence for its this particular sequence.Those skilled in the art should understand, this peptide is trimerical in the table, and it can be the subsequence of 10,9,8,7,6,5 and 4 aggressiveness, and listed this peptide of table 1 is six aggressiveness, then it can be the subsequence of 10,9,8 and 7 aggressiveness, rather than the subsequence of 5 or 4 aggressiveness.In addition, the present invention includes sequence SEQ.ID.NO.44 and 54 greater than 10 aggressiveness.Further, wherein major metabolite is that the peptide of the peptide of table 1 is also included within the scope of the present invention.The purposes of peptide of the present invention comprises that wherein active metabolite is the purposes of the peptide of one or more peptides of the present invention.The immunocompetent modified peptides that those keep peptide of the present invention comprises within the scope of the invention.Following obvious equivalent or basically by SEQ.ID.NO:1-90, or SEQ.ID.NO 1,3-16,18-90, or these peptides of forming by SEQ.ID.NO 1,3-16,18-43,45-53 or 55-90 in one embodiment, be also included within the scope of the present invention.

[0056] " the tangible chemical equivalent " of peptide of the present invention used further herein is that peptide a kind of and as herein described has for example immunocompetence of identical required activity, and show the molecule of a small amount of different chemical character or a kind of molecule that under mild conditions, is converted to peptide of the present invention (for example ester of peptide of the present invention, ether, reduzate and mixture).In one embodiment, the present invention comprises the peptide with sequence of the present invention and motif, or comprises the peptide of comparing the active same material that reduces of DPPIV with saline control.In one embodiment, this DPPIV activity is about 75% with respect to salt solution.In another embodiment, this DPPIV activity is 70% with respect to salt solution.Wherein this paper is employed refers to DPPIV active relevant " pact "+/-5%.

[0057] in addition, " conservative replace " used herein be those or owing to have similar polarity or three-dimensional arrangement, or owing to belong to same class (for example hydrophobic, tart or alkalescence) with substituted residue, and with the aminoacid replacement of substituted amino-acid residue functional equivalent.Term defined herein " the conservative replacement " comprises the little replacement of activity influence that peptide of the present invention is strengthened congenital immunity.The conservative example that replaces comprises that a kind of polarity (hydrophilic) residue replaces another kind of polarity (hydrophilic) residue (arginine/Methionin for example, glutamine/l-asparagine, or Threonine/Serine), a kind of nonpolar (hydrophobic) residue (Isoleucine for example, leucine, methionine(Met), phenylalanine, tyrosine, or Xie Ansuan) replace another kind of nonpolar (hydrophobic) residue, a kind of acidic residues (for example aspartic acid or L-glutamic acid) replaces another kind of acidic residues, a kind of alkaline residue (arginine for example, Histidine, Methionin or ornithine) replace another kind of alkaline residue.

[0058] term used herein " analogue " comprises any peptide with aminoacid sequence identical with sequence essence as herein described, wherein has a residue at least by the conservative replacement of the proximate residue of function." analogue " comprises the functional variant and the obvious chemical equivalent of table 1 aminoacid sequence.The peptide that term used further herein " functional variant " refers to show raising congenital immunity as herein described or reduces the active ability of DPPIV." analogue " comprises the amino acid whose variant of table 1, and it has homology 3 D stereo conformation." analogue " further comprises any pharmacy acceptable salt of analogue described herein." variant " further comprises any pharmacy acceptable salt of variant described herein.

[0059] " derivative " used herein is meant a kind of peptide of the present invention, and it has one or more reactions by the functional side group by the amino acid of chemical derivatization.Representational molecule by derivatize includes but not limited to, by adding ethanoyl, and the hydrochloride of amine, carbobenzoxy-(Cbz), chloracetyl, formyl radical, p-toluenesulfonyl, or tertbutyloxycarbonyl, the free amine group in the peptide molecule is obtained salt or acid amides by derivatize.Free hydroxyl group can be generated oxygen ethanoyl or oxyalkyl derivative by derivatize.Free carboxy can be generated salt by derivatize in addition, ester (for example methyl and ethyl ester) or hydrazides.Therefore " derivative " further comprises any pharmacy acceptable salt of derivative as herein described.

[0060] in an embodiment of the present invention, isolated peptides of the present invention has modified C end and/or modified N end.For example, isolated peptides has amidated carbon teminal.For example, aminoterminal can be acetylation (Ac) or carboxyl terminal can be by amidation (NH ₂).Yet in an embodiment of the present invention, if this modification of acetylize can cause required immunocompetent forfeiture, peptide of the present invention preferably is not acetylation.Aminoterminal modify and to comprise and methylating (promptly--NHCH ₃Or--NH (CH ₃) ₂), acetylize adds the benzyloxy acyl group, or seals N-terminal with any blocking groups that contains the carboxylic acid functional that is defined as RCOO--, wherein R is selected from naphthyl, acridyl, steroidal base, and similar group.C-terminal is modified and to be comprised with carbonylamino group and replace free acid, or forms cyclic lactames to introduce structural restriction at carboxyl terminal.

[0061] in one embodiment, can realize as from NH to NCH ₃Skeleton replace.Isolated peptides can also be or comprise the modification (for example point mutation as inserting or deletion, or is blocked) of table 1 aminoacid sequence.For instance, as long as required immunocompetence is kept, then described peptide can comprise table 1 aminoacid sequence that at least one site insertion D type amino acid is modified.Especially its proline residue proline analogs of becoming 4,6 or 7 yuan of rings from 5 yuan of rings is desirable.Cyclic group can be saturated or unsaturated, if undersaturated, can be fragrance or non-fragrance.

[0062] can have the side chain of natural generation of the amino acid (or D type amino acid) of described 20 genes encodings of side substitution of similar quality with other, for example use alkyl, low alkyl group, 4,5,6 to 7 yuan of cycloalkyl, acid amides, the acid amides low alkyl group, acid amides two low alkyl groups, lower alkoxy, hydroxyl, carboxyl and its lower member ester derivative, and 4,5,6 to 7 yuan of heterocyclic substituted.

[0063] this replacement can comprise but need not and is limited to: (1) off-gauge positively charged amino acid, as: ornithine, Nlys (N-(the amino butyl of 4-)-glycine), its lysine side-chain is connected in " N end "; With and aminopropyl or amino-ethyl be connected to the compound of glycine amino.(2) the no net charge of non-natural generation and the amino acid of similar arginine side chain are as Cit (citrulline) and Hci (citrulline that has one or more methylene radical); (3) have the amino acid (as Serine) that the non-standard non-natural of hydroxyl generates, as hSer (homoserine) (many methylene radical), Hyp (oxyproline), Val (betaOH) (beta-hydroxy Xie Ansuan), Pen (penicillamine), Val (betaSH) (β-trolovol); (4) proline derivative, as D-Pro, as 3, the 4-dehydroproline, Pyr (Pyrrolidonecarboxylic acid has C=O on the ring of proline(Pro)) has fluorine to replace on the proline(Pro) ring, 1,3-thiazoles alkane-4-carboxylic acid (having sulphur to replace on the proline(Pro) ring); (5) histidine derivative is as Thi (β-(2-thienyl)-L-Ala); Or (6) alkyl derivative, as Abu (the 2-aminobutyric acid has ethyl on the α carbon), Nva (norvaline, on the α carbon propyl group is arranged), Nle (nor-leucine has butyl on the α carbon), Hol (homoleucine, on the α carbon propyl group is arranged), Aib (α-An Jiyidingsuan, Xie Ansuan are not with methylene radical).Those skilled in the art is to be understood that these replacements have kept the immunocompetence of female peptide or auxiliary sequence.

[0064] in another embodiment, with the amino of N end respectively the ester of inner substituted hydroxy or carboxyl (OR) or ester, then the ester of carbon teminal carboxyl or carbon teminal can be by cyclisation, thereby forms a cyclic peptide.For example, after synthetic and the acid of fracture generation peptide, free acid is converted into the activatory ester by suitable carboxyl activator such as the dicyclohexylcarbodiimide in the solution (DCC), wherein solution such as methylene dichloride (CH ₂Cl ₂) and the mixture of dimethyl formamide (DMF).Replace the activatory ester with N end amine inside then, thereby generate cyclic peptide.Use extremely rare solution can promote the inside cyclization opposite with polyreaction.This method is known in the field.

[0065] also can cyclisation peptide of the present invention, or introduce at the end of peptide and to deaminize or the residue of decarboxylate, so just do not have terminal amino group or carboxyl, thereby reduce the susceptibility of proteolytic enzyme or the formation of restriction peptide.The C end functional groups of compound of the present invention comprises acid amides, acid amides low alkyl group, acid amides two low alkyl groups, lower alkoxy, hydroxyl, carboxyl, and lower member ester analog derivative and pharmacy acceptable salt.

[0066] also can add a N end and/or C end halfcystine, by reacting between disulfide linkage connection or other side chains, with the peptide cyclisation.

[0067] also can introduce one and deaminize or the decarboxylate residue, so just not have terminal amino group or carboxyl, thereby reduce the susceptibility of proteolytic enzyme or the formation of restriction peptide at the end of peptide.

The preparation method of peptide

[0068] peptide of the present invention's consideration comprises peptide analogs, derivative and variant, is synthetic preparation, and reorganization generates or be isolating from nature cell.Peptide of the present invention can be synthetic (for example at Modern Techniques of Peptide and Amino AcidAnalysis (New York:John Wiley with the method that well known to a person skilled in the art; Sons, 1981) and Bodansky, M., described in the Principles of Peptide Synthesis (New York:Springer-Verlag N.Y., Inc., 1984)).The example of adoptable method includes but not limited to that solid-phase peptide is synthetic in peptide of the present invention synthetic, and solution or liquid phase process peptide are synthetic, and adopt the synthetic of any commercial peptide synthetics of buying.In one embodiment, peptide of the present invention for example passes through the translation of chemical process or external mRNA external synthetic.In another embodiment, peptide of the present invention is to adopt the cDNA reorganization of conventional art and encoded peptide to prepare.Aminoacid sequence of the present invention can comprise further and be used for peptide sequence synthetic coupling agent and protecting group that this is well known to a person skilled in the art.

[0069] peptide analogs of the present invention, derivative can be by well known to a person skilled in the art that various different induced-mutation technique makes with variant.These technology can obtain in any Molecular Biology Lab handbook, comprise for example Sambrook et al., Molecular Cloning-ALaboratory Manual, 2 ^NdEd. (Plainview, NY:Cold Spring Harbor Press, 1989) or Ausubel et al., Current Protocols in Molecular Biology (John Wiley ﹠amp; Sons).Mutagenesis kit also can obtain from many commercial molecular biology suppliers.Have and in initial aminoacid sequence, produce fixed point, specific region, or the method for random mutation.As described herein, making analogue, behind derivative or the variant, can make the required activity that can strengthen congenital immunity to it and detect.

Material (agent) with reactive polypeptide

[0070] the present invention further provides a kind of can with the peptide that comprises table 1 aminoacid sequence, the material of its analogue, derivative or variant reaction." reaction " used herein refers to that this material has avidity to peptide of the present invention, can combine with peptide of the present invention, or at peptide of the present invention." material " used further herein should comprise antibody, Fab fragment, the F (ab ') that protein, polypeptide, peptide, nucleic acid (comprising DNA or RNA), non-natural generate ₂Fragment, molecule, compound, microbiotic, medicine, and any its composition.The Fab fragment is the monovalent antigen binding fragment of antibody, is generated by papain digestion.F (ab ') ₂Be the divalence Fab of antibody, generate by gastric pepsin digestion.Preferably, material of the present invention is by a kind of detectable mark or label institute mark.The antibody that the antibody that non-natural produces refers to use the peptide that is associated as two C end glycine residues and MAPS with another compound to produce.MAPS antigen is two amino-acid residues by the one of carbon tip of inserting peptide, on peptide of the present invention.This construct can be applied to a kind of animal such as rabbit subsequently, and uses approach well known to gather antibody.

