CN1798839A - Compositions and methods for diagnosing and treating asthma or other allergic or inflammatory diseases - Google Patents

Abstract

The present invention provides compositions and methods useful for detecting or treating asthma or other allergic or inflammatory diseases. In one aspect, the methods of the present invention include inhibiting the activity or expression of a component of an arginine metabolic pathway in tissues affected by asthma or other allergic or inflammatory diseases. In many embodiments, the component being inhibited is a cationic amino acid transporter, an arginase, or a component downstream of the arg nase. In many other embodiments, the activity or expression of the component is i bited by an agent that binds to the component. In still many other embodiments, the activity or expression of the component is inhibited by an agent that binds a polynucleotide encoding the component.

Description

Be used to diagnose and treat the composition and the method for asthma or other allergy or inflammatory phlegm disease

Technical field

The present invention relates to be used to diagnose or treat the composition and the method for asthma or other allergy or inflammatory phlegm disease.

Background

Asthma is the chronic inflammatory diseases phlegm disease of respiratory tract, it is characterized in that the outbreak repeatedly and the respiratory tract hyperergy (AHR) of reversible respiratory tract obstruction.General clinical manifestation comprises shortness of breath, pants, cough and uncomfortable in chest, and it can become dangerous life or fatal.Although existing therapy concentrates on mitigation symptoms bronchospasm and pneumonia, more and more understand long-term respiratory tract and reinvent the effect of the lung that in asthma, quickens in worsening.Respiratory tract is reinvented and is referred to many pathological characteristicses, comprises epithelium unstriated muscle and myofibroblast hyperplasia and/or metaplasia, goes up subcutaneous fibrosis and apposition.For mortality asthma, these processes cause respiratory tract about 300% thicken at most jointly.Although obtained huge advance made in illustrating the physiopathology of asthma, popular, the M ﹠ M of this disease have increased in 20 years in the past.Nineteen ninety-five only just has near 1,800,000 routine emergency rooms in the U.S. and goes to a doctor, 466,000 examples be in hospital and 5,429 routine death directly owing to asthma.

It has been generally acknowledged that atopic asthma is by airborne allergenic unsuitable inflammatory reaction is caused.The lung of asthmatic patient shows a large amount of infiltrations of lymphocyte, mastocyte and eosinophilic granulocyte.A large amount of verified these immunne responses of evidence are by expressing T _HThe CD4 of 2 cytokines spectrums (profile) ⁺The T cellular driven.A kind of mouse model of asthma comprises the sensitization of animal to ovalbumin (OVA), sends OVA then in the tracheae and attacks.This method produces T in mouse lung _H2 immune responses are also simulated four kinds of main pathology physiologic response seeing in human asthma, comprise SERUM IgE (atopy), hypereosinophilic syndrome, Polyblennia and the AHR of rise.The cytokine IL-13 of basophilic granulocyte, mastocyte, activated T cell and NK cell expressing plays an important role in the inflammatory response at OVA in mouse lung.Directly lung instillation mouse IL-13 causes the pathology that all four kinds of asthma are relevant, and on the contrary, and OVA-is blocked in the existence of solubility IL-13 antagonist (sIL-13R α 2-Fc) fully, and to attack inductive goblet cell mucus synthetic and at the AHR of vagusstoff.Thereby the IL-13 Mediated Signal Transduction enough causes the pathologic, physiologic phenotype that all four kinds of asthma are relevant and is mucous too much secretion and induce AHR required in the mouse model.

Low affinity marriage chain IL-13R α 1 of biological activity IL-13 specific combination and the high-affinity polymer complex body of being made up of IL-13R α 1 and IL-4R, described IL-4R is the common component of IL-4 signal transduction complex body.The high-affinity complex body is expressed in the various kinds of cell type, and described cellular type comprises monocyte-scavenger cell colony, basophilic granulocyte, eosinophilic granulocyte, mastocyte, endotheliocyte, inoblast, airway smooth muscle and airway epithelial cell.The connection of the IL-13 mediation of functional receptor complex body causes the activation of JAK1 and JAK2 or Tyk-2 kinases and the proteic dependence phosphorylation of IRS1/2.The raising of the activation triggers transcriptional activator Stat6 of IL-13 approach cascade, phosphorylation and final nuclear translocation.Many Physiologic Studieses show that lung OVA attacks and can not cause main diseases relevant phenotype of science, comprise Stat6-/-mouse that amorphs isozygotys in eosinophilic granulocyte infiltration, Polyblennia and respiratory tract hyperergy.Nearest genetics research has shown between specific people's allelotrope of IL-13 and signal transduction component thereof and asthma and the atopy chain, shows the keying action of this approach in this human diseases.

IL-13 does not also determine the receptor chain IL-13R α 2 of biological function as yet in conjunction with other of expressing in people and the mouse.Mouse IL-13R α 2 is in conjunction with IL-13, to make it possible to make up strong solubility IL-13 antagonist sIL-13R α 2-Fc than IL-13R α 1 high about 100 times avidity (Kd is 0.5 to 1.2nM).IL-13 forms at schistosomicide inductive hepatic fibrosis and granuloma sIL-13R α 2-Fc to illustrate, tumour immunity monitors as the antagonist in the multiple phlegm disease model, and OVA-attacks the effect in the asthmatic model.

Chronic obstructive pulmonary disease (COPD) is blanket property term, and it is used to describe the main airflow obstruction relevant with pulmonary emphysema and chronic bronchitis.Air bag is weak in the lung causes irreversible injury of lung with breaking to pulmonary emphysema by making.As a result, the loss of elasticity of lung tissue causes taking place collapse of airway and airflow obstruction.Chronic bronchitis is beginning and develop into the inflammatory phlegm disease of bigger respiratory tract gradually in the less respiratory tract in lung.Mucus in its increase respiratory tract and the infectation of bacteria in the segmental bronchus, they can hinder air-flow again.

COPD attacks tens million of Americans and is the serious health problem of the U.S..Prevalence rate investigation in 1998 shows that 300 ten thousand Americans have been diagnosed as the invasion and attack that pulmonary emphysema and 900 ten thousand are subjected to chronic bronchitis.COPD was the 4th major causes of death of the U.S. in 1998, and caused 112,584 examples dead in 1998.668,362 examples that COPD in 1998 also the accounts for estimation case of leaving hospital.

Current treatment to asthma and COPD comprises uses bronchodilator, reflunomide and leukotriene inhibitors.These treatments have identical therapeutic goal, promptly bronchiectasis, reduce inflammation and promote expectoration.Yet many these treatments lose validity after comprising undesirable side effect and using for some time.In addition, only limited treatment is intervened reagent and be can be used for the respiratory tract remodeling process that reduces to take place in the asthmatic patient.Therefore, still need to increase the understanding on molecular level, need also to identify that new therapeutic strategy is to resist these complicated phlegm diseases asthma and COPD.

Summary of the invention

The present invention has identified the several genes of comparing differential expression in the asthma lung tissue with non-asthma lung tissue.Institute's genes identified comprises the member of arginine pathways metabolism, as cationic amino acid transporter albumen 2 genes (CAT2) and arginase I type gene (ARG1).These genes are potential drug targets of treatment asthma or other allergy or inflammatory diseases.

On the one hand, the invention provides the method for treatment allergy or inflammatory phlegm disease.These methods comprise the reagent to the administration treatment significant quantity of suffering from allergy or inflammatory diseases, and wherein said reagent suppresses component active of arginine pathways metabolism in the tissue of described affect or expresses.Described downtrod component is not nitric oxide synthase (NOS).In many embodiments, downtrod component is arginase (for example, I type arginase) or its downstream albumen.The proteic example in downstream includes, but not limited to ornithine decarboxylase, ornithine aminotransferase, ornithine transcarbamylase, spermidine synthase and spermine synthase.In an example, can also suppress S adenosylmethionine decarboxylase, it participates in the biosynthesizing of polyamines.In another embodiment, downtrod component is cationic amino acid transporter albumen (for example, cationic amino acid transporter albumen 2).

Be applicable to that allergy of the present invention or inflammatory phlegm disease include, but not limited to that asthma, respiratory tract hyperergy, chronic respiratory are reinvented, chronic obstructive pulmonary disease (COPD) and sacroiliitis.Also can treat with dysfunction in the arginine metabolism or unusual relevant other diseases by the present invention.In many embodiments, allergy or inflammatory phlegm are sick for breathing the phlegm disease.Use the active or expression of arginine pathways metabolism component in the therapeutical agent inhibition lung tissue of the present invention, thus improvement or the elimination syndrome relevant with described disease.

Being suitable for therapeutical agent of the present invention includes but not limited to, can by RNA interfere or antisense mechanism suppress polynucleotide that the target component expresses, with the inhibitor of the biological function of the antibody of target component reaction, target component or can be (for example in conjunction with the polynucleotide of the target component or the target component of encoding, mRNA or genome sequence comprise the adjusting sequence of 3 ' or 5 ' untranslated) other conditioning agents.In many embodiments, suppress active or expression in transcriptional level, post-transcriptional level, translation skill or translation back level.In many other embodiments, inhibitor the activity of target component or expression are compared with initial activity or expression level be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than.

In one embodiment, therapeutical agent of the present invention coding or contain siRNA sequence, other genes of the downstream component of the arginase of perhaps encoding at CAT2, ARG1.In another embodiment, therapeutical agent is expressed from gene therapy vector.In many examples, gene therapy vector is under the control of tissue or cell-specific promotor.In an example, promotor is that lung is special.The example of the promotor that lung is special includes, but not limited to the special tensio-active agent protein B gene promoter of pulmonary epithelial cells and carat draws the promotor CC10 of (Clara) cell-specific.In another example, promotor is that monocyte or scavenger cell are special.The example of the promotor that scavenger cell is special includes, but not limited to people such as the proximal promoter of people's ethanoyl-ldl receptor (SRA) gene and Ross, J.Biol.Chem., 273:6662-6669, those promotors of describing in 1998.

In a further embodiment, therapeutical agent of the present invention is selected from Methionin, poly-L-Lysine, poly--L-arginine or can suppresses proteic other cationic polypeptides of cationic amino acid transporter.In another embodiment, therapeutical agent is an alpha-difluoromethyl ornithine, and it suppresses the function of ornithine decarboxylase.In a further embodiment, therapeutical agent is that IL-13 antagonist or antagonism resist-IL-13 antibody.In an example, therapeutical agent is a solubility IL-13 acceptor.

Can prepare therapeutical agent of the present invention with compatible with the route of administration of their plans.The example of route of administration includes, but not limited to parenteral, through intestines and topical application.For example, therapeutical agent of the present invention can be through intracutaneous, upper epidermis (epicutaneous), suction, per os, rectum, intravenously, intra-arterial, intramuscular, subcutaneous, intracutaneous, through skin, stride mucous membrane or other suitable approach are used.In one embodiment, by sucking the administering therapeutic agent.For example, therapeutical agent can since send from the form of the aerosol spray that contains the pressure-vessel of suitable propelling agent (for example, carbonic acid gas) or divider or atomizer.

In one embodiment, the Mammals of being treated is the people who suffers from asthma or other allergy or inflammatory diseases.

On the other hand, the invention provides the method that can be used for identifying or assessing the medicine of treatment asthma or other allergy or inflammatory diseases.Described method comprises candidate molecules is contacted with the tissue that is subjected to the invasion and attack of asthma or another kind of allergy or inflammatory diseases, and determines whether described candidate molecules can improve or eliminate disease syndrome or the phenotype in this tissue.Candidate molecules suppresses the active or expression of the non--NOS component of arginine pathways metabolism in the described tissue.Exemplary non--the NOS component includes, but not limited to arginase or cationic amino acid transporter albumen.Being suitable for use in tissue of the present invention comprises, but be not limited to, tissue/cell in the animal model of described disease, from the isolating tissue/cell of the animal model of this phlegm disease, perhaps simulate the cell culture of some aspect (for example, express spectra) of the tissue/cell of affect.In an example, assess the curative effect of candidate molecules by people's clinical trial.

In one embodiment, according to rational drug design alternative or generation candidate molecules based on structure.Identified can with the molecule of the non--NOS component interaction of arginine pathways metabolism.Then these molecules are contacted with the tissue that is subjected to the invasion and attack of asthma or other allergy or inflammatory diseases to determine whether they improve or eliminate a disease syndrome or phenotype.In another embodiment, identify drug candidate with high-throughput screening method or library of compounds.

The present invention has also described the method that is used to detect, diagnose or monitor asthma or other allergy or inflammatory phlegm disease.These methods comprise and detect at least a expression of gene spectrum in the mammalian biological sample, and whether the reference expression profile comparison of this express spectra and this gene suffered from definite this Mammals or dangerously suffer from allergy or inflammatory diseases.In many cases, the non-NOS component of described genes encoding arginine pathways metabolism.

In one embodiment, allergy or inflammatory diseases are asthma or COPD.Described biological sample can be the lung sample.Can also use the sample of mucus, blood or other types.In another embodiment, reference expression profile is the average express spectra of arginine metabolic gene in the tissue of no disease.Reference expression profile can also be the express spectra that is subjected to arginine metabolic gene in the sick tissue of attacking of phlegm.In a further embodiment, the arginine metabolic gene is selected from ARG1 or CAT2.Be used for detecting or diagnosing the material of asthma or other allergy or inflammatory diseases can be included in test kit.

More on the one hand, the invention provides the pharmaceutical composition that can be used for treating asthma or other allergy or inflammatory phlegm disease.Described pharmaceutical composition comprises a kind of reagent of pharmaceutically acceptable carrier and treatment significant quantity, and described reagent can suppress the active or expression of the non-NOS component of arginine pathways metabolism.In many embodiments, described reagent is in conjunction with non-NOS component, the polynucleotide of this non-NOS component of perhaps encoding.In an example, non-NOS component is by ARG1 or CAT2 coding.

According to following detailed, other purposes of the present invention, feature and advantage will become apparent.Although described detailed description and particular instance are pointed out embodiment preferred, but provide just to illustrating, because can obtain many changes and modification in the scope of the invention according to this detailed description, these changes and to modify be conspicuous for those skilled in the art.In addition, these Examples set principle of the present invention and should not expect specifically to illustrate all examples that the present invention is applied to infect, wherein it is useful for technician in the prior art obviously.

The accompanying drawing summary

Provide accompanying drawing to be used to illustrate rather than limit.The application is incorporated herein by reference Fig. 1,6,8 and 9 that the name of submitting on March 4th, 2004 is called the U.S. Patent application (Michael R.Bowman waits the people) of " Compositions and Methods for Diagnosing and TreatingAsthma or other Allergic or Inflammatory Diseases ".

Fig. 1 is graphic representation, and it shows coinduction CAT2 and I type arginase (ARG1) in the Balb/c mouse by allergen (OVA) and reorganization mouse IL-13 (IL-13).In brief, by making the Balb/c mouse to the OVA sensitization, attack by (IT) injection carrier (phosphate-buffered saline (PBS)) or OVA in the 14th and 25 day tracheae at the 0th day peritoneal injection, and at the 28th day results lung.In addition, with the Balb/c mouse that is used to first test with handling 3 days continuously in PBS or the IL-13 tracheae, and at the 72nd hour results lung.As described in embodiment 1, separate total lung RNA and analyze the mRNA expression with GeneChip technology (Affymetrix).The mRNA frequency is expressed as ppm.

Fig. 2 is graphic representation, and it shows by allergen (OVA) and reorganization mouse IL-13 (IL-13) induces ARG1 to express in the Balb/c mouse.In brief, by making the Balb/c mouse to the OVA sensitization, attack by (IT) injection carrier (phosphate-buffered saline (PBS)) or OVA in the 14th and 25 day tracheae at the 0th day peritoneal injection, and at the 28th day results lung.In addition, with the Balb/c mouse that is used to first test with handling 3 days continuously in PBS or the IL-13 tracheae, and at the 72nd hour results lung.As described in embodiment 1, separate total lung RNA and analyze the mRNA expression with GeneChip technology (Affymetrix).The mRNA frequency is expressed as ppm.

Fig. 3 is graphic representation, and it shows to be expressed in by OVA or adenovirus mediated IL-13 induces the ARG1 gene in the Balb/c mouse.In brief, the 0th day to Balb/c mouse tracheae in the inoculation recombinant adenovirus express mouse IL-13 or mouse excretory alkaline phosphatase (SEAP), and at the 3rd day results lung.Handle control mice as what describe among Fig. 1 with PBS, OVA or IL-13.The mRNA frequency is expressed as ppm.

Fig. 4 is graphic representation, and it shows to be expressed in by adenovirus mediated IL-13 induces the ARG1 gene in the C57b1/6 mouse.In brief, the 0th day to C57b1/6 mouse tracheae in the inoculation recombinant adenovirus express mouse IL-13 or mouse excretory alkaline phosphatase (SEAP), and at the 3rd day results lung.The mRNA frequency is expressed as ppm.

Fig. 5 is graphic representation, and it shows that arginine is taken in and is induced by LPS/IL-13 the best among the mouse macrophage clone RAW264.7.In brief, by handling the CAT2 that induced the RAW264.7 scavenger cell to express multiple level in 24 hours with LPS and/or IL-13.As describing among the embodiment 3, arginic transhipment when there is or does not exist competitive L-Methionin in assessment in 3 minute time period, final arginine concentration is 400 μ M.Specific arginine is taken in (CPM/mg protein cleavage thing) and be expressed as the percentage ratio of measuring in unprovoked cell.

Fig. 6 is graphic representation, and it shows in mouse macrophage clone RAW264.7 by lipopolysaccharides (LPS)/IL-13 coinduction CAT2 and ARG1.In brief, the RAW264.7 cell is handled with 10ng/ml IL-13 and/or 1 μ g/ml LPS.Handle after back 24 hours total RNA, and analyze ARG1 and the special mRNA expression of CAT isotype by the TaqMan real-time quantitative RT-PCR that uses as describe among the embodiment 2 from cellular segregation.Use people's such as Fink people such as (, Nat.Medicine, 4:1329-1333,1998) Fink method to count glucose estimator-normalized mRNA frequency of 6-phosphate dehydrogenase (GAPDH) and it is expressed as the copy of GAPDH from threshold cycle.

Fig. 7 is graphic representation, and it shows by 20mM Methionin suppressed the arginine absorption in the RAW264.7 mouse macrophage that LPS/IL-13 handles.In brief, the RAW264.7 cell only being exposed to medium (contrast) or CAT2-inductive condition (LPS/IL-13) 24 hours, is the arginine transport in 3 minute time period of assessment under the 100 μ M conditions in final arginine concentration then.In transport buffer, add competitive CAT inhibitor 20mM Methionin and eliminated all saturable arginine transports in the cell of contrast and LPS/IL-13 processing.

Fig. 8 is graphic representation, and it shows the generation that suppresses urea among the mouse macrophage clone RAW264.7 by 20mM Methionin.In brief, the RAW264.7 scavenger cell only was exposed to medium (contrast) or CAT2-inductive condition (LPS/IL-13) 24 hours, then with described cell balance 2 hours in the arginine transport damping fluid.In the arginine transport damping fluid of the final arginine concentration of 400 μ M competitive L-Methionin exist or not in the presence of incubation after 24 hours, produce as the evaluation urea of describing among the embodiment 4.Urea produces and is expressed as μ g urea in the supernatant liquor/mg lysis albumen.

Fig. 9 is graphic representation, and it shows that 100mM Methionin suppresses carbechal inductive rat tracheae and shrinks.In brief, with rat tracheae explant with medium or contain the medium preincubation 15 to 20 hours of 100mM L-Methionin.Wash tracheae then and when existing or not having 100mM L-Methionin, in Krebs-Henseleit solution, measure contraction.Tension force is calculated as mg tension force/mg tracheae and is expressed as the % maximum collapse (when promptly not having Methionin 10 ^-5The contraction that the M carbechal causes) mean value and standard error.

Figure 10 is graphic representation, and it shows the derived need IL-4 acceptor that ARG1 expresses.In brief, describe as Fig. 1, with IL-4 acceptor gene knock-out mice (IL4R-/-) and IL-4 knock out mice (IL4-/-) to the OVA sensitization, perhaps with PBS or IL-13 processing.Separate total lung RNA and use GeneChip technology (Affymetrix) analysis mRNA to express as describing among the embodiment 1.The mRNA frequency is expressed as ppm.

The tracheae that Figure 11 has compared in CAT-2 knock out mice and the wild-type mice shrinks.GATS2-KO represents the CAT2 knock out mice.

Figure 12 is graphic representation, and it shows that the ovalbumin allergen is attacked back ARG1mRNA expression enhancing in direct instillation rIL-13 of lung or the tracheae.Suppressed 67% inductive Arg1 mRNA and expressed by using IL-13 signal transduction that sIL13R α 2.Fc blocked.Having suppressed allergen inductive mucus with soluble receptors sIL13R α 2.Fc blocking-up IL-13 produces and respiratory tract hyperergy (AHR).

