CN1957085A - Therapeutic peptides - Google Patents

Abstract

The present invention provides a ligand isolated from Moraxella catarrhalis outer membrane protein which binds to CEACAM receptors, said ligand comprising a receptor binding domain comprising an amino acid sequence selected from the group disclosed, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. The invention also provides a medicaments and vaccines comprising said ligand, and their use in the treatment or prophylaxis of infection. Also provides ligand, and their use in the treatment or prophylaxis of infection. Also provided is a screening method for the identification of novel therapeutic compounds.

Description

Therapeutic peptide

The present invention relates to therapeutic peptide, relate in particular to the other treatment thing that is applicable to preparation vaccine or infection and screen the therapeutic peptide that in the treatment of infecting, has the active compound of potential drug.

Inventor of the present invention found in the past, the cell adhesion molecule relevant with carcinomebryonic antigen (CEACAM) is the mucosal disease substance, respiratory pathogen particularly is as the acceptor of Neisseria meningitidis (Neisseria meningitidis), hemophilus influenzae (Haemophilusinfluenzae) and morazella catarrhalis (Moraxella catarrhalis).CEACAM belongs to carcinomebryonic antigen (CEA) family, and this family is a member of immunoglobulin superfamily.The CEA gene family comprise the subfamily (CEA) of surface expression and secretor type subfamily (glycoprotein that gestation is special, PSG).The subfamily relevant with film is defined as CEACAM (with CEA relevant cell adhesion molecule) again ²⁰, this subfamily comprises many relevant glycoprotein, it is the most extensive that wherein CEACAM1 expresses ground in different tissues ¹²Main Chinese hamster ovary (CHO) cell that used CEACAM1 (being named as CD66a and BGPc in the past) transfection in these researchs of inventor of the present invention report, CEACAM1 comprise the tenuigenin tail (expression formula of its molecule is NA1BA2-TM-S or L) of four extracellular domains, TM district and a weak point (S) or length (L).In addition, also used the construct of the solubility brachymemma that comprises one or more extracellular domain.In the past studies show that Neisseria meningitidis and hemophilus influenzae are target with the N end structure territory of number of C EACAM mainly ^7,9,10This target location can cause the invasion and attack of adhering to of pair cell surface and pair cell ⁹In addition, bacterium can be in conjunction with on the phagocytic cell and the CEACAM on T and the bone-marrow-derived lymphocyte.This interaction can cause bacterial cell death ⁸, target cell death or immunologic function inhibition, for example at Nai Seshi micrococcus gonococcus (N.gonorrhoeae) (very relevant) with Neisseria meningitidis when CEACAM on T and the bone-marrow-derived lymphocyte combines, cause the inhibition of the immunologic function of T and bone-marrow-derived lymphocyte ^21,22

Since CEACAM be considered to for a long time with Neisseria in the Opa protein binding relevant with the adventitia impermeability, and hemophilus influenzae and morazella catarrhalis do not produce Opa albumen, are an accident so find to exist the CEACAM binding partner in hemophilus influenzae and morazella catarrhalis for inventor of the present invention.In the following description of this invention, the infection of mucous membrane will be particularly related to, the particularly infection of respiratory tract film, or the infection of ear (particularly otitis media), but it should be understood that relating to other site infections of CEACAM acceptor, for example the infection in sexual organ mucous membrane or urethra or other its receptor, perhaps the infection at other positions of the mankind that may scatter bacterium from mucomembranous surface, the present invention has same effectiveness.

Mucosal disease substance Neisseria meningitidis (Nm), hemophilus influenzae (Hi) and morazella catarrhalis (Mx) all are human special biologies, and are present in the upper respiratory tract, and they can scatter therefrom, cause serious infection.May carry the meningococcus bacterial strain of different serogroupss at the most in the nasopharynx of 25% healthy individual ¹But in many individualities, this biological attack mucosal barrier causes one of with the fastest developing speed and very serious disease.The increase host also imperfectly understands for the definite factor of meningococcal infection susceptibility.And; the non-immunogenic of limited protection that the special vaccine of colony provides and B group polysaccharide becomes need carry out fundamental research; with the susceptibility of understanding the host and the basis of differentiating feature under the outstanding tunicle, feature can be used as antimeningococcic common target under this tunicle.The research that inventor of the present invention carries out provides molecular basis, meningococcus that meningococcus is built the group to build the group and the understanding of human barrier cell (epithelial cell and endotheliocyte) and phagocytic cell interactive property.For some years recently, the mucous membrane of always studying the symbiosis eisseria is built group's basis, with understand a large amount of harmless groups of difference and accidental occur but the serious pathogenic agent feature of Neisseria meningitidis for example.In addition, the carrier whether the symbiosis Neisseria can be used as the possible vaccine antigen of Neisseria meningitidis has been measured in these researchs.

75% healthy individual may be carried the bacterial strain that belongs to the hemophilus influenzae species at the most ²Although as the result of Hib vaccine, at the sickness rate of west b type disease significant reduction has been arranged, the disease that is caused by the Hi bacterial strain (NTHi) of somatotype not remains a subject matter.NTHi causes the infection of part and diffusion, comprises epiglottitis, otitis media, cellulitis, pneumonia, endocarditis, microbemia and meningitis.Otitis media is one of paediatric subject matter, and the reason of the NTHi morbidity more than 20% in life 1 year that is children ^2,3NTHi also infects relevant with acute relapse with chronic obstructive pneumonia (COPD) and cystic fibrosis patient and persistence.Be what has determined the recurrent infection that caused by NTHi among these patients, perhaps otitis media is repeatedly shown effect among the children, and this is not clear.

Morazella catarrhalis is the another kind of bacterial parasite in the human airway, isolates from the local infection case with Hi usually.These two kinds of biologies are all relevant with the deterioration of sinusitis paranasal sinusitis and asthma ^5,6Mx causes the third most commonly encountered diseases of children's otitis media because of (being annual 300 to 4,000,000 cases according to estimates).It also causes the lower respiratory infection among the grownup, particularly in the patient who suffers from COPD ⁵Under rare situation, it infects relevant with diffustivity ⁵Hi and Mx cause persistent infection, and think that they can escape from host's immunologic mechanism and antibiotic effect by the tissue infiltration ⁴After deliberation the sticking property of many outer membrane proteins of Mx.But almost do not find the cell receptor of Mx, and the details of many pathogenicity bo mechanism require study ^4,5,6

A basic need of respiratory mucosa pathogenic agent is to set up firm getting in touch with airway epithelial cell.The target of these human pathogens is human special molecules, and research must have the tissue and the organ culture of vitro culture.Adhere to usually and mediate by bacterium phase and the variable structure of antigenicity.In addition, known clearly further that the adhesion of pathogenic agent is many-sided, and environmental adaptation plays great function for adherent mode.Although many nearest researchs have begun to determine the different steps of complicated cell-targeting mechanism, the details or the interchange between host and the microorganism of environmental adaptation is still waiting description.

Inventor of the present invention carries out recently studies show that Nm ^7-9And Hi ^10,11, have the mechanism of some uniquenesses and other total mechanism, be used for the CEACAM of some human cell's surface receptor of target ¹²

And the clinical separation strain of inventor's recent findings morazella catarrhalis of the present invention is a target with the mankind's CEACAM molecule also.In addition, the high molecular outer membrane protein that has been found that a kind of Moraxella now combines with this receptor.Proved that CEACAM is at different tissues ¹², be included in the expression in the airway epithelial cell ²⁴These find that prompting is a special favourable condition to the special target of CEACAM molecule for the respiratory tract bacterium, and it may be that result owing to convergent evolution occurs.

Pili (pilus) is arranged in the adhesion factor of Neisseria meningitidis ^13,14,16And adventitia does not see through (opacity) albumen, Opa and Opc ^15,16The Nm pili is long silk-like proteins structure, is made up of a plurality of pilin subunits.It is generally acknowledged that they are that the most important adhesin of tunicle bacterium is arranged ^13,14,16, reason is the some or all of adventitia part of having covered of tunicle, cause the function of part to render a service reduction, and pili passes tunicle in complete tunicle bacterium, still has function.Opa belongs to the variable protein family of antigenicity, all exists in Neisseria meningitidis and neisseria gonorrhoeae.In meningococcus, have 3-4 opa locus coding relevant, have 4 transmembrane proteins that are exposed to the ring on surface, wherein 3 rings have sequence variations ^16,17Another transmembrane protein Opc is not variation basically ^15,16In in the past 12 years, inventor of the present invention studies the proteic virulence of structure-function relationship, Opa and Opc of Nm pili, has found that neisseria does not see through proteic two human receptors.And the inventor studies with the effect of other factors in cytotoxicity the function and the LPS of surperficial sialic acid in bacterium and human neuron target cell interaction.

Present inventors have identified isolated high molecular part in conjunction with the CEACAM acceptor from the Moraxella outer membrane protein, thereby cause the present invention.

The character of part can be described by its migration model in SDS-PAGE, and its SDS-PAGE migration model shows the characteristics of USP family protein, and promptly part is split into the monomer of molecular weight between about 60 to 150kD after boiling for a long time.

Part in Mx strains A TCC 25238 (MX2) is accredited as UspA1, and has measured its aminoacid sequence.The characteristics of this part are further studied, to determine the zone or the structural domain of receptors bind, the i.e. peptide of bind receptor or the character relevant with peptide.

Therefore, the invention provides that from the morazella catarrhalis outer membrane protein, separate and the part CEACAM receptors bind, wherein said part is a polypeptide, this polypeptide comprises the receptors bind structural domain or is made up of the receptors bind structural domain, the this receptor binding domains comprises and is selected from following aminoacid sequence or forms by being selected from following aminoacid sequence: the 463rd to 863 residue of sequence shown in Figure 6,527 to 623 residues, 527 to 668 residues, 527 to 863 residues, 427 to 623 residues, 427 to 668 residues and 427 to 863 residues, perhaps its fragment, homologue, function equivalent, derivative, degeneracy thing (degenerate) or hydroxylation, sulfonation or glycation product, perhaps other secondary processed products.

In a preferred embodiment, part is a peptide species, this polypeptide comprises and is selected from following aminoacid sequence or forms by being selected from following aminoacid sequence: the 527th to 623 residue of sequence shown in Figure 6,527 to 668 residues and 427 to 623 residues, perhaps its fragment, homologue, function equivalent, derivative, degeneracy thing or hydroxylation, sulfonation or glycation product, perhaps other secondary processed products.

Use part one speech here, both be meant and the whole molecule of receptors bind, be meant that again any part of the receptors bind structural domain that comprises part of above-mentioned molecule makes it keep receptors bind character.Therefore " part " comprised the molecule of only being made up of the receptors bind structural domain, i.e. one or several required peptide zones of receptors bind.

