KR101094974B1 - Novel Biomarkers For the Serodiagnosis of Crohn?s disease - Google Patents

Abstract

The present invention relates to a specific protein that can be used for the serological diagnosis of Crohn's disease by analyzing the serological relationship between Crohn's disease patients and human-derived M. paratuberculosis.

More specifically, it is an antigen derived from Mycobacterium avium subsp. Paratuberculosis isolated from humans, and related to the discovery and identification of the Crohn's disease diagnosis antigen that specifically reacts to the serum of Crohn's disease patients. will be.

M. Paratuberculosis, Crohn's disease, diagnostic antigens, serological markers

Description

Novel Biomarkers For the Serodiagnosis of Crohn ’s Disease

The present invention relates to a novel marker for diagnosing Crohn's disease. More specifically, the present invention relates to Mycobacterium avium subsp. Paratuberculosis antigens isolated from humans, specifically to the serum of Crohn's disease patients. It relates to a reactive antigen for diagnosing Crohn's disease.

Diseases of pathogenic mycobacteria, including M. tuberculosis and M. leprae, are still typical bacterial diseases that threaten human health. Recently, the importance of mycobacterial infectious diseases has been reemerged due to an increase in infectious diseases caused by non-tuberculosis acid bacilli (NTM). Pathogenic mycobacteria are generally known to have similar physiological, biochemical and genetic characteristics, and antigenic commonality, but the etiology, immunity and relationship with the host are not clear. Pathogenic mycobacteria are difficult to prevent, less effective, and cause more serious illness in people with reduced immunity. The recent increase in the number of AIDS patients, the emergence of new viruses, organ transplantation, cell therapy, and other immunosuppressive diseases have caused existing bacterial diseases to be reactivated or cause more serious diseases through opportunistic infections. The most important pathogenic mycobacteria are the M. tuberculosis complex, and recently, an increase in lung disease caused by the M. avium complex has been reported worldwide, and the WHO has already defined it as an emerging pathogen.

Mycobacterium avium subsp. Paratuberculosis (M. paratuberculosis) is one of the most important M. avium associated with acquired immunodeficiency syndrome, the most damaging M. avium in Korea. It is a bacterium belonging to the same species as intracellulare. M. paratuberculosis causes Johne's disease, characterized by chronic granulomatous inflammation, in animals such as cattle, sheep, goats, and deer, and has been reported to be very closely associated with Crohn's disease in humans since 2000. It is reported mainly in developed countries. The potential of M. paratuberculosis to cause common infectious diseases in humans and the inability to eat because people leave dairy products have recently attracted attention as an emerging pathogen that threatens food safety in the United States. It invests a lot of material resources to control. For example, since 2000, M. paratuberculosis has emerged as the causative agent of Crohn's disease, and has invested a huge amount of about 60 billion won from 2002 to 2005 to control the disease. In the past, when acid-fast staining bacilli were observed from the intestine, it was judged to be intestinal tuberculosis caused by M. tuberculosis or M. bovis. However, Crohn's disease is known to be very different from intestinal tuberculosis. M. paratuberculosis is an important issue.

Crohn's disease is a disease that can occur in any part of the digestive system, from the oral cavity to the anus, and usually appears at the distal end of the ileum. Crohn's disease is characterized by chronic inflammatory bowel disease and is a refractory disease among the digestive diseases, usually occurring at a young age of 15-30 years old.There is no established cause or treatment for it, and once symptoms occur, anti-TNF-a, steroid Immunosuppressants such as drug-based drugs are used. The prevalence varies from country to country, but there are reports of one out of every 750 people in the United States and one out of every 500 people in Canada who suffer from Crohn's disease. In the past, it was recognized as an autoimmune disease, but the occurrence of diseases caused by microbiological triggering is prevalent in people with genetic predisposition. The genetic predisposition to patients with susceptibility to Crohn's disease is the best studied mutations in CDRD15, and recently there have been reports of mutations in IL23r and ATG16L1. However, this genetic predisposition is found not only in Crohn's disease but also in other chronic inflammatory diseases, and pathological and physiological aspects of the lesions are known to be most similar to Yone's disease, a chronic granulomatous inflammation caused by M. paratuberculosis.