Detailed Description Of The Invention

The present invention relates to composition and method for the diagnosis and treatment of asthma or other allergia or inflammatory disease. On the one hand, method of the present invention comprises the active of arginine metabolism pathway component in the tissue that suppresses to be subjected to asthma or other allergia or inflammatory disease invasion and attack or expresses. In many embodiments, downtrod component is cationic amino acid transporter albumen, arginase, the perhaps downstream component of arginase. Active or the inhibition that express of these components reduces or has eliminated disease syndrome or phenotype in affected tissue. The present invention also provides the method for the identification of the therapeutic agent for the treatment of asthma or other allergia or inflammatory phlegm disease.

In trifle below also more detailed description many aspects of the present invention. These trifles are not for restriction the present invention; These trifles can be applicable to any aspect of the present invention. In this application, unless otherwise noted, " perhaps " refer to " and/or ".

CAT2, ARG1 and inflammatory phlegm are sick

In the mouse of sensitization by tracheae in OVA attack can produce T in lung _H2 immune responses, some physiologic characters of its simulation people allergic asthma. There is important evidence to show the important function of the signal transduction of IL-13 mediation in this animal disease model. Describe after OVA in tracheae attacks or the direct lung of IL-13 instils the variation of transcribing in the mouse lung tissue with oligonucleotide arrays. As shown in Fig. 1-4, the mRNA frequency of CAT2 and/or ARG1 significantly increases when with OVA or IL-13, processing mouse. Equally, coinduction CAT2 and ARG1 (Fig. 5 and 6) in the mouse macrophage RAW264.7 of lipopolysaccharides (LPS) and IL-13 Combined Treatment. The CAT2 that induces and ARG1 express relevant with the increase of arginine transport albumen (Fig. 7), still also with the RAW264.7 cell in the production increase relevant (Fig. 8) of urea, show the activation of arginase approach. Other researchs disclose the competitive inhibitor lysine of using CAT2 can suppress the increase (Fig. 7 and 8) of arginine absorption and urea production. In addition, the tracheae that carbaminoylcholine is induced shrinks the inhibition (Figure 11) of the genetic defect that also is subject to lysine (Fig. 9) or CAT2 gene, shows that further CAT2 participates in the pathologic, physiologic of inflammatory disease. In addition, the derived need IL-4 acceptor of ARG1 expression is illustrated in Figure 10. Finally, use solubility IL13R α 2.Fc and blocked the IL-13 signal transduction, this has suppressed again the ARG1 mrna expression.

Arginine is half-essential amino acid, and it is metabolized to important Molecular regulator. Arginine is transported to VSMC (SMC) by cationic amino acid transporter albumen (CAT) family of albumen, and arginine is metabolised to nitrogen oxide (NO), polyamines or proline therein. Inflammatory mediator, growth factor and hemodynamic educational level are by inducing CAT gene expression to stimulate arginic transhipment in vascular SMC. Inflammatory cytokine also induces the expression of derivable NO synthase (iNOS) to and guide arginine metabolism to become antiproliferative gas NO. On the contrary, circulation machinery stress be blocked iNOS and ODC activity and stimulate arginase I gene expression, thereby instructs arginine metabolism to form L-PROLINE and collagen.

In can not recycling arginic non-hepatic tissue, the CAT2 transport protein of rise provides sufficient substrate arginine for the arginase activity that strengthens. The enhancing of this arginase activity is that these pathogenic courses are found usually in inflammatory disease for the bio-chemical pathway of the pathogenic course key such as fibrillatable, respiratory tract high response, goblet cell hyperplasia, the oxidative stress relevant to Apoptosis and respiratory inflammation. Therefore, suppress arginic CAT2 transhipment and will block the non-extractum hepatis propylhomoserin enzymatic pathway of inducing and do not affect the heparin pumps circulation, described heparin pumps circulates and also utilizes arginine but can recycle arginine as substrate.

The biochemistry of CAT2 and biological property

Nucleotides and the amino acid sequence of people CAT2 (also referred to as SLC7A2) provide respectively in SEQ ID NOS:1 and 2. The nucleotides of mouse CAT2 and amino acid sequence provide respectively in SEQ ID NOS:3 and 4.

Separate people CAT2 cDNA (SEQ ID NO:1) from mankind's intestines cDNA library. The nucleotide sequence of code area is predicted as 658 amino acid whose albumen (SEQ ID NO:2), and it calculates molecular weight is 71,669. Residue due to 91% is identical with the residue of mouse CAT2, so people CAT2 people's homologue of mouse CAT2 seemingly. In rna blot analysis, single (9.0kb) people CAT2 mRNA transcript is present in Various Tissues. Observe the highest expression in skeletal muscle, observe floor level in kidney. Hydropathic profile shows that the albumen of translating has 14 membrane spaning domains according to measuring tool, has three potential N-glycosylation sites on it. People CAT2 gene is allocated in human chromosome 8p21.3-p22.

The analytical table person of good sense CAT2 of genomic organization is comprised of extron and 2 untranslated extrons of most probable of 12 translations. Two kinds of albumen isotype CAT2A of CAT2 gene code and CAT2B, they derive from the alternative splicing (being exon 7 in CAT2A, is exon 6 in CAT2B) of mutual repulsion. The structure of people CAT2 gene structure and people CAT1 is closely related, shows that they belong to common gene family.

In mouse, the CAT2 gene is from being scattered in 5 different promoter transcriptions on 18kb, and this causes because transcription initiation has produced several different CAT2 mRNA isotypes in different promoters. The institute of the expression mouse CAT2 that introduces in front finds and isotype that the distal-most end promoter is close in a organized way with in cellular type, and other 5 ' UTR isotypes have more tissue specificity in their expression. On the books showing utilized some or all of 5 kinds of promoters of inferring in lymphoma cell clone, liver and skeletal muscle. Contain TATA and be rich in (G+C) and the less promoter of TATA and show as and can control mouse CAT2 gene expression. The multiple isotype that someone proposes CAT2 mRNA allows the transcriptional regulatory flexibly of this cationic amino acid transporter GFP.

Because CAT2 plays an important role in NO produces, and NO is the height reactive free radical relevant with the various diseases that comprises cancer, so proposed by suppressing the disease of CAT2 treatment take non-required NO level as feature. For example, the U.S. Patent number 5,866,123 of MacLeod has been described by producing the antibody suppression CAT2 expression of anti-mouse CAT2 albumen. International Patent Application WO 00/44766 has also been described by antisense and antibody technique and has been suppressed the method that CAT2 expresses.

The biochemistry of ARG1 and biological property

The nucleotides of people ARG1 and amino acid sequence provide respectively in SEQ ID NOS:5 and 6. The nucleotides of mouse ARG1 and amino acid sequence provide respectively in SEQ ID NOS:7 and 8.

Arginase catalysis arginine is hydrolyzed into ornithine and urea.Have at least two kinds of Mammals arginase isotypes (I type and II type), they are different in tissue distribution, Subcellular Localization, immune cross-reactivity and physiological function.ARG1 coding I type isotype, its be the kytoplasm enzyme and main in liver the component as ornithine cycle express.ARG1 works as the tripolymer of three same subunit.The hereditary defect of this enzyme causes argininemia, and it is a kind of autosomal recessive phlegm disease that is characterized as hyperammonemia.

Under 2.1-A resolving power, determined the structure (people such as Kanyo, Nature 383:554-55,1996) of tripolymer rat ARG1.The key feature of this structure is the new S-shape oligomerization motif of this proteic C-terminal, and it mediates between about 54% monomer and contacts.The Arg-308 that is positioned at this oligomerization motif makes sat linkage nucleation between a series of monomers and monomer.As determined by gel-filtration and analytical ultracentrifugation, opposite with the tripolymer wild-type enzyme, R308A, R308E and the R308K variant of rat ARG1 exist as monomeric species, and the sudden change that shows Arg-308 has made the dissociated balanced deflection of tripolymer at least 10 ⁵Doubly.These monomer arginase variants have catalytic activity, its K _Cat/ K _mValue is the 13-17% of wild-type enzyme value.The feature of rat ARG1 variant is to reduce with respect to the wild-type enzyme temperature stability.In the crystalline structure of having determined mixture between the arginic boric acid analogue 2 of human arginase and L-(S)-amino-6-boron caproic acid (ABH) under the 1.7A resolving power (people such as Cox, Nat.Struct.Biol.6:1043-1047,1999).It is crucial for the function of people ARG1 that mutation analysis has disclosed amino-acid residue Lys-141, Glu-256 and Gly-235.

Clone people ARG1 gene and determined its structure.People ARG1 gene length is 11.5kb and is divided into 8 exons.Determine cap site by s1 nuclease mapping and primer extension." TATA frame " similar sequence is positioned at 28 base places of cap site upstream, is similar to transcription factor CTF/NF1---and the sequence of " CAAT box " protein-bonded binding site is positioned at 72 base places, upstream.The sequence that has similar glucocorticosteroid response element in 5 ' end region---cAMP response element and enhanser core sequence.People ARG1 gene go up most have to-105 the 5 ' flanking region that is right after and the corresponding sections of rat gene 84% identical.In this zone of people ARG1 gene, by footprint analyzing and testing to a DNA enzyme I-protective belt and several super quick cleavage site.The sequence of the binding site that is similar to CTF/NF1 and also overlapping with the sequence of similar glucocorticosteroid response element is contained in shielded zone.

Many measuring methods have been developed to measure the arginase activity.For example, Greenberg is at Arginase, The Enzymes 4, and P.Boyer, H.Lardy and K.Myrback write, Academic Press, NY has described enzyme assay in 257,1960.People such as Geyer have described the assay method (people such as Geyer, Anal.Biochem.39:412,1971) of arginase in the tissue homogenate thing.Nishibe and Makino have reported the automatic assay method (people such as Nishibe, Anal.Biochem.43:357,1971) of red corpuscle arginase.Micrometering fixed people such as (, Anal.Biochem.35:60,1970) Hirsch-Kolb has also been described.The colorimetric estimation of the blood urea nitrogen that majority method discharges during based on the arginine hydrolysis.

In suffering from the patient of colorectal carcinoma, detected the overexpression (people such as Porembska, Cancer 94:2930-2934,2002) of arginase.The active nitrogen oxide deficiency of the arginase that increases relevant with the respiratory tract hyperergy people such as (, Br.J.Pharmacol.136:391-398,2002) Meurs with allergen inductive cNOS-source.

The active inhibition of arginase

The arginase activity can be by many amino acid, as Xie Ansuan, Methionin, leucine, Isoleucine, proline(Pro) and Threonine, and arginine analog and derivative, suppress as L-canavanine (Can) and L-ornithine (Orn).All these amino acid all work as competitive inhibitor.Catalytic reaction product ornithine of arginase and urea also work as the competitive inhibitor of arginase.The competitive inhibition of product ornithine and urea shows the quick balance chance mechanism of this enzyme.

Arginase activity and the Mn that combines closely ⁺⁺Relevant, by adding looser bonded Mn ⁺⁺Can stimulate Mn ⁺⁺Katalysis, to produce complete activatory enzyme form.Yet although need to add divalent cation with the activation arginase, metal chelator does not suppress this enzyme as EDTA and Citrate trianion.Thereby showing the melts combine site is not easy by solvent approaching.On the other hand, the arginase activity is subjected to borate and suppresses, borate work as S-hyperbolic line I-hyperbolic line uncompetitive inhibitor and to this enzyme and competitive inhibitor L-ornithine (Ki=2+/-0.5mM), L-Methionin (Ki=2.5+/-0.4mM) and the chlorination guanidine (Ki=100+/-10mM) interaction do not influence.Proposed borate very near-earth in conjunction with loose bonded Mn ⁺⁺, and disturb its hormesis.Also propose borate and suppress to come from double-core Mn ⁺⁺Center Mn ⁺⁺Chelating, thereby replace to be responsible for water molecules people such as (, J.Inorg.Biochem.77:163-167,1999) Carvajal to the melts combine of the nucleophillic attack of guanidine radicals carbon.Other experiments also show borate and urea in the mode of mutual repulsion in conjunction with arginase, yet L-ornithine and borate can be simultaneously in conjunction with this enzymes.

After deliberation negatively charged ion, as N ₃ ^-, NO ₂ ^-, BO ₄ ^3-, SCN ^-, CH ₃COO ^-, SO ₄ ^2-, ClO ₄ ^-, H ₂PO ₄ ^-, CN ^-, I ^-, Br ^-, Cl ^-And F ^-To by rats'liver arginase (RLA) to the restraining effect of L-arginine (L-Arg) hydrolysis people such as (, J.Inorg.Biochem.88:397-402,2002) Pethe.In all these negatively charged ion, only F-demonstrates the obvious suppression effect on the mM level.F-to the inhibition of RLA be reversible and to L-Arg in conjunction with being uncompetitive, 7.4 times K of pH (i) value for 1.3+/-0.5mM.This effect depends on pH, this be because when pH when 7 are increased to 10 F-to the IC of RLA ₅₀Value is increased to 19mM from 1.2.Another research has confirmed that fluorochemical is the uncompetitive inhibitor of rats'liver arginase.Fluorochemical caused the substrate of rats'liver arginase to suppress (Tormanen CD, J.Inorg.Biochem.93:243-246,2003) when this research was further illustrated in concentration of substrate and is higher than 4mM.

N (ω)-hydroxyl-L-arginine (L-NOHA) is one of the strongest arginase inhibitor of reporting up to now (Ki=150 μ M).Other products of NO synthase are to arginase or do not have influence (NO ₂ ^-, NO ₃ ^-) or very weak inhibitor (L-Cit and NO).Deriving from the possible hydrolysate (L-Orn and urea) of L-Arg and the possible hydrolysate (L-Cit, hydroxyurea and azanol) of L-NOHA also is non-activity in the concentration that is up to 2mM to arginase.The configuration of L-NOHA is important, because the D-NOHA activity is much lower, and its free-COOH and α-NH ₂Function is that identification extractum hepatis propylhomoserin enzyme is required.L-NOHA also is the active potent inhibitor (IC of the arginase of rat liver homogenate thing and mouse macrophage ₅₀Be respectively 150 and 450 μ M) (people such as Buga, Am.J.Physiol., 271:H1988-1998,1996).

Another kind of special inhibitor N-(ω)-hydroxyl-L-of arginase just-arginine (nor-NOHA) is to the strong about 40 times of (IC of the rejection ratio L-NOHA that is hydrolyzed to the L-ornithine by the catalytic L-arginine of the mouse macrophage that is irritated ₅₀Value is respectively 12+/-5 and 400+/-50 μ M).(IFN-γ+LPS) irritates that mouse macrophage causes the obvious expression of derivable NOS (iNOS) and arginase is active increases with IFN-and lipopolysaccharides.Nor-NOHA also is the potent inhibitor (IC50 value 10+/-3 μ M) of arginase in the scavenger cell that irritates of IFN-γ+LPS-.Compare with NOHA, nor-NOHA is neither the substrate of iNOS neither inhibitor, and becomes interactional useful tool between research arginase and the NOS.(people such as Tenu, Nitric Oxide, 3:427-438,1999).

Other arginase inhibitors that find recent years comprise: N-ω-hydroxyl-D, L-indospicine and 4-hydroxyl amidino groups-D, the L-phenylalanine, their suppress the arginase (people such as Hey in extractum hepatis propylhomoserin enzyme and the pulmonary alveolar macrophage, Br.J.Pharmacol.121:395-400,1997); 2 (S)-amino-6-boron caproic acid (ABH), find its arginase in suppressing inner anal sphincter active aspect than L-NOHA strong about 250 times people such as (, J.Pharmacol.Exp.Ther.290:1409-1416,1999) Baggio; And alpha-difluoromethyl ornithine (DFMO), it is usually as the irreversible inhibitor of special ornithine decarboxylase, but also show the L-arginine by the (people such as Selamnia of arginase mobile restraining effect in the intact cell, Biochem.Pharmacol.55:1241-1245,1998).These arginase inhibitors with the active diseases associated of the arginase that rises in have important pathological physiology and treatment implication.

The alternatives of the active direct inhibition of arginase is the inhibition of signal transduction pathway, the activation that causes arginase activity or arginase to be expressed.For example, can improve by using IL-13 acceptor (IL-13R) with the active relevant pathogeny of the arginase that raises.As described previously, IL-13 is main immunomodulating cytokines by activated TH2 emiocytosis.In the past few years, known very that IL-13 is the crucial medium in the pathogeny of alterative inflammation.In mouse, the IL-13 Mediated Signal Transduction enough causes the pathologic, physiologic phenotype that all four kinds of asthma are relevant and is that Polyblennia and inductive AHR are required.Consider the importance of IL-13 action effect molecule, the adjusting on its receptor level may be the important mechanism that adjusting IL-13 replied and therefore strengthened allergic response.

IL-13 and IL-4 have the common receptor subunit, i.e. the α subunit of IL-4 acceptor (IL-4R α).IL-13 deficient mice, IL-4 deficient mice and IL-4 acceptor α defective (IL-4R α (/-)) sign of mouse shown the nonredundancy effect of IL-13.IL-13 by with its effect of mediation that interacts of a kind of receptor system of complexity, described receptor system is made up of IL-4R α and two kinds of conjugated protein IL-13R α 1 of IL-13 and IL-13R α 2.The IL-13 acceptor is expressed on human B cell, basophilic granulocyte, eosinophilic granulocyte, mastocyte, endotheliocyte, inoblast, monocyte, scavenger cell, respiratory epithelium cell and smooth muscle cell.

Yet, also on people or mouse T cell, do not illustrate function IL-13 acceptor.Different with IL-4, IL-3 shows inessentially in the initial differentiating into T H2 of CD4 T cell type cell, but is important in the effector phase of alterative inflammation.This assessment is by now examining further confirmation in many bodies, described observation comprises uses that IL-13 causes alterative inflammation, the organizing specific overexpression of IL-13 causes respiratory inflammation and Polyblennia in the lung of transgenic mice, the alterative inflammation that is independent of IL-4 has been eliminated in the IL-13 blocking-up, and IL-13 shows more importantly than IL-4 in Polyblennia.Therefore, IL-13 is an attracting new treatment target during the pharmacy of allergy phlegm disease is intervened.Use IL-13R can potential inhibition or even blocking-up IL-13 signal transduction pathway, stop IL-13 inductive ARG1 to express the pathology relevant with alleviating asthma.

ARG1 and CAT2 are as the mark of inflammatory phlegm disease

The present invention determines CAT2 and ARG1 overexpression in the lung tissue of asthma animal model.Therefore, these genes or their expression product can be as the marks of inflammatory diseases such as asthma or COPD.These expression of gene levels can be by use for example, RT-PCR, nucleic acid array, and perhaps immunoassay detect.The example of immunoassay form comprises, but be not limited to, latex or other particle agglutinations, electrochemiluminescence, ELISA, RIA, sandwich assay or immunizing dose are measured (immunometric assay), time resolved fluorescence, lateral flow assay method, fluorescence polarization, flow cytometry, immunohistochemistry assay method, Western blot and protein groups chip.Can detect CAT2 and ARG1 albumen or mRNA level in body fluid or the tissue sample.

Mark diagnosis or prognosis information is provided providing in concrete experimenter's sample or is used to assess the therapeutic efficiency of inflammatory diseases.For example, relatively the CAT2 of the different steps of disease progression and the expression level of ARG1 can provide long-term prognosis, comprise the method for survival.CAT2 and ARG1 gene pleiomorphism also can be indicated the susceptibility of experimenter to inflammatory diseases.

In another example, can assess the effect of concrete treatment plan, comprise whether concrete medicine can improve the long-term prognosis among the concrete patient.Asthma, COPD and sacroiliitis are complex diseases, and their clinical manifestation is various and changeable.The patient is different to replying of sick process of phlegm and available treatment, and these differences have reflected the difference of the sick type of phlegm probably.Therefore, extra practicality of the present invention provides the method for identifying that most probable is replied the patient of therapeutic process.

Although in mouse model, implement initial expression of differentiation analysis, in animal, cause the dysfunction gene of disease when in human body, being dysfunction, can cause similar syndrome as everyone knows.Therefore this is that the present invention wishes especially and is interpreted as the present invention particularly including people CAT2 and ARG1 gene.CAT2 and ARG1 homologue from other biological also can be used for animal model with research asthma, COPD or other inflammatory diseasess.By using any means as known in the art can obtain ARG1 and CAT2 homologue from other biological.