In another preferred embodiment, described polypeptide comprises the conserved sequence in the 427th to the 623 residue zone of at least a sequence shown in Figure 6, or form by the conserved sequence in the 427th to the 623 residue zone of at least a sequence shown in Figure 6, it can be identified in sequence contrast shown in Figure 27.Therefore, in this embodiment, this polypeptide comprises at least a of following aminoacid sequence or is made up of at least a of following aminoacid sequence:

QHSSDIKTLK，

NVEEGLLDLSGRLIDQKADLTKDIK，

NVEEGLLDLSGRLIDQKADIAKNQA，

DIAQNQT，

DIQDLAAYNELQD，

QTEAIDALNKASS，

TAELGIAENKKDAQIAKAQANENKDGIAK，

NQADIQLHDKKITNLGILHSMVARAVGNNTQGVATNKADIAK，

NQADIANNIKNIYELA，

NQADIANNI，

NIYELA。

To be appreciated that, polypeptide ligand of the present invention can comprise the receptors bind structural domain of the sequence of enumerating here, its by in the sequence of enumerating here N-terminal, C-terminal or simultaneously N-terminal and C-terminal add amino-acid residue or from the sequence of N end enumerated here, C-terminal or simultaneously N-terminal and the C-terminal deletion amino-acid residue modify, these adorned polypeptide keep the ability in conjunction with the CEACAM acceptor.Therefore, the present invention further provides the part that comprises polypeptide or form by polypeptide, in this polypeptide 50,40,30,20,10,5,3 or 1 amino-acid residues are added to the N-terminal of the aminoacid sequence of enumerating here, C-terminal, perhaps in while N-terminal and the C-terminal, perhaps from the N-terminal of the aminoacid sequence enumerated here, C-terminal, perhaps N-terminal and C-terminal have deleted 50 simultaneously, 40,30,20,10,5,3 or 1 amino-acid residues, said here adorned polypeptide keep in conjunction with the ability of CEACAM acceptor and/or cause immunoreactive ability at non-modified peptides.Preferably, the amino acid of 560 positions is retained in the peptide of modification.

About the fragment of polypeptide of the present invention, if fragment keeps the ability in conjunction with the CEACAM acceptor, the fragment of any size can be used among the present invention.It can be desirable separating the minimum peptide that only comprises those receptors bind desired zones.

Can be by at N-terminal, C-terminal or simultaneously in N-terminal and C-terminal brachymemma according to polypeptide ligand of the present invention, and obtain from known morazella catarrhalis UspA1 albumen.Therefore, the present invention further provides from N-terminal and lack at least (perhaps just) 20,30,40,50,60,70,80,100,120,140,160 520 amino acid whose wild-type UspA1 sequences by the time, and/or lack at least (perhaps just) 20,30,40,50,60,70,80,100,120,140,160,180 or 200 amino acid whose wild-type UspA1 sequences from C-terminal.Preferably, this truncate keeps the CBACAM combined function.Possible truncate can be selected from down those shown in the tabulation, and all these within the scope of the invention.

Table I. the possible brachymemma combination of wild-type UspA1 protein N terminal and C-terminal

The amino acid number that lacks, at least or just:
															From N-terminal	From C-terminal
0	X	20	30	40	50	60	70	80	100	120	140	160	180	200
															20	0	20	30	40	50	60	70	80	100	120	140	160	180	200
30	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															40	0	20	30	40	50	60	70	80	100	120	140	160	180	200
50	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															60	0	20	30	40	50	60	70	80	100	120	140	160	180	200
70	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															80	0	20	30	40	50	60	70	80	100	120	140	160	180	200
100	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															120	0	20	30	40	50	60	70	80	100	120	140	160	180	200
140	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															160	0	20	30	40	50	60	70	80	100	120	140	160	180	200
180	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															200	0	20	30	40	50	60	70	80	100	120	140	160	180	200
220	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															240	0	20	30	40	50	60	70	80	100	120	140	160	180	200
260	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															280	0	20	30	40	50	60	70	80	100	120	140	160	180	200
300	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															320	0	20	30	40	50	60	70	80	100	120	140	160	180	200
340	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															360	0	20	30	40	50	60	70	80	100	120	140	160	180	200
380	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															400	0	20	30	40	50	60	70	80	100	120	140	160	180	200
420	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															440	0	20	30	40	50	60	70	80	100	120	140	160	180	200
460	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															480	0	20	30	40	50	60	70	80	100	120	140	160	180	200
500	0	20	30	40	50	60	70	80	100	120	140	160	180	200
															520	0	20	30	40	50	60	70	80	100	120	140	160	180	200

The known wild-type UspA1 sequence of brachymemma is strains A TCC25238 (MX2 in this way; GenBank sequence number AAD43465), P44 (AAN84895), O35E (AAB96359), TTA37 (AAF40122), O12E (AAF40118), O46E (AAF 36416), V1171 (AAD43469), those of TTA24 (AAD43467) (seeing embodiment 10, Table II).

Ideally, the UspA1 truncate of this embodiment comprises and is selected from following aminoacid sequence or forms by being selected from following aminoacid sequence: 463 to 863 residues, 527 to 623 residues, 527 to 668 residues, 527 to 863 residues, 427 to 623 residues, 427 to 668 residues and 427 to 863 residues in the sequence shown in Figure 6, perhaps its fragment, homologue, function equivalent, derivative, degeneracy thing or hydroxylation, sulfonation or glycation product, perhaps other secondary processed products; Perhaps comprise the conserved sequence in 427 to the 623 residue zones at least a sequence shown in Figure 6, or be made up of the conserved sequence in 427 to the 623 residue zones at least a sequence shown in Figure 6, this can identify in sequence shown in Figure 27 contrast, for example:

QHSSDIKTLK，

NVEEGLLDLSGRLIDQKADLTKDIK，

NVEEGLLDLSGRLIDQKADIAKNQA，

DIAQNQT，

DIQDLAAYNELQD，

QTEAIDALNKASS，

TAELGIAENKKDAQIAKAQANENKDGIAK，

NQADIQLHDKKITNLGILHSMVARAVGNNTQGVATNKADIAK，

NQADIANNIKNIYELA，

NQADIANNI，

NIYELA。

Production comprises can be very convenient as the fusion rotein of polypeptide ligand disclosed herein.Therefore, in further embodiment, the invention provides the fusion rotein that comprises according to polypeptide ligand of the present invention.Preferably, the full length sequence according to the fusion rotein of this embodiment is identical less than 50% on its whole length with any known full length sequence.

The homologous polypeptide can relatively be differentiated by sequence.Homologous polypeptide full length sequence and peptide sequence disclosed herein or their fragment are preferably at least 60% identical on full length sequence, and more preferably at least 70%, 80%, 90%, 95% or 99% is identical, and identical many more preferred more.Preferably, the homologous polypeptide keeps in conjunction with the ability of CEACAM acceptor and/or causes at peptide sequence disclosed herein or their segmental immunoreactive ability.Preferably, 560 positions or be Methionin with the amino acid of source position.

Figure 20 shows the contrast of the peptide sequence of different sources, shows to be modified the sequence area of reservation function (being the CEACAM binding ability) simultaneously.

The inventor after measured, the CEACAM binding ability of peptide part is relevant with the conformation based on alpha-helix by circular dichroism (CD) spectroscopy detection, just in time with the random coil inverted configuration.Therefore, in preferred embodiments, peptide part of the present invention or receptors bind structural domain or their fragment or homologue or other derivatives adopt α-Luo Xuanjiegou.Selectively, this structure is a kind of coiled coil structure.Preferably, CD spectroscopy detects according to such operation of describing among the embodiment that encloses.

In further embodiment, the invention provides structurally and peptide disclosed herein or their fragment homologous peptide.The homologous peptide is a homeopeptide as described above on the structure, and its produces circular dichroism (CD) the spectroscopy collection of illustrative plates of expression based on the conformation of alpha-helix, as shown in Figure 18.Peptide disclosed herein or their segmental Mimotopes also are conceived to out.Mimotopes can comprise D-amino acid or alpha-non-natural amino acid substitutes, but still keeps the functional character of peptide disclosed herein, comprises the CD collection of illustrative plates of expression based on the conformation of alpha-helix.

The conformation based on alpha-helix that the inventor discloses shows that peptide disclosed herein has spherical subunit structure, and this spherical subunit structure does not rely on the film that is associated and obtains and the suitable conformation of CEACAM bonded.Therefore, in embodiment further, the invention provides a kind of spherical subunit molecule that comprises peptide part of the present invention or receptors bind structural domain, it is not the UspA1 albumen of total length, and it can not need adventitia to exist in conjunction with the CEACAM acceptor.Preferred this spherical subunit molecule comprises and is less than 200 amino acid, more preferably less than 100 amino acid.

Owing to there is hybrid protein in Mx, they may contain from UspA1 and two proteic chimeric epi-positions of UspA2 ²³So receptors bind structural domain of equal value on structure and/or the function also can occur in other Usp A sample albumen.The part that comprises this receptor domain of equal value also belongs to category of the present invention.

The CEACAM binding characteristic of the peptide part meaning is meant that it is existing as antigenic effectiveness (promptly in vaccine), and the effectiveness as " microbiotic " is also arranged, and it is used to block the CEACAM combination for this reason, thereby prevents the combination of pathogenic agent and enter.

Therefore, part or receptors bind structural domain preferably are suitable for prevention or the treatment infected.

The nucleotide sequence that the present invention also provides code book invention ligandin with and homologue, fragment, polymorphic thing (polymorphism), degeneracy thing and shear variant.

Part of the present invention or its make up, and can be used to other prophylactic treatment things of vaccine or infection.

Vaccine or other infection mitigation therapeutant can comprise any known adjuvant, vehicle, vehicle, tackiness agent, carrier, sanitas etc., so that acceptable part preparation on the medicine to be provided, are used for the treatment of patient.

The present invention also provides acceptable on the medicine to be used for the preparation of the part of medical treatment.

Acceptable part preparation can be used for the treatment of or prevent the disease of any CEACAM of relating to acceptor on the medicine, for example treatment or preventing infection, respiratory tract disease, neoplastic disease and illness and the vasculogenesis relevant with neoplastic disease.

Preferably, when carrying out treatment of infection, infection is the infection of mucous membrane or particularly respiratory tract infection occurs by mucous membrane.

Most preferably, part is used as at the vaccine of Neisseria meningitidis, hemophilus influenzae and morazella catarrhalis or is used for other preventions or treatment to these bacteriums.Ideally, part is used as at the vaccine of otitis media or other preventions or the treatment of otitis media.

Aspect further, part of the present invention can also be used to differentiate novel retarding agent, and this retarding agent can be used as healing potion, avoids the infringement of many mucosal disease substances with protection susceptible colony and general public.For example part can be used to differentiate receptor analogs, and these analogues can be used for top these purposes.

Therefore the present invention also provides a kind of shaker test, be used to differentiate novel retarding agent as healing potion, this test comprises the step of screening potential healing potion, and this screening is the homology according to part among the ability of part among potential healing potion simulation the present invention or itself and the present invention.The present invention further provides healing potion, they are to differentiate out by above-mentioned shaker test.

