CN102335415A

CN102335415A - Anti-inflammatory effect, implementation method and purpose of novel membrane molecule

Info

Publication number: CN102335415A
Application number: CN2010102304696A
Authority: CN
Inventors: 曹雪涛; 韩超峰; 李楠
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2010-07-19
Filing date: 2010-07-19
Publication date: 2012-02-01

Abstract

The invention relates to an anti-inflammatory effect, implementation method and purpose of a novel membrane molecule, in particular to a purpose and application method of integrin alphaM (Itgam) in resisting bacterial infection. The invention provides the application method and a corresponding immunological principle of the molecule of the Itgam in a process of resisting the bacterial infection. The invention discloses the purpose and a strategy of the molecule in resisting the bacterial infection and particularly relates to application of the molecule to prevention and treatment of infectious shock induced by bacterial infection and endotoxic shock. According to the invention, the molecule is proved to be capable of inhibiting generation of inflammatory factors after bacterial infection, increasing the survival rate of an organism undergoing the endotoxic shock, and protecting the lung and other organs subjected to infection from inflammatory injuries.

Description

A kind of antiinflammatory action of new membrane molecule, implementation method and purposes

Technical field

The invention belongs to biotechnology and medical domain, specifically, the present invention relates to integrin alpha M (integrin α M, Itgam) effect in bacterial infection disease, mechanism of action, implementation method and purposes.

Background technology

Bacterial infection is a clinical common diseases; Can cause body infectivity local and whole body to show; Even can finally cause septic shock, endotoxin shock and multiple organ dysfunction syndrome (multiple organ dysfunction syndrome; MODS) etc. serious consequence in case severe infection causes shock or multiple organ dysfunction syndrome occurring, then causes high mortality (～50-80%) performance.Common clinical bacteria infects; All can cause the inflammatory reaction of body part and whole body like escherichia coli, Pseudomonas aeruginosa, staphylococcus aureus, Klebsiella Pneumoniae, Acinetobacter bauamnnii, enterococcus faecalis etc., cause the generation of infectious disease in various degree.

For bacterial infection patients, the doctor generally adopts heavy dose of antibiotic " bombing ", is intended to " destroying the enemy at one stroke "; Fast, thoroughly kill pathogen; But often ignored on the other hand, promptly after pathogenic bacteria death, can discharge a large amount of toxin, caused serious systemic symptom through toxin.The toxin that brings out systemic inflammatory mainly contains two types: endotoxin (the lipopolysaccharide composition of gram-negative bacteria cell wall) and superantigen (toxin such as Peptidoglycan/fat phosphorus wall complex, extracellular toxin that comprises gram-positive bacteria).The significant challenge that shock that present bacterial infection causes and multiple organ dysfunction syndrome remain clinical prevention and treatment.

People discern external microbial molecular mechanism for body and not exclusively understand; But along with the discovery of Toll appearance molecule, the cell and the molecular biology basis of the natural immunity that body produces to Institute of Micro-biology and the acquired immunity of secondary have progressively obtained understanding.

Toll appearance receptor (TLRs) is a quasi-mode identification receptor; Be distributed widely in immunocyte; Like BMDC and macrophage surface; Can discern antigen and activate corresponding signal path through the specific pathogen associated molecular pattern of pathogenic microorganism, start the natural immunity and reply, constitute the first line of defence of body opposing pathogenic microorganism invasion.TLRs is an I type transmembrane protein, belongs to TIR (Toll/IL-1 receptor) superfamily member, and Toll albumen is found the earliest in the drosophila embryos growth course, and the formation of carrying on the back veutro axon cell is played important regulating and controlling effect [Hashimoto, C. etc., Cell.1988; 52:269-279; Anderson, K.V. etc., Cell.1985; 42:791-798].

Toll albumen high conservative on spore has been found 11 kinds of Toll appearance receptors (Toll-like receptors, TLRs) [Medzhitov, R. etc., Nature.1997 mammal so far in succession; 388:394-397; Rock, F.L. etc., Proc Natl Acad Sci USA.1998; 95:588-593].The molecule of corresponding ad hoc structure in the different TLR identification different pathogens; These molecules are molecules of the common constructive expression of one type of most of pathogenic microorganism; Structure is conservative, be called the pathogen associated molecular pattern (Pathogenassociated molecular patterns, PAMPs); Corresponding receptors then for the pattern recognition receptor (Patternrecognition receptors, PRRs).Natural immune system constitutes body anti-infective the first line of defence through the conservative PAMP of PRR identification pathogen, simultaneously acquired immunity is also had important regulatory role.

Human TLR1, TLR2, TLR3 and TLR6 are positioned chromosome No. 4; Wherein TLR2 discerns compositions such as gram positive bacteria composition bacterial lipoprotein BLP; And with TLR6 synergism identification PGN molecule etc., TLR3 then discerns viruses molecule double center chain RNA (double-stranded RNA, dsRNA) [Takeuchi; O. etc., Immunity.1999; 11:443-451; Ozinsky, A. etc., Proc Natl Acad Sci USA.2000; 97:13766-13771; Alexopoulou, L. etc., Nature.2001; 413:732-738].Human TLR4 and TLR5 are positioned chromosome respectively 9 and No. 1, and (lipopolysaccharide is LPS) with bacterial flagellin molecule (flagelin) [Poltorak A. etc., Science.1998 for corresponding identification gram-negative bacteria cell wall composition lipopolysaccharide; 282:2085-2088; Hayashi F. etc., Nature.2001; 410:1099-1103]; Human TLR7 and TLR8 gene mapping are discerned single stranded RNA (single-strandedRNA, ssRNAs) [Heil, F. etc., Science.2004 in the viruses molecule in chromosome x p22; 303:1526-1529]; Human TLR9 is positioned chromosome 3p21.3, the CpG motif in identification antibacterial and the viral DNA or the few nuclear of the CpG of synthetic former times acid (CpG oligodeoxynucleotides, CpG ODN) [Hemmi, H. etc., Nature.2000; 408:740-745]; TLR11 is the newcomer of TLRs family, mainly is expressed in urinary system, and [Zhang D. etc., Science.2004 play a significant role in the process of control urinary system bacterial infection; 303:1522-1526].