According to the researches reported so far, Leprosy, tuberuculosis, paratuberculosis and Crohn's disease are related to Crohn's disease in humans. To identify the public health risks of M. paratuberculosis, identify and isolate M. paratuberculosis from people with Crohn's disease using in situ hybridization, and to detect human Crohn's disease using p35 and p36 proteins, which are antigens of M. paratuberculosis. Many studies have been conducted, including serologic studies. However, despite many studies, we are not convinced that M. paratuberculosis is the cause of Crohn's disease because studies on M. paratuberculosis have many limitations. In other words, because the pathogen grows extremely slowly (8-10 weeks) and pathogens isolated from humans exist mainly in the absence of cell walls, gene detection is possible, but culturing the cells themselves is difficult. To date, no studies on the protein bodies of M. paratuberculosis isolated from Crohn's disease have been made. Therefore, such studies are urgently needed.

Until recently, the entire genome map of many pathogenic mycobacteria has been interpreted, preparing for the post-genome era in each country. When the first human genetic map was completed, all diseases seemed to be solved, but as there are many limitations in practical application, mycobacteria are not easy to predict and direct the diseases as a whole comparison of genes. In addition, understanding the discovery and function of new pathogenic agents is essential for the development of vaccines and diagnostic techniques. For example, in 1998, the interpretation and guidance of the entire gene of M. tuberculosis H37Rv, a pathogenic tuberculosis bacterium, was completed. Many genes are suspected to be involved in immunity, but at the protein level only 341 proteins of the 3924 protein coding sequences predicted on the genome have been identified to date (8.7%). In addition, only a few of the identified proteins have been analyzed for their pathogenicity, and most of them are hypothetical. Therefore, in order to elucidate the etiology of Crohn's disease, it is necessary to analyze the function of factors related to pathogenicity based on existing studies and to use them in the development of vaccines and diagnostic techniques.

In addition, M.paratuberculosis is an infectious disease belonging to list B defined by the international OIE. This disease is a very difficult disease to eradicate on pastures once contaminated with M.paratuberculosis because of its long incubation period and the release of pathogens into feces as the disease progresses. In Korea, imports of food animals from other countries are increasing, and there is concern about the spread of common infectious diseases that did not exist in Korea. Do. There have been several reports to the WHO about the risk of M. paratuberculosis, and the United States has recently identified the bacterium as an important emerging pathogen that can be transmitted to humans through food media. However, as mentioned above, there are no domestic studies of this bacterium, and no studies related to Crohn's disease have been reported. Therefore, there is an urgent need to study the relationship between Crohn's disease and M. paratuberculosis in the control of refractory infectious diseases.

To date, there are no reports of specific antigens that can be used in certain causative agents of Crohn's disease and in serological diagnostic techniques. However, Crohn's disease has been steadily increasing both at home and abroad, and M. paratuberculosis is the most noticed as mentioned above.

In this regard, the present inventors performed bacteriological and serological analyzes of 2DE-immunoblot using sera of various patients with similar diseases and secreted antigens of human-derived M. paratuberculosis through the serological analysis of Crohn's disease and human-derived M. paratuberculosis. As a result, the relationship between M. paratuberculosis and Crohn's disease was identified, and specific proteins that could be used for serological diagnosis of Crohn's disease were identified to complete the present invention.

The present invention is to provide a specific protein that can be used in the serological diagnosis of Crohn's disease by analyzing the serological relationship between Crohn's disease and human-derived M. paratuberculosis.

The present invention relates to a specific protein that can be used for the serological diagnosis of Crohn's disease by analyzing the serological relationship between Crohn's disease patients and human-derived M. paratuberculosis.

Hereinafter, the present invention will be described in detail.

The present invention relates to an antigen derived from Mycobacterium avium subsp. Paratuberculosis and relates to a serological diagnostic antigen of Crohn's disease, characterized in that it specifically reacts to the serum of Crohn's disease patients. .