[0071] in an embodiment of the present invention, with the material of reactive polypeptide of the present invention be antibody.Antibody of the present invention used herein can be polyclone or monoclonal.In addition, antibody of the present invention can use and well known to a person skilled in the art the technology preparation.For example polyclonal antibody can be by with purified peptide of the present invention or be connected in antigenic purified peptide (for example MAPs) immune mouse, rabbit or rat and make.Can remove the spleen of immune animal then, splenocyte and myeloma cell are merged the formation hybridoma,, can generate monoclonal antibody in culture in case it is grown.Referring to for example J.G.R.Hurrel, Monoclonal Hybridoma Antibodies:Techniques and Applications (Boco Raton, FL:CRC Press Inc., 1982).

[0072] antibody of the present invention and even peptide itself can be with a detectable marker or label.The mark of antibody or peptide can be realized with one of various labeling techniques, comprises peroxidase method known in the art, chemiluminescent labeling, and the radio-labeling method.Detectable of the present invention or label can be, for example a kind of on-radiation or fluorescent marker, as vitamin H, fluorescein (FITC), acridine, cholesterol or carboxyl-X-rhodamine, it can detect with fluorescence known in the art and other imaging technique.Perhaps, marker that can detect or label can be a kind of radioactively labelled substances, comprise such as a kind of radio isotope.This radio isotope can be that any generation can detect the radiating isotropic substance, such as ³⁵S, ³²P, ¹²⁵I, ³H or ¹⁴C.The radioactivity that radio isotope produces can detect with technology well known in the art.Such as, radioisotopic gamma radiation can detect with the gamma ray imaging technique, especially the scintillation method imaging technique.Preferred material of the present invention is the high-affinity antibody with detectable label or label.

Isolated nucleic acid molecule

[0073] in addition, the invention provides the isolated nucleic acid molecule that a kind of coding comprises the peptide of table 1 aminoacid sequence, or its analogue, derivative, variant or obvious chemical equivalent, comprise a kind of peptide of puting together (for example a kind of carrier-peptidic constructs) or other peptide, or a kind of propetide, its metabolism or fracture generation table 1 have immunocompetent peptide.Because the degeneracy of genetic code, nucleic acid molecule of the present invention comprises the nucleic acid substituent of a large amount of peptides of the present invention of also encoding.The present invention further provides a kind of nucleic acid, the isolated nucleic acid molecule of itself and coding schedule 1 aminoacid sequence, or its analogue, derivative or variant hybridization.

[0074] nucleic acid molecule of the present invention can be DNA or RNA.They can prepare with the technology known to multiple those skilled in the art, include but not limited to the oligonucleotide synthesizer bought with commercial, as Applied Biosystems Model 392 DNA/RNA synthesizers, and synthetic oligonucleotide automatically.In addition, nucleic acid molecule of the present invention can be with one or more detectable markers or label.The mark of nucleic acid molecule can be with one of many methods known in the art---nick translation for example, end mark, end is filled and led up mark, the polynueleotide kinase permutoid reaction, random primer, or SP6 polysaccharase (being used for preparing rna probe) one of---and various labels realize---for example, as ³⁵S, ³²P or ³The radioactive labels of H and so on, or on-radiation label, as vitamin H, fluorescein (FITC), acridine, cholesterol, or carboxyl-X-rhodamine (ROX).

[0075] the present invention also provides a kind of nucleic acid construct of reorganization, and it comprises the nucleic acid molecule of the present invention that is operably connected on a kind of expression vector." expression vector " used herein is a kind of DNA construct, and it contains a kind of dna sequence dna that is operably connected to a kind of suitable control sequence, and this control sequence can influence the expression of described DNA in suitable host.This carrier can be, plasmid for example, and phage particle, or gene inserts fragment.Term used further herein " is operably connected " and has described functional relationship between two DNA zones.Be fit to expression vector that the present invention uses and comprise expression controlling elements at least one nucleic acid molecule that is operably connected to code book invention peptide (operon for example, promotor, lac system, leader sequence, terminator codon, and/or polyadenylation signal).In one embodiment, expression vector is a kind of carrier for expression of eukaryon that works in eukaryotic cell (for example retroviral vector, vaccinia virus vector, adenovirus carrier, herpesvirus vector, or fowlpox virus carrier).

[0076] in case is operably connected on the nucleic acid molecule of the present invention, expression vector can be suitable in the body that nucleic acid shifts or in vitro method is introduced into a recipient cell by any, described method includes, but are not limited to electroporation, the deae dextran transfection, calcium phosphate transfection, liposome transfection, single cationic-liposome merges, and the polycation liposome merges, protoplastis merges, set up the body internal electric field, the microparticle bombardment of DNA parcel, injection recombinant replication-defective C-type virus C, homologous recombination, virus vector, naked DNA shifts, or its arbitrary combination.Being fit to the recombinant viral vector that nucleic acid shifts includes but are not limited to: from such as retrovirus, and hsv, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus, the virus genomic carrier of vaccinia virus etc. and so on.

[0077] the present invention further provides at least a host cell that comprises recombinant nucleic acid construct of the present invention.Host cell of the present invention is to transform with nucleic acid construct as herein described.Host cell can be (for example intestinal bacteria) of eucaryon (for example animal, plant, insect or yeast cell) or protokaryon.

[0078] in addition, the invention provides the method that a kind of preparation comprises peptide or its analogue, derivative or the variant of table 1 aminoacid sequence.The step that this method comprises has: (a) as described herein, under the condition that allows peptide to express, cultivate at least a host cell, and it comprises a kind of nucleic acid construct of reorganization; And (b) from this at least a host cell or its substratum recovering peptide.The peptide of reorganization can be used as thick lysate and reclaims, and also can include but not limited to standard protein purification method purifying known in the art, affine and immunoaffinity chromatography, differential precipitation method, gel electrophoresis, ion exchange chromatography, isoelectric focusing method, spatial exclusion chromatography etc.

Pharmaceutical composition

[0079] the present invention further provides a kind of pharmaceutical composition, it comprises and contains aminoacid sequence of the present invention, the peptide of table 1 (SEQ ID NO:1-90) or SEQ.ID.NO.1,3-16 or 18-90 for example, or comprise the peptide of the described peptide of amino acid whose SEQ IDNO:1,3-16,18-43 or 55-90 up to 7,8,9 or 10 as the case may be, or its analogue, derivative or variant (pharmacy acceptable salt, acid salt or the ester that comprise aforementioned any material), with at least a pharmaceutically acceptable carrier, thinner or auxiliary material.With composition in other composition compatible and be not on the deleterious meaning to its recipient, pharmaceutically acceptable carrier, thinner or auxiliary material must be " acceptable ".The example of pharmaceutically acceptable carrier, thinner and auxiliary material includes but not limited to: carboxymethyl cellulose, Microcrystalline Cellulose, glycerine, gum arabic, lactose, Magnesium Stearate, methylcellulose gum, powder, salt solution, sodium alginate, sucrose, starch, talcum powder and water or the like.The prescription of pharmaceutical composition of the present invention, as described herein can the preparation with unitary dose easily.

Purposes

[0080] peptide of the present invention has proved the treatment effectiveness that strengthens congenital immunity.The treatment effectiveness that strengthens congenital immunity is by the shortage (embodiment 2) of anti-microbial activity and the anti-infective protection (embodiment 3-6 and 10) of body inner model, and the DPPIV of embodiment 7,8 and 9 detection is confirmed.Therefore, the present invention also provides the method that a kind of experimenter for the treatment of and/or preventing infects." experimenter " used herein is a kind of bird (for example chicken, turkey etc.), or a kind of Mammals (as ox, dog, people, monkey, mouse, pig, rat etc.).In one embodiment, the experimenter is the people.The experimenter may be infected or be in the infected danger.For instance, infection may be an infected by microbes.Can include but not limited to infectation of bacteria, fungi infestation, parasitic infection and virus infection with the infected by microbes of the inventive method treatment.

[0081] most of bacterial pathogens are present in general environment or host's the normal microflora.By the different mechanisms (so-called toxicity considerations will) that obtains can make bacteria planting, host tissue internal communication and invade host tissue, bacterium is evolved, and can cause serious disease.When these pathogenic factors were suppressed, bacterium can not persist in host's the tissue, thereby can not cause disease.Can include but not limited to intestinal bacteria, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella (for example Salmonella typhimurium), streptococcus aureus, streptococcus and vancomycin resistance faecalis with the representative bacterium of the inventive method treatment.

[0082] Pseudomonas aeruginosa is a kind of immanent gram-negative bacteria, because of its adaptive capacity to environment, the pathogenecity and the antimicrobial resistance of susceptible individual received publicity.It is a kind of multi-functional organism, is grown in soil, the marsh, and seashore and marine habitat, and in the plant and animal tissue.The severe complications of cystic fibrosis is the respiratory tract infection that Pseudomonas aeruginosa causes.Cancer and burn patient also generally suffer this organic severe infections, and the individuality of some other immune system defect also is like this.Different with the bacterium of many environment, Pseudomonas aeruginosa has outstanding virulence to susceptible host.

[0083] streptococcus aureus is a kind of Gram-positive spherical bacteria, about 1 micron of diameter, and the growth of microcosmic cluster is vigorous.It is one of most important human pathogen, causes the acquired and hospital infection of the community of scope from the endocarditis to the pneumonia.Though streptococcus aureus generally is listed in the extracellular cause of disease, nearest data have shown that it can infect dissimilar host cells, as full-time phagocytic cell and non-phagocytic cell, comprise endotheliocyte, inoblast and other cells.These invasion and attack are to be attached to cell surface by streptococcus aureus to cause, and the streptococcus aureus Fiberonectin has played remarkable effect in this process.The streptococcus aureus that is engulfed can be induced Apoptosis of Host Cells or survive a few days in tenuigenin---and this is considered to lack anti-streptococcus aureus effect mechanism.

[0084] streptococcus aureus grows surely in nasal passage, skin surface, mucous membrane, and oral cavity, the peripheral region of sexual organ and rectum.Streptococcus aureus can cause the shallow dermatosis, as sore, and hordeolum and furuncle.More serious infection comprises pneumonia, mazoitis, phlebitis, meningitis and urinary tract infections; Deep infection comprises osteomyelitis and endocarditis.

[0085] the representative fungi of available method of the present invention treatment, include but not limited to mould, yeast and higher fungi.All fungies all are eukaryotic, in their cytolemma sterol are arranged, but do not have peptidoglycan.Fungi infestation or mycosis are classified with the mode that enters the host according to the tissue degree of getting involved.In the host of immunologic hypofunction, normally Wen He various non-pathogenic fungi or fungi can cause potential mortality to infect.