Discover that at present TLR is not only closely related with the anti-infectious immunity of body, and have cause effect relation with the generation and the progress of a variety of diseases.Microorganism can the quick active body after getting into body natural immune system; Produce a large amount of inflammatory factors (like TNF α, IL-1 and IL-15, I type interferon etc.) by macrophage, BMDC immunocytes such as (DC) and endotheliocyte system; Directly kill and eliminate the exotic disease substance on the one hand, induce specificity acquired immunity on the one hand to pathogen.If body can not be removed pathogen timely and effectively; Then body will produce chronic inflammatory disease (like tuberculosis, chronic hepatitis, chronic nephritis and chronic gastroenteropathy); And can cause the disorder of body immune system function, secular chronic inflammatory disease can cause various clinical disease [Cook, the D.N. etc. of secondary; Nat Immunol.2004,5:975-979].Discover; Various autoimmune disease, anaphylactic disease, atherosclerosis even comprise that kinds of tumors is all closely related with chronic inflammatory disease, its pathogenesis relates to signal conduction abnormalities [Cook, the D.N. etc. of TLRs; Nat Immunol.2004,5:975-979].

The TLRs signal path is as the first road barrier of body inherent immunity; Usually remove pathogen through inducing the generation inflammatory factor with disturbing; Keep the stable of organismic internal environment; Yet the activation of TLRs is a double-edged sword, and too much inflammatory factor generation can cause various diseases associated with inflammation and autoimmune disease.The mechanism of diseases associated with inflammation such as endotoxin shock is because Gram-negative pathogen mortality in focus or the blood flow when a large amount of endotoxins that discharge get into blood, endotoxemia can take place.A large amount of effects of endotoxin are in macrophage, neutrophilic granulocyte, endotheliocyte, the platelet of body; And complement system and blood coagulation system etc., just can produce

interleukin

1,6,8 and bioactive substance such as tumor necrosis factor, histamine, five hydroxytryptamine, prostaglandin, kassinin kinin.It is microcirculation depletion, hypotension, anoxia, acidosis etc. that these materials act on that little blood vessel causes dysfunction and cause microcirculation disturbance, clinical manifestation, so cause patient to suffer a shock, severe patient can cause death and die.Its essence is the damage that the over-drastic inflammatory reaction of body of large number of biological toxin initiation causes body self, and in this process, the inflammatory reaction of the TLRs mediation on immunocyte surface is the important step of its pathogenesis.Therefore, the activation of TLR signal path must receive strict regulation and control, prevents its overactivity, especially the excessive generation of inflammatory factor.

The TLR receptor combines to distinguish MyD88 (myeloiddifferentiation factor 88) the dependency path and TRIF (Toll/IL-1receptor (TIR)-domaincontaining adaptor protein inducing IFN-β) the dependency path in activation downstream with respective ligand; MyD88 is the signaling molecule such as IRAKs, TRAF6, TAK1 and IKK and MAPK in activation downstream afterwards; The early activation that finally causes NF κ B and AP1; Mediation inflammatory factor such as TNF-α; IL-6, IL-1 β and NO production; The activation energy of TRIF activates TBK1, TRAF3 and IRF3, finally causes the generation of I type interferon and the activation of interferon-induced gene, like [Akira, S. etc., Nat.Rev.Immunol.4,499-511 (2004) such as CXCL10 and CCL5; Liew, F.Y. etc., Nat.Rev.Immunol.5,446-458 (2005)].

Present research shows that much striding in film and the born of the same parents molecule can both bear and regulate the TLR signal path, suppresses the activation of MyD88 dependency path and the generation of inflammatory factor, like A20; CYLD, DUBA, SHP-1 etc.; Also have some molecule to reach the negative purpose [Akira that regulates through reducing surface of cell membrane TLRs receptor expression; S. etc., Nat.Rev.Immunol.4,499-511 (2004); Liew, F.Y. etc., Nat.Rev.Immunol.5,446-458 (2005)].Research that the signal transduction pathway that mediates to TLR carries out and test show; Each link inflammatory reaction that control of bacterial infection caused effectively that suppresses the conduction of TLR signal, serious consequences such as the shock that prevention and treatment bacterial infection cause, organ dysfunction damage.Have and discover that the TLR antagonist can be applied to the prevention and the treatment of infectious disease; Can be applied to serious septicemia (III clinical trial phase) [Liew like TLR4 antagonist TAK-242, E5564 etc.; F.Y. etc., Nat.Rev.Immunol.5,446-458 (2005); Cook, D.N. etc., Nat Immunol.2004,5:975-979].