Preferably, the antigen is FbpC1 (Fribronectin binding protein C1), FadB1 (Fatty oxidation protein B1), PhoS2-2 ( Periplasmic phosphate - binding lipoprotein 2-2) , EchA9 (Enoyl-CoA hydratase 9), LprG (Probable conserve lipoprotein LprG), Gap (Glyceraldehyde 3-phosphate dehydrogenase), Tpi (Triosephosphate isomerase), Tal (Transaldorase), aconitate hydratase, TrpC (Indole-3-glycerol-phosphate synthase), Argine (Arginine biosynthesis bifunctional protein), FbpB (Fribronectin binding protein B), HisG (Histidine synthesis protein G), Wag31 (Conserved hypothetical protein), Mdh (Malate dehydrogenase), LppZ ( Putative lipoprotein), FecB (Probable FeIII-dicitrate-binding periplasmic lipoprotein), GabD2 (Putative succinate-semialdehyde dehydrogenase [NADP +] dependant protein) and superoxide dismutease.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the development of Crohn's disease.

As used herein, the term "Crohn's disease" is a type of inflammatory disease characterized by chronic and recurrent intestinal inflammation, consisting of four layers of mucosa, submucosa, muscle layer, and intestinal membrane from the inside. Wall of the intestine is a disease characterized by concomitant inflammation of all layers of the intestine, which can occur in all parts of the intestine, but most often at the distal end of the large intestine, which is connected to the cecum, and is often perforated due to inflammation of the intestine's entire layers .

In the present invention, the tuberculosis antigens of human Crohn's disease (n = 3) show high antibody response to M. paratuberuclosis secretory antigens isolated from human Crohn's disease patients and Yone's disease individuals. Antigens that did not respond in the sera of patients with ulcerative colitis and negative control were analyzed by 2D-immunoblot.

In addition, secretory antigens of human-derived M. paratuberculosis were identified by analyzing proteins that did not respond to animal-derived antigens but only to human-derived antigens through 2DE-immunoblot and LC-ESI-MS techniques.

In addition, using the pooling serum (n = 10) of patients with M. paratuberculosis, the secreted antigen of M. paratuberuclosis, and the cellular extract antigen, the proteins that specifically react to IgG of Crohn's disease patients were identified. The protein was detected and LC-ESI-MS was used to identify proteins that specifically react to the serum of patients with Crohn's disease. The proteins with useful diagnostic value were analyzed by matching score and bioinformatic analysis. As a result, 33 proteins that specifically respond to the patient's serum of Crohn's disease were identified by LC-ESI-MS analysis, and the BLAST search for these 33 proteins predicted the possible function of the protein, among which the decoded proteins. You could get 19.

In accordance with the present invention, the diagnosis of Crohn's disease can be performed in a biological sample by measuring the formation of an antigen-antibody complex produced by the specific reaction of the antigen-antibody by contacting the diagnostic antigen of the present invention with a serological sample. It may be by a method for confirming the presence of an antibody that specifically binds to the antigen. As used herein, the term “antigen-antibody complex” refers to the combination of the diagnostic antigen protein of the invention with an antibody that specifically recognizes it in a biological sample.

By "antibody" is meant herein a specific protein molecule directed against the antigenic site. Antibodies used in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab) ₂ fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fv molecules, Chimeric antibodies and the like. Most preferably IgG.

The Crohn's disease diagnostic antigen protein of the present invention is derived from human, and can be produced by a recombinant method based on a DNA sequence. When using a recombinant technique, a nucleic acid encoding a protein is inserted into an appropriate expression vector, the host cell is cultured so that the target protein is expressed in the transformant transformed with the recombinant expression vector, and then It can be obtained by recovering the protein.

In another aspect, the present invention provides a composition for diagnosing Crohn's disease comprising the antigen for diagnosing Crohn's disease.

The diagnostic composition of the present invention may further include reagents known in the art used for immunological analysis in addition to Crohn's disease diagnostic antigen.