Effectively vaccine component can the definite receptor target scheme information of the application of the invention prepare, and bioactive peptide mimics is for example arranged.These components can prevent that bacterium from forming bacterium colony or invasion and attack on mucous membrane, and induce have retardance, the antibody of conditioning and germicidal action.

Because the CEACAM molecule is relevant with processes such as cancer and growths, so the biological activity peptide sequence of being differentiated by part of the present invention derived from bacterium can be used for studying in cancer and the effect of growing CEACAM.These peptide sequences can also be used for controlling or the treatment vasculogenesis as the potential anticancer agent.

The information about the conformation of peptide part of the present invention that the inventor produces can be used to design the synthetic nanostructure for example from plastics.Such structure will have the antibiont degraded and be the advantage of non-immunogenic.It will be especially suitable for use as by retardance CEACAM acceptor and work to prevent the combination of pathogenic agent and " microbiotic " that enter like this.

In another embodiment, the invention provides in conjunction with the part of CEACAM acceptor preparation be used for the treatment of or prophylactic medicine in purposes, the CEACAM acceptor has participated in causing the cell-targeting of the pathogenic agent of this disease in the described disease, in this medicine, part comprises and is selected from following aminoacid sequence, or form: 463 to 863 residues in the sequence shown in Figure 6 by being selected from following aminoacid sequence, 527 to 623 residues, 527 to 668 residues, 527 to 863 residues, 427 to 623 residues, 427 to 668 residues and 427 to 863 residues, perhaps its fragment, homologue, function equivalent, derivative, degeneracy thing or hydroxylation, sulfonation or glycation product, perhaps other secondary processed products.

Other parts that are suitable for this respect of the present invention are polypeptide, this polypeptide comprises the conserved sequence in 427 to the 623 residue zones at least a sequence shown in Figure 6, or form by the conserved sequence in 427 to the 623 residue zones at least a sequence shown in Figure 6, this can determine in sequence shown in Figure 27 contrast, for example:

QHSSDIKTLK，

NVEEGLLDLSGRLIDQKADLTKDIK，

NVEEGLLDLSGRLIDQKADIAKNQA，

DIAQNQT，

DIQDLAAYNELQD，

QTEAIDALNKASS，

TAELGIAENKKDAQIAKAQANENKDGIAK，

NQADIQLHDKKITNLGILHSMVARAVGNNTQGVATNKADIAK，

NQADIANNIKNIYELA，

NQADIANNI，

NIYELA。

Preferably, disease is selected from the group of being made up of following disease: infection, respiratory tract disease, neoplastic disease and illness and the vasculogenesis relevant with neoplastic disease.

Pass through mucosal infections when pathogenic agent, when perhaps invading, above-described medicine has special effectiveness.

Medicine described herein is specially adapted to the infection (disease) treating or prevent to be caused by morazella catarrhalis.Yet part described herein is applicable to that preparation is used for the treatment of the medicine of any disease that wherein relates to the CEACAM acceptor, and this disease is the disease for being caused by Neisseria meningitidis and hemophilus influenzae for example.

In particularly preferred embodiments, described disease is an otitis media.

Part of the present invention also can be used to treat the disease that is caused by other oral cavity bacterium, for example carious tooth.

Oral cavity bacterium Fusobacterium nucleatum (Fusobacterium nucleatum) has gingival disease relevant, and with otitis media, stillbirth (still birth) with under rare cases, interrelate with microbemia.The inventor's nearest work shows, the isolate of multiple Fusobacterium nucleatum combines with CEACAMs and CEACAM1 can be suppressed by polypeptide ligand disclosed herein with combining of Fusobacterium nucleatum, and this points out part of the present invention or receptors bind structural domain to have effectiveness in treatment or in the disease of preventing to be caused by this pathogenic agent.

The effect (embodiment 7) of D-7 (according to preferred polypeptide part of the present invention) non-tunicle bacterium (not shown) of retardance and tunicle bacterium and HeLa-CC1H bonded ability (embodiment 7), retardance and multiple CEACAM bonded ability and its anti-many mucous membrane opportunistic pathogen species makes it become a kind of biocide with remarkable potential.In addition, its ability (embodiment 8) of causing retardance Mx adherent antibody response point out it as preventing otitis media or the vaccine candidate object that wherein is usually directed to the pulmonary infection of Mx (separately or as the potential of multicomponent vaccine (with other Mx antigen for example: UspA2, Hag/MID, OMPCD, Mcap) a part) ⁵Vaccine based on adhesin has been successfully used to for example inoculate anti-uropathogen intestinal bacteria (Escherichia coli) by system in mouse urocystitis model ²⁹

Obtaining therein has the risk height of bacterial strain virulence or antibiotics resistant especially and can consider to comprise that part according to the present invention is as preventive medicine by direct topical application or under the multiple situation that probiotic bacterium is sent.For the bacterium that adheres to target tissue that interacts by carbohydrate-lectin, soluble-carbohydrate has successfully prevented infection in external and animal model ^30,31,32Topical application shows the combination that suppresses streptococcus mutans among the people experimenter corresponding to the synthetic peptide of Streptococcus mutans (Streptococcusmutans) Protein S AI/II.Study and use 1mg.ml in the collutory every day ^-12 weeks of peptide, and this is enough to prevent to build the group ³³The probiotic bacterium of lactobacillus shape has been used to prevent many infection ^34,35In addition, recombinant escherichia coli strain has been used as probiotic bacterium, and wherein the LPS gene is modified the structural simulation thing with coding shiga toxin acceptor.Oral this bacterial strain demonstration can prevent with the deadly dead mouse that causes of attacking of the intestinal bacteria that produce shiga toxin ³⁶The interference peptide of topical application has further advantage, because when needs, they can be sent by using temporary transient probiotic bacterium or expression vector, and this expression vector can be controlled according to timing or expression level.The time span of therefore can control exposure giving biocide ^37,38

The interference simulation of the binding domains by receptor targeted bacterial adhesion and can not have more deleterious effect than the combination of natural symbiotic bacterium part.In addition, such specificity strategy guaranteed to wherein seldom with the tolerance of other symbiotic microorganism fauna of CEACAM bonded ^7,10,38In addition, the monomer of the peptide of the form of preponderating and unit price character is more impossible to excite undesirable signal, and this signal for CEACAMs, is seemingly induced when receptor clustering ^11,39

For participate in by further discriminating that CEACAM interacts and the key amino acid modified to reduce its size, guarantee simultaneously in conjunction with and volume lifetime improve D-7, may have leeway.This class modification will comprise mixes non-natural or D-amino acid ³³Resistance to this anti-adhesive/anti-invasion treatment can not take place because any variation of bacterium part might be them itself cause the forfeiture of function and in this case build group/infection.Expection since peptide compete mutant that receptor-specific changes fully appears and will be unlike more frequent usually, because the competition for acceptor may be to carry out between pathogenic agent under natural situation.Therefore, D-7 has antitack agent that is used as the multiple pathogenic agent of opposing and the potential that is used as vaccine candidate object.

By non-restrictive example embodiment of the present invention are described fully now, these embodiment are with reference to following accompanying drawing.

Fig. 1 has shown that (every milliliter of 1 microgram) the soluble receptors construct that has only CEACAM1-Fc (the long rod in white, left side) exists or at CEACAM1 N-structural domain specific antibody YTH71.3 (grey, the long rod of intermediary) when existing, and is fixed on the relative to level of Mx bacterial strain on the nitrocellulose with three.The combination of CD33-Fc can be ignored (black, the long rod on right side) in each case. Bacterial strain 1,2,3:MX2 (ATCC25238), MX3, MX4 (clinical separation strain).Bonded determines to use the method for Dot blot upper strata covering, and the intensity of reaction uses NIH Scion image forming program to carry out photodensitometry quantitatively.

Fig. 2 is presented at the condition of non-dissociating (heating, swimming lane 1) or boils the proteic western blotting of isolated bacterial strain MX2 under the condition of (swimming lane 2) after 10 minutes.Cover CEACAM1-Fc (every milliliter of 1 microgram) above the trace and hatch, use with the anti-people Fc antibody of horseradish peroxidase and the substrate of horseradish peroxidase and detect combining of acceptor.

Fig. 3 has shown the western blotting of the full product of cell lysis of sex change (respectively in swimming lane 1-3) of bacterial strain MX2, MX3, MX4.Cover CEACAM1-Fc (every milliliter of 1 microgram above the trace; A), anti-UspA1 peptide antibody (every milliliter of 10 microgram; B) and anti-UspA2 peptide antibody (every milliliter of 10 microgram; C) hatch.Attention conjugated protein and anti-UspA1 of CEACAM1-Fc in three bacterial strains is conjugated protein to have similar migration model.Remaining not dissociated albumen size is about 250kD (owing to used alkaline phosphatase assay, having higher sensitivity, so be detected here), combines with CEACAM1-Fc.These are only by the faint identification of anti-peptide antibody, and the chances are because the epi-position that comprises in the synthetic peptide does not expose in unmodified albumen fully, and expose gradually along with the sex change of mixture.Anti-UspA2 antibody and apparent molecular weight greater than 200kD after boiling still not the albumen of depolymerization (this is the character that UspA2 albumen is had) combine.

Fig. 4 shows the conjugated protein peptide mass-spectrogram (4a) of using tryptic digestion after electroelution of the CEACAM1 of MX2.The data that are input in the ProFound identification of proteins database are summarized in (4b).Preceding 10 albumen of identifying with and probability numbers be shown in 4c.The candidate that makes number one, here be that a Z score value is 2.34 UspA1 albumen, its details is shown in (4d), has wherein shown number, their positions in protein of the peptide of coupling, the per-cent that this protein is capped and the inventory of the peptide of coupling not here.

Fig. 5 has shown that the UspA1 of MX2 is through the segmental western blotting after the tryptic digestion.A: the contrast trace that uses two anti-(mixtures of anti-people Fc of the goat of in B and C, using and goat antirabbit Ig respectively).B: the trace that covers CEACAM1-Fc and the anti-people Fc of goat.C: cover the rabbit antibody (ETNNHQDQKIDQLGYALKEQGQHFNNR (SEQ ID NO:1)) (referring to Fig. 6) of the anti-UspA1 peptide of affinity purification, and the trace of the immunoglobulin (Ig) of anti-rabbit.*=and have a swimming lane of molecular weight marker--mark in the left side.With the indicated peptide of double-headed arrow and CEACAM1-Fc kickback (B) takes place, also with the antibody generation kickback (C) of anti-UspA1 peptide.Because combining of the peptide (arrow indication) of anti-UspA1 peptide antibody and lowest molecular weight is so the proteic N-199 of UspA1 that this CEACAM binding fragment is accredited as MX2 is to the C-terminal fragment between the K-863 (referring to Fig. 6).

Fig. 6 has shown the proteic aminoacid sequence of the UspA1 of MX2 (SEQ ID NO:2).Indicated with runic and to be used for special peptide at the immune sero-fast UspA1 of rabbit preparation.The CEACAM calmodulin binding domain CaM is included in the proteic C-terminal fragment of MX2 UspA1 of line.