Itgam claims CD11b again, belongs to the α integrin family; Integrate plain (CD18) with β 2 and form different chain receptor; Be Mac-1 (macrophage antigen-1) or CR3 (complement receptor 3), its part comprise the iuntercellular adhesion molecule (intracellular adhension molecule, ICAM) 1/2/3, complement iC3b, fiber former (fibrinogen), cellulose (fibronectin), Denatured protein etc.; High expressed is in macrophage, BMDC (dendritic cell; DC), medullary system derived cells such as NK cell and neutrophilic granulocyte, [Kinashi, T. etc. such as regulate cell adhesion, activation and engulf; Nat.Rev.Immunol.2005,5:546-559; Evans, R. etc., J.Cell Sci.2009,122:215-225.].Existing research shows that Itgam that magnesium ion activation DCs go up to express can the suppressor T cell activation, Itgam blocking antibody blocking-up DCs go up the Itgam activation then can strengthen the T cell activation [Varga G. etc., Blood 2007; 209:661-669.]; The mice of the defective of Itgam is induced enteritis more serious [Abdelbaqi, M. etc., Lab.Invest.2006 to DSS (dextran sodium sulfate); 86:380-390], possible mechanism is that Itgam can suppress T _H17 cell differentiations [Ehirchiou, D. etc., J.Exp.Med.2007,204:1519-1524].CD11b ⁺Gr1 ⁺SC (the myeloid-derived suppressor cells in medullary system source; MDSCs) can inducing T cell tolerance or hypoergia; MDSCs high expressed CD11b, but whether CD11b is to MDSCs inhibition function influential still unknown [Gabrilovich, D.I. etc.; Nat.Rev.Immunol.2009,9:162-174].

Immunology Inst., No.2 Military Medical Univ. discovers that the microenvironment of spleen, liver and lung stromal cell can produce DC subgroup (regulatory DC, the DCreg) [Zhang of the new modulability of a group high expressed CD11b; M. etc., Nat.Immunol.2004,5:1124-1133 (2004) .]; DCreg also can suppressor T cell react or activation, and promotes immunologic tolerance to keep, and suppresses asthma and hepatitis [Tang; H. etc., Blood 2006,108:1189-1197; Xia, S. etc., Blood 2008,112:3175-3185; Li, Eur.J.Immunol.2008 such as Q., 38:2751-2761].More than research shows that CD11b can acquired immunity, but the effect of CD11b in the natural immunity is not clear and definite as yet.

In sum, this area presses for and develops a kind of immunologic competence material that can effectively resist bacterial infection, suppress the inflammatory factor generation.

Summary of the invention

The present invention is just providing Itgam albumen or its coded sequence is effectively being resisted bacterial infection, suppressed the aborning new purposes of inflammatory factor.

In first aspect of the present invention, provide Itgam or its coded sequence to be used for preventing and/or treating the purposes of the medicine of bacterial infection relevant disease and/or symptom in preparation.

In an embodiment of the invention, said bacterial infection relevant disease and/or symptom are to be selected from down one or more that organize: the excessive generation of inflammatory factor behind the bacterial infection; Endotoxin shock or death; The inflammatory damage of organ; And, multiple organ dysfunction syndrome.

In yet another embodiment of the present invention, said bacterial infection be by be selected from down group one or more are bacterial: escherichia coli, Pseudomonas aeruginosa, staphylococcus aureus, Klebsiella Pneumoniae, Acinetobacter bauamnnii, enterococcus faecalis.

In another preference, said inflammatory factor is to be selected from down one or more that organize: TNF α, IL-1, IL-6, I type interferon, preferred TNF α, IL-1 or IL-6.

In another preference, said organ is selected from: lung, liver, spleen, brain or kidney.

In yet another embodiment of the present invention, said Itgam is selected from:

(a) aminoacid sequence of SEQ ID NO:2;

(b) with SEQ ID NO:2 amino acid sequence homologous, it has the bacterial infection relevant disease of inhibition and/or the active albumen of symptom;

(c) (a) or in the aminoacid sequence (b) through replacement, lack or add one or several aminoacid and have prevention or treatment bacterial infection relevant disease and/or symptom active by (a) or (b) deutero-protein or polypeptide.

In a preference, Itgam is: the product of natural purified proteins, chemosynthesis or use recombinant technique produce from protokaryon or eucaryon host, preferably are people Itgam.

In another preference, said aminoacid sequence is the coded sequence of nucleotide sequence of Gen Bank No:NM_008401.

In a preference, said host is selected from: antibacterial, yeast, higher mammal, insecticide and mammalian cell.

In yet another embodiment of the present invention, the encoding gene of said Itgam is selected from:

(i) sequence of SEQ ID NO:1; Or

(ii) under stringent condition with the sequence hybridization that (i) limits and have prevention or the molecule of treatment bacterial infection relevant disease and/or symptom.

In yet another embodiment of the present invention, the medication of said medicine is selected from: directly naked DNA injection, liposome dna direct injection, gold encapsulate that DNA particle gun blast technique, breeding unsoundness antibacterial carry the DNA method, replication defective adenoviral carries target DNA method or the coded protein of genes of interest.

In second aspect of the present invention, a kind of pharmaceutical composition is provided, it comprises:

(A) Itgam and/or its coded sequence of treatment effective dose; And

(B) acceptable carrier or excipient pharmaceutically or on the immunology.

In a preference, before giving pharmaceutical composition of the present invention, simultaneously or afterwards, have prevention or treatment bacterial infection relevant disease and/or active other active substance of symptom.Said have prevention or treatment bacterial infection relevant disease and/or active other active substance of symptom are selected from: one or more of clinical common antibiotics (comprising beta-lactam (PCs and cephalosporins), aminoglycosides, Tetracyclines, chloromycetin, macrolide, antifungal antibiotic, tuberculosis class antibiotic).

In an embodiment of the invention, Itgam and coded sequence thereof account for 0.001～99.9wt% of pharmaceutical composition gross weight in the said pharmaceutical composition.

In a preference, Itgam and coded sequence thereof account for 1～95wt% of pharmaceutical composition gross weight in the said pharmaceutical composition, are preferably 5～90wt%, more preferably 10～80wt%.