Immunological analysis in the above may include any method that can measure the binding of the antigen and the antibody. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunosorbent assay (ELISA), immunoblotting, and PAR assays. Farr assay), immunoprecipitation, latex aggregation, erythrocyte aggregation, non-turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence.

Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

The label capable of generating a detectable signal enables qualitatively or quantitatively measuring the formation of an antigen-antibody complex, examples of which include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and redox molecules. And radioisotopes can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidase, hexokinase, malate dehydrogenase, and glucose-6 Phosphate dehydrogenase, invertase and the like can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, fluorine isothiocyanate, etc. may be used. Ligands include biotin derivatives, and luminescent materials include acridinium esters, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, and the redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone and hydroquinone. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. However, any of those that can be used in immunological assays other than those exemplified above may be used.

On the other hand, the diagnostic composition of the present invention can be provided in a fixed state using a variety of methods known on a suitable carrier or support to increase the speed and convenience of diagnosis ( Antibodies : A Labotory Manual, Harlow & Lane Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polyesters, companion rocks, filter papers, ion exchange resins, plastic films, plastic tubes , Glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible form, for example spherical (bead), cylindrical (test tube or inner surface of the well), planar (sheet, test strip).

Therefore, preferably, the composition for diagnosing Crohn's disease of the present invention may be provided in the form of a diagnostic kit.

As described above, the present invention identifies human-derived M. paratuberculosis antigens capable of serologically diagnosing Crohn's disease through 2DE-immunoblot and LC-ESI-MS techniques and does not respond to other similar diseases. Only new proteins that react specifically are identified. In the future, these antigens could be used as a useful technique for the serological diagnosis of Crohn's disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

< Example  1> individual antibody response Of diagnostic antigen  excavation

1-1: Secretory  Ready

To date, proteins with significance in serological diagnosis of mycobacterium, including the diagnosis of tuberculosis, have been found mainly in secretory antigens. Based on this, secretory antigens of M. paratuberculosis derived from humans and animals were collected, and then ELISA was performed using serum of each patient.

Secretory antigens are described by Cho D et al. (Cho D, Sung N, Collins MT. Identification of proteins of potential diagnostic value for bovine paratuberculosis. Proteomics. 2006, 6 (21): 5785-5794). Once, M. paratuberculosis derived from humans and animals was grown in a modifide Watson-Reid (WR) medium containing Mycobatin J, and then centrifuged (30,000 × g, 10 min) to obtain a culture. The obtained culture was passed through a 0.2 μm filter and concentrated using Centricon Plus 80 (molecular weight> 5kDa). Finally, the culture secreting antigen was prepared by dialysis with 10 mM PBS (pH 6.8) and measuring the concentration.

1-2: Observing Individual Differences in Antibody Responses

As shown in Figure 1, using the secretion antigen of various isolates of M. paratuberculosis (standard strain VS. UCF human isolate), Crohn's disease, Crohn's-like ulcerative colitis patients, mycobacterium patients with Mycobacterium tuberculosis patients, individuals using serum Antibody reactions were varied according to the isolates of humans and animals.

This means that specific strains (human-derived strains) and specific antigens are important for the serological diagnosis of Crohn's disease, and this example was used as a basis for identifying antigens important for developing the serological diagnostic kit for Crohn's disease. .

1-3: 2 DE - immunoblot Derived from human using M. paratuberculsosis of Secretory antigen  Specific antigen discovery

As shown in Figures 2-4, other similar diseases centered on secretory antigens of M. paratuberuclosis isolated from human Crohn's disease patients (UCF-4, UCF-5) and animal Yone disease (B236, B238) individuals. 2D-immunoblot, which does not respond to the serum derived from the patient, specifically reacts with the serum (n = 3) of the patients with high antibody titer among the Crohn's disease patients in the ELISA technique mentioned in Example 1-2 above. The protein was analyzed by 2DE-immunoblot and LC-ESI-MS.

As a result, a total of eight specific M. paratuberculosis antigens could be identified in the secreted antigen of human-derived M. paratuberculosis (see FIG. 4). The molecular weight and function of the identified antigen are shown in the table below.