By suppressing CAT2 or ARC1 active treatment inflammatory diseases

CAT2 or ARG1 genome sequence, promotor, exon, intron, rna transcription thing or encoded protein can be the targets of treatment or therapeutical agent.They also can be used to produce the gene therapy vector that suppresses CAT2 and/or ARG1 expression or CAT2 and/or ARG1 protein-active.

Mechanism is not made restriction, the present invention's part promptly suppresses CAT2 and/or ARG1 expression or activity and can improve inflammatory diseases such as asthma or COPD based on following principle.CAT2 and/or ARG1 inhibitor also can effectively be treated fibrosis, respiratory tract hyperergy, goblet cell hyperplasia, respiratory inflammation and oxidative stress.Described inhibition can occur in and transcribe, transcribe on back, translation or the translation back level.For example, can target deciding CAT2 or ARG1 promotor or mRNA transcribes or translates suppressing respectively.Also can decide CAT2 or the proteic translation post-treatment of ARG1 by target, as glycosylation and Dimerized.

Find that in the mouse model of asthma CAT2 and ARG1 expression pattern make it possible to filler test reagent, its objective is and regulate CAT2 and/or ARG1 expression or CAT2 and/or ARG1 activity.The agent of being had a try can perhaps be screened CAT2 and/or the active influence of ARG1 gene product by them by them to the influence that CAT2 and/or ARG1 express on mRNA or protein level.

In another embodiment, CAT2 and/or ARG1 expression or CAT2 and/or the active conditioning agent of ARG1 can be used as the therapeutical agent of asthma, COPD and other inflammatory phlegm diseases.Conditioning agent can be polynucleotide, as ribozyme or RNAi, polypeptide has CAT2 and/or ARG1 mutant, virus or the non-viral gene treatment carrier of dominance negative interaction or can suppress CAT2 and/or any other small molecules or the biomolecules of ARG1 activity or CAT2 and/or ARG1 genetic expression as the activity to wild-type CAT2 and/or ARG1.This conditioning agent can be mixed with and be used for pharmaceutical composition of the present invention.

Probe, primer, antisense and RNAi sequence

An aspect of of the present present invention relates to and is used for detecting or the quantitatively polynucleotide probes or the primer of biological sample CAT2 or ARG1 gene product.CAT2 or ARG1 probe/primer can derive from the arbitrary portion of CAT2 or ARG1 gene.Described probe/primer can have required length arbitrarily.For example, probe can have 15,20,25,50,75,100,125,150,175,200,225,250,275,300,325,350,400 or more continuous nucleotides.In many embodiments, probe can be under strict or height stringent condition and rna transcription thing or its complementary sequence hybridization of CAT2 or ARG1 gene.

The example of different hybridization stringency is listed in table 1.The height stringent condition is the same with condition A-F at least strict condition, and stringent condition is the same with condition G-L at least strict condition; The stringent condition that reduces is the same with condition M-R at least strict condition.As used in the table 1, under given hybridization conditions, implement hybridization at least about 2 hours, washed twice under corresponding wash conditions then, each 15 minutes.

Table 1 stringent condition

Stringent condition	The multi-nucleotide hybrid body	Crossbred length (bp) 1	Hybridization temperature and damping fluid H	Wash temperature and damping fluid H
					A	DNA:DNA	＞50	65 ℃; 1 * SSC-or-42 ℃; 1 * SSC, 50% methane amide	65℃；0.3×SSC
B	DNA:DNA	＜50	T B *；1×SSC	T B *；1×SSC
					C	DNA:RNA	＞50	67 ℃; 1 * SSC-or-45 ℃; 1 * SSC, 50% methane amide	67℃；0.3×SSC
D	DNA:RNA	＜50	T D *；1×SSC	T D *；1×SSC
					E	RNA:RNA	＞50	70 ℃; 1 * SSC-or-50 ℃; 1 * SSC, 50% methane amide	70℃；0.3×SSC
F	RNA:RNA	＜50	T F *；1×SSC	T F *；1×SSC
					G	DNA:DNA	＞50	65 ℃; 4 * SSC-or-42 ℃; 4 * SSC, 50% methane amide	65℃；1×SSC
H	DNA:DNA	＜50	T H *；4×SSC	T H *；4×SSC
					I	DNA:RNA	＞50	67 ℃; 4 * SSC-or-45 ℃; 4 * SSC, 50% methane amide	67℃；1×SSC
J	DNA:RNA	＜50	T J *；4×SSC	L J *；4×SSC
					K	RNA:RNA	＞50	70 ℃; 4 * SSC-or-50 ℃; 4 * SSC, 50% methane amide	67℃；1×SSC
L	RNA:RNA	＜50	T L *；2×SSC	T L *；2×SSC
					M	DNA:DNA	＞50	50 ℃; 4 * SSC-or-40 ℃; 6 * SSC, 50% methane amide	50℃；2×SSC
N	DNA:DNA	＜50	T N *；6×SSC	T N *；6×SSC
					O	DNA:RNA	＞50	55 ℃: 4 * SSC-or-42 ℃; 6 * SSC, 50% methane amide	55℃；2×SSC
P	DNA:RNA	＜50	T P *；6×SSC	T P *；6×SSC
					Q	RNA:RNA	＞50	60 ℃: 4 * SSC-or-45 ℃; 6 * SSC, 50% methane amide	60℃；2×SSC
R	RNA:RNA	＜50	T R *；4×SSC	T R *；4×SSC

¹: crossbred length is desired to the hybridization zone of hybridization polynucleotide.When the target polynucleotide of polynucleotide and unknown nucleotide sequence is hybridized, suppose that crossbred length promptly is the length of hybridization polynucleotide.When the multi-nucleotide hybrid of known array, the sequence alignment by polynucleotide also identifies that the one or more zones with optimal sequence complementarity can determine crossbred length.

^H: in hybridization and lavation buffer solution, (1 * SSPE is 0.15M NaCl to SSPE, 10mMNaH ₂PO ₄With 1.25mM EDTA, pH 7.4) can replace SSC (1 * SSC is 0.15M NaCl and 15mM Trisodium Citrate).

T _B*-T _R*: expection length should be than the melting temperature(Tm) (T of this crossbred less than the hybridization temperature of the crossbred of 50 base pairs _m) little 5-10 ℃, wherein determine T according to following equation _mFor the crossbred of length less than 18 base pairs, T _m(℃)=2 (#A+T base)+4 (#G+C base).For length is the crossbred of 18 to 49 base pairs, T _m(℃)=81.5+16.6 (log10Na ⁺)+0.41 (%G+C)-(600/N), wherein N is the base number in the crossbred, Na ⁺Be the sodium ion volumetric molar concentration (Na of 1 * SSC in the hybridization buffer ⁺=0.165M).

Another aspect of the present invention relates to coding and contains the CAT2 of amino-acid residue change and the polynucleotide of ARG1 mutant.These mutant can and suppress wild-type CAT2 and the proteic activity of ARG1 with wild-type CAT2 and ARG1 protein competition.Replace, add or lack in the nucleotide sequence of polynucleotide by importing one or more Nucleotide, can produce the isolating polynucleotide molecule of encoding mutant type CAT2 and ARG1 gene, replace, add or disappearance thereby in coded albumen, import one or more amino acid.These technology are to know in this area.By standard technique, can in CAT2 and ARG1 gene, import sudden change as site-directed mutagenesis and PCR mediated mutagenesis.Alternatively, in all or the partial sequence as the encoding sequence that sudden change can be imported at random CAT2 and ARG1 gene or cDNA by saturation mutagenesis, and the biological activity that can screen the gained mutant can suppress the active mutant of wild-type protein (dominance is born mutant) to identify.After the mutagenesis, can recombinant expressed coded albumen and can determine this proteic activity.

Can further modify polynucleotide to increase the body internal stability.Possible modification includes, but not limited at 5 ' and/or 3 ' the terminal flanking sequence that adds; In main chain, use thiophosphatephosphorothioate or 2-O-methyl rather than phosphodiesterase key; And/or comprise unconventional base, as inosine, queosine and wybutosine, and the ethanoyl of VITAMIN B4, cytidine, guanine, thymus pyrimidine and uridine-, methyl-, sulfo--and other modified forms.

Another aspect of the present invention relates to isolating polynucleotide molecule, and itself and CAT2 or ARG1 gene or their transcript are antisense." antisense " polynucleotide comprise " justice is arranged " polynucleotide complementary nucleotide sequence with proteins encoded, for example, with the coding strand of double-stranded cDNA molecule complementary or with mRNA sequence complementary nucleotide sequence.Therefore, antisense polynucleotides can form hydrogen bond with adopted polynucleotide are arranged.Antisense polynucleotides can with the complete coding strand of CAT2 of the present invention or ARG1 gene or only its part complementation.In one embodiment, " coding region " of the coding strand of antisense polynucleotides molecule and nucleotide sequence of the present invention is antisense.In another embodiment, " non-coding region " of the coding strand of antisense polynucleotides molecule and nucleotide sequence of the present invention is antisense.

Can design antisense polynucleotides of the present invention according to Watson and Crick base pairing rules.The antisense polynucleotides molecule can with the complete coding region complementation corresponding to the mRNA of gene of the present invention.It can also be the oligonucleotide with the part antisense of coding region or non-coding region.Antisense oligonucleotide length can be for for example, about 5,10,15,20,25,30,35,40,45 or 50 Nucleotide.Use method as known in the art to use chemosynthesis and enzyme ligation can make up antisense polynucleotides of the present invention.For example, use naturally occurring Nucleotide or multiple modified Nucleotide can the chemosynthesis antisense polynucleotides (for example, antisense oligonucleotide), described modified Nucleotide is designed for the biological stability that increases described molecule or increases antisense and the physical stability of the duplex that forms between the adopted polynucleotide is arranged, for example, the Nucleotide that can use phosphorothioate derivative and acridine to replace.The example that can be used to produce the modified Nucleotide of antisense polynucleotides comprises 5 FU 5 fluorouracil; 5-bromouracil; the 5-chlorouracil; 5-iodouracil; xanthoglobulin; xanthine; 4-ethanoyl cytosine(Cyt); 5-(carboxyl hydroxymethyl) uridylic; 5-carboxymethylamino methyl-2-sulphur uridine; 5-carboxymethylamino 6-Methyl Uracil; dihydrouracil; β-D-galactosyl queosine; inosine; the N6-isopentenyl gland purine; the 1-methyl guanine; the 1-methylinosine; 2; the 2-dimethylguanine; the 2-methyladenine; the 2-methyl guanine; the 3-methylcystein; 5-methylcytosine; the N6-VITAMIN B4; the 7-methyl guanine; 5-methylamino 6-Methyl Uracil; 5-methoxyl group amino methyl-2-thiouracil; β-D-mannose group queosine; 5 '-methoxyl group carboxymethyl uracil; the 5-methoxyuracil; 2-methyl sulfo--N6-isopentenyl adenosine; uridylic-the 5-oxyacetic acid (v); wybutoxosine; pseudouracil; queosine; 2-sulfo-cytosine(Cyt); 5-methyl-2-sulfo-uridylic; 2-sulfo-uridylic; 4-sulfo-uridylic; methyl uracil; uridylic-5-hydroxy methyl acetate; 3-(3-amino-3-N-2-carboxyl propyl group) uridylic; (acp3) w and 2,6-diaminopurine.Alternatively, use expression vector to produce antisense polynucleotides by biological method, wherein antisense orientation with the polynucleotide subclone in the described expression vector (promptly, the RNA that transcribes from the polynucleotide that inserted will be an antisense orientation with the target polynucleotide, further describe in the trifle below).

Antisense polynucleotides molecule of the present invention is applied to the experimenter usually or original position produces, thereby they are hybridized with encode CAT2 and the proteic cell mRNA of ARG1 and/or genomic dna or combine, thereby for example, suppress described proteic expression by suppressing to transcribe and/or to translate.Hybridization can be by conventional Nucleotide complementation with form stable duplex or, for example, for antisense polynucleotides molecule, by the specific interaction in the double-helical major groove in conjunction with DNA duplex.The example of the route of administration of antisense polynucleotides molecule of the present invention is included in tissue site (for example, intestines or blood) direct injection.Alternatively, can modify the antisense polynucleotides molecule with target fixed selected cell, systemic administration then.For example, for systemic administration, thereby can modify acceptor or antigen that their specific combination of antisense molecule are expressed on selected cell surface, described specific combination can be for example by being connected to this antisense polynucleotides molecule and cell surface receptor or antigen bonded peptide or antibody.Also the antisense polynucleotides molecule can be delivered to cell with the carrier of describing in the literary composition.In order to realize enough ICs of antisense molecule, can use the vector construction body, wherein the antisense polynucleotides molecule places under the control of strong polII or polIII promotor.

Another aspect of the present invention relates to α-anomer polynucleotide molecule.α-anomer polynucleotide molecule can form special double-stranded crossbred with CAT2 and ARG1 RNA, and wherein β-the unit with common is opposite, and described chain is parallel to each other.α-anomer polynucleotide molecule can also contain 2-o-methyl ribonucleotides or chimeric RNA-DNA analogue.

In another embodiment, isolating polynucleotide are ribozymes.Ribozyme is the catalytic RNA molecule with RNA enzymic activity, and it can cut the strand polynucleotide that have complementary region with them, as mRNA.Thereby ribozyme can be used for the mRNA transcript of catalytic cutting CAT2 and/or ARG1 gene, thereby suppresses the translation of described mRNA.Can have ribozyme based on the nucleotide sequence design of CAT2 and ARG1 gene to CAT2 and ARG1 gene specific.Can be used for selecting catalytic RNA from the mRNA of CAT2 and ARG1 genetic transcription with special RNA enzymic activity from the RNA library of molecules.Alternatively, CAT2 and ARG1 expression of gene can prevent that with formation the triple helix structure that described gene is transcribed from suppressing by regulatory region (for example, promotor and/or enhanser) complementary nucleotide sequences fixed by target and these genes in target cell.

Interfere (RNAi) also can suppress CAT2 and ARG1 expression of gene with RNA.This technology is the technology that is used for PTGS (" PTGS "), wherein uses homoduplex RNA (" dsRNA ") can specially remove the target gene activity.In many embodiments, will and observe sequence-specific reducing in the gene activity with the dsRNA transfered cell of about 21 Nucleotide of target gene homologous.RNA interferes the mechanism that gene silencing on the mRNA level is provided.It provides effective and widely used method for gene knockout and therapeutic purpose.

Can interfere the sequence of inhibition of gene expression can have any required length by RNA.For example, this sequence can have at least 15,20,25 or more continuous nucleotides.This sequence can be the polynucleotide of dsRNA or any other type, and precondition is that this sequence can form functional reticent mixture with degraded said target mrna transcript.

In one embodiment, described sequence contains short intervening rna (siRNA) or is made up of this weak point intervening rna (siRNA).SiRNA can have the dsRNA of 19 to 25 Nucleotide for for example.Can endogenous generation siRNA by the long dsRNA molecule of RNA enzyme III associated nucleic acid enzyme liberating that is called Dicer.Also can be with the transfered cell of transcribing of outer seedbed of siRNA or logical overexpression construct.SiRNA is in case formation just is assembled into the complex body that contains endoribonuclease with protein ingredient, and it is called RNA inductive silencing complex (RISCs).Untwisting that the ATP of siRNA produces activates RISC, and it decides complementary mRNA transcript by Watson-Crick base pairing target successively, thus cutting and destruction mRNA.The cutting of mRNA takes place near the centre in the zone of siRNA chain combination.This sequence-specific mRNA degraded causes gene silencing.

At least two kinds of methods can be used for realizing the gene silencing of siRNA mediation.At first, can external synthetic siRNA and transfered cell with temporary transient inhibition of gene expression.Synthetic siRNA provides and has realized the easy of RNAi and valid approach.SiRNA is the duplex of the mixing oligonucleotide of weak point, and it can comprise for example, having 19 Nucleotide of symmetrical dinucleotides 3 ' overhang.Use synthetic 21bp siRNA duplex (for example, being UU or dTdT 3 ' overhang after 19 RNA bases), can in mammalian cell, realize sequence-specific gene silencing.These siRNAs can hit fixed gene translation and not activate the protein kinase (PKR) that relies on DNA by longer dsRNA by special inhibition mammalian cell, and described activation may cause non-special the preventing of many protein translations.

Secondly, can be from carrier expression in vivo siRNA.This method is used in stably express siRNA in cell or the transgenic animal.In one embodiment, siRNA expression vector through engineering approaches is transcribed to drive from the siRNA of polymerase III (pol III) transcription unit.PolIII transcription unit is suitable for hair clip type siRNA expresses, because they use the short Transcription Termination site of being rich in AT, it causes, and (for example, UU), this feature helps the siRNA function sending out centre type siRNA adding 2bp overhang.The polIII expression vector also can be used to produce the transgenic mice of expressing siRNA.

In another embodiment, siRNA can express in tissue-specific mode.In the method, Chang double-stranded RNA (dsRNA) tissue-specific promoter expression in the nucleus of selected clone or transgenic mice at first.Long dsRNA is processed to siRNA (for example, passing through Dicer) in nucleus.SiRNA comes out and the special silence of mediated gene from nucleus.Similarly method can be used in combination with the organizing specific promotor to produce tissue-specific knocking down (knockdown) mouse.Any 3 ' dinucleotides overhang as UU, can be used for the siRNA design.In some cases, because siRNA might be cut by the RNA enzyme at strand G residue place, so avoided the G residue in the overhang.For siRNA sequence self, have been found that the siRNA that has 30-50%GC content in some cases has more activity than the siRNA with higher G/C content.In addition, because 4-6 Nucleotide polythymidylic acid sequence can be used as the termination signal of RNA polIII, so when design during, can avoid in some cases in the target sequence＞one section sequence of 4T or A from the sequence of RNA polIII promoter expression.In addition, can highly structural or is conditioned protein binding in some zone of mRNA.Thereby usefully the different positions on gene order length is selected siRNA target locating point.At last, potential target locating point can with suitable genome database (people, mouse, rat, or the like) relatively.Consider from some situations, can remove and have and any target sequence that other encoding sequence homologous 16-17 above in abutting connection with base pair.

In one embodiment, siRNA is designed to have two inverted repeats of cutting apart by short intervening sequence and finishes with a string T as the Transcription Termination site.This design has produced predicts the rna transcription thing that is folded into short hair clip type siRNA.The length of the intervening sequence of the length of the inverted repeats of the hairpin stem that the selection of siRNA target sequence, coding are inferred, the order of inverted repeats, coding hairpin loop and the existence or the shortage of composition and 5 '-overhang can change to obtain required result.

Tightly can select the siRNA target by scanning mRNA sequence with searching AA dinucleotides and record at 19 Nucleotide in AA downstream.Additive method also can be used for selecting the siRNA target.In one embodiment, the selection of siRNA target sequence is fully rule of thumb determined (to see, for example, people such as Sui, Proc.Natl.Acad.Sci.USA 99:5515-5520,2002), as long as target sequence begins with GG and as do not have remarkable sequence homology with other genes by the blast search analysis.In another embodiment, select the siRNA target sequence with more detailed method.This method is utilized following argument, and promptly any come-at-able site of endogenous mRNA can be fixed with by synthetic oligodeoxyribonucleotide/RNA enzyme H method degraded people such as (, Nature Biotechnol.20:500-505,2002) Lee by target.

In another embodiment, making up hair clip type siRNA expression cassette to contain the sense strand of target, is thereafter the antisense strand of short intervening sequence, target and as 5-6 T of transcription terminator.The order of sense strand and antisense strand can change and not influence the active for gene silencing of hair clip type siRNA in the siRNA expression construct.In some cases, the part that can cause active for gene silencing of putting upside down of order reduces.

As the length of the nucleotide sequence of siRNA expression cassette stem can, for example, be 19 to 29.The ring size can be 3 to 23 Nucleotide.Also can use other length and/or ring size.

In another embodiment, can use 5 ' overhang in the hair clip type siRNA construct, precondition is that hair clip type siRNA has function.In one embodiment, 5 ' overhang comprises about 6 nucleotide residues.

In a further embodiment, the target sequence of RNAi is the about 21 aggressiveness sequence fragments that are selected from CAT2 and ARG1 encoding sequence, as SEQ ID NOS:1 and 5.Target sequence can be selected from ORF district or non-ORF district.5 ' end of each target sequence all has dinucleotides " NA ", and wherein " N " can be any base, and " A " represents VITAMIN B4.Remaining 19 aggressiveness sequences have 30% to 65% GC content.In many examples, remaining 19 aggressiveness sequences do not comprise any four successive A or T (that is, AAAA or TTTT), three successive G or C (that is, GGG or CCC), seven " GC " that perhaps are in line.Use the example of the target sequence of above-mentioned standard (" not strict standard (Relaxed Criteria) ") preparation in table 2, to illustrate.Each target sequence has SEQ ID NO:3n in the table 2; Corresponding siRNA sense strand and antisense strand have SEQ ID NO:3n+1 and SEQ ID NO:3n+2 respectively, and wherein n is a positive integer.For each CAT2 and ARG1 encoding sequence (for example, being respectively SEQ ID NOS:1 and 5), can select a plurality of target sequences.