Fig. 7 has shown separating of the trypsin hydrolyzing peptide that reacts with CEACAM.Use 1 milligram every milliliter trypsin treatment morazella catarrhalis bacterial strain MX2, handled 10 minutes at 37 degrees centigrade.The sample of tryptic digestion carries out SDS-PAGE.After the dyeing, electroelution is carried out in the zone of 50kD spend the night.The albumen that electroelution comes out is by freeze-drying, with last second glue in the resuspended back of damping fluid.The part of glue is transferred to and is carried out western blotting on the nitrocellulose filter.On trace, cover the peptide band (Blot) that CEACAM1-Fc determines and CEACAM reacts.

" * ": refer to and sent to the peptide of doing the N-terminal sequential analysis.

Fig. 8 shows the aminoacid sequence (SEQ ID NO:3) of the peptide of UspA1 albumen after tryptic digestion of MX2.Shown 50kD trypsin hydrolyzing peptide (amino acid 463 to 863) is in conjunction with the antiserum(antisera) of CEACAM and anti-UspA1 peptide (amino acid 753 to 780, line).The N-terminal sequence that this molecular weight is approximately the CEACAM binding peptide of 50kD be " ALESNVEEGL" (SEQ ID NO:4), this sequence appears at after No. 462 amino acid of trypsinase cleavage site.

Fig. 9 is a width of cloth synoptic diagram, having shown that amplification produces the position that the uspA1 gene fragment is used to carry out the primer of expression of recombinant proteins.The dna encoding CEACAM1 binding site that amplifies by primer P4 and P7, also design and use other primers (alphabetical A to I) that should the zone.

Figure 10 has shown the sequence (SEQ ID NO:5) of recombinant fragment 4-7.The zone of line is the N-terminal zone with the reactive trypsin hydrolyzing peptide of CEACAM1.The predicted molecular weight of fragment 4-7 approximately is 26kD.The segmental predicted molecular weight that adds histidine mark is about 28kD.The position of the peptide of brachymemma marks (referring to Figure 13) with " T ".

Figure 11 is a width of cloth synoptic diagram, has shown general clone, expression and the purifying strategy of the UspA1 peptide that is used to recombinate, and is represented as the pQE30 system.

Figure 12 is the figure of carrier pQE30.

Figure 13 shown CEACAM1-Fc in the trace cover butter with the combining of recon 4-8 (swimming lane 3), with the combining of 4-T (swimming lane 4) and with combine (swimming lane 5) of 4-7 polypeptide.Swimming lane 1 contains treponema contrast recombinant peptide, and swimming lane 2 contains the split product that inductive not comprises the M15 of 4-8 construct.

The western blotting of Figure 14 shown recombinant peptide and anti-histidine mark antibody (on) and with the reactivity of CEACAM-Fc (descending).Swimming lane 2 to 4 comprises the 6-8 peptide, and swimming lane 1 comprises the 4-8 peptide in contrast.The peptide migration position of prediction is shown in the right side.Two peptides all with anti-histidine mark antibodies.Although but 4-8 combines with CEACAM1-Fc, 6-8 is quite different.

Figure 15 shows that the D-8 polypeptide combines (swimming lane 1) with CEACAM1-Fc, but with the CD33-Fc that compares does not combine (swimming lane 2).The source of this peptide is by itself and anti-histidine mark antibody be confirmed (swimming lane 3) that react.

Figure 16 is a width of cloth synoptic diagram, has shown segmental relative size of reorganization rUspA1 and position.Used the 4-7 of reorganization to be used for the bacterium blockade test---referring to Figure 17.

Figure 17 shows, the CHO-CEACAM1 transfectant is hatched under the condition that does not have peptide (A), perhaps hatch (B) with the control peptide (treponema peptide) of reorganization, perhaps with UspA1 r4-7 peptide (sequence and MX2 bacterial strain corresponding, C and D) hatch together, add the concentration of peptide and bacterium as shown in the figure, the time of hatching is 2 hours.Hatching when finishing, rinse unconjugated bacterium, in conjunction with last bacterium use anti-morazella catarrhalis polyclonal antiserum and with TRITC (TRITC) link coupled two anti-detections.When the concentration of morazella catarrhalis UspA1 peptide of reorganization is every milliliter of 1 microgram, allogenic morazella catarrhalis strain (MX1) has been had significant inhibition, and concentration is combined with significant inhibition for hemophilus influenzae when being every milliliter of 10 microgram.

Figure 18 is the circular dichroism spectrographic figure that shows reorganization D-7 peptide and have the D-7 of K 560 I sudden change.This spectrum shows that D-7 has α-Luo Xuanjiegou, and D-7 (K 560 I) adopts the random coil structure.

Figure 19 shows the linear peptides in the leap D-T zone of a series of debond CEACAM1.What underscore was arranged is K residue corresponding to the K560 of D-7.

Figure 20 is the sequence contrast in D-7 zone of the proteinic aminoacid sequence of UspA1 of ten bacterial strain Mx:TTA24, TTA37, p44, O12E, O35E, O46E, MX2, V1171, MX3 and MX4.Last end line shows most sequences.

Figure 21 is the manual sequence contrast of O35E D-7 and MX2D-7.Last end line shows most sequences.

Figure 22 shows according to MegAlign ^TMThe table of the sequence identity in the Mx isolate D-7 zone that (DNASTAR Inc.) measures.

Figure 23 shows that recombinant peptide D-7 (rather than reorganization 6-8 be the residue E659-K863 of the UspA1 of MX2) suppresses the combining of transfection CHO cell of homology and heterology bacterial strain and expression CEACAM1.At no peptide (the 1st hurdle), control peptide (2 μ g.ml are arranged ^-1The 2nd hurdle) or D-7 (2 μ g.ml are arranged ^-1The 3rd hurdle) under the situation behind the pre-cultured cell, adds bacterium and by removing non-adhesion bacterium cultivating 1 hour after scouring.Use the antiserum(antisera) of anti-different strains then and put together two of rhodamine to resist, detect and cell bonded bacterium.Mx: morazella catarrhalis, Nm. Neisseria meningitidis; Hi: hemophilus influenzae.

Figure 24 shows that D-7 suppresses the interactional a series of figure of bacterium-CC1-Fc.(a) be presented at peptide concentration scope 0.001-0.1 μ g.ml ^-1In, with homology Mx bacterial strain MX2 (grey hurdle) and with the dose-dependent inhibition of heterology bacterial strain MX1 (black hurdle) bonded acceptor.C represents control peptide D-8 Δ.Show mean value n=3, * P＜0.015 is with respect to independent CC1-Fc.(b) reorganization D-7 (black hurdle) rather than D-8 Δ (grey hurdle) suppress homology and a plurality of bacterial strains (shown in top every section) of heterology species and combining of CC1-Fc.The full cellular lysate point sample of the different isolates that will belong to from difference is to soluble cotton and load CC1-Fc separately or at described peptide (each 2 μ g.ml ^-1) exist down.Use NIH Scion Image software, the per-cent inhibiting value when obtaining in the presence of described peptide not exist with respect to them by photodensitometry.Two to three independent experiments of data representation.

Figure 25 shows that D-7 suppresses bacterium and combines with the clone of a series of expression CEACAM.(a) as shown at no peptide or peptide (2 μ g.ml are arranged ^-1) situation under behind the pre-cultured cell, bacterium and the interactional immunofluorescence analysis of transfection CHO cell of expressing CEACAM1.In the presence of D-7, observed inhibition by the bacterial adhesion of rhodamine flagrate.(b) use viable count test quantitative analysis bacterium and CHO-CEACAM1 cell bonded to suppress.As shown, with target individual layer and control peptide (2 μ g.ml ^-1C) or a series of D-7 cultivate in advance together.Shown the mean value (n=3-6, * P＜0.05) of comparing the % inhibition of using D-7 with control peptide.The bacterial strain that uses among a and the b is: (THi, Rd) and Hi-aeg (A3), (c) D-7 suppresses with different CEACAMs bonded Nm (C751D) for Mx (MX1), Nm (C751D), Hi.At no peptide, control peptide (2 μ g.ml are arranged ^-1The grey hurdle) or D-7 (2 μ g.ml ^-1The black hurdle) (HeLa) cell is drawn in the sea of pre-culture expression CEACAM1 (CC1), CEA or CEACAM6 (CC6) under the situation.Use immunofluorescence analysis and, obtain the average quantity of each cell adhesion Nm in all cases by 20 cells of direct census.(d) tunicle and the fimbriate derivative h18.18 with the bacterial strain MC58 that expresses Opa and Opc adhesin infects the hela cell line (HeLa-CC1H) with high-level CEACAM1-expression, and this hela cell line adheres to by pili and the support of Opa albumen.As shown, formerly (pre) or (sim) interpolation D-7 rather than D-8 Δ have simultaneously reduced cell adhesion.(e) as shown, at no peptide,, control peptide (2 μ g.ml are arranged ^-1) or D-7 (2 μ g.ml ^-1) situation under behind the pre-cultured cell, the interactional inhibition of A549 pulmonary epithelial cells of bacterium and known expression CEACAMs.The bacterial strain that uses be Mx (MX1), Nm (C751D) and Hi (Hi-aeg, A3).

Figure 26 is that the rabbit antibody that shows the affinity purification of anti-D-7 had both suppressed homology Mx and combines with CEACaM1-Fc (CC1-Fc), suppresses heterology Mx and CEACaM1-Fc (CC1-Fc) bonded figure again.In the experiment of Dot blot coverture, use NIH Scion Image software to measure the combination that contrasts with respect to no antibody by photodensitometry.Mx bacterial strain for test has been observed good inhibition, but comprises Hi (THi Rd ﹠amp for other bacterial species of test; Hi-aeg A3), Nm (C751A ﹠amp; C751D) and Ng (P9-13) then be not like this.Three independently experiments of data representation.

Figure 27 shows Neisseria meningitidis and independent HMEC1 cell (A) or the adhesion in the presence of control peptide (B) or retardance peptide D-7 (C).Notice the almost completely inhibition in the presence of D-7.

Figure 28 is corresponding to the segmental multiple contrast of the segmental known UspA1 protein sequence of MX2 sequence 4-T.The residue that to guard in the sequence of all analyses is drawn black shade.Will be conservative in all sequences that they exist, but corresponding position has the residue of disappearance to draw the Dark grey shade in one or more sequences.When there is variation in the residue that exists in described position, will has residue and draw light grey shade.

Embodiment 1. morazella catarrhalis strains combine with people CEACAM1-Fc by UspA1 albumen

The reference strain (ATCC25238 a, cloned culture of this bacterial strain is named as MX2) that the morazella catarrhalis strain of using in this research (Mx) comprises clinical isolates (MX3 and MX4) and buys from American type culture collection.