Identified protein Molecular mass  ( kDa ) Possible function ModD 45 Fibronectin attachment
Culture filtrate glycoprotein MAP1018c 35 Enoyl-CoA hydroratase PepA 32 Serin protease MAP2950c 20 Hypothetical protein DegQ 8 Serine protease htrA-like protein MAP1420 45 Hypothetical protein SodA 21 Superoxide dismutase MAP4056c 13 Hypothetical protein

< Example  2> Crohn's disease  M. for serological diagnosis paratuberculosis  Diagnostic value factor discovery

In the present embodiment, as shown in Fig. 4 (A, B), the secreting antigen and cellular extraction of pooling serum (n = 10) and M. paratuberuclosis isolated from humans in patients with M. paratuberculosis among Crohn's disease patients ) Antigen was used to detect the antigen specifically responding to IgG of Crohn's disease. The detected antigens were identified by LC-ESI-MS method as shown in the table below and analyzed for antigens with useful diagnostic value through matching score and bioinformation analysis.

The experimental method used in this Example is as follows. Finally, protein samples collected according to each condition were analyzed by 2D electrophoresis and LC-ESI-MS technique.

200 ug of each sample (7 cm IPG strip) or 400 ug (11 cm IPG strip) was rehydrated in buffer [8 M urea, 2% CHAPS, 80 mM DTT, 0.001% bromophenol blue, 0.2% Amphorite (Biolyte, Bio-Rad)], IPG strips (Bio-Rad) have a pH range of 3-10, pH 4-7, pH 3.9-5.1, pH 4.7-5.9, and their length Was used 7 cm or 11 cm. The PROTEAN IEF cell (Bio-Rad) with a default cell temperature of 20 ° C and a maximum current per strip of 50 μA was used.

First, the prepared sample was loaded into a rehydration tray and rehydrated for 12-16 hours, and then an electrode wick pre-soaked with tertiary distilled water to remove salt or impurities from the sample was placed on the electrode. The plate was placed on top of the strip, covered with sufficient mineral oil, and the excess salt was removed at 250 V for 30 minutes.

After the desalination process, the voltage was increased from 250 V to 4000 V for 2 hours at 7 cm and focused at 4000 V to 20,000 VHr, from 250 V to 8000 V at 11 cm, and Focusing was performed at 8000 V for 35,000 VHr. After focusing, a hold step was placed at 500 V to prevent overfocusing or drift of the sample.

After the IEF was terminated, the strips were stored at -20 ° C until secondary separation, the IPG strips stored at -20 ° C were dissolved at room temperature, and the strips were equilibrated in a buffer buffer I [6 M urea so that the proteins in the strip could bind to the SDS. , 0.375 M Tris-Cl (pH 8.8), 2% DTT (w / v), 2% SDS, 20% glycerol] and reacted for 20 minutes. After the first reaction, the solution was added to equilibration buffer II [6 M urea, 0.375 M Tris-Cl (pH 8.8), 2.5% iodoacetamide (w / v), 2% SDS, 20% glycerol] and subjected to a second reaction for 20 minutes. The anode side of a sufficiently equilibrated strip was inserted to the left of the SDS-PAGE gel (marker direction). Electrophoresis was performed using 15% polyacrylamide gel or 10-20% gel (Bio-Rad), and the gel was dyed with Coomassie brilliant blue R250 (Bio-Rad), followed by 30% It was decolorized with a solution of methanol and 10% glacial acetic acid.

Significantly increased protein spots separated by 2-DE were identified by LC-ESI-MS analysis, and the identified proteins are shown in Table 1 below.

A large number of protein spots reacted with anti-human IgG were observed, of which only 13 protein spots (CF) in culture filtrate (CF) and 4 protein spots (CE) in cellular extract (CE) were detected in human IgG. Specifically, no IgA-specific reaction spots were observed.

Finally, 33 proteins out of 40 irradiated protein spots were identified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis. The BLAST search for 33 proteins predicted the possible function of the protein, of which 14 open reading frames (ORFs) were considered hypothetical proteins and 19 were decoded.