Can use other standard design RNAi target sequence.In an example, the GC content that remains 19 aggressiveness sequences is limited to 35% to 55%, and gets rid of and anyly have three continuous A or T (that is, AAA or TTT) or have 5 or 19 aggressiveness sequences of the palindromic sequence of polybase base more.In addition, can select 19 aggressiveness sequences to have low sequence homology with people's gene with other.In one embodiment, by BLASTN to NCBI people UniGene bunch of search of sequence database potential target sequence.People UniGene database contains the nonredundancy group of directed bunch of gene.Each UniGene bunch comprises the sequence of representing a unique gene.Can be chosen in the 19 aggressiveness sequences that the BLASTN search is not hit other people gene down.In this search, the e value can be set at strict value (as " 1 ").In addition, target sequence can be selected from the ORF district, and from the initiator codon to the terminator codon 75bp at least.Use the example of the target sequence of these standards (" strict standard (Stringent Criteria) ") preparation in table 2, to illustrate (SEQID NO:3n, wherein n is a positive integer).Also provide the siRNA of each target sequence (SEQ ID NO:3n) that justice and antisense sequences (being respectively SEQ ID NO:3n+1 and SEQ ID NO:3n+2) are arranged.

Table 2.RNAi target sequence and siRNA sequence

SEQ ID NO (encoding sequence)	Strict standard (target: SEQ ID NO:3n not; SiRNA has justice: SEQ ID NO:3n+1; SiRNA antisense: SEQ ID NO:3n+2)	Strict standard (target: SEQ ID NO:3n; SiRNA has justice: SEQ ID NO:3n+1; SiRNA antisense: SEQ ID NO:3n+2)
			SEQ ID NO：1	SEQ ID NOS：9-725	SEQ ID NOS：1,332-1,409
SEQ ID NO：4	SEQ ID NOS：726-1,331	SEQ ID NOS：1,410-1,517

Use several different methods as known in the art can assess the validity of siRNA sequence.For example, siRNA sequence of the present invention can import the cell of overexpression CAT2 or ARG1 gene.Can detect polypeptide or the mRNA level of CAT2 in the cell or ARG1.Before importing, the siRNA sequence shows the validity of siRNA sequence in suppressing CAT2 or ARG1 genetic expression with the remarkable change of the expression level of LRG afterwards.In an example, also monitor the siRNA sequence import before and other expression of gene levels afterwards.Can select that CAT2 or ARG1 expression are had the effect of inhibition but the siRNA sequence of other expression of gene of not remarkably influenced.In another example, multiple siRNA or other RNAi sequences can be imported in the identical target cell.These siRNA or RNAi sequence specific suppress CAT2 or ARG1 genetic expression does not still suppress other genetic expressions.In another example, can use the siRNA or other RNAi sequences that suppress CAT2 or ARG1 gene and other one or more genetic expressions.

In a further embodiment, can be at base portion, sugar moieties or phosphoric acid ester backbone modifications polynucleotide molecule of the present invention, for example to improve the stability of this molecule, hybridization or solvability.For example, can modify the deoxyribose phosphate ester main chain of polynucleotide molecule to produce the peptide polynucleotide.Term " peptide polynucleotide " or " PNAs " used in the literary composition are meant the polynucleotide stand-in, for example, dna analog, wherein deoxyribose phosphate ester main chain is replaced and only keeps four kinds of natural nucleus bases (nucleobase) by false peptide main chain.The neutral main chain that has shown PNAs allows specific hybridization DNA and RNA under conditions of low ionic strength.Use the standard solid phase peptide synthetic schemes can implement the synthetic of PNA oligomer.

PNAs can be used in treatment and the diagnostic use.For example, PNAs can for example pass through, and the antisense reagent that can suppress as the sequence specific of CAT2 or ARG1 expression is duplicated in inducible transcription or translation retardance or inhibition.The PCR that the PNAs of polynucleotide molecule of the present invention also can for example instruct by PNA clamps (clamping), be used for the single base-pair mutation of analyzing gene, when with other enzymes (for example, the S1 nuclease) unites when using, as artificial restriction enzyme or as the probe or the primer of dna sequencing or hybridization.

In another embodiment, (for example can modify PNAs, take in the stability or the cell that strengthen them), this described modification can be by being attached to PNA with lipophilic or other auxiliary groups, perhaps, perhaps realize by the other technologies of using liposome or medicine as known in the art to send by forming the PNA-DNA mosaic.For example, can produce the PNA-DNA mosaic of polynucleotide molecule of the present invention, it can make up the favourable character of PNA and DNA.These mosaics allow DNA identification enzymes (for example, archaeal dna polymerase) and DNA partly to interact and the PNA part will provide high binding affinity and specificity.Can use according to the number of key between base stacking, the nuclear base and the joint of the suitable length of direction selection and be connected the PNA-DNA mosaic.It is synthetic to implement the PNA-DNA mosaic.For example, use standard phosphoramidite coupling chemical process can be on solid support the synthetic DNA chain, and modified nucleoside analog, for example, 5 '-(4-methoxyl group trityl) amino-5 '-deoxidation-thymidine phosphoramidite can be as the transcribed spacer between the 5 ' end of PNA and DNA.Then with the PNA monomer progressively coupling have the chimeric molecule of 5 ' PNA sections and 3 ' DNA sections with generation.Alternatively, can synthesize chimeric molecule with 5 ' DNA sections and 3 ' PNA sections.

CAT2 and ARG1 polypeptide

Aspects more of the present invention relate to the sudden change CAT2 and the ARG1 polypeptide that can suppress normal CAT2 or ARG1 polypeptide active, and suitable as immunogen anti-to produce-polypeptide fragment of CAT2 or anti--ARG1 antibody.In one embodiment, produce the CAT2 and the ARG1 polypeptide (for example, the negative mutant of dominance) of sudden change by recombinant DNA technology.Alternatively, use CAT2 and the ARG1 polypeptide that the standard peptide synthetic technology can the chemosynthesis sudden change.

The invention still further relates to CAT2 or ARG1 variant polypeptides, its antagonist as CAT2 or ARG1 polypeptide works.In one embodiment, the antagonist of CAT2 or ARG1 polypeptide or agonist are as therapeutical agent.For example, the antagonist of CAT2 or ARG1 polypeptide can reduce CAT2 or the proteic activity of ARG1 and improve inflammatory phlegm disease among the experimenter, CAT2 or ARG1 albumen overexpression in this phlegm disease.By mutagenesis, for example, the discrete point mutation of CAT2 or ARG1 gene or brachymemma can produce CAT2 or ARG1 variant polypeptides.

In certain embodiments, the antagonist of CAT2 or ARG1 polypeptide for example passes through, and competitiveness is regulated the activity of CAT2 or ARG1 polypeptide, can suppress one or more activity of the natural existence form of CAT2 or ARG1 polypeptide.Thereby, can cause the particular biological effect by variant treatment with limited function.

Can identify or the CAT2 that works as CAT2 or ARG1 polypeptide agonist or as CAT2 or ARG1 polypeptide antagonist or the mutant of ARG1 polypeptide by the combinatorial library of screening mutant.In certain embodiments, these variants can for example be used as treatment albumen of the present invention.By for example, thereby the degeneracy group that synthetic oligonucleotide mixture enzymatic is connected to gene order potential CAT2 or ARG1 peptide sequence be can be used as independent polypeptide to be expressed, perhaps alternatively, (for example express as the one group of bigger fusion rotein that wherein contains this group CAT2 or ARG1 peptide sequence, be used for phage display), the diversified library that can produce CAT2 or ARG1 polypeptide variants.There is several different methods to can be used for producing potential CAT2 or ARG1 polypeptide variants library from degenerate oligonucleotide sequence.Can in the automatization dna synthesizer, carry out the chemosynthesis of degeneracy gene order, then the synthetic gene is connected in the suitable expression vector.Use gene degeneracy group to make it possible in a kind of mixture, to provide all sequences of required group of the potential CAT2 of coding or ARG1 peptide sequence.The method of synthetic degenerate oligonucleotide is as known in the art.

In addition, can be used for producing the diversified colony of CAT2 or ARG1 polypeptide fragment, with screening with select CAT2 or ARG1 variant polypeptides subsequently corresponding to the fragment library of the albumen coded sequence of CAT2 or ARG1 gene.In one embodiment, can produce encoding sequence fragment library by the following method, promptly the double-stranded PCR fragment of handling CAT2 or ARG1 gene coded sequence under the condition of once cutting with nuclease only takes place to make an appointment with in each molecule therein, this double-stranded DNA sex change, renaturation DNA are formed double-stranded DNA, it can comprise having justice/antisense right from different cleaved products, remove the strand part by handling from the duplex that forms again, and gained fragment library is connected to expression vector with the S1 nuclease.By this method, can obtain expression library, N-end, C-end and the interior segments of the multiple size of its coding CAT2 or ARG1 polypeptide.

Several technology known in the art can be used for screening the gene product of the combinatorial library that produces by point mutation or brachymemma and the cDNA library that screening has the gene product of selected character.Be used to screen big gene library, the most widely used technology that can carry out high throughput analysis generally comprises is cloned into gene library in the reproducible expression vector, gained library with carrier transforms suitable cell, and express combination gene under certain condition, the active detection of purpose helps separating the carrier of the detected gene of its product of coding under the described conditions.The whole mutagenesis of recurrence (REM) is a kind of technology that strengthens function mutation body frequency in the library, it can unite use to identify CAT2 or ARG1 polypeptide variants (people such as Delgrave with the screening assay method, Protein Engineering 6:327-331,1993).

Have and be less than about 100 amino acid, can pass through synthetic method less than the part of about 50 amino acid whose CAT2 or ARG1 polypeptide or CAT2 or ARG1 variant polypeptides usually, the technology of using those skilled in the art to know produces.For example, use any one to pass through the obtainable solid phase technique of commercial sources, can synthesize these polypeptide as the Merrifield solid phase synthesis process, in the Merrifield solid phase synthesis process, amino-acid sequence is added to the amino acid chain of growth.The equipment of automated peptide synthesis can (FosterCity Calif.), and can visit according to manufacturer's operation instruction and do from supplier such as Perkin Elmer/Applied BioSystems Division by commercial sources.

The method and composition that is used to screen protein inhibitor or activator is as known in the art.See U.S. Patent number 4,980,281,5,266,464,5,688,635 and 5,877,007, incorporate them into this paper as a reference.

Antibody

According on the other hand, can prepare the proteic specific antibody of CAT2 or ARG1.In many embodiments, antibody of the present invention can be to be not less than 10 ⁵M ^-1Binding affinity in conjunction with CAT2 or ARG1 albumen.As restriction, these antibody can be monoclonal, polyclonal, chimeric, humanized, scFv, Fv, Fab ', Fab or F (ab ') ₂

Can use total length CAT2 or ARG1 albumen, perhaps alternatively, the invention provides as immunogenic CAT2 or the proteic antigenic peptide fragment of ARG1.In many embodiments, the proteic antigen peptide of CAT2 or ARG1 contains at least 8 amino-acid residues, and comprises CAT2 or the proteic epi-position of ARG1, thus the antibody and CAT2 or ARG1 albumen formation specific immune complex body that produce at this peptide.In many other embodiments, antigen peptide contains at least 8,12,16,20 or amino acids residue more.

Use technique known can identify immunogenicity part (epi-position).These technology comprise the ability of screening polypeptide and antigen-specific antibody, antiserum(antisera) and/or T clone or cloning reaction.Can prepare these antiserum(antisera)s and antibody as the technology of describing in the literary composition and use is known.The proteic epi-position of CAT2 or ARG1 is the part that reacts with the level of the reactivity that is not less than full-length polypeptide basically (for example, in ELISA and/or t cell responses mensuration) and these antiserum(antisera)s and/or T cell.These epi-positions can be in these be measured with similar in appearance to or react greater than the reactivity of full-length polypeptide.Usually the method for using those skilled in the art to know can be implemented these screenings.For example, polypeptide can be fixed on the solid support and contact with the patients serum to allow intraserous antibodies institute fixed polypeptide.For example remove unconjugated serum and use then, ¹²⁵The A Protein Detection bonded antibody of I mark.

The exemplary epi-position that antigen peptide comprises is CAT2 or the proteic zone of ARG1 that is positioned at the surface of this polypeptide, for example, and hydrophilic area, and have high antigenic zone.

CAT2 or ARG1 immunogen are (for example, CAT2 or ARG1 albumen, its fragment, perhaps CAT2-or ARG1-fusion rotein) be generally used for by with suitable experimenter (for example, rabbit, goat, mouse or other Mammalss) the preparation antibody of described immunogen immune.Suitable immunogen preparation for example can contain the CAT2 of recombinant expressed CAT2 or ARG1 immunogen or chemosynthesis or ARG1 immunogen.Said preparation can also comprise adjuvant, the complete or not exclusively position agent as Fu Shi, perhaps similar immune stimulant.Induce anti--CAT2 or anti-ARG1 antibody response with the suitable experimenter of immunogenic formulation immunity.Preparation, separate and use mono-clonal and polyclone anti--technology of CAT2 or anti--ARG1 antibody is well known in the art.

Therefore, another aspect of the present invention relates to mono-clonal or polyclone anti--CAT2 or anti--ARG1 antibody.The example of the immunocompetence of immunoglobulin molecules part comprises F (ab) and F (ab ') ₂Fragment, it can be by producing with enzyme such as pepsin antibody.The invention provides in conjunction with CAT2 or proteic polyclone of ARG1 and monoclonal antibody.

By standard technique, as using enzyme-linked immunosorbent assay (ELISA), use immobilization CAT2 or ARG1 albumen or CAT2 or the proteic fragment of ARG1 can make progress in time monitoring anti--CAT2 or anti--ARG1 antibody titer among the immune experimenter.If wish, can separate at CAT2 or the proteic antibody molecule of ARG1 and by well-known technology from Mammals (for example) from blood, be further purified as the A protein chromatographic, to obtain the IgG fraction.Suitable time after immunity, for example, as anti--CAT2 or anti--when the ARG1 antibody titer is the highest, can obtain producing the cell of antibody and be used for preparing monoclonal antibody from the experimenter by standard technique, described standard technique is for for example, hybridoma technology, human B cell hybridoma technology, EBV-hybridoma technology, perhaps trisome knurl (trioma) technology.The technology that is used to produce monoclonal antibody hybridoma is well-known.

Any of many well-known schemes that is used to merge lymphocyte and immortalized cell system can be used for producing anti--CAT2 or anti--ARG1 monoclonal antibody.Usually, immortalized cell system (for example, myeloma cell line) can derive from the mammalian species identical with lymphocyte.For example, lymphocyte and the fusion of immortalization mouse cell lines by in the future personal immunogenic formulation mice immunized of the present invention can produce murine hybridoma.The example of immortalized cell system comprises the responsive mouse myeloma cell line of the substratum that contains xanthoglobulin, aminopterin and thymidine (" HAT substratum).Any one of many myeloma cell lines can be used as the fusion partner according to standard technique, for example, and P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp210-Ag14 myelomatosis strain system.These myelomatosis strain systems can obtain from ATCC.Usually, use polyoxyethylene glycol (" PEG ") that responsive murine myeloma cell of HAT and mouse boosting cell are merged.The hybridoma that uses the HAT substratum select to merge then to obtain, described substratum kill that merge and myeloma cell unproductive fusion (the splenocyte of Rong Heing death after a couple of days because transformed).By antibody to hybridoma culture supernatants screening specific combination CAT2 or ARG1 polypeptide, for example, use the standard ELISA assay method, detect the hybridoma that produces monoclonal antibody.

The alternative approach of the hybridoma of preparation secrete monoclonal antibody is by (for example making up the immunoglobulin (Ig) library with CAT2 or the reorganization of ARG1 protein screening, the antibody phage display libraries) thus the proteic immunoglobulin library member of separation and combination CAT2 or ARG1 can identify and separate monoclonal anti-CAT2 or anti--ARG1 antibody.The test kit that produces and screen phage display library can obtain (for example, PharmaciaRecombinant Phage Antibody System, catalog number (Cat.No.) 27-9400-01 by commercial sources; With Stratagene SurfZAp ^TMPhage Display Kit, catalog number (Cat.No.) 240612).

Anti--CAT2 or anti--ARG1 antibody also comprise " strand Fv " or " scFv " antibody fragment.The scFv fragment contains the V of antibody _HAnd V _LStructural domain, wherein these structural domains are present in the single polypeptide chain.Usually, the Fv polypeptide also comprises V _HAnd V _LPeptide linker between the structural domain, it makes scFv form antigen in conjunction with desirable structure.

In addition, also comprise in the scope of the invention reorganization that contains people and inhuman part anti--CAT2 or anti--ARG1 antibody, as chimeric and humanized monoclonal antibody, described antibody can be by the preparation of standard recombinant dna technology.These chimeric and Humanized monoclonal antibodies can be by recombinant DNA technology generation as known in the art.

Humanized antibody is desirable in people experimenter's the therapeutic treatment.The humanization form of inhuman (for example, mouse) antibody is that chimeric molecule, immunoglobulin chain or its fragment of immunoglobulin (Ig) is (as Fv, Fab, Fab ', F (ab ') ₂Perhaps other antigens of antibody are in conjunction with subsequence), it contains the minmal sequence that derives from non-human immunoglobulin.Humanized antibody comprises human normal immunoglobulin (receptor antibody), and the residue of complementary determining region (CDR) that wherein forms acceptor is by replacing as the residue of the CDR of the required specificity of having of mouse, rat or rabbit, affinity and ability from inhuman species (donor antibody).In some cases, the Fv framework residue of human normal immunoglobulin is replaced by corresponding inhuman residue.Humanized antibody can also contain neither the residue of also not finding at receptor antibody in the CDR of input or frame sequence.Usually, humanized antibody will contain basically all or at least one, and common two variable domains, wherein all or basically all CDR districts corresponding to those CDR districts of non-human immunoglobulin, and all or basically all constant regions are those constant regions of human normal immunoglobulin consensus sequence.Humanized antibody also can comprise at least a portion constant region for immunoglobulin (Fc), as the constant region of human normal immunoglobulin.

These humanized antibodies can use transgenic mice to produce, and described transgenic mice can not be expressed endogenous heavy chain immunoglobulin and light chain gene, but can expressing human heavy chain and light chain gene.Use selected antigen, for example, CAT2 or ARG1 are proteic all or part of with normal way immune transgenic mouse.Can obtain at this antigenic monoclonal antibody with conventional hybridization knurl technology.The human normal immunoglobulin transgenosis that this transgenic mice contains can be reset during the B cytodifferentiation, and experiences classification conversion and somatic mutation subsequently.Thereby, use this technology, may produce treatment and go up useful IgG, IgA and IgE antibody.

Can produce the humanized antibody of the selected epi-position of identification with the technology that is called " guiding is selected ".In the method, selected non-human monoclonal antibodies, for example, murine antibody is used to guide the selection of the humanized antibody of discerning identical epi-position.

In one embodiment, can weaken or remove CAT2 or the proteic biological function of ARG1 at CAT2 or the proteic antibody of ARG1.In many cases, can obtain in the activity at least 25% reduction.In many other situations, can realize in the activity reducing at least 50%, 60%, 70%, 80%, 90%, 95% or more than.

Anti--CAT2 or anti--ARG1 antibody can be used for by standard technique, as affinity chromatography or immunoprecipitation separation of C AT2 or ARG1 albumen or CAT2 or the proteic mutant of ARG1.CAT2 or ARG1 albumen that the reorganization that anti--CAT2 or anti--ARG1 antibody help expressing the CAT2 of or sudden change natural from cell purification or ARG1 albumen and the purifying host cell produces.In addition, anti--CAT2 or anti--ARG1 antibody can be used to detect CAT2 or ARG1 albumen (for example, in the cell lysate on cell surface or in the cell conditioned medium liquid) so that the abundance and the pattern of assessment CAT2 or ARG1 protein expression.Anti--CAT2 or anti--ARG1 antibody diagnosticability are used for monitoring the part of tissue protein level as the clinical detection step, for example to be used for, determine the effect of given treatment plan.The detectable material of antibody coupling (for example, physical connection) can be promoted to detect.The example of detectable substance comprises plurality of enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material, perhaps radio active material.The example of suitable enzyme comprises horseradish peroxidase, alkaline phosphatase, tilactase, perhaps acetylcholinesterase; The example of suitable prothetic group complex body comprises streptavidin/vitamin H and avidin/biotin; The example of suitable fluorescent material comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine base amine fluorescein, dansyl chloride or phycoerythrin; The example of luminescent material comprises luminol,3-aminophthalic acid cyclic hydrazide; The example of bioluminescent material comprises luciferase, luciferin and aequorin; The example of suitable radio active material comprises ¹²⁵I, ¹³¹I, ³⁵S and ³H.