The acceptor cover butter shows that Mx combines with CEACAM1-Fc acceptor construct

In order to estimate the interaction of morazella catarrhalis strain and CEACAM1, with bacterium (about 4-8 * 10 ⁶Individual) be added on the nitrocellulose filter, use 3%BSA-PBST to seal non-specific combination site after the dry air.On the nitrocellulose bar, cover CEACAM1-Fc (every milliliter of 1-2 microgram) or add the N end structure domain antibodies YTH71.3 of CEACAM1 simultaneously.CD33-Fc (every milliliter of 1-2 microgram) is as negative control.According to former described preparation chimeric protein construct ¹¹Combination by the anti-people Fc of the goat of coupling horseradish peroxidase or alkaline phosphatase antibody test Chimerical receptor.Chromogenic substrate is used diaminobenzidine and hydrogen peroxide or nitroblue tetrazolium and 5-bromo-4-chloro-3-indoles phosphoric acid respectively.All bacterial strains all combine with CEACAM1-Fc, but do not combine (Fig. 1) with CD33-Fc.The combination of acceptor is suppressed when monoclonal antibody YTH71.3 exists, and this points out this bacterial strain to combine with the N-terminal structural domain of acceptor.Therefore, all bacterial strains all have the combination (not display result) that equates with the N-Fc construct of the brachymemma of the CEACAM1 that only comprises N end structure territory.

The protein-bonded evaluation of CEACAM1-Fc in the morazella catarrhalis

With not through in advance heat treated or at 100 degrees centigrade of full product of cell lysis (about 3 * 10 that boil 10 minutes Mx ⁷Individual cell) joins in each swimming lane of 10%bis-tris polyacrylamide (Invitrogen) gel, 180 volts of electrophoresis 45 minutes.The trace condition of use standard is transferred to the albumen on the gel on the nitrocellulose filter.According to top description, solubility chimeric construct body is covered on the film.All observe single albumen in each case with the CEACAM1-Fc specific combination.The migration of albumen on gel shows the UspA1 albumen of Mx, because the split product of bacterium when last sample if do not have heat denatured then CEACAM1 conjugated protein＞migration of the apparent molecular weight of 200kD, if split product is at first through heating, then CEACAM1 is conjugated protein moves (Fig. 2) with the molecular weight (approximately 92kD) that reduces.

The antibody of anti-UspA1 but not the antibody of anti-albuminoid UspA2 combine with the CEACAM1-Fc of Mx bacterial strain is conjugated protein

The proteic sequences Design peptide of UspA according to the morazella catarrhalis of announcing.Be ETNNRQDQKIDQLGYALKEQGQHFNNR (SEQ ID NO:1; The UspA1 peptide) and KDEHDKLITANKTAIDANKAS (SEQ ID NO:6; The UspA2 peptide).Cysteine residues and KLH coupling that peptide is integrated by N-terminal, the link coupled peptide is used for immunize rabbit (every rabbit 200 microgram peptides), immunity 14 days at interval.Bring into use complete Freund's adjuvant, use incomplete Freund's adjuvant afterwards.The 0th day and the immunity after took a blood sample every 14 days.Use the peptide that suits with AminoLinkPlus post (Pierce) link coupled to come the purifying polyclonal antibody.Use the special antibody of UspA in the western blotting cover butter, concentration is every milliliter of 1-10 microgram, and detection of antibodies uses the goat antirabbit two of coupling alkaline phosphatase anti-.The colour developing of trace as previously mentioned.The conjugated protein migration on SDS-PAGE of the CEACAM1-Fc of three Mx bacterial strains is identical with the migration of UspA1 antibody binding proteins, but different with the migration of UspA2 antibody binding proteins (Fig. 3).

Be accredited as UspA1 with the morazella catarrhalis part of CEACAM1-Fc co-precipitation

The overnight culture of bacterium is resuspended with the PBSB of the octyl group β D glucopyranoside that contains 100mM, wherein contains protease inhibitor cocktail (pic; 1mM PMSF, 1mM E-64,1 μ M pepstatin A, 6nM bestatin and 100 μ M EDTA).Sample is 4 degrees centigrade of constantly mixed spending the night.Simultaneously 100 microlitres and Sepharose CL-4B link coupled albumin A (Sigma) are together hatched with the CEACAM1-Fc or the CD33-Fc (using as contrast) of 20 micrograms, spend the night at 4 degrees centigrade.Use the PBSB rinsing then 3 times, remove any unconjugated acceptor.15000g removed insoluble bacterial components in centrifugal 30 minutes.The extract of solubility is hatched 2 hours with one of two kinds of acceptor-albumin As-Separose mixture at 4 degrees centigrade respectively, and (ratio is 5 * 10 ⁸The acceptor construct of the every microgram of individual bacterium).At the octyl group β D glucopyranoside that uses 50mM and PBSB repeatedly after the rinsing, under the sex change condition with SDS-PAGE electrophoresis and western blot analysis sample.

In testing with the co-precipitation of CEACAM1-Fc, MX4 has produced a strong painted protein band (about 97kD), and MX3 produces a weak relatively painted protein band, about 92kD.Be consistent (in Fig. 3, showing) by the proteic molecular weight of co-precipitation and those observed proteic molecular weight in acceptor covering experiment.Two albumen not with the CD33-Fc co-precipitation.Because by the antibodies of the albumen of co-precipitation and anti-UspA1 peptide, so they further are defined as UspA1 albumen.In addition, MX4 albumen after gel scales off, is analyzed (face as follows) with the MALDI-TOF mass spectrum to it.

Identify the CEACAM1 part by the MALDI-TOF mass spectrum

(a) Western covers sample.The full product of cell lysis of MX2 and MX3 carries out SDS-PAGE in Trench glue.Method with electroelution will be come out with the corresponding protein band wash-out of CEACAM1-Fc binding partner.With sample concentration, add in the swimming lane in second glue, carry out electrophoresis, then suitable albumen after the electrophoresis is carried out the digestion of glue endotrypsin.The peptide that obtains uses (MALDI-TOF) mass spectrum of substance assistant laser desorpted/ionization-flight time to analyze.Fig. 4 a has shown an example of the conjugated protein mass spectrum spectrum of the CEACAM of the MX2 that obtains.In the peptide molecular weight input ProFound Identification of Fusion Protein site that obtains, the result who obtains such as Fig. 4 b are shown in the c.Have the predicted molecular weight of the peptide that produces behind the UspA1 albumen process tryptic digestion of 10 proteic molecular weight and morazella catarrhalis to match at this, this fragment accounts for proteic about 18% (Fig. 4 d) of UspA1.The Z score value of estimation is 2.34, and effectively pointing out this albumen is that (it is exactly to be higher than 95% that the Z score value is higher than 1.65 to UspA1; Http: // 129.85.19.192/profound_bin/webProFound.exe).In addition, through similar analysis, the uncombined high molecular band of another of MX2 is confirmed as UspA2.For the MX3 bacterial strain, CEACAM1 combination and non-binding albumen are defined as UspA1 and UspA2 respectively similarly.

(b) sample of co-precipitation: for MX4, also once be confirmed as UspA1 through the MALDI-TOF mass spectrum in the full classified inquiry with the albumen (as mentioned above) of CEACAM1-Fc co-precipitation, the Z score value is 2.27.12 peptides are mated, covered this proteic 21%.

Therefore originally determine in described reference and clinical strains morazella catarrhalis by high-molecular-weight protein UspA1 target people CEACAM1.

Enzyme to UspA1 and recombinant peptide is cut

(a) UspA1 combines with CEACAM1-Fc through the peptide after the tryptic digestion

Use the trypsin Sigma of every milliliter of 0.1-1 milligram) processing MX2 bacterial suspension (10 ¹⁰Every milliliter in individual cell), hatched 1-4 hour at 37 degrees centigrade.Postdigestive split product dissociates with the SDS-PAGE damping fluid, boils and goes up sample and carry out electrophoresis.After being transferred on the nitrocellulose filter, use the anti-UspA1 peptide antibody of acceptor construct CEACAM1-Fc or affinity purification to cover trace.Also combine (Fig. 5) with the small segment of receptor response with the specific antibody of anti-UspA1.

(b) catarrh does not draw the location of the proteic CEACAM binding domains of MX2 bacterial strain UspA1

Use 1 milligram every milliliter trypsin treatment MX2 bacterial suspension, handled 10 minutes for 37 degrees centigrade.

The sample of tryptic digestion carries out the SDS-PAGE gel electrophoresis.After the dyeing, to the 50kD zone electroelution that spends the night.The albumen that the freeze-drying electroelution goes out, resuspended with damping fluid, last second glue.The part of glue is transferred on the nitrocellulose filter, covers by the western blotting that uses CEACAM1-Fc, has determined the peptide band (Fig. 7) with the CEACAM reaction.

Peptide corresponding to the about 50kD of molecular weight and about 150kD carries out the N-terminal order-checking.The sequence of N-terminal is ALESNVEEGL (SEQ ID NO:4) (the approximately peptide of 50kD) and ALESNV (SEQ ID NO:7) (the approximately peptide of 150kD).The albumen of 150kD obviously is the proteic tripolymer of 50kD, because they have identical N-terminal sequence.The N-terminal sequence of this MX2 UspA1 peptide is shown in Fig. 8.

(c) recombinant peptide

The N-terminal reorganization MX2 peptide of being made up of 1-449 amino acid in the sequence shown in Figure 6 that makes up does not combine with CEACAM.Peptide (being made up of the 463-863 amino acid in the sequence shown in Figure 8) behind the tryptic digestion of the about 50kD of this further demonstration has the N-terminal sequence of ALESNVEEGL (SEQ ID NO:4), comprises the CEACAM binding domains.

Embodiment 2. determines the receptors bind structural domain on the multiple virulence determinant of mucosal disease substance

Neisseria meningitidis and hemophilus influenzae by with CEACAM on overlapping site bonded part target people CEACAM molecule.Mx part of the present invention is target N structural domain also.These observationss have shown an infusive possibility, and promptly receptor target may relate to the similar features on many mucosal disease substance parts.

Because the interaction of Nm and Hi and CEACAM is subjected to the constitutional features influence in the variable domains of surface expression, this points out them may comprise similar key amino acid, these amino acid whose sterie configurations make they can with receptors bind, these key amino acids may be guarded in variable protein.Really, two hypermutation rings (HV1 and HV2) of our research and other people research prompting Opa may participate in the CEACAM target ^9,18A strong technology can be determined interactional possibility determinant between part and the acceptor, and this technology is a phage display, and it can be used for determining mimic epitopes (stochastic sequence of simulation binding domains) ¹⁹And fit (aptamer) (with suppressing the more approaching sequence of part bonded original texture) ²⁰

The present invention also makes following situation become very feasible, promptly can combine the stand-in of the mucosal disease substance of CEACAM as other, and the constitutional features of this part can help to determine the required important feature in N end structure territory at the part target CEACAM of Nm and Hi with CEACAM bonded Mx ligand structure territory.The antibody of Mx structural domain may have the potentiality of determining other identical, similar or parts that close positions is regional in can receptor targeted.