Serological diagnostic markers of human-derived M. paratuberculosis that specifically respond to IgG in Crohn's disease patients Protein name spot  # M.W.
( Da ) predicted function MAP 0047c hypothetical protein 14, 27 41100 hypothetical protein MAP 0217 FbpC1
(Fibronectin binding protein C1) 19 30929 Putative esterase MAP 0494 hypothetical protein 38 36215 hypothetical protein (26-301: WcaG-Nucleoside diphosphate sugar epimerases) MAP 0630c hypothetical protein 15 28618 hypothetical protein MAP 0790 FadB1
Fatty oxidation protein B1 28 76177 1-709: multifunctional fatty acid oxidation complex subunit alpha MAP 0872 PhoS2-2
(Periplasmic phosphate-binding lipoprotein 2-2) 11, 12 37211 50-358: PstS -ABC type phosphate transport system MAP 0948 hypothetical protein 3 61155 hypothetical protein MAP 1018c EchA9
(Enoyl-CoA hydratase 9) 9 36852 7-350: enoyl-CoA hydratase MAP 1138c LprG
(Probable conserve lipoprotein LprG) 20, 21 24721 DUF1396- This family consists of several putative lipoproteins (unknown function) MAP 1164 Gap
(Glyceraldehyde 3-phosphate dehydrogenase) 29 36095 2-337: Glyceraldehyde-3-phosphate dehydrogenase / erythrose-4-phosphate dehydrogenase MAP 1166 Tpi
(Triosephosphate isomerase) 17, 18 27545 Triosephosphate isomerase-the key enzymes of the glycolytic pathway MAP 1177c Tal
(Transaldorase) 7 40338 17-359: Transaldolase-like proteins from plants and bacteria MAP 1210c Aconitate hydratase One 101546 aconitate hydratase (82-379: Aconitase A catalytic domain) MAP 1305 TrpC
(Indole-3-glycerol-phosphate synthase) 33 28125 45-258: Indole-3-glycerol phosphate synthase (IGPS); an enzyme in the tryptophan biosynthetic pathway, catalyzing the ring closure reaction of 1- (o-carboxyphenylamino) -1-deoxyribulose-5-phosphate (CdRP) to indole-3- glycerol phosphate (IGP), accompanied by the rel MAP 1339 hypothetical protein 39 15437 hypothetical protein (6-143: Usp: Universal stress protein family.Usp is a small cytoplasmic bacterial protein whose expression is enhanced when the cell is exposed to stress agents MAP 1362 ArgJ
Arginine biosynthesis bifunctional protein 22, 36, 37 40929 38-404: Ornithine acetyltransferase (OAT) family; also referred to as ArgJ MAP 1609c FbpB
(Fribronectin binding protein B) 5 34706 Fibronectin binding protein MAP 1659 hypothetical protein 16 26333 hypothetical protein MAP 1718c hypothetical protein 24 15518 hypothetical protein (31-149: DUF1942-Domain of unknown function, beta-sandwich structure) MAP 1846c HisG
(Histidine synthesis protein G) 31 30541 48-207: ATP phosphoribosyltransferase MAP 1889c Wag31
(Conserved hypothetical protein) 40 28050 4-215: Cell division initiation protein MAP 1981c hypothetical protein 32 27294 hypothetical protein (3-245: Zn-ribbon protein, possibly nucleic acid-binding) MAP 2120c hypothetical protein 2 61155 hypothetical protein MAP 2121c hypothetical protein 10 33671 hypothetical protein MAP 2541c Mdh
(Malate dehydrogenase) 13 34631 1-326: malate dehydrogenase MAP 2770 hypothetical protein 23, 25, 26 19316 hypothetical protein MAP 3041 LppZ
(Putative lipoprotein) 8 41254 39-400: Glucose / sorbosone dehydrogenases MAP 3092 FecB
(Probable FeIII-dicitrate-binding periplasmic lipoprotein) 12 36983 a protein involved in the transport of iron from ferric citrate in Escherichia coli MAP 3567 hypothetical protein 34, 35 30184 hypothetical protein (1-271: short chain dehydrogenase, 1-2-2: 3-ketoacyl- (acyl-carrier-protein) reductase) MAP 3673c GabD2
(Putative succinate-semialdehyde dehydrogenase) 4 49979 role of the formimg an alternative pathway from alpha-ketoglutarate to succinate (in M.TB) MAP 3956 hypothetical protein 6 45659 hypothetical protein (2-402: dihydrolipoamide dehydrogenase) MAP 4308c hypothetical protein 30 33645 hypothetical protein MAP 0187c Superoxide dismutease 20 23030 Superoxide dismutease