[0086] parasite is the organism that obtains nutrition and protection from other organisms (being called the host) of living.They can be from animal to the mankind, from the mankind to the mankind, or from the mankind to zoochory.Several parasites have been considered to the major reason through food and water-borne disease.By edible contaminated food and water, or by taking in the material of the ight soil that has touched infected human or animal, they can be propagated to the host from the host.Parasite is life and breeding in infected human or animal host's tissue and organ, and usually through defecate.Dissimilar parasites is arranged, and it differs in size, from small, and single celled microbial organisms (protozoon), bigger to what need not that microscope just can see, the many cells worm.Can include but not limited to giardia duodenalis with the general parasitic example of method treatment of the present invention, Cryptosporidium parvum, Cayenne ring sporozoite and toxoplasma gondii.

[0087] virus is different from fungi and bacterium, lacks a lot of attributes of free living cell.The single virus particle is a kind of static structure, and quite stable can't change or change its integral part.Have only when being associated with cell, virus is reproducible and obtain some attribute of living system.The disease that virus causes is a lot, comprises the upper respiratory tract infection (URTIs) as common cold and pharyngitis (throat pain).Can include but not limited to virus of AIDS, avian influenza virus, varicella virus, cold sore virus, common cold virus, gastroenteritis virus (particularly children), gland fever virus, influenza virus, Measles virus, mumps virus, pharyngitis virus, pneumonitis virus, rubella virus, atypical pneumonia virus, lower respiratory infection virus (for example respiratory syncytial virus or RSV) with other examples of the virus of method of the present invention treatment).

[0088] inventor proves in this article, comprise or form by table 1 aminoacid sequence or SEQ.ID.NO.1,3-16 or 18-90 basically, or the peptide that comprises in another embodiment or form by SEQ.ID.NO.1,3-16,18-43,43-53 or 55-90 basically, or its analogue, derivative, variant, or its obvious chemical equivalent, infect prevent and/or treat aspect effective in cure.Therefore, treat and/or prevent the present method that the experimenter infects, comprise and will comprise the peptide of table 1 aminoacid sequence or SEQ.ID.NO.1,3-16,18-90, or its analogue, derivative, variant, or its obvious chemical equivalent is applied to the experimenter.Peptide of the present invention can be connected to another material or share with other materials such as microbiotic (such as penicillin, methicillinum or vancomycin), treat and/or prevent the effect of infection with increase, and/or increasing the target effect, these are all within the scope of the invention.

[0089] in an embodiment of the present invention, peptide of the present invention comprises table 1 aminoacid sequence or SEQ.ID.NO.1,3-16 or 18-90, or comprise SEQ.ID.NO.1,3-16,18-43,43-53 or 55-90 in another embodiment, or its analogue, derivative, or variant, or its obvious chemical equivalent.In another embodiment, peptide of the present invention is regulated experimenter's congenital immunity, thereby treats and/or prevents experimenter's infection.Innate immune response is the front reaction to the cause of disease that is run into.It comprises the mechanism of the multiple development that keeps off infection.A kind of such mechanism relates to the startup of immune effector cell and raises.

[0090] in one embodiment, peptide of the present invention can also strengthen congenital immunity or innate immune response in inflammation-inhibiting.

[0091] in another embodiment, peptide of the present invention has been proved to be the active conditioning agent of DDPIV.They have been proved can reduce the DPPIV activity.Like this, they can be used for screening can be from treating the experimenter who is benefited the specific Immunological diseases with peptide, comprise experimenter's sampling from the under a cloud or known DPPIV of having relative disease, sample and peptide of the present invention and a kind of DPPIV substrate are hatched jointly, monitor peptide of the present invention then compared with the control to the active influence of DPPIV, wherein active the minimizing then shows peptide is applied to the potential benefit of experimenter with treatment DPPIV relative disease.In another embodiment, compare with control group, the active adjusting of DPPIV (for example active reduction of DPPIV) can be represented a kind of DPPIV relative disease under the situation that has peptide to exist.Peptide of the present invention like this can be used for diagnosing the DPPIV relative disease.On the other hand, the peptide of this aspect can be used for treating many Immunological diseases such as DPPIV relative disease, virus of AIDS/acquired immune deficiency syndrome (AIDS) for example, Graves disease, cancer (as lung cancer and colorectal carcinoma), diabetes, autoimmune disorder such as rheumatoid arthritis and multiple sclerosis.

Administration

[0092] according to method of the present invention, peptide of the present invention as herein described can infect to treat and/or prevent the experimenter, and/or the significant quantity of treatment or prevention DPPIV relative disease, as the treatment significant quantity, directly is applied to the experimenter.Similarly, by will allow the nucleic acid of the mode encoded peptide that peptide expresses in the experimenter, give in the experimenter with the significant quantity that treats and/or prevents infection, peptide of the present invention can be applied to the experimenter indirectly.

[0093] in addition, the nucleic acid molecule of peptide of the present invention or this peptide of encoding can be applied to the experimenter with the significant quantity of treatment experimenter's infection or inflammation or DPPIV or congenital immunity relative disease." treatment infection or inflammation or DPPIV or congenital immunity relative disease are effective " used herein is meant and effectively relaxes slow or alleviate by the clinical lesion or symptom and the related inflammation that infect due to (being caused by bacterium, fungi, parasite, virus etc.).For example; the experimenter has infected microorganism; effectively the amount of the peptide (or nucleic acid of this peptide of encoding) of treatment infected by microbes is meant; can relax slow or alleviate or the amount of minimized infected by microbes symptom, this symptom includes but not limited to have a headache, variation performance, fever and the thrombopenia of Wry Neck, poor appetite, nausea,vomiting,diarrhea, abdominal discomfort, acute renal failure, many internal organs ischemia injury.Effectively the amount of the peptide (or nucleic acid of this peptide of encoding) of treatment experimenter infection or inflammation can be different because of the specific factor of every example, comprises experimenter's the weight and the severity of experimenter's disease.Those of skill in the art can easily determine the sufficient quantity of peptide (or nucleic acid of this peptide of encoding).Similarly, the significant quantity of treatment DPPIV relative disease can be different because of some similar factors well known by persons skilled in the art.

[0094] similarly, in the method for the invention, the nucleic acid of peptide of the present invention or this peptide of encoding also can be applied to the experimenter that may develop into infection with the amount of effective prevention experimenter infection." effectively preventing infection, inflammation or DPPIV or congenital immunity relative disease " used herein comprises the development or the performance of clinical lesion due to effective inhibition or the preventing infection (being caused by bacterium, fungi, parasite, virus etc.) or symptom.Effectively the prevention experimenter infect the amount of peptide (or nucleic acid of this peptide of encoding) can be different because of the specific factor of every example, comprise severity, the disease of experimenter's sex, weight and experimenter's disease character, infect position and administering mode.Those of skill in the art can easily determine the sufficient quantity of peptide (or nucleic acid of this peptide of encoding).

[0095] in one embodiment, the present invention includes described peptide with microbiotic such as the infection of vancomycin administering therapeutic.If like this, microbiotic can be with independent preparation, or administration simultaneously in a kind of preparation or pharmaceutical preparation.Perhaps, microbiotic can administration before or after peptide of the present invention is used.Comprise microbiotic and peptide of the present invention, and the pharmaceutical preparation of pharmaceutically acceptable carrier is considered also to comprise within the scope of the invention.

[0096] nucleotide sequence of peptide of the present invention or this peptide of encoding, as disclosed herein, can be applied to human or animal experimenter with currently known methods, include but not limited to oral administration, parenteral admin (for example on the manadesma, in the capsule, in intracutaneous, intracutaneous, intramuscular injection, the socket of the eye, in abdominal cavity, the canalis spinalis, in the breastbone, blood vessel, intravenous injection, thin-walled or subcutaneous administration), topical, Transdermal absorption administration, intranasal administration, pulmonary administration (for example administration in the tracheae) and osmotic pump administration.In one embodiment, medication is by vein or subcutaneous injection, parenteral admin.

[0097] when use the part, peptide of the present invention can be suitably and other components, and carrier as known in the art and/or auxiliary material or penetration enhancer are used in combination.The character of this other components must be pharmaceutically acceptable except them and is effectively to its expection administration without limits, and can not reduce the activity of composition active ingredient.In one embodiment, they are non-irritating to its skin used or mucous membrane.The example of appropriate carriers comprises ointment, emulsion, gelifying agent, suspensoid, comprises the colloidal powder that has or do not have collagen purification.Composition can also be saturated transdermal patch, plaster and bandage, preferred liquid or semi-liquid form.

[0098] for oral administration, the preparation of peptide (or nucleic acid of this peptide of encoding) can be capsule, tablet, pulvis, granule or suspensoid or solution.Preparation can have traditional additive, as lactose, N.F,USP MANNITOL, W-Gum or yam starch.Preparation also can contain tackiness agent, as Microcrystalline Cellulose, derivatived cellulose, gum arabic, W-Gum or gelatin.In addition, preparation can contain disintegrating agent, as W-Gum, yam starch or Xylo-Mucine.Preparation also can further contain calcium phosphate dibasic anhydrous or sodium starch glycolate.At last, preparation can contain lubricant, as talcum powder or Magnesium Stearate.

[0099] for parenteral admin, peptide (or nucleic acid of this peptide of encoding) can share with aseptic aqueous solution, preferably with the isoosmotic aseptic aqueous solution of experimenter's blood.A kind of like this preparation of preparation can be: solid active agent is dissolved in contains material compatible on the physiology, as sodium-chlor, glycine etc., and make the aqueous solution in the water with buffer pH value compatible with physiological condition, then make described solution aseptic.Preparation can the unit of being placed on or multi-dose container in, as sealed ampoule or bottle.The also available any injection system administration of preparation comprises those any ways as herein described.

[0100] for transdermal administration, peptide (or nucleic acid of this peptide of encoding) can share with transdermal enhancer such as propylene glycol, polyoxyethylene glycol, Virahol, ethanol, oleic acid, N-Methyl pyrrolidone etc., transdermal enhancer has increased the percutaneous permeability of peptide or nucleic acid, makes peptide or nucleic acid transdermal enter blood.The composition of promotor and peptide or nucleic acid also can be further share with polymer substance as ethyl cellulose, hydroxypropylcellulose, ethylene/vinyl acetate, polyvinylpyrrolidone etc., obtains gel-form composition.This gel-form composition is dissolved in the solvent as methylene dichloride, is evaporated to required viscosity, coat then on the material of mounting, make patch.Peptide or nucleic acid can or the position transdermal administration that infects near the experimenter.Perhaps peptide or the nucleic acid position transdermal administration beyond can be in the infected zone, thus realize being administered systemically.

[00101] for intranasal administration (as nasal spray) and/or pulmonary administration (inhalation), the preparation of peptide or nucleic acid comprises the aerosol agent, can be according to well known to a person skilled in the art the method preparation.The aerosol agent can comprise or solid particulate or solution (aqueous solution or non-aqueous solution).Atomizer (as jet-type atomizer, ultrasonic sprayer etc.) and spraying gun can be used for preparing aerosol (for example using as the alcoholic acid solvent) from solution, metered-dose inhaler and Diskus can be used for generating the aerosol of small-particle.The size of ideal aerosol particles can include but not limited to comminution by gas stream, spraying drying and stagnation point condensation by adopting any one acquisition in some methods known in the art.

[00102] pharmaceutical composition of intranasal administration can be solid preparation (a for example meal), can contain auxiliary material (as lactose).The solid preparation administration can be lifted the container that is placed with medicinal powder to the nose place, sucks fast and passes through nasal passage.The composition of intranasal administration also can comprise the water-based or the oily solution of nasal spray or nasal drop.For using atomizer, the preparation of peptide or nucleic acid can contain the aqueous solution and additive, comprises for example vehicle, buffer reagent, isotonic agent, sanitas or tensio-active agent.Nasal spray can, for example by the suspension of peptide or nucleic acid or solution are produced by nozzle.