Of the present invention resisting-CAT2 or anti--ARG1 antibody can also be used for that the therapeutical agent target is had the CAT2 of rising or the cell or tissue that ARG1 expresses.For example, therapeutical agent such as small molecules CAT2 or ARG1 antagonist can be connected to anti--CAT2 or anti--ARG1 antibody so that this therapeutical agent target is had the CAT2 of rising or the cell or tissue that ARG1 expresses.

Therapeutical agent directly or indirectly (for example by the joint group) coupling (for example, covalently bound) arrives suitable monoclonal antibody.When each of reagent and antibody all had substituting group with another kind of reaction, the direct reaction between this reagent and the antibody was possible.For example, nucleophilic group on reagent and the antibody one, as amino or sulfydryl can with the group that contains carbonyl on reagent and the antibody another kind, as acid anhydrides or acid halide, or contain the alkylation reaction of good leavings group (for example, halogenide).

Alternatively, can wish by the joint group therapeutical agent and antibody coupling.The joint group can be used as transcribed spacer and works, so that antibody and reagent are away from so that avoid interference binding ability.The joint group also can be used to increase substituent chemical reactivity on reagent or the antibody, thereby increases coupling efficiency.Chemically reactive increase can promote reagent, the perhaps use of functional group on the reagent, itself otherwise will be impossible.

Those skilled in the art it is evident that multiple congenerous or exclusive-OR function is difunctional or poly functional reagent (as Pierce Chemical Co., Rockford, those that describe in the catalogue of Ill.) can be used as the joint group.For example, can realize coupling by the saccharide residue of amino, carboxyl, sulfydryl or oxidation.There are many reference to describe these methods, for example, people's such as Rodwell U.S. Patent number 4,671,958.

When therapeutical agent is more effective when not having the antibody moiety of immunoconjugates of the present invention, can wish to use the joint group, its in internalization to cutting between cell stage or afterwards.Many different joint groups that cut have been described.The mechanism that reagent discharges in these joint group cells by disulfide bond reduction (for example comprises, the U.S. Patent number 4,489,710 of Spitler), irradiation by the photo-labile key (for example, people's such as Senter U.S. Patent number 4,625,014), hydrolysis by the derivatize amino acid side chain (for example, people's such as Kohn U.S. Patent number 4,638,045), hydrolysis by serum complement mediation (for example, people's such as Rodwell U.S. Patent number 4,671,958), with acid catalyzed hydrolysis (for example, people's such as Blattler U.S. Patent number 4,569,789) cutting of carrying out.

May wish more than one reagent is coupled to antibody.In one embodiment, a kind of a plurality of molecules of reagent are coupled to an antibody molecule.In another embodiment, the reagent of more than one types is coupled to an antibody.No matter specific embodiments how, can have the immunoconjugates of an above reagent with the several different methods preparation.For example, more than one reagent can directly be coupled to antibody molecule, perhaps can use the joint in a plurality of sites that are provided for adhering to.

Carrier

Another aspect of the present invention relates to the carrier of the polynucleotide that contain coding CAT2 and ARG1 polypeptide or its part.Carrier can be plasmid or virus vector.

Expression vector of the present invention can be designed to express CAT2 and ARG1 polypeptide in protokaryon or eukaryotic cell.For example, CAT2 and ARG1 polypeptide can be at bacterial cells, as expressing in intestinal bacteria (E.coli), insect cell (use rhabdovirus expression vector), yeast cell or the mammalian cell.In certain embodiments, these albumen can be used as treatment albumen for example of the present invention.Alternatively, for example, use the T7 promotor to regulate sequence and T7 polysaccharase, expression vector can be in in-vitro transcription and translation.

In another embodiment, use the mammalian expression vector that comprises the organizing specific regulatory element to express herbicide-tolerant polynucleotide.The organizing specific regulatory element is as known in the art and can comprises the promotor that epithelial cell is special.Promotor, neuron-specific promotor that other limiting examples of suitable tissue-specific promotor comprise the special white protein promotor of liver, lymph sample specific promoter, TXi Baoshouti and immunoglobulin (Ig) are (for example, the neurofilament promotor), pancreas specific promoter, with mammary gland specific promoter (for example, whey promotor).Also comprise and grow the promotor of regulating, for example, afp promoter.

The present invention also provides recombinant expressed promotor, and it contains with antisense orientation is cloned into the coding CAT2 in the expression vector or the polynucleotide of ARG1 polypeptide.That is, dna molecular can be visited in a certain way and make ground and connect and regulate sequence, and described mode allows to express (by transcribing of dna molecular) and RNA molecule corresponding to the mRNA antisense of CAT2 of the present invention or ARG1 gene.Can select to be operably connected with the adjusting sequence of antisense orientation clone's polynucleotide, to instruct this antisense rna molecule continuous expression in the various kinds of cell type.For example, can select viral promotors or enhanser, perhaps regulate sequence with the composing type that instructs sense-rna, tissue-specific or expression that cell type is special.Antisense expression vector can be the form of recombinant plasmid, phagemid or attenuated virus, wherein produces antisense polynucleotides under the control of efficient regulatory region.Can determine the activity of promotor/enhanser by the cell type that carrier imported.

The present invention also provides gene delivery vector, is used for that polynucleotide are delivered to cell, tissue or Mammals and expresses.For example, polynucleotide sequence of the present invention can be in gene delivery vector part or systemic administration.These constructs can with in the body or turn in (exvivo) form utilize virus or non-virus carrier method.Use endogenous Mammals or allogeneic promoter can induce the expression of these encoding sequences.The expression in vivo of encoding sequence can be composing type or regulated.The present invention includes the gene delivery vector of the polynucleotide that can express expection.Gene delivery vector can be for for example, and virus vector is as retrovirus, slow virus, adenovirus, adeno associated virus (AAV), simplexvirus or Alphavirus carrier.Virus vector can also be Astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus or batch film virus vector.

Cytotropic the sending of gene therapy construct of the present invention is not limited to above mentioned virus vector.Can also use other delivering methods and medium, for example, the deposition of the DNA that connects of nucleic acid expression vector, the DNA that connects or be free of attachment to the polycation condensation of the adenovirus self of killing, part, liposome-dna complex, eukaryotic cell delivery vector cell, photopolymerisable hydrogel material, hand held transgenosis particle gun, ionizing rays, nuclear charge neutralization or with the fusion of cytolemma.Can use the transgenosis of particle mediation.In brief, sequence can be inserted in the conventional carrier that contains the conventional control sequence that is useful on high level expression, this carrier and synthetic transgenosis molecule then, as polymerization DNA-in conjunction with positively charged ion, picture polylysine, protamine and white protein be incubation together, described transgenosis molecule is connected to cellular targets and decides part, as asialoorosomucoid, Regular Insulin, semi-lactosi, lactose or transferrin.Also can use naked DNA.Use biodegradable latex bead can improve absorption efficient.The latex bead that the endocytosis of pearl starts back DNA bag quilt can effectively be transported in the cell.Thereby promote the destruction of endosome and DNA to be discharged in the tenuigenin can further to improve this method to increase hydrophobicity by handling pearl.

Another aspect of the present invention relates to the adjustable expression system of use and expresses CAT2 or ARG1 gene.These systems include, but not limited to Tet-open/close system, moulting hormone system, progesterone system and rapamycin system.

Another aspect of the present invention relates to the purposes of host cell, and described host cell is encoded or contain carrier conversion, transfection or the transduction of CAT2 or ARG1 polypeptide or its part.Host cell can be protokaryon or eukaryotic cell.These host cells can be used to express the CAT2 or the ARG1 polypeptide of any hope.

Detection method

As discussed earlier, CAT2 or ARG1 expression of gene level can be as the marks of inflammatory phlegm disease.Can detect and measure the relative quantity of CAT2 or ARG1 product (polynucleotide or polypeptide) by any means as known in the art.It can be qualitative or quantitative detecting or measure.

The general method of the polynucleotide of detection through transcribing comprises from cell or tissue sample extraction RNA, then the RNA of label probe and extraction is hybridized and certification mark probe (for example, RNA trace or nucleic acid array).

The general method that is used for the peptide detection comprises from cell or tissue sample extraction albumen, the special antibody of target protein is combined with protein sample, and detect described antibody.For example, the detection of CAT2 or ARG1 polypeptide can use anti--CAT2 or anti--ARG1 polyclonal antibody to finish.Usually the second antibody by applying marking detects antibody.Mark can be radio isotope, fluorescent chemicals, enzyme, enzyme cofactor or part.These methods are well known in the art.

In another embodiment, the detection of CAT2 or ARG1 protein expression is finished by using the small molecules that CAT2 or ARG1 protein product are had a high binding affinity.In many examples, small molecules normally detects easily.In many other examples, by the direct or indirect mark small molecules of other detectable materials.The example of these detectable substances includes, but are not limited to enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radio active material, particulate material, perhaps colloidal metal.

In certain embodiments, CAT2 or ARG1 gene self can be used as the mark of inflammatory diseases.For example, the increase or the minimizing (as duplicating or lacking by gene) of the genome of CAT2 or ARG1 gene copy may be relevant with inflammatory diseases.

Can also be by gel electrophoresis, column chromatography, perhaps directly order-checking, quantitative PCR, RT-PCR, nested PCR or other technologies as known in the art are assessed the detection of specific CAT2 or ARG1 polynucleotide molecule.

Use any means as known in the art can detect all or part of existence or the copy number of CAT2 or ARG1 gene.In one embodiment, with the existence of southern blotting technique analysis and evaluation CAT2 or ARG1 gene and/or the amount of genome copy.Be used for DNA detection and/or quantitative other useful method include, but not limited to direct order-checking, gel electrophoresis, column chromatography, quantitative PCR, the additive method that perhaps those skilled in the art will know that.

Screening method

The present invention also provides the method that is used for identifying conditioning agent (literary composition is also referred to as " screening assay method "), described conditioning agent (for example promptly contains the treatment part, peptide, peptide mimics (peptidomimetic), the class peptide, polynucleotide, small molecules or other drug) candidate or test-compound or reagent, its (a) is in conjunction with CAT2 or ARG1 albumen, perhaps (b) is inhibited to CAT2 or the proteic activity of ARG12, perhaps, more specifically, (c) (for example to CAT2 or ARG1 albumen and its one or more natural substrates, peptide, albumen, hormone, cofactor or polynucleotide) interaction have regulating effect, or (d) inhibited to CAT2 or ARG1 expression of gene.These assay methods generally include the reaction between CAT2 or ARG1 gene and one or more mensuration components.Other components can be test-compounds self, perhaps the combination of test-compound and CAT2 or the proteic binding partners of ARG1.

Test-compound of the present invention is generally inorganic molecule, little organic molecule and biomolecules.Biomolecules includes, but are not limited to amino acid, nucleic acid, lipid, sugar, steroid, polypeptide, polynucleotide, polysaccharide, and in Mammals any naturally occurring or synthetic organic compound of biologically active.In one embodiment, test-compound is little organic molecule.In another embodiment, test-compound is a biomolecules.

Test-compound of the present invention can obtain from any available source, and described source comprises the system library of natural and/or synthetic compound.Can obtain test-compound by any of many methods in the combinatorial library method as known in the art, described method comprises: biological library; Class peptide library (but but have the peptide function have the new non-peptide main chain of enzyme liberating resistance still keep bioactive molecular library, see for example, people such as Zuckermann, J.Med.Chem.37:2678-85,1994); Addressable parallel solid phase in space or liquid phase library; The synthetic library method that need deconvolute; " pearl one compound " library method; With the synthetic library method of using affinity chromatography to select.Biological library and class peptide library method are confined to peptide library, and other four kinds of methods can be applicable to the library (Lam, Anticancer Drug Des.12:145,1997) of peptide, non-peptide oligomer or micromolecular compound.

Term as used herein " binding partners " refers to biologically active agent, and it is as CAT2 or the proteic substrate of ARG1, perhaps alternatively, and as the part that CAT2 or ARG1 albumen is had binding affinity.As mentioned above, biologically active agent can be any of multiple naturally occurring or synthetic compound, amino acid, polypeptide, polysaccharide, Nucleotide or polynucleotide.

The screening of CAT2 or ARG1 protein inhibitor

The invention provides screening as the test-compound of CAT2 or the proteic inhibitor of ARG1 and the method for screening the pharmaceutical composition that contains described test-compound.Described screening method comprises that the aliquots containig that will express the cell sample of CAT2 or ARG1 contacts with one of multiple test-compound, and relatively in each aliquots containig the expression of CAT2 to determine with respect to the sample of other test-compounds processing or with respect to untreated sample or control sample, whether any test-compound provides expression level or the active remarkable reduction of CAT2 or ARG1.In addition, thus can design screening method by combination test-compound and CAT2 or ARG1 albumen and definite test-compound to CAT2 or the proteic influence of ARG1.

In addition, the invention still further relates to screening and can regulate the method for the bonded test-compound of CAT2 or ARG1 albumen and binding partners, described method realizes by test-compound, CAT2 or ARG1 albumen and binding partners are made up together and determine whether to take place the proteic combination of binding partners and CAT2 or ARG1.Test-compound can be small molecules or biomolecules.As hereinafter discussing, can provide test-compound from multiple library well known in the art.

The additive method and the composition that are used to screen protein inhibitor are as known in the art.See U.S. Patent number 4,980,281,5,266,464,5,688,635 and 5,877,007, they are incorporated herein by reference.

The inhibitor of CAT2 or ARG1 expression, activity or binding ability can be used as therapeutic composition of the present invention.One of inhibitor of the arginine transport of CAT2-mediation is a Methionin.These inhibitor can be mixed with pharmaceutical composition, as described below.

The high flux screening assay method

The invention provides the method for the test-compound active or that express that can suppress CAT2 or ARG1 being carried out high flux screening.In one embodiment, high-throughput screening method comprises combination test-compound and CAT2 or ARG1 albumen and detects test-compound to CAT2 or the proteic effect of ARG1.The functional examination method is as the little physiograph of cell sensor (cytosensormi crophysiometer), calcium flux assay method, as FLIPR ^(CA), perhaps the TUNEL assay method can be used to measure cytoactive for Molecular Devices Corp, Sunnyvale, as hereinafter discussing.

Multiple high-throughput functional examination method is well-known in the art and can unites the reactivity that is used to screen and/or study dissimilar activation test-compounds.Because coupling system is difficult to prediction usually, so need the many assay methods of configuration to detect multiple coupling mechanism.Multiple technology based on fluorescence is well known in the art and can high-throughput and ultra-high throughput screening activity, includes but not limited to BRET ^Or FRET ^(all by Packard Instrument Co., Meriden, CT produces).Can also operate BIACORE ^System is to detect combining of test-compound and the independent component for the treatment of target.

By combination test-compound and CAT2 or ARG1 albumen and determine the activity that combines between them, can carry out diagnositc analysis to illustrate coupling system.Use the general assay method of the little physiograph of cell sensor also to can be used for measuring metabolism activation, and the variation of calcium motion can be by using the technology based on fluorescence, as FLIPR ^(Molecular Devices Corp, Sunnyvale CA) detects.In addition, can determine that by the TUNEL assay method existence of apoptosis cell, described assay method utilize the free 3-OH end that the cutting of genomic dna causes during the Flow cytometry apoptosis.As mentioned above, multiple functional examination method well known in the art can unite be used to screen and/reactivity of the dissimilar activation test-compound of research.In one embodiment, high flux screening assay method of the present invention is utilized as BIACORE ^The plasma resonance technology of the meta-tag that system (Biacore International AB, Uppsala, Sweden) provides.When the resonance of no plasma body takes place when metal/liquid surface excites surface plasma wave.Cause reflection from surperficial point-blank light by the contact sample, surface plasma body resonant vibration causes the upper layer change of refractive.The refraction index changing of the given variation of upper layer mass concentration is similarly for many biologically active agents (comprising albumen, peptide, lipid and polynucleotide), and because BIACORE ^Sensor surface can functionalization with in conjunction with multiple these biologically active agents, so can detect multiple test-compound.

Therefore, the invention provides the test-compound high flux screening is suppressed CAT2 or the proteic active ability of ARG1, this by with test-compound and CAT2 or ARG1 albumen in the high throughput assay method, as BIACORE ^, perhaps in assay method, as BRET based on fluorescence ^Middle combination realizes.In addition, the high throughput assay method can be used for identifying that in conjunction with CAT2 or the proteic specificity factor of ARG1, perhaps alternatively, evaluation prevents the test-compound of CAT2 or ARG1 albumen and binding partner binds.In addition, the high flux screening assay method can be used for determining that test-compound whether can be in conjunction with CAT2 or ARG1 albumen or in conjunction with CAT2 or the proteic binding partners of ARG1 through improvement.

The diagnostic assay method

The illustrative methods of the existence of the polynucleotide of CAT2 or ARG1 or coding CAT2 or ARG1 comprises from the experimenter and obtains biological sample in the detection of biological sample, and (for example with biological sample and the polynucleotide that can detect described albumen or coding CAT2 or ARG1, mRNA, genomic dna) compound or reagent contact, thereby detect the existence of CAT2 or ARG1 polynucleotide in this biological sample.Detection corresponding to the exemplary agents of CAT2 or ARG1 gene or CAT2 or proteic mRNA of ARG1 or genomic dna be can with the polynucleotide probes through mark of CAT2 or ARG1 mRNA or genomic dna hybridization.The suitable probe that is used for diagnostic assay method of the present invention is described in this article.Detecting CAT2 or the proteic exemplary agents of ARG1 is specific recognition CAT2 or the proteic antibody of ARG1.

The diagnostic assay method also can be used for expression amount or the activity of CAT2 in the quantitative biological sample or ARG1.This for example quantitatively can be used for, determine inflammatory diseases, as asthma, COPD and arthritic progress or seriousness.Thisly quantitatively for example also can be used for, determine the seriousness of treatment back inflammatory diseases.

The method of using the pre-packing diagnostic kit for example contain the probe polynucleotide described at least a literary composition or antibody reagent to implement to describe in the literary composition, for example, described test kit can be advantageously used in demonstrating with diagnosis in the clinical setting experimenter of inflammatory diseases such as asthma, COPD and arthritic symptom or family history.

In addition, wherein expressing the arbitrary cell type of CAT2 or ARG1 or tissue can be used in the prognosis or diagnostic assay method that literary composition describes.

Determine the seriousness of inflammatory diseases

In diagnostic assay method field, the present invention also provides the method for definite inflammatory diseases such as asthma, COPD and arthritic seriousness, this method is passed through from experimenter's sample separation, detection is with respect to second sample from normal specimens or control sample, existence, amount and/or the activity of CAT2 or ARG1 in described experimenter's sample.In one embodiment, compared the expression level of CAT2 in two kinds of samples or ARG1, and the expression that CAT2 or ARG1 improve in the given the test agent is indicated as inflammatory diseases such as asthma, COPD and sacroiliitis.

The exemplary agents that detects CAT2 or ARG1 is can be in conjunction with the antibody of CAT2 or ARG1.In some cases, antibody can be directly or is coupled to detectable label indirectly.Antibody can be polyclone or monoclonal.Can use complete antibody, perhaps its fragment (for example, Fab or F (ab ') ₂).About probe or antibody, term " mark " with the detectable substance coupling (for example is intended to comprise, physical connection) come this probe of direct mark or antibody to probe or antibody, and by with the reactive indirect labelling probe or the antibody of another reagent of direct mark.The fluorescently-labeled second antibody of use that comprises the example of indirect labelling detects first antibody and thereby it can detect with fluorescently-labeled streptavidin with vitamin H end mark dna probe.Term " biological sample " is intended to comprise from the isolating tissue of experimenter, cell and biofluid, and the intravital tissue of experimenter, cell and fluid.That is, detection method of the present invention can be used for CAT2 or ARG1 mRNA, albumen or the genomic dna in the detection of biological sample in external and the body.For example, the ex vivo technique that is used to detect CAT2 or ARG1 mRNA comprises RNA blot hybridization and in situ hybridization.The ex vivo technique that is used to detect CAT2 or ARG1 comprises enzyme-linked immunosorbent assay (ELISAs), Western blot, immunoprecipitation and immunofluorescence.The ex vivo technique that is used to detect CAT2 or ARG1 genomic dna comprises southern blotting technique hybridization.In addition, be used to detect that technology comprises anti--CAT2 or the anti-ARG1 antibody that imports mark to the experimenter in the body of CAT2 or ARG1.For example, can come traget antibody with radio-labeling, existence and the position of this radio-labeling in the experimenter can be detected by the standard imaging technique.