Aspect the 3rd of the present invention, a kind of pharmaceutical composition is provided, it comprises:

(A) Itgam and/or its coded sequence of treatment effective dose;

(B) prevention or treatment bacterial infection relevant disease and/or active other active substance of symptom; And

(C) acceptable carrier or excipient pharmaceutically or on the immunology.

In an embodiment of the invention, said have prevention or treatment bacterial infection relevant disease and/or active other active substance of symptom are clinical common antibiotics.

In a preference, said clinical common antibiotics be selected from down the group in one or more: beta-lactam (PCs and cephalosporins), aminoglycosides, Tetracyclines, chloromycetin, macrolide, antifungal antibiotic, tuberculosis class antibiotic.

In fourth aspect of the present invention, the method for a kind of prevention or treatment bacterial infection relevant disease and/or symptom is provided, said method comprises: the Itgam and the coded sequence thereof that need the object effective dose of prevention or treatment.

In a preference, said bacterial infection relevant disease and/or symptom are one or more disease and/or symptoms that cause because of bacterial infection that are selected from down group: the excessive generation of inflammatory factor behind the bacterial infection; Endotoxin shock or death; The inflammatory damage of organ; And, multiple organ dysfunction syndrome.

In another preference, said bacterial infection be by be selected from down group one or more are bacterial: escherichia coli, Pseudomonas aeruginosa, staphylococcus aureus, Klebsiella Pneumoniae, Acinetobacter bauamnnii, enterococcus faecalis.

In another preference, said inflammatory factor is to be selected from down one or more that organize: TNF α, IL-6, I type interferon, preferred TNF α, IL-1 or IL-6.

In another preference, said organ is selected from: lung, liver, spleen, brain or kidney, preferred lung, liver or spleen.

In others of the present invention; A kind of transgenic cell, organ, tissue or individuality are provided; Said cell, organ, tissue or individuality obtain through conventional transgene method and have changed Itgam gene of the present invention over to, to stablize high expressed Itgam albumen or polypeptide.

In others of the present invention; A kind of transgenic cell, organ, tissue or individual method of preparing also is provided; Said cell, organ, tissue or individual high expressed Itgam albumen or the polypeptide stablized; Wherein said method comprises Itgam gene of the present invention is changed in cell, organ, tissue or the individuality, and selects and obtain to stablize cell, organ, tissue or the individuality of high expressed Itgam albumen or polypeptide.

In an embodiment of the invention, said method also comprises makes individual and other the individual hybridization of the same race of gained, to obtain to stablize the filial generation of high expressed Itgam albumen or polypeptide.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1: the Itgam deficient mice is more responsive to endotoxin shock.Wherein, Fig. 1 a is an elisa assay, Fig. 1 b rate analysis for survival, and Fig. 1 c is lungs HE dyeing pathological analysis.(" * ", P＜0.01), (contrast is Itgam ^-/-The Itgam that deficient mice is brood ^+/-Mice).

Fig. 2: the Itgam deficient mice is to Gram ^-Infect more responsive and to Gram ⁺Infect more tolerance.Fig. 2 a rate analysis for survival, Fig. 2 b, e are elisa assay, Fig. 2 c, d are the bacterial clone counting.(" * ", P＜0.01), (contrast is Itgam ^-/-The Itgam that deficient mice is brood ^+/-Mice).

Fig. 3: the macrophage of Itgam defective produces strong reaction to the TLR ligand stimulation.Fig. 3 a is an elisa assay, and Fig. 3 b and c analyze for Western Blot.(contrast is Itgam ^-/-The Itgam that deficient mice is brood ^+/-Mouse macrophage).

Fig. 4: Itgam crosses expression can suppress the generation of macrophage inflammatory cytokine.Fig. 4 is elisa assay (" * ", P＜0.05; " * * ", P＜0.01), (contrast is the empty carrier plasmid);

Fig. 5: Itgam blocking-up can promotion TLR signal and inflammatory cytokine produce.Fig. 5 a is an elisa assay, and Fig. 5 b analyzes for Western Blot.(" * ", P＜0.05; " * * ", P＜0.01) (contrast is IgG).

Embodiment

The inventor is through a large amount of research and animal model test, finds Itgam in bacterial infection disease, the generation of the inflammatory factor that can effectively suppress to be caused behind the bacterial infection, improves the organ dysfunction state, improves patient's survival rate.At this, the inventor has accomplished the present invention on the basis.

Particularly; Carry out the focus that applied research is inflammation molecular biology and RESEARCH ON CELL-BIOLOGY to the inflammation related gene; Prevention and treatment that the nucleotide and the protein of inflammation suppressor gene is applied to inflammation are the effective technologies of manual intervention bacterial infection; Therefore no matter be, or inflammation gene therapy aspect relatively all have application prospect extensively at functional genome research.

The inventor is through discovering: the Itgam defective obviously promotes inflammatory reaction.At LPS, Gram ^-And Gram ⁺Induce in the model of endotoxin shock, observe the Itgam defective and can significantly promote preceding inflammatory cytokine and I type interferon to produce, the Itgam deficient mice is more obvious to the inductive lung injury of LPS, to Gram ^-Bacterial infection is more responsive, and removes Gram ⁺The antibacterial ability is then stronger, and prompting Itgam possibly possess the application prospect of treating diseases associated with inflammation such as endotoxin shock.Thus, the invention provides the Itgam anti-inflammatory molecular is applied to the prevention of bacterial infection disease and method and the strategy in the treatment, particularly can be used for endotoxin shock, infect the lungs and the hepar damnification that are caused.