[00103] peptide of pulmonary administration or nucleic acid preparation can occur with suitable form by the suction apparatus administration, and the particle that can effectively arrive the size of lung's lower respiratory tract or nasal sinus can be arranged.For absorbing by the mucomembranous surface that comprises the lung mucous membrane, preparation of the present invention can comprise emulsion, and this emulsion comprises as biologically active peptides, the submicron particle of diversification, adhesivity polymer, and/or water continuous phase.Absorption via mucomembranous surface can be achieved by the adhesion of emulsion particles.

[00104] pharmaceutical composition that is used for quantitative suction apparatus can contain the fine powder of peptide or nucleic acid, makes the suspension in the non-aqueous media.For example peptide or nucleic acid can be suspended in the propellent under the help of tensio-active agent (as sorbitol olein, soybean lecithin or oleic acid).Metered-dose inhaler uses the propellent gas (as Chlorofluorocarbons (CFCs), HCFC, fluorocarbon, or hydrocarbon polymer) that is stored in the container (as metal tin) to make mixture (as making the pressurized gas of liquefaction) usually.Sucker needs to drive in the breathing process.For example, metering valve drives and mixture can be discharged as aerosol.Diskus adopts the respiration drive of mixed powder.

[00105] peptide of the present invention or nucleic acid also can be from osmotic pressure Micropump or other regularly releasing device release or administrations.The rate of releasing drug of primary osmotic pump can respond gel fast and regulates by being arranged on micropore in the drug release hole.The osmotic pressure Micropump can be used for the control release or the target administration of peptide or nucleic acid.

[00106] according to method as herein described, peptide of the present invention is applied to the experimenter can be by giving the experimenter peptide itself, or giving the experimenter with the nucleic acid that allows the mode encoded peptide that peptide expresses.Therefore in an embodiment of the present invention, treatment or prevention experimenter infect can be by being applied to the experimenter to a certain amount of peptide of the present invention.In another embodiment of the present invention, treatment or prevention experimenter infect, can be by the nucleotide sequence of the peptide of the present invention of encoding in the mode that allows peptide to express the experimenter is applied to the experimenter.

[00107] peptide of the present invention is for example injected and is transfused blood by being used to import the known technology of peptide and other drug, comprising, can be applied to or give the experimenter.Tried the local infection of a certain specific part of body, it is desirable to treat peptide and directly import this zone by injection or some other modes (as by peptide being imported blood or other body fluid).The amount of used peptide is meant, effectively treats and/or prevents the experimenter as defined above and infects, and the definite easily amount of those of skill in the art.

[00108] in the method for the invention, by the nucleic acid of the mode encoded peptide of expressing with the permission peptide is imported in abundant experimenter's cell, also peptide can be applied to or import the experimenter.The amount of the nucleic acid of coding treatment peptide should be able to generate as defined above, treats and/or prevents the peptide of the significant quantity of experimenter's infection.Those of skill in the art can must determine this amount easily.

[00109] nucleic acid of coding peptide of the present invention can import the experimenter with ordinary method known in the art, includes but not limited to electroporation, the deae dextran transfection, calcium phosphate transfection, liposome transfection, single cationic-liposome merges, the polycation liposome merges, and protoplastis merges, and sets up the body internal electric field, DNA wraps by microprojectile bombardment methods, the injection of recombinant replication-defective virus, homologous recombination, the vivo gene treatment, outer-gene treatment, virus vector, naked DNA shifts, or its any combination.The recombinant viral vector that is suitable for gene therapy includes but are not limited to, and comes the carrier of the viral chromosome group of retrovirus, hsv, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus and vaccinia virus freely.

[00110] scope of the present invention also comprises, the nucleic acid of the peptide of the present invention of encoding can import in the external suitable cell with ordinary method, thereby at cell inner expression treatment peptide.The cell of expression of peptides can import the experimenter subsequently, with treatment/or prevention infect in the body.In a kind of so indirect vivo gene methods of treatment, cell preferably must obtain from the experimenter, integrates the nucleic acid that coding is treated peptide with dna technique, then introduces the experimenter again.

[00111] scope of the present invention also comprises, contains the preparation of the nucleic acid of peptide of the present invention or this peptide of encoding, can be further and pharmaceutically acceptable carrier, and thinner or vehicle share, thereby constitute pharmaceutical composition.Pharmaceutical composition of the present invention, representative carrier, thinner and vehicle are as indicated above.

[00112] preparation of the present invention can prepare with the pharmaceutical field known method.For example, the nucleic acid of peptide of the present invention or this peptide of encoding can mix mutually with carrier, thinner or vehicle, makes suspensoid or solution.Optional, also can add one or more auxiliary materials (as buffer reagent, seasonings, tensio-active agent etc.).Route of administration is depended in the selection of carrier.Pharmaceutical composition can be used for the nucleic acid molecule of peptide of the present invention or this peptide of encoding is applied to the experimenter, to treat and/or prevent infection.The peptide that is provided or the amount of nucleic acid can effectively treat and/or prevent the experimenter's who accepts pharmaceutical composition infection.As indicated above, this amount is to determine easily to those skilled in the art.

Diagnosis and screening detect

[00113] the invention provides a kind of method, be used to diagnose the congenital immunity disease that has under a cloud, or the experimenter of DPPIV relative disease, whether the experimenter is to using peptide of the present invention in prediction, list in table 1, SEQ ID.NO.1,3-16 or 18-90 or in another embodiment as those, the peptide of SEQID.NO.1,3-16,18-43,45-53 or 55-90, or its analogue, derivative, the treatment that variant or obvious chemical equivalent carry out is replied, and screening can be regulated the material of the immune effect of (as improving, suppress or simulation) peptide of the present invention.In another embodiment, the method that the invention provides to be screening peptide of the present invention, or the listed peptide of table 1 or immunocompetence analogue, derivative and the variant of its immunocompetence modifier.

[00114] in one embodiment, a kind of prediction has immunologic function disorder, whether the treatment of peptide of the present invention there is the method for replying as the patient of congenital immunity relative disease, it comprises from the experimenter and obtains biological specimen, peptide of the present invention is applied to described sample, and is reference with the positive or negative contrast, monitoring can be indicated the predetermined labels thing of this disease, as be used to indicate the DDPIV of DDPIV relative disease, indicate the level of the immune biomarker of infection, cytoactive or bacterial load.Positive control can be a sample of taking from the experimenter that known Immunological diseases are arranged.Negative control can be a sample of taking from the same subject of not using this peptide.If compare with control group, activity, the level of peptide aignment mark thing, or cell survival rate, then the experimenter has such immunologic function disorder, and may benefit from the treatment of peptide.

[00115] more specifically, in one aspect of the present invention, if the experimenter has or the DPPIV of having relative disease under a cloud, it is suitable so the DPPIV activity being detected as the mark of this disease.On the one hand, compare with control group, the active minimizing of DPPIV shows that the experimenter replys peptide treatment.Optional, if the experimenter is infected or under a cloud the infection, then fetch sample from patient, with the sample from patient is reference, detect pathogenic agent load, cell survival rate or cytokine-expressing or the producing method of sample behind the administration for peptides, wherein patient's cause of disease load reduces or cell survival rate uprises or cell expressing/generations changes behind the administration for peptides, shows that then the experimenter will benefit from peptide and treat or immunologic derangement arranged.

[00116] in another embodiment, whether has identical immunocompetence if observe modifier or other materials of peptide or table 1 peptide with peptide of the present invention, can compare with the reference peptide of regulating effect known to having, monitor described peptide to the active influence of DPPIV, sample (is taken from or infected with material, perhaps infect the mouse of known DPPIV relative disease) influence, or in order to monitor preventive effect, peptide or material are applied to sample, in described sample, cause described infection or DPPIV relative disease, monitor peptide then and whether regulate or suppress described infection or DPPIV relative disease or immunoreactive development.Described sample can be an animal model, and wherein the initiation of disease or infection is to finish in the animal model accepted that meets the ethics guide, then uses the effect of this animal or its suitable biology sample detection peptide.

[00117] whether treatment has the method for replying to infected by microbes to have the present invention further provides a kind of experimenter of prediction, wherein treatment comprises that its analogue, derivative, variant or obvious chemical equivalent are applied to the experimenter the peptide that comprises aminoacid sequence of the present invention, for example table 1 or SEQ ID.NO.1,3-16 or 18-90 or SEQ ID.NO.1,3-16,18-43,45-53 or 55-90.This method comprises the one or more biomarkers (as the inflammation biomarker) that detect experimenter's diagnostic sample, and wherein the existence of at least one biomarker (as the inflammation biomarker) shows that the experimenter has response to treatment.

[00118] " biomarker " used herein or " marker " are the biomarkers that is fit to arbitrarily, known or have recognized that itself and described disease-related (Immunological diseases for example, infect, inflammatory disease, the DPPIV relative disease, the congenital immunity disease), this marker comprises any molecule (for example genetic transcription) that is derived from gene, meaningful (coding) or antisense (non-coding) probe sequence from gene, the perhaps partial-length of gene or total length translation product, perhaps its antibody, it can be used for monitoring and immune response, innate immune response, inflammation, and/or DPPIV-relative disease associated state, imbalance, or disease.

[00119] according to the inventive method, experimenter's diagnosis sample can detect in external or body.Check is carried out external, and experimenter's diagnostic sample available standards method is separated and obtained.Diagnostic sample can be a tissue, comprises any muscle tissue, skin tissue or soft tissue, and its available standards biopsy method separation obtains.In addition, this diagnostic sample can be a body fluid, comprises blood, saliva, serum, or urine.Experimenter or patient can be known have infected by microbes or other immunologic function disorders, for example DPPIV relative diseases; Under a cloud have infected by microbes or other Immunological diseases, as congenital immunity disease or DPPIV relative disease; Or be considered to not have infected by microbes or other Immunological diseases, as congenital immunity disease or DPPIV relative disease.

[00120] the method according to this invention can detect the expression of one or more required markers of experimenter's diagnostic sample." expression " used herein dactylitis disease marker gene is transcribed at least a mRNA transcription product, and perhaps at least one mRNA translates into marker protein.Therefore, by certification label thing albumen, marker cDNA or marker mRNA, can detect the marker representation of diagnostic sample.Based on particular technology discussed in this article, the suitable form of marker is conspicuous.

[00121] protein to be detected can be used standard method known in the art, separation and purification obtains from experimenter or patient's diagnosis sample, this standard method includes but not limited to: extract (for example with the purification agent that makes protein dissolution) from required tissue, then carry out column chromatography (as FPLC and HPLC) affinity purification, immunoprecipitation (with antibody and the reaction of the markers of inflammation thing be concerned about), and precipitation (for example use Virahol and such as the material of Trizol).Can then carry out electrophoresis (for example using the SDS-polyacrylamide gel) behind the protein separation.Expectation can detect the proteic expression of any or all formal notation (comprising precursor, the form that the proteolysis inscribe is handled, and other form of posttranslational modification generation) of diagnostic sample.Nucleic acid can separate from diagnostic sample with the standard technique known to those skilled in the art and obtains.