Using at present known protein matter engineering method and recombinant DNA technology to carry out the work of determining minimum CEACAM1 binding domains among the MX2 UspA1.Can be by acceptor cover butter described above at the in-vitro screening recombinant peptide, with the MX2 structural domain of detection with receptors bind.The peptide of histidine mark can separate on the nickel post, and cuts histidine mark as required, and is used for immunize rabbit and mouse, to obtain antibody, is used for further research.

Can for example to the retardance of receptors bind, determine the peptide that has suitable length for biologic applications by checking its immunostimulatory properties and other functions.

Embodiment 3. acceptors and the required key character of ligand interaction

Because the N end structure territory of CEACAM is enough to make CEACAM and Opa protein-interacting, inventor of the present invention is using display technique of bacteriophage, also the adhesion epi-position in CEACAM N end structure territory is studied.Inventor of the present invention has used the information of the alanine scanning mutagenesis 9 of acceptor having been studied the ligand binding region on the acceptor, and this research is helpful for this research.Also have for this situation, also available part cover butter carries out biological elutriation (affine concentrating) for the chimeric phage that carries receptor sequence.

Receptor analogs has the potential of the multiple bacterial strain of retardance, and this is irrelevant with the Opa albumen type that produces, although have antigenic variation because our research has shown Opa albumen, for elementary adhesion ⁹, have identical character on the different Opa protein requirement acceptors.What is interesting is in addition, notice that CEA antigen comes off from intestinal mucosa, and might block the adhesion of coli strain, and also target CEACAM of known intestinal bacteria.This has been suggested the mechanism as pathogenic agent in the congenital immunity reply intestines.Therefore these receptor analogs are as healing potion ¹²

Embodiment 4. summarizes with the preparation of CEACAM bonded reorganization morazella catarrhalis UspA1 peptide

Total length along UspA1 goes out many fragments by pcr amplification, uses primer as shown in Figure 9.Following description obtains recombinant peptide.In the first round, the dna encoding that discovery and CEACAM1 bonded recombinant peptide are obtained by P4 and P8 primer amplification, rather than by the DNA regional code between P1 and the P5, and P4 combines with CEACAM1 to P7 zone recon, and P6 then can not to the P8 zone.Also used other primer in the P7 zone at P4.The D-7 zone has kept and CEACAM bonded character.The 4-7 fragments sequence is shown in Figure 10.

General clone, expression and purifying strategy for reorganization UspA1 peptide

Use pBAD systems produce UspA1 fragment 1-5.Required PCR product is cloned in the pBAD carrier with TA, and uses the TOP10 coli strain to increase.Remaining step as shown in figure 11, unique difference is to use pectinose to induce pBAD.Use pBAD and two systems produce fragments of pQE30 (Figure 11) 4-7, obtain similar combining the result with CEACAM.Remaining fragment uses the strategy among Figure 11 to be prepared.

Carrier and pQE30 expression system

Use pQE30 carrier (Figure 12) and coli strain M15.M15 comprises plasmid pREP4, and this plasmid-encoded supressor, this factor have suppressed to be cloned into transcribing of DNA in the pQE30 carrier.Add IPTG concentration and reach the coding that 1mM has suppressed this supressor, the fragment of then being cloned among the pQE30 is transcribed.

Clone's strategy

At first pcr amplification product is connected among the pCR2.1.Produced a stable host like this, amplified production can reclaim by restriction enzyme digestion (BamHI/pstI), and these two ends that also guaranteed gene fragment all are cut.PQE30 carries out also that similar enzyme is cut and reclaims carrier after enzyme is cut with gel-purified, and this has guaranteed that also two restriction sites all are cut.The pQE30 that cuts of enzyme uses the T4 dna ligase to be connected at 16 degrees centigrade with the UspA1 amplified production to spend the night, connect product and be transformed in the intestinal bacteria M15 competent cell of handling with calcium chloride.On the LB agar plate that contains penbritin (every milliliter of 100 microgram) and kantlex (every milliliter of 25 microgram), screen transformant.A picking 4-8 bacterium colony is cultivated in being added with antibiotic LB liquid nutrient medium.The centrifugal thalline that obtains of the culture of 3-4 milliliter extracts plasmid in a small amount with the method for alkaline lysis.Purified carrier carries out enzyme as mentioned above to be cut, and checks that uspA1 inserts fragment.The bacterium that comprises the pQE30 carrier with correct insertion clip size grows in 50 milliliters of substratum, and culture is induced (face as follows) with IPTG.Use western blotting screening Recombinant Protein Expression then.In addition carrier is checked order, insert with definite whether fragment is correct DNA zone, and check whether any sequence errors is arranged.

Express and purifying

The M15 that comprises the pQE30/uspA1 construct is grown in the LB liquid nutrient medium that contains penbritin (every milliliter of 100 microgram) and kantlex (every milliliter of 25 microgram), and shaking culture is until OD600=0.5, add then IPTG to concentration be 1mM.Culture was further cultivated 3-4 hour, centrifugal recovery thalline.The bacterial sediment thing buffer B (8M urea, 50mMTris, 10% ethanol, 2%Tween, the 5mM imidazoles, pH7) in solubilising 1-3 hour, 20,000g removed membrane substance in centrifugal 20 minutes.Supernatant was hatched on shaking table 1-2 hour with the nickel resin, then by the polypropylene post.With the remaining resin of 5-10 milliliter buffer B rinsing, use the albumen on 0.5 milliliter elution buffer (in the buffer B the add 100mM imidazoles) elution of bound.The albumen of wash-out is checked with SDS-PAGE, dialyses then and removes urea and other salt.

Recombinant fragment

A: fragment 1-5 and 4-8

These show by fragments of pBAD systems produce that 1-5 do not combine with CEACAM1 and 4-8 combines with CEACAM1.

B: the fragment 4-8,4-8T, 4-7 and the 6-8 that use the pQE30 systems produce

Fragment 4-8 is the reorganization UspA1 fragment of first preparation, finds that in the trace cover butter it combines with CEACAM1 highly affinely.Except the 4-8 reorganization UspA1 peptide of total length, also observe an albumen less, brachymemma.This peptide (called after 4-T) also combines with CEACAM1, seems that its expression level is than 4-8 low (Figure 13).PQE30/4-T is carried out sequential analysis find, because a mispairing (CAA is to TAA) causes in a residue Q624 place appearance terminator codon (referring to Figure 10).

Preparation peptide 4-7 and 6-8 are to get rid of the possibility that occurs second CEACAM binding site in 6-8.As being predicted from peptide 4-T, 4-7 has shown combine (Figure 13) with CEACAM, and 6-8 does not combine (Figure 14) with CEACAM.

C: by the fragment D-8 and the D-7 of pQE30 systems produce

Find that rUspA1 fragment D-8 (Figure 15) and D-7 (not shown) all combine with CEACAM1.

The biological activity of recombinant peptide 4-7

Morazella catarrhalis bacterial strain MX1 and hemophilus influenzae bacterial strain Rd with transfection the Chinese hamster ovary of CEACAM1 (CHO) cell combine.Their combination can be suppressed by the UspA1 recombinant peptide 4-7 of morazella catarrhalis, but can not be suppressed (Figure 17) by control peptide.

Further showing in the experiment, recombinant peptide D-7 (rather than reorganization 6-8 is the residue E659-K863 of the UspA1 of MX2) suppress homology and heterology bacterial strain the two with combine (Figure 23) of the transfection CHO cell of expression CEACAM1.At no peptide (the 1st hurdle), control peptide (2 μ g.ml are arranged ^-1The 2nd hurdle) or D-7 (2 μ g.ml ^-1The 3rd hurdle) behind the pre-cultured cell, adds bacterium and after cultivating 1 hour, remove NA bacterium under the situation by washing.That uses then that the antiserum(antisera) of anti-different strains and rhodamine put together is two anti-, detects and cell bonded bacterium.Mx: morazella catarrhalis, Nm. Neisseria meningitidis, Hi: hemophilus influenzae.

The sign of embodiment 5.CEACAM1-binding peptide

Find that peptide D-7 is the strongest in conjunction with recombinant peptide.Therefore, the D-7 zone of MX2 (142 amino acid; Referring to Figure 16) contain the CEACAM1 combining information.

The peptide 4-T of brachymemma (197 amino acid) keeps faint combination (Figure 13).Therefore the zone with CEACAM1 binding ability may be contained in the D-T zone.

Find that single amino acid replaces among the D-7, K 560 I have almost abolished the CEACAM1 combination.

The disappearance in 571-632 zone among the peptide D-8 (D-8 Δ) causes the forfeiture of CEACAM1 bonded.

Prepared the linear superposition peptide (Figure 19) of crossing over the D-T zone and tested their abilities in conjunction with CEACAM1.Do not observe and the combining of CEACAM1.

Circular dichroism is learned

With circular dichroism (CD) spectroscopy analysis peptide D-7 and mutant D-7 (K 560 I).Use Jobin-Yvon CD6 spectropolarimeter at room temperature to obtain circular dichroism.Measuring concentration in quartz cuvette is the reorganization D-7 of 0.1mg/ml and the spectrum of D-7 (K 560 I).All spectrum are that 8 scanning deducts relevant nonprotein buffer reagent spectrographic mean value and uses Excel (Microsoft Inc.) mapping.

The spectrum that obtains shows that D-7 has α-Luo Xuanjiegou, and D-7 (K 560 I) adopts random coil structure (Figure 18).Do not wish to be subjected to the constraint of any particular theory, the CEACAM1 combination that proposes Mx UspA1 need may be the coiled coil structure based on the conformation of alpha-helix.

General introduction

From The above results, the inventor draws as drawing a conclusion: the CEACAM1 combination of Mx UspA1 except the conformation based on alpha-helix, may be beyond the coiled coil structure, also to need the zone among the D-7.

The CEACAM1 binding domains of Mx UspA1 adopts based on the discovery of the conformation (this conformation is that receptors bind is needed) of alpha-helix interesting especially.The careful reconstruct of Opa protein requirement is to present the CEACAM-binding domains, and this structural domain occurs in different surfaces and exposes on the ring texture.This discovery prompting, the spontaneous refolding of D-7 is to provide CEACAM-in conjunction with character.Coiled structure provides and has the spherical subunit structure that is suitable for CEACAM1 bonded conformation in D-7.This favourable character of D-7 peptide means that this peptide and derivative, homologue or fragment will have special effectiveness as subunit vaccine or therapeutical agent.Derivative with D-7 peptide of required conformation based on alpha-helix can be differentiated with unique " fingerprint " circular dichroism.

Embodiment 6. sequential analyses

In Figure 20, shown the contrast in D-7 zone of the proteic aminoacid sequence of UspA1 of 10 bacterial strains of Mx.

In 10 bacterial strains 6 correlated sequence area be identical.MX3 and MX4 bacterial strain are 100% identical and have total identity of 90.85% and 88.03% respectively on the obtained sequence in D-7 zone, consider 13 and 17 amino-acid residues of N-end of also undeterminate MX3 and MX4 respectively.