The present invention is directed to novel anti-inflammatory molecular Itgam with antiinflammatory action; Generation to the inflammatory factor of macrophage in the immunocyte under the microbe composition effect is studied, and has verified treatment and the protective effect to the endotoxin shock animals of the sequence of using this molecule.

The experiment proof:

1) the Itgam deficient mice is more responsive to the TLR endotoxin shock, produces more preceding inflammatory cytokine and I type interferon, shows more serious lungs and hepar damnification;

2) the Itgam deficient mice produces more preceding inflammatory cytokine and I type interferon to bacterial infection, to Gram ^-Antibacterial more infects sensitivity, and to Gram ⁺Infect more tolerance;

3) macrophage of Itgam defective produces stronger signal transduction reaction, more preceding inflammatory cytokine and I type interferon;

4) Itgam suppresses the generation of macrophage signal transduction and inflammatory factor;

5) blocking-up Itgam then strengthens macrophage signal transduction and inflammatory factor.

The present invention is directed to the different nucleotide and the protein of Itgam gene, in order to expression and the endotoxin shock due to the bacterial infection and the hepar damnification of the inflammation-inhibiting factor.These inventions confirm that associated products of Itgam are expected to become the effective means that treatment and prevention of bacterial sexuality are dyed.

Itgam albumen (polypeptide)

As used herein; Term " Itgam (polypeptide) ", " Itgam albumen (polypeptide) ", " Itgam ", " Itgam ", " CD11b " interchangeable use; The sequence that is meant SEQ ID NO:1 (is GenBank No.NM_001145808; Homo sapiens source) coded protein or its homologous protein (for example GenBank No.NM_008401, mice source), or these protein have the variation or the modified forms of antiinflammatory action.For example, said Itgam albumen can be selected from: (a) aminoacid sequence of SEQ ID NO:2; Or (b) in the aminoacid sequence that (a) limits through replacing, lack or adding one or several aminoacid and have and suppress the active of inflammatory factor by (a) deutero-protein or polypeptide.

Protein of the present invention or polypeptide can be the products of natural purification, or the product of chemosynthesis, or use recombinant technique from protokaryon or eucaryon host (for example, antibacterial, yeast, higher mammal, insecticide and mammalian cell), to produce.Itgam albumen or polypeptide are preferably by people Itgam gene or its homologous genes or family gene coding among the present invention.

The variant form of protein of the present invention or polypeptide comprises (but being not limited to): one or more (it is individual to be generally 1-50; Preferably 1-30, more preferably 1-20,1-10 best; For example 1,2,3,4,5,6,7,8,9 or 10) amino acid whose disappearance, insertion and/or replacement; And at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) aminoacid.For example, in the art, when replacing, can not change the function of protein or polypeptide usually with the close or similar aminoacid of performance.Again such as; Add the function that or several aminoacid can not change protein or polypeptide usually yet at C-terminal and/or N-terminal, Itgam protein for example of the present invention or polypeptide can comprise or not comprise initial methionine residues and still have the activity that suppresses inflammatory factor.

Can adopt radiation or be exposed to mutagenic agent and get off to produce random mutagenesis, also can obtain protein or polypeptide in above-mentioned (b) through direct mutagenesis method or other known Protocols in Molecular Biology.The coded sequence of code for said proteins capable of using or polypeptide makes up transgenic animal, and observe these transgenic animal to the resistance of inflammation whether to some extent improvement screen and differentiate gained protein or polypeptide.

The host used according to the recombinant production scheme, protein of the present invention or polypeptide can be glycosylated, maybe can be nonglycosylated.This term also comprises proteic active fragment of Itgam and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with coded albumen of the sequence of Itgam albumen coded sequence hybridization and polypeptide or the albumen that utilizes the proteic antiserum of anti-Itgam to obtain.The present invention also can use other polypeptide, as comprises Itgam albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of Itgam.Usually, this fragment have the Itgam protein sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

The Itgam protein coding gene

As used herein; Term " Itgam gene " or " Itgam albumen coded sequence " interchangeable use; All be meant a kind of sequence of encode Itgam albumen of the present invention or polypeptide; It can be the sequence of SEQ ID NO:1, under stringent condition with the molecule of SEQ ID NO:1 sequence hybridization or with the homologous family gene molecule of above-mentioned numberator height, said expression of gene has certain inhibitory action to the generation and the influence of inflammatory factor.Itgam gene of the present invention can be selected from: (i) sequence of SEQ ID NO:1; Or (ii) under stringent condition with the sequence hybridization that (i) limits and have the active molecule of the inflammatory factor of inhibition.

As used herein, term " stringent condition " is meant: (1) than hybridization under LIS and the higher temperature and eluting, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturant, like 50% (v/v) Methanamide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only the homogeny between two sequences at least 50%, preferred more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, be more preferably 95% and just hybridize when above.For example, said sequence can be the complementary series of the sequence that limits in (a).

Itgam gene nucleotide full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For the pcr amplification method; Can be disclosed according to the present invention about nucleotide sequence; Especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of conventional method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence than long time, usually need carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

Should understand; Itgam gene of the present invention is preferably available from the people; Available from (as having more than 50% of other animal with people Itgam gene height homology; Preferred more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more preferably more than 85% as 85%, 90%, 95% even 98% sequence homogeny) other gene also within the equivalency range that the present invention preferably considers.The Method and kit for of aligned sequences homogeny also is that this area is known, like BLAST.

Carrier, host and transgenic animal

The invention still further relates to the carrier that comprises the Itgam gene, and with the host cell of this carrier through the genetic engineering generation, and pass through the transgenic animal that transgenic obtains high expressed Itgam.