[00122] according to the inventive method, can detect the marker representation that the experimenter diagnoses sample, the technology of determining easily with known field is (as immunological technique, hybridization analysis, fluorescence imaging technology, and/or radioassay), and any detection method disclosed herein is (as the heavy fixed reaction of immunity, western blot analysis etc.), can detect marker representation in the diagnostic sample.For example can be with the marker representation of the material testing experimenter diagnostic sample that reacts with the markers of inflammation thing." reacting " used herein is meant that this material has avidity to marker, can be bonded to marker, or directly act on marker." material " used further herein comprises protein, polypeptide, peptide, nucleic acid (comprising DNA or RNA), antibody, Fab fragment, fragment, molecule, compound, microbiotic, medicine and arbitrary combination thereof.Preferred, according to the techniques described herein, material of the present invention is with detectable marker or label.In an embodiment of the present invention, the material that reacts with this marker is a kind of antibody.

[00123] material of the present invention is the antibody that reacts with required marker, the diagnostic sample of taking from the experimenter flows through affinity chromatographic column and obtains purifying, affinity column has the antibody of anti-marker, and antibody is attached to (as being pearl, gel or flat a kind of insoluble organic polymer) on the solid phase carrier as part.Being attached to the form that antibody on the solid phase carrier can pillar uses.The example of suitable solid phase carrier includes but not limited to: agarose, Mierocrystalline cellulose, dextran, polyacrylamide, polystyrene, agarose and other insoluble organic polymer.If desired, can further will resist the antibody of marker to be attached on the solid phase carrier by link molecule.Technician under this area can must determine easily, is the suitable bonding conditions (such as temperature, pH value and salt concn) of guaranteeing material and antibody linked.In a preferred embodiment of the invention, be attached on the agarose pillar such as sepharose 4B with the antibody of anti-marker.

[00124] in addition, when material is a kind of antibody, express by the immune marker that can detect experimenter's diagnostic sample in conjunction with research and standard immunoassay detection technique, this adopts and can carry out immunoreactive one or more antibody with marker in conjunction with research.For example, can use the ELISA method from the marker albumen of affinity column wash-out, western blot analysis, flow cytometer, or any other utilizes the interactional immunostaining of antigen-antibody to detect.Preferably, detect the expression of diagnostic sample marker with western blot analysis.

[00125] or, by diagnose the nucleic acid of sample to carry out hybridization analysis from the experimenter, diagnose the marker representation of sample with the check experimenter to extracting.According to this method of the present invention, the Northern engram analysis of available mRNA carries out hybridization analysis, and one or more nucleic acid of the nucleic acid hybridization of also available and coded markings thing carry out the Southern engram analysis of DNA.Nucleic acid probe can prepare with multiple technologies well known by persons skilled in the art, includes but not limited to following technology: the digestion with restriction enzyme of marker nucleic acid; Use the commercial automatic synthetic oligonucleotide of buying of oligonucleotide synthesizer as Applied BiosystemsModel 392DNA/RNA synthesizer, this oligonucleotide has the corresponding sequence of selected part with the nucleotide sequence of marker nucleic acid.

[00126] the used nucleic acid probe of the present invention can be DNA or RNA, and length can change between the total length of markers of inflammation thing nucleic acid at about 8 Nucleotide.In addition, nucleic acid probe of the present invention can come mark with one or more detectable markers or label.The mark of nucleic acid probe can be finished with one of a plurality of methods known in the art, and described method includes but not limited to any those methods described herein.Adopt as PCR or RT-PCR method, the combination corresponding to two or more nucleic acid probes (or primer) of marker nucleic acid difference or congruent region can be used to detect the marker representation of diagnostic sample.

[00127] in the method for the invention, after expression of certification mark thing or the activity, can measure or quantize the detection that the experimenter diagnoses marker representation degree in the sample.Such detection method is well known to those skilled in the art, can comprise immunohistochemistry/immunocytochemistry, flow cytometry, mass spectroscopy, western blot analysis, or ELISA method certification mark thing albumen quantity or monitoring substrate generate with certification mark thing (enzyme) active (analyzing as DPPIVF).For example use immunohistochemical method, can place tissue slice (paraffin embedding) on the slide glass, hatch with marker antibody then.Slide glass can be subsequently be hatched with two anti-(anti-at one), and this is two anti-with dyestuff or other calorimetric system (such as fluorescence dye, radioactivation agent, or the material with high electronic scanning ability) mark, so that the marker video picture in the section.

[00128] the present invention describes in the following example, and its elaboration helps to understand the present invention, and should not be annotated to being limited in the defined any scope of the present invention of the claim of enclosing.

Embodiment

Synthesizing of embodiment 1-peptide

[00129] peptide in the table 1 is synthetic with the solid-phase peptide synthetic technology

[00130] peptide required with respect to 1mmole, the amino acid of all required Fmoc-protections is with 3 times of molar excess weighings.(DMF makes the solution of 3mMol in 7.5ml) then amino acid to be dissolved in dimethyl formamide.According to the substitution value of resin, take by weighing an amount of Rink amide mbha resin.Then this resin transfer is arrived in the reaction vessel of automatic DNA synthesizer DNA, steeped in advance 15 minutes with methylene dichloride (DCM).

[00131] joins 25% piperidines DMF solution (30ml) in the resin and mixed 20 minutes, make resin go protection.Resin goes after the protection, and the first step coupling is with the amino acid solution of 3mMol and the 2-of 4mMol (1H-benzotriazole-1-yl)-1,1,3, the N of 3-tetramethyl-urea hexafluorophosphate (HBTU) and 8mMol, and N-diisopropylethylamine (DIEPA) mixes.This solution joins before the resin, can activate 5 minutes in advance.Make this amino acid coupling 45 minutes.

[00132] resin after the coupling fully washes with DMF and N,N-DIMETHYLACETAMIDE (DMA).The amino acid of the Fmoc protection in the connection goes protection with above-mentioned the same manner, and next amino acid connects up with same coupling strategy AA:HBTU:DIEPA.

[00133] after synthetic the finishing, peptide is ruptured from resin with a kind of cutting reagent that contains 97.5% trifluoroacetic acid (TFA) and 2.5% water.With resin in cutting reagent floating 1.5 hours.Solution B gravity filtration, filtrate collection is in the centrifuge tube of 50ml.With cold ether sedimentation peptide is separated.Centrifugal and pour out ether after, the crude product peptide washs once more with ether, in vacuum drier dry 2 hours then.Then peptide is dissolved in deionized water (10ml) ,-80 ℃ of freezing and freeze-drying down.The freeze-drying peptide can carry out purifying with HPLC then.

[00134] because the wetting ability of these peptides can't use ether to separate.Therefore need use chloroform extraction.Steam and remove TFA, the peptide resistates is dissolved in 10% acetate (15ml).Solution is with chloroform (30ml) washed twice, so that impurity in the acetate peptide solution and scavenging agent are removed.Then with the aqueous solution of peptide-80 ℃ of freezing and freeze-drying, obtain the peptide freeze-dried powder of available HPLC purifying.

[00135] peptide SE Q.ID.NO:33 and 34 contains the N-amino acid that methylates separately.This coupling is that N-methylamino acid, PyBroP and N-hydroxybenzotriazole * H2O (HOBt) and DIEPA solution are carried out chemical combination together containing in the RV of resin.After the coupling 45 minutes, again the N-methylamino acid is carried out a coupling, to guarantee complete coupling.Observe, the coupling of ensuing N-methylamino acid is also incomplete.Therefore, current coupling N, N, N ', N '-tetramethyl--O-(7-azepine benzotriazole-1-yl) urea hexafluorophosphate (HATU) and without HBTU.This still obtains a crude product peptide, and it typically contains two kinds of impurity, accounts for the 30-40% of total pure product.Change the HPLC condition peptide is carried out purifying, the peak of pure peptide and approaching wash-out impurity are separated.

The non-antimicrobial acivity of embodiment 2-

[00136] bacterium (streptococcus aureus 25923) is inoculated in the hole that contains peptide (200 μ M), solvent (Tris) or microbiotic (erythromycin, 120 μ g/ml).Bacterial growth 2 hours.After this, bacteria living ability WST-1 colorimetric survival detection method (catalog number 1 644 807; The Luo Shi diagnosis) detects.DMEM and DMEM+WST-1 contrast as a setting.Shown in Figure 1A and B, compare with microbiotic contrast, SEQ ID NO:5 and 47 two peptides clear show as lack active.

The effect of embodiment 3-endogenous protective

[00137] mouse peritoneal (IP) infectable infection streptococcus aureus 25923.The peptide SEQ ID NO:1,4,5,6,45 and 47 of 4 hours pneumoretroperitoneum injection 12mg/kg, the SEQ.ID.NO.1 of 24mg/kg (Fig. 2 A and 2B), 9.6mg/kg SEQ.ID.NO.5 (Fig. 2 C), 13mg/kg SEQ.ID.NO.47 (Fig. 2 D), 12mg/kg SEQ.ID.NO 4 (Fig. 2 E), 9mg/kg SEQ.ID.NO.6 (Fig. 2 F), 13mg/kg SEQ.ID.NO.45 (Fig. 2 G).Infected back 24 hours, and put to death the animal of survival, peritoneal lavage fluid is measured its remaining bacterial count (every ml colony-forming unit (CFU/ml)) under treating with the peptide treatment with without peptide with flat band method.

[00138] specify dead animal that the highest bacterial number of any animal in this research is arranged.Shown in Fig. 2 A-G, compare peptide SEQ ID NO:1,4,5,6,45 and 47 with control group and obviously demonstrated provide protection.

Preventive protection effect in the embodiment 4-body

[00139] infects the peptide SEQ ID NO.1 (Fig. 3 A) of preceding 24 hours abdominal injection 12mg/kg and the peptide SEQ.ID.No.5 (Fig. 3 B) of 11.5mg/kg.Mouse peritoneal infectable infection streptococcus aureus 25923 then.Infected back 24 hours, and put to death the animal of survival, peritoneal lavage fluid is surveyed its remaining bacterial count (every ml colony-forming unit (CFU/ml)) under treating with the peptide treatment and without peptide with flat band method.

[00140] specify dead animal that the highest bacterial number of any animal in this research is arranged.Peptide SEQID NO:1 and 5 has obviously demonstrated provide protection ((0 dead mouse (peptide treatment group) and 2 dead mouses (control group)).See also Fig. 3 A and B.

[00141] the following discussion is the result that the inventor gets in touch embodiment 1-4 experiment gained.

[00142] inventor shows, and peptide or peptide as a part of the present invention as described herein with aminoacid sequence shown in the table 1 can strengthen congenital immunity.Especially, peptide SEQ ID NO:1,4,5,6,45 and 47 can prevent and avoid to infect (Fig. 2 and embodiment 3 shown in the body inner model; Fig. 3 and embodiment 4).Yet, do not have antimicrobial acivity as embodiment 1 and peptide SEQ IDNO:5 shown in Figure 1 and 47.In view of the above, the adjusting via peptide SEQ ID NO:5 and/or 47 pairs of congenital immunities shows that these peptides can be as a kind of curative of infectious diseases treatment.

Embodiment 5 human blood infection models

[00143] effect is also used the human blood from the heparin test tube that volunteer's donor is collected by qualified medical personnel, estimates in the infection model in the body of formerly external back.In this infection model, the whole human blood of collecting in the heparin test tube is divided into 2 five equilibriums of 0.5mL.Each five equilibrium adds salt solution or peptide (0.5mM), hatches 45 minutes.After hatching, add 3.9x10 ²The streptococcus aureus of CFU/mL (ATCC bacterial strain 25923) hatched 24 hours.After 24 hours, from each hole, get five equilibrium, use colony-forming unit (CFU) counting to be used to assess infectation of bacteria.The average result of a plurality of CFU countings as shown in Figure 4.