In one embodiment, biological sample contains the protein molecular from the experimenter.Alternatively, biological sample can contain from experimenter's mRNA molecule or from experimenter's genomic dna molecule.In an example, biological sample be by ordinary method from the isolating tissue sample of experimenter, for example, examination of living tissue.

The prognosis assay method

The diagnostic method of describing in the literary composition also can be used for identifying to suffer from or dangerous developing into CAT2 or ARG1 expression or active relevant inflammatory phlegm are sick as asthma, COPD and arthritic experimenter unusually.

In addition, the prognosis assay method of describing in the literary composition can be used to determine whether that can use reagent (for example, agonist, antagonist, peptide mimics, albumen, peptide, polynucleotide, small molecules or other drug candidates) to the implementer expresses or active relevant inflammatory diseases with unusual CAT2 or ARG1 with treatment or prevention.Thereby, the invention provides and determine whether the experimenter can express or active relevant inflammatory diseases with the effective treatment of reagent and the CAT2 that increases or ARG1.

Can design method of prognosis and whether have poor long-term surviving prospect or progression of disease with the experimenter who determines the experience the treatment of inflammatory diseases.In one embodiment, can promptly, determine prognosis in a couple of days after diagnosis in the short period of time.By set up inflammatory phlegm disease from showing effect to CAT2 or ARG1 express spectra than the different steps in late period, expression pattern will show and embody the not good possibility increase of spectrum and prognosis and be associated.This prognosis can be used to design more positive therapeutic scheme and the possibility that increases long-term surviving and good order and condition then.

The detection of hereditary change

Method of the present invention also can be used for detecting the hereditary change in CAT2 or the ARG1 gene, thereby determines whether the experimenter with the gene that is changed is in the destructive danger that is characterized as unusual adjusting in CAT2 or ARG1 activity or the polynucleotide expression.In many embodiments, described method comprises that detection is from there being or not existing hereditary change in experimenter's the cell sample, described hereditary change feature is at least a change that influences the integrity of CAT2 or ARG1 gene, perhaps CAT2 or ARG1 gene abnormal expression.For example, these hereditary changes can detect below determining at least a existence of project: 1) from CAT2 or the one or more Nucleotide of ARG1 genetically deficient; 2) add one or more Nucleotide to CAT2 or ARG1 gene; 3) one or more Nucleotide of replacement CAT2 or ARG1 gene; 4) chromosome rearrangement of CAT2 or ARG1 gene; 5) change of the messenger RNA(mRNA) transcript level of CAT2 or ARG1 gene; 6) the unusual modification of CAT2 or ARG1 gene is as the unusual modification of dna methylase gene pattern; 7) existence of the non-wild-type splice mode of the messenger RNA(mRNA) transcript of CAT2 or ARG1 gene; 8) horizontal CAT2 of non-wild-type or ARG1; 9) allelotrope of CAT2 or ARG1 gene forfeiture; With 10) unfavorable posttranslational modification of CAT2 or ARG1.As describing in the literary composition, many known assay methods are arranged in this area, they can be used for detecting the change in CAT2 or the ARG1 gene.Exemplary biological sample is from experimenter's separate blood sample by ordinary method.

In certain embodiments, the detection that changes is included in polymerase chain reaction (PCR), as anchored PCR or RACE PCR, perhaps alternatively, use probe/primer in connecting chain reaction (LCR), LCR can be used for detecting the point mutation in CAT2 or the ARG1 gene.This method can may further comprise the steps: from experimenter's collecting cell sample, from the cellular segregation polynucleotide of this sample (for example, the genome polynucleotide, mRNA or both), with described polynucleotide sample with contact with one or more primers of CAT2 or ARG1 gene specific hybridization under certain condition, thereby CAT2 or ARG1 the gene hybridization and the amplification of (if existence) take place, and detect the existence of amplified production or do not exist, perhaps detect the size of amplified production and with its length and control sample comparison.Be appreciated that hope combines PCR and/or LCR as preliminary amplification step with any technology that is used for detecting the sudden change that literary composition describes.

Alternative amplification method comprises: self-sustained sequence replication, transcription amplification system, Q-β replicative enzyme or other polynucleotide amplification methods arbitrarily, use the molecule of the well-known technology for detection amplification of those skilled in the art then.These detection schemes can be used for detecting polynucleotide molecule (if these molecules exist with low-down number).

In alternative embodiment, can identify from the CAT2 of sample cell or the sudden change in the ARG1 gene by the change in the restriction enzyme cleavage pattern.For example, sample separation and contrast DNA with its amplification (randomly), digest with one or more restriction endonucleases, and determine the fragment length size and compare size by gel electrophoresis.The difference of fragment length size shows that there is sudden change in sample DNA between sample and the contrast DNA.In addition, sequence-specific ribozyme can be used for to give a mark for the existence of specific sudden change by the generation or the forfeiture of ribozyme cleavage site.

In other embodiments, the genetic mutation in CAT2 or the ARG1 gene can be by with sample and contrast polynucleotide, and for example, DNA or RNA are hybridized with the high density arrays that contains hundreds of or thousands of oligonucleotide probes and to be identified.For example, the genetic mutation in CAT2 or the ARG1 gene can be identified in the two-dimensional array of the dna probe that contains the light generation.In brief, the first hybridization array of probe can be used for the dna sequence dna of a segment length in scanning samples and the contrast, to identify the sequence change between the sequence by the linear array of generation order eclipsed probe.This step allows to identify point mutation.Be the second hybridization array after this step, it allows to use and all variants that detected or the littler specific sudden change of particular probe characterization array of sudden change complementary.Each sudden change array is made up of parallel probe groups, probe groups and wild type gene complementation, and another is organized and mutator gene complementation.

In a further embodiment, any of multiple sequencing reaction as known in the art can be used for directly checking order CAT2 or ARG1 gene and sequence and corresponding wild-type (contrast) sequence by comparative sample CAT2 or ARG1 gene detects sudden change.Also expection can be used any one of multiple automatization sequence measurement when carrying out diagnostic assay, comprises by mass spectroscopy and checking order.

Detect the additive method that suddenlys change in CAT2 or the ARG1 gene and comprise such method; prevent that wherein the protection of cutting agent influence is used for detecting the base mismatch of RNA/RNA or RNA/DNA heteroduplex (people such as Myers; Science, 230:1242,1985).Usually, the art technology of " mispairing cutting " begins by heteroduplex is provided, wherein by making (mark) RNA that contains wild-type CAT2 or ARG1 gene order or DNA and obtaining described heteroduplex from the potential mutant rna or the DNA hybridization of tissue sample.With the duplex of agent treated two strands, described reagent cuts the strand district of the duplex that exists owing to the base-pair mismatch between contrast and the sample chain.For example, the RNA/DNA duplex can be handled with enzymatic digestion mispairing district with the S1 nuclease with processing of RNA enzyme and DNA/DNA crossbred.In other embodiments, DNA/DNA or RNA/DNA duplex can be handled zone with the digestion mispairing through azanol or perosmic anhydride and piperidines.After the digestion of mispairing zone, on denaturing polyacrylamide gel, pass through size separation gained material to determine the mutational site.In one embodiment, can mark contrast DNA or RNA in order to detect.

In an embodiment again, the mispairing cleavage reaction uses one or more albumen, its in the system that determines the base mismatch in the identification double-stranded DNA to (" dna mismatch reparation " enzyme that is called) the point mutation from cell sample gained CAT2 or ARG1 cDNAs is detected and maps.For example, colibacillary mutY enzyme is cutting A in G/A mispairing place, and cuts the T of G/T mispairing place from the thymidine DNA glycosylase of HeLa cell.According to exemplary, based on CAT2 or ARG1 gene order, for example, the probe of wild-type CAT2 or ARG1 gene order is hybridized with cDNA or other DNA products from subject cell.Duplex is handled with dna mismatch repair enzyme, and cleaved products (if exist) can detect from electrophoresis scheme etc.See, for example, U.S. Patent number 5,459,039.

In other embodiments, the change of electrophoretic mobility will be used for identifying the sudden change of CAT2 or ARG1 gene.For example, single strand conformation polymorphism (SSCP) can be used for detecting the difference of electrophoretic mobility between sudden change and the wild-type polynucleotide.The single stranded DNA fragment of sample and contrast CAT2 or ARG1 polynucleotide can and allow its renaturation by sex change.The secondary structure of strand polynucleotide becomes according to sequence.Gained changes feasible can the detection even single sequence change in the electrophoretic mobility.Dna fragmentation can be used through the probe mark of mark or detection.By using RNA (rather than DNA) can strengthen the sensitivity of this mensuration, wherein secondary structure is more responsive to the change of sequence.In one embodiment, subject methods uses the heteroduplex analysis to come change based on electrophoretic mobility to separate double-stranded heteroduplex molecule people such as (, Trends Genet.7:5-7,1991) Keen.

In a further embodiment, use denaturing gradient gel electrophoresis (DGGE) to measure the motion of mutant in the polyacrylamide gel that contains the denaturing agent gradient or wild-type fragment.When DGGE when the analytical procedure, guaranteeing its incomplete sex change, this can press from both sides and realize by for example adding the GC of about 40bp that high-melting-point is rich in the DNA of GC by PCR with modifying DNA.In another embodiment, replace different (Rosenbaum and Reissner, Biophys.Chem.265:12753,1987) of denatured gradient with thermograde with the mobility of evaluation contrast and sample DNA.

The example of the other technologies of check point sudden change includes, but not limited to selectivity oligonucleotide hybridization, selective amplification or selectivity primer extension.For example, can prepare Oligonucleolide primers, wherein known sudden change places central authorities, then under certain condition with target DNA hybridization, described condition only allows to hybridize (people such as Saiki, Proc.Natl.Acad.Sci.USA when finding to mate fully, 86:6230,1989).When these allele specific oligonucleotide oligonucleotide are attached to Hybond membrane, and during with the target DNA hybridization of mark, these oligonucleotide are hybridized with target or many different sudden changes of pcr amplification.

Alternatively, the allele specific oligonucleotide amplification technique that depends on the selectivity pcr amplification can be used in combination with the present invention.Can carry targeted mutagenesis at the 3 ' end of the centre of this molecule (thereby amplification depends on different hybridization) or a kind of primer as the oligonucleotide of specific amplification primer, wherein under suitable condition, mispairing can prevent or reduce polymerase extension.In addition, wish in the sudden change zone, to import new restriction site to produce detection based on cutting.Expect that the Taq ligase enzyme that is used to increase can increase in certain embodiments.In some cases, only connect when mating fully when 3 ' the terminal existence in 5 ' sequence, making may be by seeking the existence of increasing or not having the existence that detects the specific site known mutations.

Effect during the monitoring clinical trial

Monitoring reagent (for example, medicine, small molecules, albumen, Nucleotide) is to CAT2 or ARG1 expresses or active influence not only can be applicable in the essential drugs screening, and can be applicable in the clinical trial.For example, by determine that as the screening assay method of describing in the literary composition reagent reduces CAT2 or ARG1 expression, protein level or downward modulation CAT2 or the active validity of ARG1 and can monitor in the CAT2 of the CAT2 that shows increase or ARG1 expression, protein level or rise or the active experimenter's of ARG1 clinical trial.In these clinical trials, the expression of CAT2 or ARG1 or active " reading " that can be used as particular organization's phenotype.

For example, in order to study the influence of reagent to the relevant injury of CAT2-in the clinical trial or ARG1, can isolated cell and prepare RNA and analyze the expression level of CAT2 or ARG1.By rna blot analysis, RT-PCR, GeneChip as describing in the literary composition ^Perhaps Taqman analyzes, and perhaps alternatively measures the proteic amount that is produced by one of method of describing in the multiple literary composition, perhaps by measuring the activity level of CAT2 or ARG1, and can the quantitate gene expression level.Like this, gene expression dose can be used as reading, shows the physiological response of cell to reagent.Therefore, this response behaviour can be determined at a plurality of time points during treating this individuality before the treatment and with this reagent.

In one embodiment, (for example the invention provides monitoring with reagent, antagonist, peptide mimics, albumen, peptide, polynucleotide, small molecules or other drug candidates of identifying by the screening assay method of describing in the literary composition) treatment experimenter's the method for validity, it comprises that step (i) obtains using preceding sample from the experimenter before using described reagent; (ii) detect before using the expression level of CAT2 in the sample or ARG1 albumen or mRNA; (iii) obtain one or more back samples of using from the experimenter; (iv) detect expression level or the activity use CAT2 in the sample of back or ARG1 albumen or mRNA; (the expression level of CAT2 or ARG12 albumen or mRNA or actively use the expression level of CAT2 in the sample of back or ARG1 albumen or mRNA or activity and (vi) so change this reagent using in the sample before v) relatively using to described experimenter with one or several.For example, wish using of this reagent of minimizing, that is, reduce the validity of this reagent so that the expression of CAT2 or ARG1 or activity are increased to than expression that is detected or active higher level.According to this embodiment, CAT2 or ARG1 express or actively can even reply the indicator that is used as reagent validity when not existing in observable phenotype.

Methods of treatment

The invention provides that treatment dangerously suffers from, susceptible or be diagnosed as inflammatory diseases, as the prevention and the methods of treatment of asthma, COPD and osteoarthritis and rheumatoid arthritis.Prevention and methods of treatment about treatment can customize or improve these treatments especially based on the knowledge that obtains from pharmacogenomics (Pharmacogenomics) field.Used in the literary composition " pharmacogenomics " comprises the genomics technology, is applied to the clinical development and the listing of medicine as gene sequencing, statistical genetics and gene expression analysis.More specifically, how this term gene of referring to study the experimenter has determined his or she reply (for example, experimenter " drug responses phenotype " or " the drug responses genotype ") to medicine.Thereby another aspect of the present invention provides according to the customization of the drug responses of individuality with CAT2 or ARG1 conditioning agent the prevention of this individuality or the method for therapeutic treatment.Pharmacogenomics allows the clinician or the physician will prevent or the therapeutic treatment target will be from the benefited maximum experimenter of this treatment and avoid making the experimenter to experience the treatment of the side effect that drug toxicity is correlated with.

Prevention method

On the one hand, the invention provides by the experimenter is used and regulate CAT2 or ARG1 expresses or active reagent prevents the method for the pathologic process that CAT2-among the experimenter or ARG1-are relevant.

Be in that and unusual CAT2 or ARG1 expresses or active relevant inflammatory phlegm disease, can for example pass through, any of diagnosis of describing in the literary composition or prognosis assay method or unite and identify as the experimenter in the danger of asthma.

Can before the symptom performance of CAT2 that is characterized as increase or ARG1 protein expression, use preventive, thereby this disease obtains prevention, perhaps alternatively, postpones its development.(for example depend on the unusual type of CAT2 or ARG1, the usually adjusting outside the arm's length standard deviation), can for example CAT2 or ARG1 mutain, CAT2 or ARG1 protein antagonist, anti--CAT2 or resist-ARG1 antibody or CAT2 or ARG1 antisense polynucleotides be used for the treatment of the experimenter.Can determine suitable reagent based on the screening assay method of describing in the literary composition.

Methods of treatment

Another aspect of the present invention relates to for therapeutic purpose adjusting CAT2 or ARG1 protein expression or active method.Therefore, in exemplary, be used for control method of the present invention comprise with cell with suppress that CAT2 or ARG1 express or CAT2 or ARG1 proteic one or more the active reagent relevant with this cell contact.The reagent of regulating CAT2 or ARG12 expression or protein-active can be the reagent of describing in the literary composition, as the proteic naturally occurring target molecule of polynucleotide, polypeptide or polysaccharide, CAT2 or ARG1 (for example, CAT2 or ARG1 protein substrate or acceptor), the peptide mimics of anti--CAT2 or anti--ARG1 antibody, CAT2 or ARG1 protein antagonist, CAT2 or ARG1 protein antagonist, perhaps other little organic and first machine molecules.

These control methods can carry out (for example, by the experimenter being used described reagent) in vivo.Equally, the invention provides treatment is diagnosed as and suffers from or dangerously suffer from that the enhancing that is characterized as CAT2 or ARG1 is expressed or the method for the individuality of active inflammatory diseases.In one embodiment, this method comprises and uses that downward modulation CAT2 or ARG1 express or active reagent (for example, the reagent of identifying by the screening assay method of describing in the literary composition) or combination of agents.Described treatment can further be positioned to be subjected to the tissue or the cell of the sick invasion and attack of inflammatory phlegm.

Pharmacogenomics

Be used in combination CAT2 or ARG1 modulators for treatment inflammatory diseases,, can consider pharmacogenomics (that is, the individual genotype of research and should individuality to the relation between foreign compound or the drug responses) as asthma, COPD and sacroiliitis.Difference in the therapeutical agent metabolism can cause serious toxicity or treatment failure by dosage and the relation between the haemoconcentration that changes the pharmacological activity medicine.Thereby physician or clinician can consider to use the knowledge that obtains in the research of related drugs genomics and determine whether to use CAT2 or ARG1 conditioning agent and customization dosage and/or the treatment plan with CAT2 or ARG1 modulators for treatment.

Pharmacogenomics is handled because disposition of drug that affected philtrum changes and unusual effect cause the significant clinically heritable variation in the drug responses.Usually, can distinguish two types pharmacogenetics situation.As changing drug effect in the genetics situation of the single-factor transmission of the mode (drug effect of change) of health or as changing the genetics situation that single-factor that health acts on the mode (drug metabolism of change) of medicine is transmitted.These pharmacogenetics situations can be used as rare hereditary defect or occur as naturally occurring polymorphism.For example, glucose-6-phosphate dehydrogenase (G6PD) defective (G6PD) is common hereditary enzymophathy, and wherein main clinical complication is the hemolytic action behind absorption oxidisability medicine (antimalarial drug, sulfa drugs, anodyne, itrofurans medicine) and the edible broad bean.

The high resolution graphics that a kind of pharmacogenomics method (being called " association of genome range ") of identifying the gene of prediction drug responses mainly depends on the people's gene group of being made up of known gene-correlation site (for example, by on the people's gene group 60,000-100, " diallele " genetic marker figure that 000 polymorphism or variant sites are formed, each gene has two kinds of variants).This high resolving power genetic map can compare to identify and concrete viewed drug responses or the relevant gene of side effect with each the Genome Atlas of significant quantity experimenter on the statistics that participates in II/III phase drug test.Alternatively, can be from the people's gene group this high resolution graphics of combination results of tens million of approximately known single nucleotide polymorphism (SNPs).Used in the literary composition " SNP " occurs in the common change in the single nucleotide base in the section of DNA.For example, a SNP can take place in per 1000 bases of DNA.SNP can the involved in diseases process.Yet most SNP are not disease-relateds.The given genetic map that takes place based on this SNPs, individuality can be depended on they separately in the genome SNP concrete mode packet become hereditary classification.By this way, treatment plan can be customized to heredity and go up similar individuality, consider that wherein in the individuality of these genetic resemblances may be the common proterties.

Alternatively, the method that is called " candidate gene approach " can be used to identify the gene of prediction drug responses.According to this method, if the gene of known coded medicine target (for example, CAT2 or ARG1 gene), then can in colony, quite easily identify all common variants of this gene, and can determine whether a kind of form with this gene replys relevant with certain drug to another kind of form.

As the property illustrated embodiment, the activity of drug metabolism enzyme is main definite factor of pharmaceutically-active intensity and time length.The discovery of the genetic polymorphism of drug metabolism enzyme (for example, N-acetyl-transferase 2 (NAT2) and cytochrome P 450 enzymes CYP2D6 and CYPZC19) provides about some experimenter behind the standard of drug administration and the safe dose and can not get desired effect of drugs or show the drug responses of exaggeration and the explanation of serious toxicity.With two kinds of phenotypic expressions, described phenotype is fast metabolic pattern (extensivemetablizer) and slow inactivation (poor metablizer) to these polymorphisms in colony.Slow inactivation is genotypic popular different in different groups.For example, the gene of coding CYP2D6 is highly polymorphic and has identified several sudden changes that in slow inactivation they all cause not existing of function CYP2D6.The slow inactivation of CYP2D6 and CYP2C19 is the often drug responses and the side effect of experience exaggeration when accepting standard dose.If metabolite is the active treatment part, slow inactivation does not show treatment and replys so, as illustrated in the analgesic activity of the morphine monomethyl ether of the metabolite morphine mediation that forms as CYP2D6.Another extremely is the ultrafast metabolic pattern that is called, and they do not reply standard dose.Recently, to be accredited as be because CYP2D6 gene amplification to ultrafast metabolic molecular basis.