Recombinant DNA technology (Science, 1984 through routine; 224:1431), coded sequence of the present invention capable of using can be used to express or produce the Itgam albumen of reorganization.In general following steps are arranged:

(1) with the proteic polynucleotide of coding Itgam of the present invention (or variant), or with recombinant expression carrier conversion that contains these polynucleotide or transduction proper host cell;

(2) host cell of in proper culture medium, cultivating; With

(3) separation from culture medium or cell, protein purification or polypeptide.

Among the present invention, term " carrier " and " recombinant expression carrier " interchangeable use refer to bacterial plasmid well known in the art, phage, yeast plasmid, zooblast virus, mammalian cell is viral or other carrier.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain origin of replication, promoter, marker gene and translation control element usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains Itgam coded sequence and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc.Described DNA sequence can effectively be connected on the suitable promoter in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome binding site and the transcription terminator that translation initiation is used.Preferred pcDNA3.1 carrier, pIRES2-EGFP carrier, the Adeno-X expression system of using among the present invention.

In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate dihydrofolate reductase, neomycin resistance and the green fluorescent protein (GFP) of usefulness like eukaryotic cell, or be used for colibacillary tetracycline or amicillin resistance.

Comprise the carrier of above-mentioned suitable DNA sequence and suitable promoter or control sequence, can be used to transform appropriate host cell, so that it can marking protein or polypeptide.Host cell can be a prokaryotic cell, like bacterial cell; Or eukaryotic cell such as low, like yeast cells; Or higher eucaryotic cells, like zooblast.Representative example has: escherichia coli, streptomyces, Agrobacterium; Fungal cell such as yeast; Zooblast etc.In the present invention, preferably adopt escherichia coli bacterial cell, mouse macrophage as host cell.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhancer is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promoter transcribing with enhancing gene usually.Persons skilled in the art all know how to select appropriate carriers, promoter, enhancer and host cell.

Term among the present invention " transgenic animal " or " transformed animal " interchangeable use all refer to cell, organ, tissue or the individuality that Itgam gene of the present invention is also stablized high expressed Itgam albumen or polypeptide that change over to through conventional transgene method acquisition.

The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cell membrane.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) is technological with other various LCs and the combination of these methods.

Pharmaceutical composition

The present invention also provides a kind of pharmaceutical composition, and described compositions contains Itgam albumen of the present invention or its coded sequence of effective dose, and pharmaceutically acceptable carrier.

In preferable embodiment, said pharmaceutical composition can be used for treatment and known in the state of the art the treatment or the prevention of bacterial infectious disease, the for example excessive generation of inflammatory factor behind the bacterial infection; Endotoxin shock or death; The inflammatory damage of organ; Multiple organ dysfunction syndrome.

As used herein, term " contain " or " comprising " comprised " comprising ", " basically by ... constitute " and " by ... constitute ".

As used herein, term " pharmaceutically acceptable " composition is applicable to people and/or animal and does not have excessive bad side reaction (like toxicity, stimulation and allergy), the material of rational benefit/risk ratio is promptly arranged.

As used herein, term " effective dose " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.

As used herein, term " pharmaceutically acceptable carrier " refers to be used for the carrier of therapeutic agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art." Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences, Mack Pub.Co. can find discussing fully about pharmaceutically acceptable excipient in N.J.1991).

Acceptable carrier can contain liquid on combination of Chinese medicine is learned, like water, saline, glycerol and ethanol.In addition, also possibly there is complementary material in these carriers, like filler, disintegrating agent, lubricant, fluidizer, effervescent, wetting agent or emulsifying agent, correctives, pH buffer substance etc.Usually, can these materials be formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.

Itgam albumen or its coded sequence effective ingredient account for 0.001～99.9wt% of composition total weight in the compositions of the present invention; Being preferably 1～95wt% of composition total weight, more preferably is 5～90wt%, more preferably 10～80wt%.Surplus is materials such as pharmaceutically acceptable carrier and other additive.

As used herein, term " unit dosage forms " is meant for taking convenience, becomes single to take required dosage form preparation of compositions of the present invention, includes but not limited to various solid formulation (like tablet), liquid agent, capsule, slow releasing agent.

In another preferred implementation of the present invention, said compositions is unit dosage forms or multi-pharmaceutics, and wherein the content of Itgam albumen or its coded sequence is 0.01～2000mg/ agent, preferred 0.1～1500mg/ agent, more preferably 1～1000mg/ agent.In another preference of the present invention, use 1～6 dose of compositions of the present invention every day, preferably use 1～3 dose; Most preferred, the dosage of taking every day is 1 dose.

The effective dose that should be understood that used Itgam albumen or its coded sequence can change with the order of severity of the object of waiting to use or treat.Concrete condition decides according to the individual instances (for example object body weight, age, health, the required effect that reaches) of object, and this is in the scope that skilled practitioners can be judged.

Compositions of the present invention can be solid-state (like granule, tablet, lyophilized powder, suppository, capsule, sublingual lozenge) or liquid (like oral liquid) or other suitable shape.Route of administration can adopt: (1) is naked DNA or protein injection directly; (2) cDNA, mRNA and the protein with Itgam is connected with transferrins/poly-l-lysine complex, to strengthen its biological effect; (3) cDNA, mRNA and protein and positively charged lipid form complex, to overcome the difficulty of passing through cell membrane due to the phosphoric acid skeleton negative charge; (4) get into cell with mediation behind liposome cDNA, mRNA and the protein, not only helped macromolecular smooth entering but also avoided the hydrolysis of the various enzymes in extracellular; (5) cDNA, mRNA and protein combine to make its endochylema retention time to increase by 10 times with cholesterol; (6) transport cDNA, mRNA and protein with immunoliposome and can make its unitransport to target tissue and target cell; (7) also can preferably the Itgam related drugs be written in the target cell for reprinting cell (like fibroblast) cDNA, mRNA and protein in-vitro transfection; (8) electricity punching (electroporation) promptly imports target cell by means of electric current with cDNA, mRNA and protein.