[00144] parallel with the effect that shows in the mouse infection model, this people's elder generation external back body inner model further specifies the effect of the peptide that this that use in people's treatment differentiates in using.

Embodiment 6 anti-inflammation activity

[00145] measures the effect that the peptide treatment is replied LPS stimulating cytokine in the body of earlier external back.In these researchs, SEQ ID NO 5 after 4 hours, collects the blood of every mouse with the 100mg/kg intravenously administrable.Separate PBMC, stimulate, measure the cytokine levels that obtains with LPS.Apparent from these researchs, carrying out interior therapeutic with peptide is enough to change the responsiveness (Fig. 5) of cell to the external back body internal stimulus of elder generation subsequently---cause the Inflammatory response of LPS stimulation is reduced.Similar results is found in the blood of collecting in 24 hours behind the vivo medicine-feeding.

Embodiment 7-tests plasma D PPIV activity with mouse blood

[00146] ICR mouse heart puncture obtains mouse blood, and is collected in the blood collection test tube that contains heparin.The blood of several mouse compiled and with branch packing such as 300 μ L.Peptide is dissolved in the pH5.5 acetate buffer solution to concentration 9mM.30 μ L join in the blood of 300 μ L with this storing solution, and resuspension mixes (concentration in the blood is 0.82mM).For control sample, the blank acetate buffer solution of 30 μ L is joined in the 300 μ L blood.Each peptide group is prepared three parts, and control group is prepared 6 parts.Sample was hatched in the microminiature tube of 37 ℃ of sealings two hours.Hatch the centrifugal sample of back 4000rcf, isolate blood plasma.Blood plasma is transferred to one 96 hole check-out console detect DPPIV.The DPP IV substrate gly-pro-p-Nitroaniline (16mM deionized water solution) of 5 μ L is joined in the blood plasma (plasma concentration 0.8mM) of 95 μ L, and detect the increase of uv-absorbing in 20 minutes (405 nanometer).Enzymatic lysis gly-pro-p-Nitroaniline generates the speed of p-Nitroaniline as the activity of DPP IV (Durinx C et al., (2001) " Reference valuesfor plasma dipeptidyl-peptidase IV activity and their association with otherlaboratory parameters (the active reference value of blood plasma DPP IV and with the relation of other experiment parameter) " .Clin Chem Lab Med.39 (2): 155-9).

[00147] the results are shown in Table 1.Can be observed peptide to the active influence of DPP IV.Its result is shown as with respect to physiological saline contrast (being set to 100%) normalized, average percentage activity.Any activity of 100% of being less than means the active reduction of DPPIV.

[00148] one aspect of the present invention, or in one embodiment, the active reduction of DPPIV approximately at least 25% (promptly about 75%+/-5%) has been considered to activity.Technician under this area can understand the ideal activity level and may change because of the application of polypeptide.

Discuss

[00149] II type transmembrane serine protease dipeptidyl peptidase IV (DPPIV) is also referred to as CD26 or adenosine deaminase binding protein, is a main regulatory factors that comprises the various physiological processs of immunologic function.CD26/DPP IV is the cell surface glycoprotein of a 110-kD, and it is mainly expressed on ripe thymocyte, activated T cell, B cell, NK cell, scavenger cell and epithelial cell.It has two kinds of functions at least, signal conduction function and proteolysis function (Morimoto C, Schlossman SF.The structure and function of CD26 in.-.TheT-cell immune response.Immunol.Review.1998,161:55-70.).One of effect of its cell comprises from chemokine N end check separates the activity that dipeptides is regulated chemokine.Chemokine NH ₂The adjusting of end not only to the combining and subsequent reactions of its acceptor, and all be very important to the acceptor specificity that changes treated chemokine.In addition, proved that solubility rCD26 has strengthened the endothelial migration of striding of T cell, reply [Oravecz and reduced monocytic migration, T. wait the people, (1997) Regulation of the receptor specificity andformation of the chemokine RANTES (regulated on activation, normal Tcell expressed and secreted) by dipeptydyl peptidase IV (CD26)-mediatedcleavage ((regulate active to receptor-specific and cytokine RANTES by the cutting of DPP IV (CD26)-mediation, normal T cell expressing and excretory) the adjusting of formation) .J.Exp.Med.186:1865-1872; Iwata, S. wait the people, (1999) CD26/dipeptidylpeptidase IV differentially regulates the chemotaxis of T cells andmonocytes toward RANTES:possible mechanism for the switch from innateto acquired immune response (CD26/ DPP IV differentiation is regulated the chemotaxis towards RANTES of T cell and monocyte: from congenital immunity to the immune response conversion day after tomorrow may mechanism) .Int.Immunol.11:417-426].These results show that the CD26/DPPIV differentiation is regulated T cell and monocytic chemotactic response, and participates in congenital conversion to acquired immune response.Like this, the active reduction of DDPIV has reverse effect when the time comes, promotes the reaction of natural immunity reaction and macrophage migration.Other has report, the pharmacology of DPPIV enzymic activity suppresses to reduce the arthritic progress of sacroiliitis experimental rat model (Tanaka S et a1., Anti-arthritic effects of the novel dipeptidyl peptidase IV inhibitors TMC-2A andTSL-225 (the arthritis effect of novel dipeptidyl peptidase IV inhibitor TMC-2A and TSL-225) .Immunopharmacology 1998,40:21-26; Tanaka S, et al.,: Suppression ofarthritis by the inhibitors of dipeptidyl peptidase IV (inhibitor of DPP IV is to arthritic inhibition) .Int J Immunopharmacol 1997,19:15-24), mean that the active reduction of DPP IV can amelioration of inflammation in some cases.Anti-inflammatory action makes DPPIV become the desirable molecule that screening has these active new compounds together with its regulating effect to chemotactic activity.

[00150] CD26/DPPIV has participated in the pathology of multiple disease, such as AIDS and HIV progression of disease (Blazquez etc., 1992; Vanham etc., 1993; Schols etc., 1998; Oravecz etc., 1995), Graves disease (Eguchi etc., 1989; Nishikawa etc., 1995), and cancer (Steccaet etc., 1997) and diabetes (Hinke etc., 2000; Marguet etc., 2000).

[00151] further, CD26 is as a kind of indicator of t cell activation, shown and makes parallel undulations as rheumatoid arthritis (Nakao etc., 1989) and autoimmune thyroiditis several autoimmune disorders such as (Eguchi etc., 1989).CD26 has been described to a kind of and the good markers of activity level dependency these diseases.In addition, also it is studied as the index (Constantinescu etc., 1995) of making progress in the chronic progressive multiple sclerosis course of disease.

[00152] peptide of the present invention has confirmed to reduce the activity of DPPIV.Similarly they can be used for treating some amynologic disease, such as the DPPIV relative disease, and can regulate congenital immunity and inflammation from one side, as cause the inflammation of septicemia.

The plasma D PPIV activation analysis that embodiment 8 usefulness human bloods carry out

[00153] human blood is obtained from volunteer's donor by titular medical worker, and is collected in the blood collecting test tube of heparinization.Blood is divided into 300 μ L.Peptide is dissolved in the acetate salt buffer salt solution, and the various concentration of 30 μ L of pH5.5 are added to resuspended mixing (final concentration as shown in Figure 6 in the blood) in the 300 μ L blood.For contrast, the blank acetate salt buffer salt solution of 30 μ L is added in the 300 μ L blood.Three parts of every kind of prepared at concentrations, and six parts of contrast contrast preparations.Sample was hatched in airtight microtest tube 2 hours for 37 ℃.After hatching, blood plasma 4000rcf is centrifugal from sample separation.Blood plasma is transferred to 96 hole check-out consoles and is carried out the DPPIV detection.5 μ L DPPIV substrate gly-pro-p-Nitroaniline (deionized water solution of 16mM) are added to begin in the 95 μ L blood plasma (concentration in the blood plasma is 0.8mM) to detect, monitoring UV absorbs the increase by 20 minutes of (405nm).Gly-pro-p-Nitroaniline enzymolysis obtains the preparation rate of p-Nitroaniline as the activity of DPPIV (Durinx C et al. (2001) " Reference values for plasma dipeptidyl-peptidase IVactivity and their association with other laboratory parameters (the active reference value of blood plasma DPP IV and with the relation of other experiment parameters) " .Clin Chem LabMed.39 (2): 155-9).

That [0154] measures in the activity that SEQ ID NO.5 shows in the human blood and the mouse blood is active suitable, shows that detected result is relevant with the mensuration that has the compound of human therapy possibility.

Embodiment 9 is the DPPIV dose response curve relatively

[00155] dose response of SEQ.ID.NO.7 (KSRIVPAIPVSLL) among the PCT/CA02/01830 that submits in SEQ.ID.NO.5 of the present invention and on December 2nd, 2002.The peptide of different concns was hatched 2 hours for 37 ℃ with full ICR mouse blood.The blood of hatching with acetate salt buffer salt solution (20mM) is with comparing.After hatching, centrifugal 10 minutes separated plasmas of 4000rcf.Substrate Gly-Pro-p-Nitroaniline (0.8mM) is added in the blood plasma, and the enzymolysis speed of DPPIV is measured by the increase that monitoring product p-Nitroaniline UV absorbs.The result shows the dose response of SEQ.ID.NO.5 of the present invention as shown in Figure 7, shows the increase along with peptide concentration, and the active reduction of DPPIV is more.KSRIVPAIPVSLL does not have similar dose response, show that the latter's peptide obviously works, and peptide of the present invention is represented the peptide of novel type.

Embodiment 10 strengthens the curative effect of antibiotic therapy

[00156] infected preceding 24 hours, the peptide of 60mg/kg is expelled to CD-1 mouse (N=10 animal/group through IP; Fig. 8 A; Male mice, Fig. 8 B: female mice).Mouse uses the streptococcus aureus (the MRSA strains A TCC33591 of Fig. 8 A and the UC6685 of Fig. 8 B) of methicillin-resistant through the IP injection (1.5*10 of Fig. 8 A subsequently ⁷With Fig. 8 B～4*10 ⁵) infect.The vancomycin of given dose infected back 1 hour and twice of 5 hours subcutaneous administration.Monitor the survival in 5 days (Fig. 8 A) or 8 days (Fig. 8 B).

[00157] shown in Fig. 8 A and B, SEQ ID NO1 and the treatment of SEQ ID NO5 combined with antibiotic are with respect to not having treatment (solvent) or only antibiotic therapy survival increase.

[00158] though for for the purpose of being aware and understand, aforementioned the present invention is illustrated in detail, but those skilled in the art should be understood that, by reading disclosed content, under the prerequisite that does not depart from claims true scope of the present invention, form and details can be done many variations.

Table 1 is continuous

Table 1 note 1:

X ₁Be selected from the compound based on glycine (for example Nlys), hSer, Val (betaOH) that K, R, S, O or N-end are replaced by basic functionality

X ₂Be selected from V, I, R and W

Comprise a kind of nearly 10 amino acid whose isolated peptides, it comprises SEQ ID NO 55 aminoacid sequences.