Alternatively, the method that is called " genetic expression mapping " can be used for identifying the gene of prediction drug responses.Whether for example, the genetic expression of the animal of drug administration (for example, CAT2 or the ARG1 at CAT2 or ARG1 conditioning agent expresses) can indicate the gene approach relevant with toxicity to open.

The information that produces from more than one said medicine genomics method can be used for determining the individual prevention or the optimal dose and the treatment plan of therapeutic treatment.This knowledge can be avoided adverse effect or treatment failure when being applied to administration and medicament selection, thereby strengthens treatment or prevention efficient when with CAT2 or ARG1 modulators for treatment experimenter.

Pharmaceutical composition

The invention still further relates to the pharmaceutical composition that contains CAT2 or ARG1 conditioning agent and pharmaceutically acceptable carrier.

Used term in the literary composition " pharmaceutically acceptable carrier " is intended to comprise any and all solvents, solubilizing agent, weighting agent, stablizer, tackiness agent, absorption agent, matrix, buffer reagent, lubricant, controlled release carrier, thinner, emulsifying agent, wetting agent, lubricant, dispersion medium, coating, antibacterial agent or anti-mycotic agent, isotonic agent and the absorption delay agent compatible with medicament administration, or the like.It is well known in the art being used for these media of pharmaceutically active substances and the purposes of reagent.Except with inconsistent conventional media of active compound or reagent, can consider to use it in the composition.Can also mix supplement in the composition.

It is compatible with the route of administration of its plan that pharmaceutical composition of the present invention is formulated into.The example of route of administration comprises parenteral, for example, intravenously, intracutaneous, subcutaneous, per os (for example, suction, hypogloeeis, segmental bronchus and lung), through skin (part), stride mucous membrane and rectal administration.The solution or the suspension that are used for parenteral, intracutaneous or subcutaneous application comprise following component: sterile diluent, as water for injection, salts solution, fixed oil, polyoxyethylene glycol, glycerine; Propylene glycol or other synthetics; Antibacterial agent such as phenylcarbinol or para methyl paraben; Antioxidant is as xitix or sodium pyrosulfate; Sequestrant is as ethylenediamine tetraacetic acid (EDTA); Buffer reagent is as acetate, Citrate trianion or phosphoric acid salt be used for the reagent of adjustment of tonicity, as sodium-chlor or glucose.Can use acid or alkali, example hydrochloric acid or sodium hydroxide are regulated pH.Parenteral administration can be enclosed in ampoule, disposable syringe or the multi-dose vials of glass or plastics manufacturing.

The pharmaceutical composition that is suitable for injecting use comprises aseptic aqueous solution (wherein water soluble) or dispersion agent and is used for preparing the sterilized powder of aseptic parenteral solution or dispersion agent temporarily.Use for intravenously, suitable carrier comprises physiological saline, bacteriostatic water, Cremophor EL ^TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS).In all situations, thereby Injectable composition should be aseptic and be that fluid can easily be injected to a certain extent.It must be stablized under production and condition of storage and must preserve under the condition that prevents microorganism such as bacterium and fungal contamination effect.Carrier must be solvent or dispersion medium, and it for example contains, water, ethanol, polyvalent alcohol (for example, glycerine, propylene glycol and liquid polyethylene glycol, or the like) and their suitable mixture.For example, by using coating, as Yelkin TTS, for dispersion agent by keeping required granular size and by using tensio-active agent can keep suitable flowability.By multiple antibacterial agent and anti-mycotic agent, for example, p-Hydroxybenzoate, butylene-chlorohydrin, phenol, xitix, Thiomersalate or the like can realize preventing action of microorganisms.In many cases, can comprise isotonic agent in the composition, as sodium-chlor, sugar or polyvalent alcohol (for example, mannitol, Sorbitol Powder).By comprise the delay absorption agent in composition, for example, monostearate aluminium and gelatin can prolong the absorption of Injectable composition.

By active CAT2 or ARG1 conditioning agent are mixed The suitable solvent with needed amount and required above-named a kind of composition and the combination of composition, prepare aseptic parenteral solution by filtration sterilization then.Usually, prepare dispersion agent by active compound being mixed contain in basic dispersion medium and the sterile carrier from above-named required other compositions.For the sterilized powder that can prepare aseptic parenteral solution, the illustrative preparation method is vacuum-drying and frost drying, and its generation has activeconstituents and from the powder of any extra desirable ingredients of the sterile filtration solution of front.

Oral composition generally includes natural instincts thinner or edible carrier.They can be enclosed in the gelatine capsule or be compressed into tablet.Treatment is used for per os, and active compound can mix vehicle and use with tablet, lozenge or capsular form.Can also use fluid carrier to prepare oral composition with as mouth-washes, wherein the compound per os in the fluid carrier is used and is sent swish and spue or swallow.The wedding agent of pharmaceutically compatible and Adjuvanting material can be included in the composition as its part.Composition below tablet, pill, capsule, lozenge or the like can contain arbitrarily, the compound that perhaps has similar quality: tackiness agent, as Microcrystalline Cellulose, yellow natural gum or gelatin; Vehicle is as starch or lactose; Disintegrating agent is as alginic acid, Primogel, perhaps W-Gum; Lubricant is as Magnesium Stearate or Stertes; Glidant is as colloid silica; Sweetener is as sucrose or asccharin; Perhaps seasonings, as Mentha arvensis L. syn.M.haplocalyxBrig, wintergreen oil, perhaps orange seasonings.

For using by suction, compound can be with from containing suitable propelling agent, and for example, the aerosol spray that the pressure-vessel of carbonic acid gas or divider, spraying gun, segmental bronchus sucker produce or the form of nose drops are sent.In addition, compound can be the form of liquor, gel or desciccate.Sucking preparation can be aqueous solution, and it for example contains, POE-9 LE, glycocholate and deoxycholate salt.If desired, suck preparation and can also contain vehicle, as lactose.Spraying gun can be waterborne suspension or solution, and it comprises and is used to regulate pH and/or tensile carrier or vehicle.

In one embodiment, may contain the treatment part of bioactive compounds with preparing carriers, described carrier will be protected compound in order to avoid remove fast from health, as controlled release preparation, comprise and implanting and the microencapsulation delivery system.Can use biodegradable, biocompatible polymkeric substance, as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, poe and poly(lactic acid).The method for preparing these preparations it will be apparent to those skilled in the art that.Described material can also pass through commercial sources, and for example, from Alza Corporation and NovaPharmaceuticals, Inc obtains.Liposome suspension (comprising the liposome of using at the monoclonal antibody target infected cell of virus antigen) can be used as pharmaceutically acceptable carrier.These liposome suspensions can prepare according to the method that well known to a person skilled in the art, for example, and as U.S. Patent number 4,522, described in 811.

In one embodiment, be used for easy using or dosage same with the per os of dosage unit form preparation or parenteral composition.Used dosage unit form comprises the physically discrete unit that is suitable for as the experimenter's who is treated single dose in the literary composition; Per unit contains the predetermined amount that combines the active compound that produces required result of treatment as calculated with required pharmaceutical carrier.The specification of dosage unit form of the present invention is specified these conditions that also directly depend on by the inherence restriction that this active compound of preparation in the specific characteristic of active compound and the concrete result of treatment that will reach and this area is used for individual treatment.

The toxicity of these compounds and therapeutic efficiency can be determined by standard pharmaceutical methods in cell culture or the laboratory animal, for example determine LD ₅₀(the lethal dosage of 50% colony) and ED ₅₀(the effective dosage of treatment in 50% colony).Dosage between toxicity and the result of treatment is than being therapeutic index and can being expressed as ratio LD ₅₀/ ED ₅₀Can select to show great order and treat the exponential compound.Although can use the compound of performance toxic side effect, should careful design described targeting compounds be attacked the delivery system at the position of tissue, so that minimize, thereby reduce side effect to the not potential injury of affected cell.

The data that obtain from cell culture assays method and zooscopy can be used to prepare the multiple dosage that is used for the people.In many cases, the dosage of these compounds is in the circulation composition scope, and this circulation composition scope comprises having seldom or do not have toxic ED50.Dosage can change in this scope, and this depends on used formulation and used route of administration.For any compound that is used for the inventive method, can treat effective dose according to a preliminary estimate from the cell culture assays method.Can in animal model, prepare dosage to realize comprising circulating plasma concentration as the IC50 (promptly realizing the half maximum test-compound concentration that suppresses of symptom) that in cell culture, determines.These information can be used for determining more accurately useful dosage among the mankind.For example, can measure level in the blood plasma by high performance liquid chromatography.

By the doctor in charge based on the dosage of using that can determine pharmaceutical composition of the present invention such as the various factors of seriousness, time of application and other clinical factors of disease type, pathogenic site, severity of disease, patient's age, sex and diet, inflammation.In one embodiment, suction, whole body or injectable are used and can be begun with minimum effective dose, and dosage will increase in the preset time process, up to observing positive findings.Subsequently, consider any disadvantageous effect that may occur, cumulative dosage will be limited to the level of the corresponding increase that will tell on.Add other known factors to final composition and also can influence dosage.Can monitoring development by the sick development of use standard method periodical evaluation phlegm.

Pharmaceutical composition of the present invention can be used with potion or multi-agent.Agent is inferior can use with any required interval.In one embodiment, each agent comprises about 0.1 μ g-100mg, 1 μ g-10mg, 10 μ g-1mg or 100 μ g-500 μ g active therapeutic agents.Can use the dosage that is lower than 0.1 μ g or is higher than 100mg.The volume of each agent can be for for example, and 0.1ml is to 5ml, 0.1ml to 1ml or 0.2ml to 0.5ml.

Pharmaceutical composition of the present invention can be included in container, packing or the divider with the working instructions of using.

Test kit

The present invention also comprises the test kit that is used for detection of biological sample CAT2 or the existence of ARG1 gene product.This test kit can contain the reagent of assessing the expression of CAT2 or ARG1 on Nucleotide or protein level.In one embodiment, described reagent can be antibody or its fragment, wherein said antibody or its fragment specific combination CAT2 or ARG1.For example, can prepare target antibody by method as known in the art.Randomly, described test kit can contain polynucleotide probes, and wherein said probe specific combination is corresponding to the polynucleotide through transcribing of CAT2 or ARG1 gene.This test kit can contain the device of the amount of CAT2 in the device of amount of CAT2 in the working sample or ARG1 albumen or mRNA and comparative sample and contrast or the standard or ARG1 albumen or mRNA.Compound or reagent can be packaged in the suitable container.This test kit can also contain the working instructions that use this test kit to detect CAT2 or ARG1 albumen or polynucleotide.

The present invention also provides the test kit of the suitability of inflammatory diseases among each the inhibition experimenter who assesses multiple compound.Described test kit comprises the multiple compound that will test and is used to assess CAT2 or reagent that ARG1 expresses (for example, to the special antibody of corresponding protein or probe or the primer special to corresponding polynucleotide).

Embodiment

Embodiment 1: the genetic expression in the mouse lung relevant with atopic reaction changes

Obtain Balb/c mouse (6-8 age in week) from Jackson Laboratories.All animals that are used for this research are all raised under the laminar flow hood super clean bench in the pathogen-free domestic facility of controlled environment.The Animal WelfareAct and the Department of Health that use in zoologic laboratory, the Animal Experimental Study criterion that Education and Welfare (NIH) guilding principle is listed are all abideed by in all experiments.

Be dissolved in 10 μ gOVA (Sigma, St.Louis, MO) the next immune Balb/c mouse of 200 μ l PBS by intraperitoneal (i.p.) injection at the 0th day.At the 14th and 25 day with the mixture anesthesia of mouse, and with attack in 1.5% solution of 50 μ l OVA or the equal-volume PBS tracheae with ketamine and xylazine (be respectively 45 and 8mg/kg).At the 24th, 25 and 27 day with 100 μ l PBS, hIgG (400 μ g/ml) or sIL-13R α 2-Fc (400 μ g/ml) peritoneal injection mouse.According to people such as Urban, Immunity 8 (2): 255-645,1998 implement the purifying of hIgG.Collect lung at the 28th day and freeze suddenly and be used for the RNA separation.

In order to identify the change of the mRNA concentration that depends on the IL-13 Mediated Signal Transduction, before allergy is attacked and during, the mouse that two OVA-attack is handled with 3 peritoneal injections with solvable IL-13 receptor fusion protein sIL-13R α 2-Fc.As the Fc contrast partly of receptor fusion protein, the mouse of two OVA-attacks is used hIgG with intraperitoneal carry out similar processing.Second group of 6 control mice carried out sensitization to OVA and not attack subsequently similarly, and, perhaps handle in identical time course together with peritoneal injection hIgG (n=2) or sIL-13R α 2-Fc (n=2) by using PBS damping fluid (n=2) in the tracheae separately.For the second time lung antigen is attacked that (the 28th day) back is attacked at the 78th hour results OVA and the lung tissue of the control mice handled of damping fluid only.

By to being used to the Balb/c mouse tested or Stat 6 deficient mice intratracheal instillations administered recombinant mouse IL-13 (mIL-13 once a day first with the mixture anesthesia of ketamine and xylazine (be respectively 45 and 8mg/kg); 5 μ g in the 50 μ l final volume), used three days.Use at initial IL-13 and to collect lungs in back 72 hours and on dry ice, freeze suddenly.

Mortar and pestle with cooled with liquid nitrogen are pulverized the mouse lung tissue that freezes suddenly, are suspended in 6ml4M guanidinium isothiocyanate/0.7% beta-mercaptoethanol (GTC/ME) and impulse ultrasound and handle 2 minutes.Suspensions of tissues extracts twice and uses equal-volume isopropanol precipitating nucleic acid through the phenol (Promega Total RNA test kit) of acid balance.Precipitation is resuspended among the 0.8ml GTC/ME, extracts twice again with equal-volume acidifying phenol, and with chloroform extraction 1 time.Use ethanol sedimentation RNA, be suspended in it in water that I DEPC handles and pass through OD ₂₈₀Quantitatively.

The improved Superscript test kit (BRL) that describes in detail before use has (people such as Byrne, Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (New York), 2000) from the synthetic cDNA of the total RNA of 10 μ g.Carry out at 50 ℃ that article one chain is synthetic to be caused and the t7 rna polymerase promotor that will contain polythymidylic acid primer (T7T24) is used to carry out external subsequently sense-rna (cRNA) amplification and biotin labeling from the ribosome-RNA(rRNA) mistake preventing.With the specification sheets purifying cDNA of BioMag C-terminal pearl (Polysciences) according to the manufacturer, and in the 48 μ l 10mM sodium acetates of pH 7.8 wash-out.

The responsive transcription of the external T7 polysaccharase driving of (above) enforcement is with synthetic and biotin labeling antisense cRNA, Qiagen Rneasy column spinner purifying and cRNA fragmentation as described.Describe as manufacturer's working instructions, the GeneChip hybridization mixture contains the smart DNA of 10 μ g fragmentation cRNA, 0.5mg/ml acetylize BSA, 0.1mg/ml Pacific herring that is dissolved in 1 * MES damping fluid, and its cumulative volume is 200 μ l.Reaction mixture was hybridized 18 hours with AffymetrixMullKsubA and MullKsubB oligonucleotide arrays down at 45 ℃.Remove hybridization mixture and wash array also with streptavidin R-phycoerythrin (Molecular Probes) use GeneChip Fluiditics Station 400 (Affymetrix) dyeing, and according to manufacturer's operation instruction Hewlett Packard GeneArray scanner scanning.Collect fluorescence data and change into gene specific difference mean value with MicroArray Suite 4.0 softwares.

11 members' typical curve is by forming from clone's the bacterium and the gene fragment of phage sequence, these curves are that 0.5pM forms the peak in every kind of hybridization mixture of 150pM in concentration, represent about 1,000,000/(ppm) 3.3 to 1000 RNA frequency, wherein assumed average transcript size is 2kb.By the IVT reaction synthesizing biotinylated typical curve fragment that drives from T7-polysaccharase based on the template (as preceding) of plasmid.Be marked with assessment chip susceptibility and will change into the RNA frequency of representing with ppm from the measured fluorescence difference mean value of individual gene in the biotinylated rna fragment conduct at formation peak as typical curve.Contain the coupling fully of gene specific oligonucleotide and the mean fluorecence difference between mismatch probe group and be used for determining frequency about the typical curve that forms the peak.In addition, mainly be used to assess the absolute being of gene product or do not have people such as (, Nat.Biotechnol.14:1850-1856,1996) Lockhart based on the single positive or negative second group of algorithm of replying the right fraction of probe.The sensitivity of single micro-array chip is set to half of Cmin, two existence of (spike-in) template at place, any three adjacent typical curve peaks under this concentration.The typical curve linear regression by force by 0 and the gene frequency of minimum report be set to the sensitivity of individual GeneChip .

Measured a plurality of independently repeat condition of each treatment or control experiment condition and expression data has been carried out conventional statistical analysis in the hope of removing false positive.The frequency values of measuring from the individual bulk measurement of given experimental group uses Excel software to compare at first.The mean value of treatment and control animal is compared to obtain average multiple change (AFC).Calculate two tail (two-tailed) Si Shi T checks with the equal covariance that does not wait covariance or have a frequency values that log transforms with original frequency value.In this work, only report AFC change greater than 2 times and at least a experiment condition those genes of Si Shi t check P＜0.05.The genome of determining by AFC＞2 times and t check P＜0.05 double standards subsequently through editor with remove in most test files non-existent call gene and remove since on MullKsubA and subB oligonucleotide arrays gene cover (tile) redundancy repeatedly.At last, the average expression frequency of removing the animal for the treatment of is less than those genes of 2 times of the mean values of only damping fluid contrast between experiment.

Mouse 11K subA and subB GeneChip  surpass 13,000 kinds of musculus cdnas, ESTs and control sequence between allowing to ask.The average sensitivity of oligonucleotide arrays response is respectively 13ppm and 12ppm for MullKsubA and subB oligonucleotide arrays.By relatively the frequency ratio of Actin muscle and glyceraldehyde-3-phosphate dehydrogenase calculating being monitored the quality of purified RNA and deutero-cDNA product, described Actin muscle and glyceraldehyde-3-phosphate dehydrogenase derive from independently probe groups, and the representative of described frequency ratio is from 5 ' of gene separately-terminal to 3 '-terminal probe.From the different components of contrast and treatment animal from RNA measured 5 '/3 ' than carrying out balance, mean value is 0.81, scope is 0.77 to 0.90, as reporting in the table 3.On the Mul1KsubA of combination and the subB GeneChip  altogether in 13,179 sequence coverages, the individual gene in average 5294 (+/-533) is in calling the file (table 3) that is present in separate analysis, and total variation coefficient is 10.1%.In addition, report that to each subgroup all that exist at least one file call the summation of the calculated rate of gene, mean value is 485,000, and total variation coefficient is 20.9%.The similarity of the RNA quality of chip susceptibility and independent GeneChip  experiment is reflected in the population equilibrium of measured genetic expression, thereby for each file of typical curve lattice ruleization that uses common formation peak provides support (Hill, A.A. wait the people, Genome Biol.2 (12), 2001).

Attack the overall genetic expression (as shown in table 3) that each group of three treatment groups of inductive genetic expression measures and carried out well balanced being used to identify allergen about total mRNA frequency of calling the gene number and between multiple contrast and treatment file, calculating of mRNA integrity, existence.

The general introduction of table 3.RNA balance

	Balb/C 72 hours (IL-13)		STAT6 72 hours (IL-13)		Balb/C 72 hours (OVA)
									Contrast N=5	IL-13 N＝6	Contrast N=4	IL-13 N＝5	Contrast N=6	OVA N＝4	OVA+xFc13Ra2 N＝2
Average RNA 5 '/3 ' ratio	0.90	0.77	0.88	0.80	0.76	0.78	0.80
								The gene # that exists	5396 +/-813	4984 +/-568	5553 +/-475	5422 +/-5143	5143 +/-285	5352 +/-568	5210 +/-394
Sum frequency 1	450 +/-101	581 +/-131	482 +/-80	539 +/-174	447. +/-83	414 +/-52	480 +/-89

¹All that exist at least one file are called the average sum frequency (* 10 of gene ^-3)

Intraperitoneal is used human IgG or sIL-13R α 2-Fc jointly and is not significantly changed the gene expression profile that the control mice of handling with PBS is measured, thereby will merge into a set from the frequency values of 6 control mice when calculating average untreated baseline expression values.Similarly, when calculating the average frequency value of the mRNA frequency that the lung allergen attacks, will use the mouse that four OVA that damping fluid or hIgG handle attack jointly and merge into a set with intraperitoneal.Relatively average lung mRNA frequency has been identified 246 kinds of sequence coverages between the mouse of the control mice of 6 PBS processing and four OVA attacks, and wherein APC is a twice or bigger.In this gene sets, 132 kinds are satisfied second kind of choice criteria of P＜0.05 and show in following table 4.