In addition; Also can contain other active substance that is useful on improvement and treatment bacterial infection disease in the compositions of the present invention, described other active substance is selected from down group: one or more of clinical common antibiotics (comprising beta-lactam (PCs and cephalosporins), aminoglycosides, Tetracyclines, chloromycetin, macrolide, antifungal antibiotic, tuberculosis class antibiotic).

The relevant nucleotide of Itgam of the present invention and pharmaceutical grade protein each other can Combined application, can also unite with other medicines and treatment means, are used for the prevention and the treatment of bacterial infection disease.

Advantage of the present invention

1. the present invention has disclosed the new function of Itgam and coded sequence thereof, the liver function of the expression of the inflammatory factor that promptly suppresses bacterial infection and caused, protection endotoxin shock body, improves the survival rate of infected individuals;

2. the invention provides a kind of expression, protection endotoxin shock body of the inflammatory factor that can effectively suppress bacterial infection and caused liver function, improve the newtype drug of the survival rate of infected individuals.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook " molecular cloning: lab guide " (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.

Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

Antibody among the hereinafter ELISA is all available from R&D company.

Embodiment 1:Itgam defective produces facilitation to the mice endotoxin shock

At first adopt 15mg/kg LPS (to claim lipopolysaccharide again; Be the part of TLR4, available from Sigma company), 20mg/kg Poly (I:C) (TLR3 part; Available from Sigma company); Behind 20mg/kg CpG OGN (the TLR9 part is given birth to the worker available from Shanghai) lumbar injection Itgam deficient mice and control mice (available from the U.S. Jaxson laboratory) different time, collect serum; Detect inflammatory factor protein level (measure TNF α and IL-6 after 2 hours, measure IFN-β and IP-10 after 4 hours) through ELISA (used kit is available from R&D company); Collect lungs, and section does HE dyeing, observe the micro-pathological change (primary part observation inflammatory cell infiltration, erythrocyte ooze out etc.) of lungs, after observing LPS simultaneously and stimulating, the mice survival rate.

The result of elisa assay is respectively shown in Fig. 1 a.The result shows: the Itgam defective promotes the generation of mice inflammatory factor TNFalpha, IL-6, IFN-β and IP-10.

The mice survival rate is shown in Fig. 1 b: the Itgam deficient mice is more early dead to the inductive endotoxin shock of LPS, and is promptly more responsive.

The painted result of HE is shown in Fig. 1 c: the Itgam deficient mice shows more serious lung injury in endotoxin shock.

This instance shows: the Itgam defective promotes the mice endotoxin shock.

Embodiment 2:Itgam defective makes mice to Gram ^- Bacterial infection is more early dead, in the serum in higher, the blood of the generation of inflammatory factor amount of bacteria more.The Itgam defective makes mice remove Gram ⁺ The antibacterial ability strengthens

At first adopt 1 * 10 ⁸Individual dust Xi Shi escherichia coli (E.coli O111:B4 is available from CCTCC) lumbar injection Itgam defective and control mice are got blood at the different time eyeball.Detect inflammatory factor TNF α, IL-6 and IFN-β output in (used kit is available from R&D company) serum with ELISA, bacterial cultivation detects dust Xi Shi escherichia coli quantity (CFU) in the blood, and dynamic observes the survival rate of mice.

The mice survival rate is shown in Fig. 2 a after infecting dust Xi Shi escherichia coli.The result shows: the Itgam deficient mice is more early dead.

To the elisa assay result of inflammatory factor TNF α in the serum and IL-6 output shown in Fig. 2 b.The result shows: the Itgam deficient mice produces higher inflammatory factor TNF α, IL-6 and IFN-β after infecting dust Xi Shi escherichia coli.

To the dust Xi Shi escherichia coli quantity in the blood shown in Fig. 2 c.The result shows: dust Xi Shi escherichia coli have the capable diffusion of stronger blood in Itgam deficient mice body.This result shows: the expression of Itgam can suppress the generation of inflammatory factor in the serum.

Tail vein injection 1 * 10 then ⁴With 1 * 10 ⁵Individual Listerella (Listerial monocytogenes is available from CCTCC) obtained mouse liver and spleen after 2 days, counted the bacterial population of each internal organs; Simultaneously collect serum after 1 day and 2 days and measure cytokine production with ELISA at bacterial infection.

Bacterial number is shown in Fig. 2 d in mouse liver and the spleen behind the infection Listerella, and inflammatory cytokine levels is shown in Fig. 2 e in the serum.The result shows: the interior Listerella of Itgam deficient mice liver and spleen quantity still less, TNF α and IL-6 output are higher in the serum.

This instance shows: the Itgam deficient mice is to Gram ^-Bacterial infection is more responsive, and removes Gram ⁺The antibacterial ability then strengthens.

The mice source peritoneal macrophage of embodiment 3:Itgam defective produces more inflammatory factor

Give Itgam deficient mice lumbar injection 1ml meat soup (available from Sigma), obtain macrophage with PBS flushing abdominal cavity after three days.