Table 1 note 2

X wherein ₁Be selected from the compound based on glycine (for example Nlys), hSer, Val (betaOH), wherein X that K, H, R, S, T, O or N-end are replaced by basic functionality ₂Be selected from A, I, L, V, K, P, G, H, R, S, O, Dab, Dpr, Cit, Hci, Abu, Nva, Nle, wherein X ₂Can be that N-is methylated, X wherein ₃Be selected from I, V, P, G, H, W, E, wherein in one embodiment, X ₃Do not methylated by N-.In one embodiment, isolated peptides can be to reach 10 amino acid whose aminoacid sequences, but is not SEQ.ID.NO.2 or 17.

Table 1 note 3

X wherein ₁, X ₂And X ₃As what define in SEQ ID NO:56, wherein " a " is selected from S, P, I, R, C, T, L, V, A, G, K, H, R, O, C, M and F or a kind of nearly 10 amino acid whose isolated peptides, and it comprises described sequence.

Table 1 note 4

X wherein ₁X ₂X ₃P is as what define in SEQ ID NO:56, and " b " is selected from A, A ^*, E, G, S, L, F, K, W, C, I, V, T, D, Y, R, H, O, Q, N, P and M, but be not P in embodiment.In one embodiment, isolated peptides is a kind of nearly 10 amino acid whose peptides, and it comprises SEQ ID NO 58 rather than SEQ ID NO 17.

Table 1 note 5

X wherein ₁, X ₂And X ₃As what in SEQ ID NO 56, define, " a ₁" be selected from K, I, R, H, O, L, V, A and G, " a ₂" be selected from S, P, R, T, H, K, O, L, V, A, G, S and I.In one embodiment, a ₁Not acetylizad, perhaps a wherein ₁Be K, K is not acetylizad, perhaps is not SEQ ID NO 2.In one embodiment, isolating peptide comprises nearly 10 amino acid, and it comprises SEQ.ID.NO.59.

Table 1 note 6

X wherein ₁, X ₂And X ₃As what in SEQ ID NO 56, define, wherein " a " is selected from S, R, K, H, O, T, I, L, V, A and G, wherein " b " is selected from A, V, I, L, G, K, H, R, O, S, T and F or a kind of nearly 10 amino acid whose peptides, and it comprises SEQID NO 60.

Reference

Blazquez MV, Madueno JA, Gonzalez R, Jurado R, Bachovchin WW, Pena J, Munoz E.Selective decrease of CD26 expression in T cells from HIV-1-infected individuals (CD26 expresses in the T cell of selectivity reduction HIV-1-infected individuals) .J Immunol.1992 Nov 1; 149 (9): 3073-7.

Vanham G, Kestens L, De Meester I, Vingerhoets J, Penne G, Vanhoof G, Scharpe S, Heyligen H, Bosmans E, Ceuppens JL, et al.Decreasedexpression of the memory marker CD26 on both CD4+and CD8+Tlymphocytes of HIV-infected subjects (expression of the reduction of memory marker CD26 on the CD4+ of HIV infected subjects and the CD8+T lymphocyte) .J Acquir ImmuneDefic Syndr.1993 Jul; 6 (7): 749-57.

Schols D, Proost P, Struyf S, Wuyts A, De Meester I, Scharpe S, Van DammeJ, De Clercq E.CD26-processed RANTES (3-68), but not intact RANTES, has potent anti-HIV-1 activity (RANTES rather than the complete RANTES that are CD26-processing have potent anti-HIV-1 activity) .Antiviral Res.1998 Oct; 39 (3): 175-87.Erratum in:Antiviral Res 1999 Jan; 40 (3): 189-90.

Oravecz T, Roderiquez G, Koffi J, Wang J, Ditto M, Bou-Habib DC, Lusso P, Norcross MA.CD26 expression correlates with entry, replication andcytopathicity of monocytotropic HIV-1 strains in a T-cell line (CD26 express with close monocytic HIV-1 strain enter T clone, therein duplicate fall ill relevant with cell) .Nat Med.1995 Sep; 1 (9): 919-26.Comment in:Nat Med.1995 Sep; 1 (9): 881-2.

Nishikawa Y, Nakamura M, Fukumoto K, Matsumoto M, Matsuda T, TanakaY, Yoshihara H.[Adenosine deaminase isoenzymes in patients with Graves ' disease (Robert Graves patient's adenosine deaminase isomerase)] Rinsho Byori.1995Oct; 43 (10): 1057-60.[Article in Japanese]

Eguchi K, Ueki Y, Shimomura C, Otsubo T, Nakao H, Migita K, KawakamiA, Matsunaga M, Tezuka H, Ishikawa N, et al.Increment in the Tal+cells inthe peripheral blood and thyroid tissue of patients with Graves ' disease (increase of Ta1+ cell in Graves disease peripheral blood of patients and the parathyroid tissue) .J Immunol.1989 Jun 15; 142 (12): 4233-40.

Stecca BA, Nardo B, Chieco P, Mazziotti A, Bolondi L, Cavallari A.Aberrant dipeptidyl peptidase IV (DPP IV/CD26) expression in humanhepatocellular carcinoma (unusual DPP IV (DPPIV/CD26) is expressed in the human hepatocellular carcinoma) .J Hepatol.1997 Aug; 27 (2): 337-45.

Hinke SA, Pospisilik JA, Demuth HU, Mannhart S, Kuhn-Wache K, Hoffmann T, Nishimura E, Pederson RA, McIntosh CH.Dipeptidylpeptidase IV (DPIV/CD26) the degradation of glucagon.Characterization ofglucagon degradation products and DPIV-resistant analogs (DPP IV of hyperglycemic-glycogenolytic factor (DPP IV/CD26) degraded.The evaluation of hyperglycemic-glycogenolytic factor degraded product and DPIV-resistance analogue) .J Biol Chem.2000 Feb 11; 275 (6): 3827-34.

Marguet D, Baggio L, Kobayashi T, Bernard AM, Pierres M, Nielsen PF, Ribel U, Watanabe T, Drucker DJ, Wagtmann N.Enhanced insulin secretionand improved glucose tolerance in mice lacking CD26 (glucose tolerance of enhanced insulin secretion and raising in the mouse of shortage CD26) .Proc Natl Acad Sci U SA.2000 Jun 6; 97 (12): 6874-9.

Nakao H, Eguchi K, Kawakami A, Migita K, Otsubo T, Ueki Y, ShimomuraC, Tezuka H, Matsunaga M, Maeda K, et al.Increment of Tal positive cellsin peripheral blood from patients with rheumatoid arthritis (increase of the peripheral blood T al positive cell of patient with rheumatoid arthritis) .J Rheumatol.1989 Jul; 16 (7): 904-10.

Constantinescu CS, Kamoun M, Dotti M, Farber RE, Galetta SL, Rostami A.A longitudinal study of the T cell activation marker CD26 in chronicprogressive multiple sclerosis (research of the differentiation of T cell activation marker CD26 in the chronic progressive multiple sclerosis) .J Neurol Sci.1995 Jun; 130 (2): 178-82.

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Sequence table

<110>Inimex?Pharmaceuticals，Inc.

＜120〉NOVEL PEPTIDES FOR TREATING and PREVENTINGIMMUNE-RELATED

DISORDERS, INCLUDING TREATING and PREVENTING INFECTION BY

MODULAT?INGINNATE?IMMUNITY

<130>190476-388607

<140>To?be?Assigned

<141>2007-04-03

<150>PCT/CA2006/001650

<151>2006-10-04

<150>US?60/722,962

<151>2005-10-04

<150>US?60/722,958

<151>2005-10-04

<150>US?60/722,959

<151>2005-10-04

<160>90

<170>PatentIn?version?3.3

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＜223〉immune peptide

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＜223〉Xaa equals acetylizad K.

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<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>11

Arg?Ala?Val?Pro?Ala

1 5

<210>12

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>12

Arg?Arg?Ile?Val?Pro?Ala

1 5

<210>13

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>13

Arg?Lys?Val?Pro?Ala

1 5

<210>14

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>14

Arg?Ile?Val?Pro?Lys

1 5

<210>15

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>15

Arg?Pro?Val?Pro?Ala

1 5

<210>16

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>16

Arg?Ile?Pro?Pro?Ala

1 5

<210>17

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>17

Arg?Ile?Val?Pro?Pro

1 5

<210>18

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>18

Arg?Ile?Val?Pro?Gly?Gly?Ala

1 5

<210>19

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>19

Gly?Gly?Ile?Val?Pro?Ala

1 5

<210>20

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>20

Gly?Ile?Val?Pro?Ala

1 5

<210>21

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>21

Arg?Gly?Val?Pro?Ala

1 5

<210>22

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>22

Arg?Ile?Val?Pro?Gly

1 5

<210>23

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>23

Arg?Ile?Val?Pro?Ser

1 5

<210>24

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>24

Arg?Ile?Val?Pro?Leu

1 5

<210>25

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>25

Arg?His?Val?Pro?Ala

1 5

<210>26

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>26

Arg?Ile?Pro?Val?Ala

1 5

<210>27

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>27

Arg?Val?Ile?Pro?Ala

1 5

<210>28

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>28

Arg?Ile?Ile?Pro?Ala

1 5

<210>29

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>29

Ala?Val?Pro?Ile?Arg

1 5

<210>30

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>30

Ala?Pro?Val?Ile?Arg

1 5

<210>31

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>31

Arg?Ile?Val?Pro?Ala

1 5

<210>32

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>32

Cys?Arg?Ile?Val?Pro?Ala?Cys

1 5

<210>33

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa equals to have the I. of NMe skeleton

<400>33

Arg?Xaa?Val?Pro?Ala

1 5

<210>34

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa equals to have the methylate A. of skeleton of N-

<400>34

Arg?Ile?Val?Pro?Xaa

1 5

<210>35

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>35

Arg?Ile?Val?Pro?Phe

1 5

<210>36

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>36

Cit?Ile?Val?Pro?Ala

1 5

<210>37

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>37

Arg?Leu?Val?Pro?Ala

1 5

<210>38

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>38

His?Ile?Val?Pro?Ala

1 5

<210>39

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>39

Ile?Arg?Arg?Val?Pro?Ala

1 5

<210>40

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>40

Ala?Arg?Val?Pro?Ala

1 5

<210>41

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>41

Ile?Arg?Val?Pro?Ala

1 5

<210>42

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>42

Orn?Ile?Val?Pro?Ala

1 5

<210>43

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>43

Ser?Ile?Val?Pro?Ala

1 5

<210>44

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>44

Val?Ser?Ile?Ile?Lys?Pro?Ala?Arg?Val?Pro?Ser?Leu?Leu

1 5 10

<210>45

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>45

Lys?Pro?Ala?Arg?Val?Pro?Ser

1 5

<210>46

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>46

Arg?Val?Pro?Ser?Leu?Leu

1 5

<210>47

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>47

Lys?Pro?Arg?Ala?Val?Pro

1 5

<210>48

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>48

Pro?Ala?Arg?Val?Pro

1 5

<210>49

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>49

Ile?Arg?Val?Pro

1

<210>50

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>50

Arg?Val?Pro?Ser

1

<210>51

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>51

Arg?Val?Pro

1

<210>52

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>52

Pro?Ser?Val?Pro?Gly?Ser

1 5

<210>53

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>53

Gly?Leu?Lys?His?Pro?Ser

1 5

<210>54

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>54

Arg?Ile?Val?Pro?Ala?Ile?Pro?Val?Ser?Leu?Leu

1 5 10

<210>55

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa is selected from K, R, and S, O, or the N end is substituted with the compound based on glycine of basic functionality.

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa is selected from V, I, R, and W.

<400>55

Xaa?Xaa?Pro

1

<210>56

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa is selected from K, H, and R, S, T, O, or the N end is substituted with the compound based on glycine of basic functionality.