In the above, a preferred embodiment of the present invention has been described, but this is only an example and various modifications and changes to the present invention are possible. In addition, the fact that all such modifications and changes belong to the scope of the present invention will become more apparent through the following claims.

As described above, the antigen for diagnosing Crohn's disease derived from Mycobacterium avium subsp. useful. These antigens are expected to be useful in useful techniques for the serological diagnosis of Crohn's disease.

1 shows individual antibody responses using secretory antigens of UCF human isolates and bovine isolates of M. paratuberculosis. However, UC: ulcerative colitis (patient ulcerative colitis), CD: crohn's disease (patient Crohn's disease), TB: tubercle bacillus (patient tuberculosis bacteria).

However, S / P = (OD sample ㅡ OD negative control) / (Mean OD positive control ㅡ Mean OD negative control)

Figure 2 is the result of 2DE-immunoblot sera of Crohn's disease patients secreted antigen of UCF human isolate of M. paratuberculosis. Protein spots reacting only with human-derived antigens were identified to identify the proteins in FIG. 3.

FIG. 3 shows the secreted antigens of M. paratuberuclosis isolated from human Crohn's disease patients (UCF-4, UCF-5) and Johne's disease (B236, B238) individuals. 2D-immunoblot analyzes antibodies that specifically react with the serum of patients (n = 3), and reacts only with human-derived antigens, but not with animal-derived antigens, via 2DE-immunoblot and LC-ESI-MS techniques. This is an analysis. In the figure shows a total of eight specific M. paratuberculosis antigens identified in the secreted antigen of human-derived M. paratuberculosis.

Figure 4 pooling serum of patients with M. paratuberculosis (n = 10) using a (secretory antigen) and b (cell antigen) of UCF-4 strains of M. paratuberculosis isolated from patients with Crohn's disease. In other words, proteins that specifically react to human IgG are labeled.

Claims (4)

A marker for diagnosing Crohn's disease consisting of EchA9 (Enoyl-CoA hydratase 9), an antigen derived from Mycobacterium avium subsp. Paratuberculosis isolated from humans and specifically reacting to Crohn's disease serum Crohn's disease diagnostic composition comprising a. The method according to claim 1, wherein the marker is an antigen derived from Mycobacterium avium subspecies paratuberculosis isolated from humans and specifically responds to Crohn's disease serum, FbpC1 (Fibronectin binding protein C1), FadB1 (Fatty oxidation protein B1), PhoS2-2 (Periplasmic phosphate-binding lipoprotein 2-2), LprG (Probable conserve lipoprotein LprG), Gap (Glyceraldehyde 3-phosphate dehydrogenase), Tpi (Triosephosphate isomerase), Tal (Transaldorase), aconitate hydratase, TrpC (Indole-3-glycerol-phosphate synthase), Argine (Arginine biosynthesis bifunctional protein), FbpB (Fibronectin binding protein B), HisG (Histidine synthesis protein G), Wag31 (Conserved hypothetical protein), Mdh (Malate dehydrogenase), LppZ ( One selected from the group consisting of putative lipoprotein), FecB (Probable FeIII-dicitrate-binding periplasmic lipoprotein), GabD2 (Putative succinate-semialdehyde dehydrogenase [NADP +] dependant protein) and superoxide dismutase Crohn's disease diagnostic compositions including the antigen further characterized in that the marker formed. delete Crohn's disease diagnostic kit comprising the marker of claim 1.

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