Use 100ng/ml LPS then respectively, 1 μ M CpG ODN, 10 μ g/ml Poly (I:C), 10 μ g/mlpoly (I:C) handle peritoneal macrophage (1 * 10 ⁵Individual cell/ml RPMI 1640 culture medium).Collecting cell is ELISA (used kit is available from R&D company) and is measured cytokine production (measure TNF α and IL-6 after 8 hours, measure IFN-β after 12 hours) analysis.

Collect whole-cell protein simultaneously and detect the activation of TLRs signal transduction with Western Blot.

The influence that the Itgam defective produces the Turnover of Mouse Peritoneal Macrophages inflammatory factor shown in Fig. 3 a, to the influence of TLRs signal transduction shown in figure b and c.The result shows: Itgam deficient mice peritoneal macrophage produces higher inflammatory factor after receiving the TLR ligand stimulation; The activation of synchronous signal transduction molecule is strong (being mainly reflected in NF-κ B and IRF3) more.

This result shows: the Itgam defective promotes the generation of the peritoneal macrophage inflammatory factor in mice source.

Embodiment 4:CD11b (Itgam) crosses the expression inhibiting Turnover of Mouse Peritoneal Macrophages and produces inflammatory cytokine

Distinguish transfection control plasmid (empty carrier plasmid) then, wild type CD11b and continuous activation type CD11b plasmid are (according to bibliographical information Susannah, E. etc.; Biochem.Biophys.Res.Commun.2005; The activated form mutant of 337:142-148 design), use 100ng/ml LPS after 48 hours, 1 μ M CpG ODN; 10 μ g/ml Poly (I:C), 10 μ g/ml poly (I:C) handle peritoneal macrophage (1 * 10 ⁵Individual cell/ml RPMI1640 culture medium).Collecting cell is ELISA (used kit is available from R&D company) and is measured cytokine production (measure TNF α and IL-6 after 8 hours, measure IFN-β after 12 hours) analysis.

CD11b crosses influence that expression produces the Turnover of Mouse Peritoneal Macrophages inflammatory factor shown in Fig. 4 a.The result shows: cross expression wild type CD11b and can suppress Turnover of Mouse Peritoneal Macrophages generation inflammatory factor, the depression effect of continuous activation type CD11b is stronger.

This result shows: the peritoneal macrophage that activatory CD11b suppresses the mice source produces inflammatory factor.

Embodiment 5:CD11b (Itgam) blocking-up promotes Turnover of Mouse Peritoneal Macrophages to produce inflammatory cytokine

Give wild-type mice lumbar injection 1ml meat soup (available from Sigma), obtain macrophage with PBS flushing abdominal cavity after three days.

With CD11b blocking-up type antibody (available from eBioscience, 10 μ g/ml) pretreatment wild type macrophage, blocking-up CD11b activation.Use 100ng/ml LPS then, 1 μ M CpG ODN, 10 μ g/ml Poly (I:C), 10 μ g/ml poly (I:C) handle peritoneal macrophage (1 * 10 ⁵Individual cell/ml RPMI 1640 culture medium).Obtain wild-type mice intraperitoneal injection LPS (available from Sigma company, 15mg/kg) with LPS instance 3.Collecting cell is ELISA (used kit is available from R&D company) and is measured cytokine production (measure TNF α and IL-6 after 8 hours, measure IFN-β after 12 hours) analysis.

Blocking-up CD11b activation produces the inflammatory cytokine influence shown in Fig. 5 a to macrophage, and blocking-up CD11b activation influences shown in Fig. 5 b macrophage TLRs signal transduction.The result shows: blocking-up CD11b activation promotion wild type macrophage produces inflammatory cytokine, promotes TLRs signal transduction (being mainly reflected in NF-κ B and IRF3).

All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims

1.Itgam or its coded sequence is used for preventing and/or treating the purposes of the medicine of bacterial infection relevant disease and/or symptom in preparation.

2. purposes as claimed in claim 1 is characterized in that, said bacterial infection relevant disease and/or symptom are to be selected from down one or more that organize: the excessive generation of inflammatory factor behind the bacterial infection; Endotoxin shock or death; The inflammatory damage of organ; And, multiple organ dysfunction syndrome.

3. purposes as claimed in claim 1; It is characterized in that, said bacterial infection be by be selected from down group one or more are bacterial: escherichia coli, Pseudomonas aeruginosa, staphylococcus aureus, Klebsiella Pneumoniae, Acinetobacter bauamnnii, enterococcus faecalis.

4. purposes as claimed in claim 1 is characterized in that, said Itgam is selected from:

(a) aminoacid sequence of SEQ ID NO:2;

5. purposes as claimed in claim 1 is characterized in that, the encoding gene of said Itgam is selected from:

(i) sequence of SEQ ID NO:1; Or

6. purposes as claimed in claim 1; It is characterized in that the medication of said medicine is selected from: directly naked DNA injection, liposome dna direct injection, gold encapsulate that DNA particle gun blast technique, breeding unsoundness antibacterial carry the DNA method, replication defective adenoviral carries target DNA method or the coded protein of genes of interest.

7. pharmaceutical composition, it comprises:

(A) Itgam and/or its coded sequence of treatment effective dose; And

(B) acceptable carrier or excipient pharmaceutically or on the immunology.

8. pharmaceutical composition as claimed in claim 7 is characterized in that Itgam and coded sequence thereof account for 0.001～99.9wt% of pharmaceutical composition gross weight in the said pharmaceutical composition.

9. pharmaceutical composition, it comprises:

(A) Itgam and/or its coded sequence of treatment effective dose;

(C) acceptable carrier or excipient pharmaceutically or on the immunology.

10. pharmaceutical composition as claimed in claim 9 is characterized in that, said have prevention or treatment bacterial infection relevant disease and/or active other active substance of symptom are clinical common antibiotics.