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa is selected from A, I, and L, V, K, P, G, H, R, S, O, Dab, Dpr, Cit, Hci, Abu,

Nva, Nle, wherein to can be N-methylated for Xaa.

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa is selected from I, V, P, G, H, W, E.

<400>56

Xaa?Xaa?Xaa?Pro

1

<210>57

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa is selected from S, P, I, R, C, T, L, V, A, G, K, H, R, O, C, M, and F.

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa is selected from K, H, and R, S, T, O, or the N end is substituted with the compound based on glycine of basic functionality.

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa is selected from A, I, and L, V, K, P, G, H, R, S, O, Dab, Dpr, Cit, Hci, Abu,

Nva, Nle, wherein to can be N-methylated for Xaa.

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa is selected from I, V, P, G, H, W, E.

<400>57

Xaa?Xaa?Xaa?Xaa?Pro

1 5

<210>58

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa is selected from K, H, and R, S, T, O, or the N end is substituted with the compound based on glycine of basic functionality.

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa is selected from A, I, and L, V, K, P, G, H, R, S, O, Dab, Dpr, Cit, Hci, Abu,

Nva, Nle, wherein to can be N-methylated for Xaa.

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa is selected from I, V, P, G, H, W, E.

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa is selected from A, A*, and E, G, S, L, F, K, W, C, I, V, T, D, Y, R, H, O, Q,

N, P and M, wherein A* represents the A. of D amino acid form

<400>58

Xaa?Xaa?Xaa?Pro?Xaa

1 5

<210>59

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa is selected from K, I, R, H, O, L, V, A, and G.

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa is selected from S, P, R, T, H, K, O, L, V, A, G, S, I.

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa is selected from K, H, and R, S, T, O, or the N end is substituted with the compound based on glycine of basic functionality.

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa is selected from A, I, and L, V, K, P, G, H, R, S, O, Dab, Dpr, Cit, Hci, Abu,

Nva, Nle, wherein to can be N-methylated for Xaa.

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa is selected from I, V, P, G, H, W, E.

<400>59

Xaa?Xaa?Xaa?Xaa?Xaa?Pro

1 5

<210>60

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa is selected from S, R, K, H, O, T, I, L, V, A, and G.

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa is selected from K, H, and R, S, T, O, or the N end is substituted with the compound based on glycine of basic functionality.

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa is selected from A, I, and L, V, K, P, G, H, R, S, O, Dab, Dpr, Cit, Hci, Abu,

Nva, Nle, wherein to can be N-methylated for Xaa.

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa is selected from I, V, P, G, H, W, E.

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa is selected from A, V, I, L, G, K, H, R, O, S, T, and F.

<400>60

Xaa?Xaa?Xaa?Xaa?Pro?Xaa

1 5

<210>61

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>61

Arg?Ile?Val?Pro?Ala?Cys

1 5

<210>62

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa equals r.

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa equals r.

<400>62

Xaa?Xaa?Val?Pro

1

<210>63

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa equals A-NHOH.

<400>63

Arg?Ile?Val?Pro?Xaa

1 5

<210>64

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>64

Arg?Ile?Val?Pro?Pro?Ala

1 5

<210>65

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>65

Arg?Ile?Gly?Pro?Ala

1 5

<210>66

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa equals Pip.

<400>66

Arg?Ile?Val?Xaa?Ala

1 5

<210>67

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa equals Thz.

<400>67

Arg?Ile?Val?Xaa?Ala

1 5

<210>68

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa equals Fpro.

<400>68

Arg?Ile?Val?Xaa?Ala

1 5

<210>69

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa equals Dhp.

<400>69

Arg?Ile?Val?Xaa?Ala

1 5

<210>70

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>70

Arg?Ile?His?Pro?Ala

1 5

<210>71

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>71

Arg?Ile?Trp?Pro?Ala

1 5

<210>72

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>72

Arg?Ile?Val?Pro?Trp

1 5

<210>73

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>73

Ser?Pro?Val?Ile?Arg?His

1 5

<210>74

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>74

Cys?Pro?Val?Ile?Arg?His

1 5

<210>75

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>75

Arg?Ile?Glu?Pro?Ala

1 5

<210>76

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>76

Arg?Ile?Val?Pro?Glu

1 5

<210>77

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>77

Arg?Ile?Val?Pro?His

1 5

<210>78

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>78

Arg?Ser?Val?Pro?Ala

1 5

<210>79

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>79

Glu?Arg?Ile?Val?Pro?Ala?Gly

1 5

<210>80

<211>5

<212>PRT

<213>Ariticial

<400>80

Lys?Val?Ile?Pro?Ser

1 5

<210>81

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>81

Lys?Val?Val?Pro?Ser

1 5

<210>82

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>82

Lys?Pro?Arg?Pro

1

<210>83

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>83

Arg?Ile?Pro

1

<210>84

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>84

Orn?Val?Pro

1

<210>85

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>85

Ser?Val?Pro

1

<210>86

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>86

Lys?Val?Pro

1

<210>87

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>87

Arg?Arg?Pro

1

<210>88

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>88

Gly?Val?Pro

1

<210>89

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<400>89

Lys?His?Pro

1

<210>90

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immune peptide

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa equals the Tyr. of D-amino acid form

<400>90

Arg?Ile?Val?Pro?Ala?Xaa

1 5

Claims (45)

1. the isolating peptide of forming by any one the aminoacid sequence of SEQ.ID.NO.1-90 basically, or its analogue, derivative or functional variant, or its obvious chemical equivalent, or its pharmacy acceptable salt.

2. the described isolating peptide of claim 1, it has C-end of modification and/or the N-end of modifying.

3. the described isolating peptide of claim 2, it has C-end of modification.

4. the described isolating peptide of claim 3, it has amidated C-end.

5. the described isolating peptide of claim 1, it comprises: the aminoacid sequence of the SEQ.ID.NO.1-90 that is modified by at least one D type aminoacid replacement.

6. material, itself and the described peptide of claim 1 react.

7. the described material of claim 6, it is an antibody.

8. the described material of claim 7, it is a monoclonal antibody.

9. isolated nucleic acid molecule, the described peptide of its coding claim 1.

10. recombinant nucleic acid construct, it comprises: be operably connected to the described nucleic acid molecule of claim 9 on the expression vector.

11. at least a host cell, it comprises: the described recombinant nucleic acid construct of claim 10.

12. one kind prepares the peptide that comprises the listed aminoacid sequence of SEQ.ID.NO.1-90, or the method for its analogue, derivative or variant, described method comprises step:

(a) under the condition that allows this peptide to express, cultivate the described at least a host cell of claim 11; With

(b) reclaim this peptide from this at least a host cell or its substratum.

13. a pharmaceutical composition, it comprises: the described peptide of claim 1 and pharmaceutically acceptable carrier, thinner or vehicle.

14. the described pharmaceutical composition of claim 13, it further comprises microbiotic.

15. treat and/or prevent the method that the experimenter infects, it comprises: will comprise the peptide of the listed aminoacid sequence of SEQ.ID.NO 1-90, or its analogue, derivative, variant or tangible chemical equivalent are applied to the experimenter.

16. the described method of claim 15, wherein said peptide have the aminoacid sequence of SEQ ID NO:1,3-16,18-90.

17. claim 15 or 16 described methods, wherein said peptide is regulated experimenter's congenital immunity, infects thereby treat and/or prevent the experimenter.

18. claim 15,16 or 17 described methods, wherein said infection is an infected by microbes.

19. the described method of claim 18, wherein said infection is selected from infectation of bacteria, fungi infestation, parasitic infection and virus infection.

20. the described method of claim 19, wherein said bacterium are Gram-positive or Gram-negative bacteria.

21. the described method of claim 20, wherein said bacterium is selected from the faecalis of intestinal bacteria, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella, streptococcus aureus, suis and drug resistance of vancomycin.

22. the described method of claim 19, wherein said fungi are selected from mould, yeast and high-grade fungi more.

23. the described method of claim 19, wherein said parasite are unicellular or cellulous.

24. the described method of claim 23, wherein said parasite are selected from giardia duodenalis, Cryptosporidium parvum, Cayenne ring sporozoite and toxoplasma gondii.

25. the described method of claim 19, wherein said virus be selected from following disease-related: AIDS, bird flu, varicella, cold sore, Common Cold, gastroenteritis, gland fever, influenza, lower respiratory infection, measles, parotitis, pharyngitis, pneumonia, rubella, severe acute respiratory syndrome and upper respiratory tract infection.

26. the described method of claim 25, wherein said virus are respiratory syncytial virus (RSV).

27. claim 15 or 16 described methods, wherein said experimenter is infected or is had the risk of infection.

28. the described method of claim 27, wherein said peptide are with oral administration, parenteral administration, transdermal administration, intranasal administration, topical, respiratory tract administration or the administration of osmotic pump mode.

29. predict that whether the experimenter has the method for replying to the treatment of infected by microbes or immune correlated disease for one kind, wherein said treatment comprises: the peptide that will comprise the aminoacid sequence of SEQ.ID.NO1-90, or its analogue, derivative or variant are applied to the experimenter, this method comprises: in the presence of the DPPIV substrate, under the condition that allows DPPIV and substrate reactions, by peptide of the present invention is applied to sample, detect the DPPIV activity of experimenter's diagnostic sample, wherein than the control group that does not have peptide, the active minimizing of described DPPIV shows that the experimenter replys treatment.

30. isolating peptide, it comprises: the aminoacid sequence that is selected from SEQ.ID.NO.1-90.

31. the described isolating peptide of claim 30, wherein said aminoacid sequence is selected from SEQ.ID.NO.1,3-16,18-90.

32. the described isolating peptide of claim 31, wherein said aminoacid sequence is selected from SEQ.ID.NO.1,3-16,18-54,61-90.

33. the described isolating peptide of any one of claim 30 or 31, it comprises immunocompetence.

34. the described isolating peptide of claim 33, wherein said aminoacid sequence is selected from SEQ.ID.NO.1,3-16,18-43,45-53 and 61-90.

35. reach 10 amino acid whose isolating peptides most, it comprises the described peptide of claim 34.

36. reach 7 amino acid whose isolating peptides most, it comprises the described peptide of claim 33.

37. the purposes of the described isolating peptide of any one of claim 31 to 36 in treatment DPPIV relative disease.

38. the purposes of the described peptide of any one of claim 30 to 37 in treatment infection or innate immunity relative disease or inflammation related disease.

39. the described isolating peptide of any one of claim 1-5 or 30-36, it has compares about 75% or DPPIV activity still less with saline control.

40. any one of claim 1-5 or 30-36 be described to be had and compares about 75% or the active isolating peptide of DPPIV still less with saline control, treatment infect or the innate immunity relative disease in purposes.

41. described method of any one of claim 15-26 or claim 37, any one described purposes of 38 or 40, it further comprises antibiotic administration in the experimenter.

42. the described method of claim 41, wherein said microbiotic is used with described peptide.

43. the described method of claim 42, wherein said microbiotic is used after described peptide administration.

44. described method of any one of claim 15-26 or 41-43 or claim 37, any one described purposes of 38 or 40, in case wherein said peptide is that metabolism is that the propetide of peptide of SEQ.ID.NO.1-90 or the form of material are applied to the experimenter with administration.

45. the described method of claim 44, wherein said propetide or material are fusion roteins, and it comprises claim 1-5 or described peptide of 30-36 and drug administration carrier at least.

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