Disappearance (Figure 20) shown in remaining two bacterial strain TTA37 and O35E have, it occurs in the D-T zone.Comprise that total identity of breach is respectively 70.4% and 50% among the D-7.When manual contrast, TTA37 is identical at 100 amino acid regions of residue, and O35E 71 identical (Figure 21) in 72 amino acid.Known O35E does not combine with CEACAM1.TTA37 is not suitable for test.

The adhesion retardance character of embodiment 7.D-7

The result

At first use soluble receptors to investigate the potential of D-7 as the antitack agent of the homology of effective anti-Mx, Nm, Ng and Hi and heterology bacterial strain.Whole cell lysates of two Mx bacterial strains are got ready be coated onto on the soluble cotton before, cultivate CEACAM1-Fc (CC1-Fc) and D-7 in advance.Show reorganization D-7 with the dose-dependently mode significantly suppress CC1-Fc and heterology bacterial strain MX1 and with homology bacterial strain MX2 combine that (Figure 24 a).Inhibition is significant and seems at 0.01 μ g.ml ^-1More than reached platform.The previous control peptide (D-8 Δ) that does not combine (embodiment 5) with CEACAM1 that shows does not show such inhibition.In addition, measured the inhibition of a plurality of bacterial strains.The most of bacterial strains that use have been represented worldwide clinical isolates (referring to method).D-7 has shown significantly and specificity suppresses, but the D-8 Δ does not have (Figure 24 b).Use transfection that Chinese hamster ovary (CHO) cell (CHO-CEACAM1) of CEACAM1 is arranged then, investigated the retardation of D-7.When using soluble receptors, observing Mx, Nm and Hi and CHO-CEACAM1 cell bonded inhibition (Figure 25 a and b) after pre-the cultivation with D-7 but without control peptide.Bacterium and CHO-CEACAM1 cell bonded suppress be dose-dependently and be equal to or greater than 0.1 μ g.ml for Mx and Hi ^-1The time and for Nm at 0.2 μ g.ml ^-1The time be significant (Figure 25 b).At about 2 μ g.ml ^-1Concentration under obtained bacterium and CHO-CEACAM1 bonded near abolishing (Figure 25 a and b) fully.The value of the inhibition that obtains is pointed out potentially, and the order of the interactional affinity of CEACAM of the bacterium of using in this research is Nm＞Mx＞Hi.Except CEACAM1, shown also other member of target people CEACAM family of Neisseria meningitidis, comprise epithelium CEA and CEACAM6 (NCA) ⁹, some Mx bacterial strains also are so, the Hi bacterial strain is tending towards the CEACAM1 (data not shown) of target as preferred acceptor.With D-7 (2 μ g.ml ^-1) after pre-the cultivation, to express CEACAM1,6 and the HeLa cell of the transfection of CEA observed the Nm bonded and suppressed (Figure 25 c).

Use HeLa-CC1H (a kind of be used for expressing the clone of high-caliber CEACAM1), the effect of further having tested D-7 with inflammatory conditions in the possible body of partial simulation epithelial cell.High Rd allows to express the bacteria adherence of Opa to target cell, although there is tunicle in this target cell ⁷Therefore, this model allows us also to use the phenotype (h18.18) of the Nm that can find in vivo, and is promptly tunicate, fimbriate and expression outer membrane protein Opa and Opc ¹⁵, ²⁵At first, tested the Opa-disappearance derivative of h18.18 and combining of HeLa-CC1H, found its low relatively (each cell is in conjunction with 10-20 bacterium) and be reason because of the expression pili.As expected, the adhesion of h18.18 and HeLa-CC1H strengthened (each cell adhesion 100-150 bacterium) greatly although and fimbriate contribution, formerly or simultaneously with the cell adhesion (Figure 25 d) of D-7 processing the minimizing in a large number h18.18.Except above-mentioned character, be A549 (known expression CEACAMs also at people's pulmonary epithelial cells ²⁶) on tested D-7.Why select this clone, because it also expresses the acceptor of other Mx part ²⁷, therefore can test the effect of D-7 in reducing bacterial load.Under the situation that has D-7 (2 μ g.ml-1) rather than D-8 Δ, observed the remarkable reduction (Figure 25 e) of Nm and Hi and A549 cell interaction.Also observed the bonded minimizing for the Mx bacterial strain, although and relatively low by comparison, it is significant (Figure 25 e).

Method

Bacterial isolates and cultivation

Mx and Nm grow on the BHI agar that contains 10% thermal treatment horse blood, and Hi grows on the brain heart infusion that contains the Levinthal base (BHI) agar.The Ng bacterial strain is cultivated on GC agar.The 37 ℃ of cultivations in the 5%CO2 incubator of all bacteriums reach 16h.The Mx bacterial strain is the clinical isolates that obtains from otitis media and patient COPD and the representative isolate from several countries.Eagan, c1 and f1 are the typical Hi that has b, c and f type tunicle respectively, and Rd is a kind of no tunicle derivative of d type bacterial strain.Strains A 930065 and NT1 are that NTHi. strains A 3, F2087, F3035 and F1947 are Egyptian (aegyptius) secretory product that belongs to of Hi biological group.Nm strain isolated C751A, C751B and C751D are that the Opa of three kinds of uniquenesses of serologic group A bacterial strain C751 expresses strain isolated.Other strain isolateds are from following serologic group: PMC17 (A), C311 and MC58 (B), and C114 (C) PMC2 (29E), PMC4 (W135) and PMC10 (Y).Ng strain isolated P9-13,16 and 35 is the interior Opa mutation of the bacterial strain of bacterial strain P9, and other clinical separation strains are worldwide.Further described in the former research of most bacterial strains that this research is used ^10,26,28

Antibody

Anti--polyhistidyl mouse monoclonal antibody is available from Qiagen, by 0.2 μ g.ml ^-1Concentration is used.The polyclonal antiserum of anti-Mx, Nm and Hi bacterial strain is to prepare with rabbit according to standard program, with the full cell pyrolysis liquid of multiple bacterial strain as antigen.Anti--UspA1 antibody (R38) that this research is used is embodiment 1 He ²⁶The middle description.The polyclonal antiserum of anti-D-7 is in conjunction with Ni-NTA resin (Qiagen with peptide; Each immunity is with 100-200 μ g peptide) immunize rabbit produces.By coming the deactivation complement at 56 ℃ of coctoantiserum 30min.Anti--D-7 antibody is with the peptide D-7 that links AminoLink Plus post, comes protein affinity purification according to the program of manufacturers (Pierce).

Soluble receptors Gou Jianti ﹠amp; Clone

Be used for this research transfection solubility CEACAM1-Fc and the former research of Chinese hamster ovary celI of CEACAM1 describe ^11,9The HeLa cell of expressing a series of CEACAM molecules is by Wolfgang Zimmerman professor (Munich university, German) and doctor ScottGray-Owen (Toronto university, Canada) give, these HeLa cells are grown in containing RPMI 1640 substratum of 10% foetal calf serum (FCS).A549 human lung carcinoma cell (Flow laboratory) is grown in containing the F12 Ham substratum of 10% foetal calf serum (FCS).The HeLa cell of expressing high-level CEACAM molecule is by using Tet-On ^TM(Clontech) gene expression system produces.The ceacaml gene clone is gone into pTRE-2hyg reaction plasmid (Ciontech) and is transfected into the HeLa cell that has regulatory gene (Ciontech) with Fugene-6 (Roche).Transfectional cell is with 400 μ g.ml ^-1Hygromycin selection.Those CEACAM express the male transfectional cell and screen with FACS and limiting dilution.At 0.25 μ g.ml ^-1HeLa-CC1H clone when doxycycline exists produces the acceptor of highest level.

The acceptor cover butter

These experiments are based on previously described method ⁹, following except.For suppressing research, at room temperature with CEACAM1-Fc (0.1 μ g.ml ^-1) and D-7 or control peptide (0.001-2 μ g.ml ^-1) cultivated in advance 1 hour.Use CEACAM1-Fc (0.1 μ g.ml then separately ^-1) cover spot or cultivate in advance with peptide as described.Perhaps using acceptor (0.1 μ g.ml ^-1) before the covering, with rabbit anti--D-7 antibody (10 μ g.ml ^-1) covered spot 1 hour.In either case, use NIH ScionImage program, measure the level of receptors bind by photodensitometry.

The D-7 peptide suppresses bacterium and expresses the adhesion of the cell of CEACAM

In 37 ℃ of 199 substratum that adding 2% foetal calf serum, with confluent monolayer and the peptide D-7 or control peptide (the 0.1-2 μ g.ml of cell ^-1) cultivated in advance 30-60 minute.After the pre-cultivation of peptide, check that individual layer is to guarantee not having deleterious effect to take place to described cell.Subsequently, the ratio with the about 100-200 of each a cell bacterium was cultivated individual layer 1 hour in 37 ℃ of 199 substratum that adding 2% foetal calf serum.By removing NA bacterium 4 times, handle then that individual layer carries out that immunofluorescence detects or with 1% saponin(e cracking, and the diluent bed board of bacterium is used for according to previously described method mensuration colony-forming unit (cfu) with 199 substratum washings ²⁵For immunofluorescence detects, with cell fixation in anhydrous methanol 10 minutes, with the washing of 1% bovine serum albumin among the PBS that contains 0.05% tween with blocked 1 hour.The bacterium that the two anti-detections of using antibacterial antiserum(antisera) and rhodamine to put together are depended on.By using Olympus IX70 microscope, amplify the X400 direct census, obtained to adhere to the number of bacteria of the cell of expressing HeLa-CEACAM.To after the bacterium of 20 cell adhesions of selection is counted at random from the double experiment, obtained mean value in conjunction with bacterium.According to the method described above, by the cfu assay determination with the adherent bacterium of HeLa-CC1H.

Embodiment 8. anti-D-7 antibody suppress Mx-CEACAM1 and interact

The result

The rabbit anti-serum of the anti-D-7 that produces contains antibody, in the western blotting of WCL covers, and this antibody and UspA1 cross reaction (data not shown) from a plurality of Mx bacterial strains.Use the antibody of affinity purification, do not observe combine (data not shown) of anti--D-7 and NmOpa or Hi P5 by western blotting.Before CC1-Fc covers, the WCL of a series of Mx bacterial strains and anti--D-7 (10 μ g.ml ^-1) cultivation cause the inhibition of significant receptors bind, most of bacterial strains show the inhibition (Figure 26) greater than 80%.Yet the bacterial strain for Hi, Nm and Ng in similar experiment is only observed low-level inhibition.Therefore the antibody of anti-D-7 can provide the protection that anti-Mx infects, and D-7 can be used as more broad spectrum antimicrobial peptide, is used for many different CEACAM target bacteriums.