Table 4. genetic expression changes general introduction

	72 hours IL-13 of Balb/C are to PBS	72 hours IL-13 of STAT6 are to PBS	72 hours OVA of Balb/C are to PBS	PBS STAT6 is to Balb/C
					The gene that exists #·	5306+/-615	5480+/-536	5224+/-386	5472+/-622
Sum frequency	522+/-130	514+/-136	441+/-72	466+/-86
					＞2X AFC 	279	28	246	43
P＜0.05 §	288	5	344	54
					2XAFC+P＜0.05	136	0	132	1

The sum of the mRNA transcript that calls of 13,179 sequence coverage existence.

All that exist at least one file are called the average sum frequency (* 10 of gene ^-3)

^Satisfy the gene number that 2 times or bigger average multiple change standard

^§Satisfy the gene number of Si Shi t inspecting standard P＜0.05.

^‖Satisfy the gene number of the double standards of 2 times of AFC and Si Shi t check P＜0.05.

This allergen inductive gene sets is filtered subsequently to remove non-existent gene and redundant some genes that cover oligonucleotide arrays of calling in most experiments is measured.The mouse that mouse of attacking for the control mice of only using damping fluid, OVA and the OVA that uses the IL-13 antagonist jointly attack is reported average mRNA frequency values.Relative AFC between expressing and express by the contrast lung that background colour is represented with the OVA inductive classifies to gene by functional annotation.Find that the lung allergic response raises the expression of multiple gene sets, only has three statistics significantly to reduce.Many members of institute's inductive allergic effect response gene set are from correlation function family, comprise Fc acceptor, proteolytic enzyme, proteinase inhibitor, complement, chitinase associated protein, immunoglobulin (Ig) and some excretory signal transducers, comprise the chemokine and three leaves (trefoil) factor.Some genes and gene family can be reinvented asthma pathology with respiratory tract hyperergy (AHR) to related with epithelial cell metaplasia and Polyblennia, hypereosinophilic syndrome, respiratory tract.

Pathological research show contain Stat6-/-mouse of amorphs in OVA attack cause that the lung eosinophilic granulocyte soaks into, inhibition (Kuperman, people such as D., J.Exp.Med.187:939-948,1998 of the excessive generation of mucus and AHR; Akimoto, people such as T., J.Exp.Med.187:1537-1542,1998; Miyata, people such as S., Clin.Exp.Allergy 29：114-123，1999)。Use and the scheme identical the Balb/C wild-type mice, make Stat6-/-backcross Balb/C genetic background and contrast (n=4) and handle of amorphs with instil mIL-13 (n=5) or PBS damping fluid of lung.Multi-agent mIL-13 lung instil the back lung tissue express spectra Stat6-/-identified that AFC is 28 kinds of genes of 2 to 3.2 times in the mouse, but these genes do not satisfy T inspecting standard (p＜0.05) and can not think statistics significant (table 4).In addition, these 28 kinds of genes are not corresponding to mIL-13 inductive gene in the Balb/C wild-type background.Expression in the mouse lung tissue that preceding 25 kinds of significant genes of statistics of selecting in the Balb/C background by AFC are attacked by OVA classify and to from similar processing contain Stat-/-frequency values that allelic mouse obtains compares.These data show the gene induced Stat of the needs function of the mIL-13 mediation of all measurements in the Balb/C wild-type, this with for Stat-/-invalid mouse in the no physiologic response of allergen attack consistent.

In contrast, in the Balb/C wild-type (n=4) that the PBS damping fluid is handled measured lung cdna express with damping fluid only handle contain Stat-/-mouse (n=4) of amorphs compares.This has identified that relatively AFC is 54 kinds of genes (table 4) of 2 times or bigger 43 kinds of genes and Si Shi T check P＜0.05.Yet, these gene frequencies relatively in, only a kind of gene satisfies this double selection standard.Serum albumin D-box binding protein Stat-/-reduced by 3.2 times (P=0.03) in the invalid mouse.Except an exception of serum albumin D-box binding protein, these data show when not existing immunity to irritate, from Stat-/-mouse lung of amorphs in the difference of overall genetic expression very limited.Except Stat-relatively/-mIL-13 handle with the damping fluid control mice, these results also point out the dual AFC that is used for filtering data and statistics standard effectively to remove false positive and call.

Embodiment 2: change by genetic expression in the IL-13 lung instillation inductive mouse lung

In order to identify the change of IL-13 mediation in the lung cdna statement, with 6 Balb/c mouse (Jackson Laboratories, Bar Harbor ME) handles with multi-agent 5 μ g (0 hour, 24 hours and 48 hours) lung instillation recombined small-mouse IL-13 (mIL-13).Contrast Balb/C mouse second group (n=4) only instils with identical scheme with damping fluid.In addition, with Stat6-/-invalid mouse instils with multi-agent mIL-13 (n=4) or PBS damping fluid (n=5) lung and carries out same treatment, gathers in the crops all lungs afterwards to determine express spectra in the time of 78 hours.Relatively by intratracheal instillation mIL-13 with only identified that with the gene expression profile data of the Balb/c mouse of damping fluid control treatment average 5306 kinds of existing call in the gene average multiple difference greater than 279 kinds of genes of 2 times in individual file.In these 279 kinds of genes, 136 kinds are satisfied second kind of standard, i.e. Si Shi t check P＜0.05 (going up table 4).

In attack the genetic expression that mediates with direct IL-13 instillation by allergen, exist obviously overlapping.The gene most probable ground of observing unidentified IL-13 rise in OVA inductive model has reflected the difference of the strength of signal that direct instillation cytokine provides.Because all pathologic, physiologics that the interior IL-13 of tracheae uses or the special transgenosis overexpression of lung of IL-13 causes seeing in the mouse model of atopic asthma are replied, so the gene most probable that IL-13 regulates has reflected the set of the expansion of disease related gene.Fig. 1 shows that CAT2 and ARG1 gene are raised in the Balb/c mouse of accepting OVA or IL-13 processing.Fig. 2 has shown the mRNA frequency of ARG1 in the Balb/c mouse that OVA or IL-13 handle.

Embodiment 3: pass through OVA or adenovirus mediated IL-13 induced expression ARG1 gene in the Balb/c mouse

In brief, Balb/c mouse intranasal vaccination recombinant adenovirus is explained 5 * 1010 particles of mouse IL-13 (Ad-IL13) or mouse excretory alkaline phosphatase (Ad-SBAP).Handle control mice with PBS, OVA or IL-13 as described in example 1 above.Inoculate back 72 hours and put to death animal and gather in the crops lung and be used for the RNA extraction.Prepare RNA according to manufacturer's recommendation from lung tissue with RN-easy Mini test kit (Qiagen).Determine that with Affymetrix Mu U74Av2 oligonucleotide arrays ARG1 expresses.The result shows in Fig. 3.The mRNA frequency is expressed as ppm.

Embodiment 4: by IL-13 induced expression ARG1 gene adenovirus mediated in the C57b1/6 mouse

In brief, with 5 * 1010 particle intranasal vaccination Balb/c mouse of Ad-IL13 or Ad-SEAP.Handle control mice with PBS as described in example 1 above.Inoculate back 72 hours and put to death animal.Separate total lung RNA and analysis ARG1 expression as describing among the embodiment 2.The result shows in Fig. 4.The mRNA frequency is expressed as ppm.

Embodiment 5: express with CAT2 and ARG1 among the mouse macrophage clone RAW264.7 of LPS and IL-13 processing

The RAW264.7 cell that will converge is with in the Eagle substratum (cDME) of assigning to the complete Dulbecco improvement of the 20ml that has added 4mM L-glutaminate (CTS), 10% foetal calf serum (JRH Biosciences), non-essential amino acid (Gibco) and 10mM HEPES (Gibco) at 1: 5.With 100ng/ml recombined small-mouse IL-13 (R﹠amp; D Systems) and/or 1 μ g/ml (Sigma) irritated branch junction cell 24 hours from the lipopolysaccharides (LPS) of Pseudomonas aeruginosa (Pseudomonas aeruginosa) serotype 10.After irritating in 24 hours, scrape from culturing bottle and to get cell, with cold PBS washing 1 time, and with cell precipitation containing 600 μ l damping fluid RLT of 10 μ l/ml beta-mercaptoethanols (RN-easy Mini test kit, Qiagen) in cracking.Lysate is-80 ℃ of preservations.

Use recommendation the RAW264.7 cell preparation RNA from handle of RN-easy Mini test kit (Qiagen) according to the manufacturer.By the quantitative RNA of the absorption of 260nm, manufacture 1: 6 typical curve from the sample that LPS/IL-13 handles, begin with the 150ng/ reactant.Use TaqManEZ RT-PCR test kit (Applied Biosystems) that all remaining samples are measured Arg1, CAT1, CAT2A, CAT2B, CAT3 and CAT4 with the 50ng/ reactant, and GAPDH mRNA expression is used to standardize.With Primer Express software (Applied Biosystems) design primer.Arg1, CAT1, CAT3 and CAT2 are input as complete mRNA encoding sequence from GenBank, and only use CAT2A-and the special exon of CAT2B-for CAT2.With primer sequence public database is carried out the BLAST retrieval to guarantee specificity.Below Wyeth is synthetic the primer of sequence (5 '-3 ') and FAM-mark /probe oligonucleotides of TAMRA-cancellation: table 5 primer

Gene	5 ' primer	The FAM probe	3 ' primer
				CAT1	SBQ ID NO：1,518	SEQ ID NO：1,519	SEQ ID NO：1,520
CAT2A	SEQ ID NO：1,521	SEQ ID NO：1,522	SEQ ID NO：1,523
				CAT2B	SEQ ID NO：1,524	SEQ ID NO：1,525	SEQ ID NO：1,526
CAT3	SEQ ID NO：1,527	SEQ ID NO：1,528	SEQ ID NO：1,529
				CAT4	SBQ ID NO：1,530	SEQ ID NO：1,531	SEQ ID NO：1,532
ARG1	SEQ ID NO：1,533	SEQ ID NO：1,534	SEQ ID NO：1,535

Upward use the standard 40 of recommending in the EZ RT-PCR test kit to take turns loop parameter at ABI 7700 Sequence Detector (Applied Biosystems) and carry out pcr amplification.Use people's such as Fink people such as (, Nat.Medicine, 4:1329-1333,1998) Fink method, produce with the threshold cycle number and express indication.Carry out real-time quantitative RT-PCR behind the laser-assisted cell selection.

Fig. 5 shows that CAT2A, CAT2B and ARG1 express and is slightly induced by independent LPS, but significantly induced by the combination of LPS and IL-13.

Arginine in the RAW264.7 cell that embodiment 6:LPS and IL-13 handle is taken in

To be dissolved in 1 * 10 among the 0.5ml cDME ⁶Individual RAW264.7 cell is layered in the 24 hole tissue culturing plates.After adherent 2 hours, add and contain LPS (Sigma) and/or rhIL13 (R﹠amp; D Systems) 0.5ml substratum, its final concentration is respectively 1 μ/ml and 10ng/ml.At 5%CO ₂With in the atmosphere of 95% air in 37 ℃ of incubations after 20 hours, with cell with Arg lavation buffer solution #1 (140mM choline chloride 60,5mM KCl, 0.9mMCaCl ₂, 1mM MgSO ₄, 5.6mM glucose and 25mM HEPES, pH 7.4) washing 3 times, use Arg transport buffer (137mM choline chloride 60,5.4mM KCl, 1.8mM CaCl then ₂, 1.2mM MgSO ₄, 10mM HEPES, be adjusted to pH 7.4) wash again 4 times.Add 5mM L-leucine (Sigma) and 38nM L-[2,3,4,5-in the transport buffer (0.5ml) ³H] mixture to the final concentration of arginine (Amersham) and L-arginine (Sigma) is 400 μ M L-arginine or 100 μ M L-arginine and envrionment temperature incubation 3 minutes.Contain the bipartite hole of each processing of the arginic transport buffer incubation of 5mM L-with quantitative unsaturation combination by using.By in transport buffer, adding 20mM L-Methionin (Sigma) extra duplicate sample is carried out the CAT2 blocking-up.By ending transhipment 4 times with ice-cold Arg lavation buffer solution #2 (10mM HEPES, pH 7.4 for 137mM NaCl, 10mM Tris) washing.Cracking among 500 μ l, 1.0% SDS of cell in being dissolved in 10mM HEPES (pH 7.4).(Pierce) carries out protein quantification with the micro-BCA test kit, and 400 μ l lysates are suspended in the 10ml scintillation solution, the glass scintillation bottle of packing into, and carried out radiometer several 1 minute.Specific arginine absorbs and is calculated as saturated combination (CPM/mg albumen 400 μ M Arg)-unsaturation in conjunction with (CPM/mg albumen 5mM Arg).

As shown in Figure 6, by induce arginine to absorb with combined treatment RAW264.7 cell the best of LPS and IL-13.Yet the arginine of increase absorbs the inhibition (Fig. 7) that is subjected to Methionin, and Methionin is the competitive inhibitor that is used for the CAT2 of arginine transport.

Embodiment 7: produce with the urea in the RAW264.7 cell of LPS and IL-13 processing

For arginine transport research (above), in 24 orifice plates, irritate the RAW264.7 scavenger cell.After irritating 20 hours, use Arg lavation buffer solution #1 washed cell three times, wash again 4 times with the Arg transport buffer then.Cell is at 5%CO ₂With in the atmosphere of 95% air contain 5mM L-leucine, 400 μ M L-arginine ,+/-the Arg transport buffer of 20mM L-Methionin in 37 ℃ of following incubations 24 hours.By with 12, centrifugal 10 minutes clarified supernatant of 000rpm, and get 100 μ l and use UV absorption reagent cassette method (R-Biopharma) to measure urea in triplicate according to manufacturer's operation instruction, just in the 96 hole assay plate in cumulative volume 300 μ l/ holes, carry out with 1/10 scale.Cell is dissolved in 1.0%SDS cracking among the 10mM HEPES (pH 7.4) with 500 μ l, and uses quantitatively albumen of Micro-BCA test kit (Pierce).Urea produces and is expressed as μ g urea/mg protein cleavage thing.

Fig. 8 shows that LPS/IL-13 handles the urea that has increased in the RAW264.7 cell and produces.Take in data consistent with the arginine that shows in Fig. 6 and 7, the urea that the competitive inhibitor Methionin that is used for the CAT2 of arginine transport suppresses to increase produces.

The derived need IL-4 acceptor that embodiment 8:ARG1 expresses

As describing among Fig. 1, IL-4 acceptor gene knock-out mice (IL4R-/-) and IL-4 knock out mice (IL4-/-) are used the OVA sensitization, perhaps handle with PBS or IL-13.Separate total lung RNA and analysis ARG1 expression as describing among the embodiment 2.The result shows in Figure 10.The mRNA frequency is expressed as ppm.

Embodiment 9: the influence that Methionin shrinks carbechal inductive tracheae

The rat in age in 8-10 week is used for this experiment.Tracheostomize is also removed the reticular tissue that adheres to fast.Each tracheae is cut into long 3-4mm, cultivates 15-20 hour in the RPMI-1640 that contains carrier, leucine (25mM), Methionin (100mM) and DMEM (v/v) substratum or both mixtures then.The composition of substratum (mM) comprises 0.1 non-essential amino acid, 4%FBS, 2.0 glutamine, 0.05 beta-mercaptoethanol, 100U/ml penicillin/100 μ g/ml Streptomycin sulphates.

With the excellent longitudinal ligament of tracheae with stainless steel needle, this rod inserts pedestal double-deck, the glass organ bath, and this glass organ bath has been full of 15ml Krebs-Henseleit (K-H) solution (37 ℃), and it has following composition (mM): 118NaCl; 4.7KCl; 1.2KH ₂PO ₄11.1 glucose; 1.2MgSO ₄2.8CaCl ₂And 25NaHCO ₃At each experimental session to this solution 5%CO ₂And 95%O ₂The mixed gas continuous charge.Top upholder is connected to the TSD125 force transducer by the silk thread ring.The tensile of tracheal ring changes (BIOPAC Systems, Inc.) synchronous recording and being presented on the PC computer by the MP150 system.

The tracheae of treated with medicaments is washed 3 times with 10 minutes at interval with K-H solution.Produce carbechal (10 ^-2To 10 ^-5M) response curve.Only when being stablized, the contraction of front concentration increases the concentration of reagent.

When each experiment finished, all tracheaes are trace and weighing on gauze pad all.Tension force is calculated as a milligram tension force/milligram weight (mg/mg) and is expressed as the individual per-cent (%) of the power that the 10-5M carbechal of tracheae when not having medicine causes.All values all is expressed as mean value ± SE.In this result, use the paired t check of Si Shi.Think that less than 0.05 p value be significant.

As shown in Figure 9, carbechal inductive rat tracheae shrinks the inhibition that is subjected to Methionin.

Embodiment 10: reduce carbechal inductive tracheae by disappearance CAT2 gene and shrink

As the processing CAT2 knock out mice described among the embodiment 9 (CAT2-/-).As shown in Figure 11, also suppressed carbechal inductive tracheae by disappearance CAT2 gene and shunk, shown that further CAT2 participates in the pathologic, physiologic of inflammatory diseases.

Getting in touch of inhibition that embodiment 11:ARG1mRNA expresses and the blocking-up of IL-13 signal transduction

Balb/C is handled with untreated mouse to the OVA sensitization, handle with PBS, rIL-13 or sIL13Ra2.Fc, as describing among Fig. 1.As the total lung RNA of separation that describes among the embodiment 2 and analyze ARG1 and express.The result shows in Figure 12.The mRNA frequency is expressed as ppm.

Although sets forth in detail and described the present invention in the specification sheets of accompanying drawing and front; but correspondingly should think described accompanying drawing and this specification sheets only be the property illustrated and be not restrictive, should be appreciated that only show and described in the preferred embodiment and the scope of the invention change and modify all and be protected.

Claims (20)

1. method, it comprises the reagent to the administration treatment significant quantity of suffering from allergy or inflammatory diseases, wherein said reagent suppresses to be subjected to component active of arginine pathways metabolism in the tissue of described affect or expresses, and described component is not nitric oxide synthase (NOS).

2. the process of claim 1 wherein that described disease is a respiratory disease.

3. the method for claim 2, wherein said respiratory disease is that asthma, chronic respiratory are reinvented or chronic obstructive pulmonary disease (COPD).

4. the method for claim 3, wherein said reagent can be in conjunction with the polynucleotide of the described component or the described component of encoding.

5. the method for claim 4, wherein said component is an arginase.

6. the method for claim 4, wherein said component is a cationic amino acid transporter albumen.

7. the method for claim 4, the downstream of wherein said component arginase in described approach.

8. the method for claim 2, wherein said reagent are interfered by RNA or antisense mechanism suppresses the expression of described component.

9. the method for claim 8, wherein said reagent coding or contain to interfere and suppress the siRNA that ARG1 expresses in the described tissue by RNA.

10. the method for claim 8, wherein said reagent coding or contain to interfere and suppress the siRNA that CAT2 expresses in the described tissue by RNA.

11. the method for claim 2, wherein said reagent is alpha-difluoromethyl ornithine.

12. the method for claim 2, wherein said reagent are Methionin or cationic polypeptide.

13. the process of claim 1 wherein that described Mammals is the people.

14. the method for claim 13, wherein said people suffers from asthma or COPD, and described component is arginase or cationic amino acid transporter albumen, and wherein said reagent can be in conjunction with the polynucleotide of the described component or the described component of encoding.

15. identify the compositions and methods that to treat allergy or inflammatory diseases, it comprises: molecule is contacted with the tissue that is subjected to the invasion and attack of asthma or another kind of allergy or inflammatory diseases, and wherein said molecule can be in conjunction with the non--NOS component of arginine pathways metabolism or the polynucleotide of the described component of encoding; With determine whether described molecule can improve or the syndrome or the phenotype of elimination and described asthma or disease-related.

16. the method for claim 15, wherein said molecule is according to selecting or generation based on the rational drug design of structure or according to the SCREENED COMPOUND library.

17. the method for claim 15, wherein said component are arginase or cationic amino acid transporter albumen.

18. a method, it comprises:

Detect at least a expression of gene spectrum in the mammiferous biological sample; With

Whether the reference expression profile of more described express spectra and described at least a gene determining described Mammals and suffer from or dangerously to suffer from allergy or inflammatory diseases,

Non--NOS the component of wherein said a kind of genes encoding arginase metabolism approach.

19. the method for claim 18, wherein said disease is an asthma.

20. pharmaceutical composition, the reagent that it contains the active of pharmaceutically acceptable carrier and the non--NOS component that can suppress arginase metabolism approach or expresses.

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