Embodiment 9.D-7 peptide suppresses bacterium and expresses the adhesion of the human endothelial cell of CEACAM

In order to assess the effect of D-7, use the confluent monolayer of HMEC-1 cell to endotheliocyte.(the two all is 1 μ g.ml with human microvascular endothelial cell (mvec) and D-7 or reorganization contrast molecule ^-1) cultivated in advance 60 minutes.Cultivated 1 hour with the infection rate interpolation bacterium (strain isolated of the expression OpaD of Neisseria meningitidis bacterial strain C751) of 100 bacteriums of each cell and at 37 ℃ then.After this, remove NA bacterium by washing and at room temperature individual layer was fixed in the methyl alcohol 10 minutes.By the rabbit polyclonal antiserum(antisera) with anti-Neisseria meningitidis, and two anti-detections of puting together with anti-rabbit TRITC subsequently adhere to bacterium.

Under the situation that does not have peptide, each cell of most endotheliocyte has 30 bonded bacteriums at the most.Exist under the situation of control peptide, do not observe tangible bacterium bonded and suppress.Yet, exist under the situation of D-7, in conjunction with having been abolished in fact, there is the cell (Figure 27) that depends on l-2 bacterium once in a while.

Therefore D-7 can suppress Neisseria meningitidis and the endotheliocyte and the epithelial adhesion of Opa-CEACAM mediation.

The conservative property of embodiment 10. sequence of fragment " 4-T " (427-623 the amino acid of MX2 UspA1) in known UspA1 protein sequence

Table II. the bacterial strain of use and sequence

The GenBank accession number	Bacterial strain	Length (aa)
			AAN84895	P44	913
AAB96359	O35E	832
			AAF40122	TTA37	873
AAF40118	O12E	922
			AAF36416	O46E
	892
		AAD43469	V1171	912
AAD43467	TTA24				941
		AAD43465	ATCC25238(MX2)	863

Carry out the multiple sequence contrast with all full length protein sequences, to differentiate the respective segments in other bacterial strain sequence.

Table III. the location of respective segments

Bacterial strain	From following amino acid	Till following amino acid	Length
				ATCC25238	427	623	197
O12E	515	682	168
				O35E	495	592	98
O46E	456	652	197
				P44	477	673	197
TTA24	505	701	197
				TTA37	486	633	148
V1171	476	672	197

These segmental multiple sequence contrasts have been shown among Figure 28.Shown relevant identity per-cent below.

Table IV. the fragments sequence identity per-cent that defines in the Table III

	O12E	O35E	O46E	P44	TTA24	TTA37	V1171
								ATCC25238	95	85	96	99	96	97	97
O12E		92	95	96	95	79	94
								O35E			90	86	91	89	89
O46E				97	98	98	98
								P44					97	97	97
TTA24						98	98
								TTA37							98

As among the embodiment 6 about shown in the D-7 zone, The above results shows that the 4-T zone is a high conservative in different bacterial strains, for all tested bacterial strains (except the O35E (85% identity)), sequence identity is 95% or greater than 95%.As previously described, O35E does not combine with CEACAM.Therefore, comprise as the conserved regions of the sequence 4-T (427-623) that shows among Figure 28 or be preferred peptide of the present invention by the peptide of forming as the conserved regions of the sequence 4-T (427-623) that shows among Figure 28, it is in treatment or preventing disease, especially wherein relates in the disease of CEACAM acceptor having effectiveness.

Reference:

1.Cartwright，K. et al.1995.In MeningococcalDisease.John Wiley & Sons.

2.van Alphen，L.& van Ham，S.M..1994.Rev MedMicrrobiol 5：245.

3.Foxwell，A.R.et al.1998.Microbiol Mol Biol Rev62：294.

4.van Alphen，L.et al.1995.Am J Respir Crit CareMed 151：2094.

5.Karalus，R.et al.2000.Microbes & Infection 2：5

6.Kraft.M.2000.Clin Chest Med 21：301.

7.Virji，M.et al.1996.Mol Microbiol 22：929.

8.Virji，M.et al.1996.Mol Microbiol 22：941.

9.Virji，M.et al.1999.Mol Microbiol 34：538.

10.Virji，M.et al.2000.Mol Microbiol 36：784.

11.Hill，D.et al.2001.Mol Microbiol 39：850.

12.Hammarstrm，S.1999 Cancer Biol 9：67.

13.Stephens，D.S.1989.Clin Micrrobiol Rev 2：S104.

14.Virji，M.et al.1991.Mol Microbiol 5：1831.

15.Achtman，M.1995 Trends Microbiol 3：186.

16.Merz，A.J.& So，M.2000 Annu Rev Cell Dev Biol16：423.

17.Woods，J.P.& Cannon，J.G.1990 Infect Immun58：569.

18.Wang，L-F & Yu，M.1996 In Methods Mol Biol 66：269(Morris，G.E.ed).Humana Press.

19.Colman-Lerner.2000.Trends Guide p.56(Wilson，E.ed.).

20.Beauchemin，N.，et al.，1999.Exp Cell Res 252：243-249.

21.Boulton I.C.& Gray-Owen S.D.2002.Nat Immunol3(3)：229-36.

22.Chen T.et al.，2001.J Leukoc Biol 70(2)：335-40.

23.Lafontaine，E.R.，et al.，2000.J Bacteriol 182：1364-1373.

24.Virji，M.2001.Tirends in Microbiology，9：258-259.

25.Virji，M.et al.1995.Mol Microbiol 18：741-754.

26.Hill，D.J.& Virji， M.2003.Mol Microbiol 48：117-129.

27.Holm，M.M.，et al.2004.Infect Immun 72：1906-1913.

28.Virji，M.et al.1990.Microb Pathog 9：441-450.

29.Langermann，S.et al.1997.Science 276：607-611.

30.Ofek，I.et al.2003.FEMS Immunol Med Microbiol38：181-191.

31.Simon，P.M.et al.1997.Infect Immun 65：750-757.

32.Idanpaan-Heikkili，I.et al.1997.J Infect Dis176：704-712.

33.Kelly，G.C.et al.1999.Nat Biotechnol 17：42-47.

34.Gan，B.S.et al.2002.J Infect Dis 185：1369-1372.

35.Reid，G.& Burton，J.2002.Microbes Infect 4：319-324.

36.Pinyon，R.A.et al.2004.J Infect Dis 189：1547-1555.

37.Reid，G.et al.2001.Trends Microbiol 9：424-428.

38.Toleman，M.et al.2002.Cell Microbiol 3：33-44.

39.Budt.M.et al.2002.Biol Chem 383：803-812.

Claims (24)

1. isolated polypeptide, it comprises the aminoacid sequence that is selected from following sequence, or is made up of the aminoacid sequence that is selected from following sequence: the conserved sequence of differentiating in the 527th to 623 residue of sequence shown in Figure 6 and the sequence shown in Figure 28 contrast.

2. according to the isolated polypeptide of claim 1, wherein said conserved sequence is selected from the group of being made up of following sequence:

QHSSDIKTLK，

NVEEGLLDLSGRLIDQKADLTKDIK，

NVEEGLLDLSGRLIDQKADIAKNQA，

DIAQNQT，

DIQDLAAYNELQD，

QTEAIDALNKASS，

TAELGIAENKKDAQIAKAQANENKDGIAK，

NQADIQLHDKKITNLGILHSMVARAVGNNTQGVATNKADIAK，

NQADIANNIKNIYELA，

NQADIANNI and

NIYELA。

3. according to the isolated polypeptide of claim 1 or claim 2, it is the UspA1 protein of brachymemma.

4. according to each isolated polypeptide of claim 1 to 3, it and CEACAM receptors bind.

5. medicine, it comprises according to each polypeptide and one or more pharmaceutically acceptable adjuvants, vehicle, vehicle, tackiness agent, carrier or sanitas of claim 1 to 4.

6. be used for the treatment of according to the medicine of claim 5 or the purposes of preventing infection.

7. the medicine according to claim 5 is used for the treatment of or prophylactic purposes, and the participation of CEACAM acceptor causes the cell-targeting of the pathogenic agent of this disease in this disease.

8. according to the purposes of claim 7, be used for the treatment of or illness, vasculogenesis, dental caries tooth and gingival disease that preventing infection, respiratory disease, neoplastic disease and neoplastic disease are relevant.

9. according to each purposes of claim 6 to 8, wherein said infection is a mucosal infections, or takes place through mucous membrane.

10. according to the purposes of claim 9, wherein said infection is caused by Neisseria meningitidis, hemophilus influenzae or morazella catarrhalis.

11. according to the purposes of claim 9, wherein said infection is caused by Fusobacterium nucleatum.

12., be used for prevention or treatment otitis media according to the purposes of the medicine of claim 5.

13. differentiating, comprise that they simulate the ability of described polypeptide to the screening of potential therapeutical agent, or the homology of they and described polypeptide as the purposes in the shaker test of the new retardance reagent of therapeutical agent according to each polypeptide of claim 1 to 4.

14. a vaccine, it comprises according to each polypeptide and one or more pharmaceutically acceptable adjuvants, vehicle, vehicle, tackiness agent, carrier or sanitas of claim 1 to 4.

15. the method that infects in the individuality that treatment or prevention need treat or prevent, it comprises the medicine according to claim 5 that gives significant quantity.

16. described and illustrated as the front in fact polypeptide as accompanying drawing 6 with reference to accompanying drawing 6.

17. medicine in fact as previously described, it comprises the polypeptide of claim 16.

18. in conjunction with the part of CEACAM acceptor preparation be used for the treatment of or prophylactic medicine in purposes, the participation of CEACAM acceptor causes the cell-targeting of the pathogenic agent of this disease in this disease, wherein this part comprises the aminoacid sequence that is selected from following sequence, or form by the aminoacid sequence that is selected from following sequence: the conserved sequence of differentiating in the 527th to 623 residue of sequence shown in Figure 6 and the sequence shown in Figure 28 contrast, or keep its fragment or the functional equivalents of CEACAM binding ability.

19. according to the purposes of claim 18, wherein said conserved sequence is selected from following sequence:

QHSSDIKTLK，

NVEEGLLDLSGRLIDQKADLTKDIK，

NVEEGLLDLSGRLIDQKADIAKNQA，

DIAQNQT，

DIQDLAAYNELQD，

QTEAIDALNKASS，

TAELGIAENKKDAQIAKAQANENKDGIAK，

NQADIQLHDKKITNLGILHSMVARAVGNNTQGVATNKADIAK，

NQADIANNIKNIYELA，

NQADIANNI, and

NIYELA。

20. according to the purposes of claim 18 or claim 19, wherein said disease is selected from infection, respiratory disease, neoplastic disease and neoplastic disease relevant illness, vasculogenesis, dental caries tooth and gingival disease.

21. according to the purposes of claim 18 to 20, wherein said pathogenic infection mucous membrane, or enter through mucous membrane.

22., cause that wherein the pathogenic agent of described disease is selected from Neisseria meningitidis, hemophilus influenzae and morazella catarrhalis according to each purposes of claim 18 to 21.

23. according to each purposes of claim 18 to 21, the pathogenic agent that wherein causes described disease is a Fusobacterium nucleatum.

24. according to the purposes of claim 22 or 23, wherein said disease is an otitis media.

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