KR20120131532A - Protein for dianosing porcine proliferative enteritis and application thereof - Google Patents

Abstract

PURPOSE: A protein for diagnosing porcine proliferative enteritis is provided to effectively diagnose Lawsonia intracellularis infection in pigs. CONSTITUTION: An antigen protein fragment for diagnosing porcine proliferative enteritis contains an amino acid sequence of sequence number 1. A composition for diagnosing porcine proliferative enteritis contains a protein fragment of sequence number 1. A kit for diagnosing porcine proliferative enteritis contains the protein fragment or an antibody which specifically recognizes the protein fragment. A method for detecting an antibody for Lawsonia intracellularis in a biological sample comprises a step of providing a biological sample; a step of reacting the biological sample with proteins; and a step of detecting the presence of a complex in the biological sample and proteins.

Description

Protein for diagnosing swine proliferative ileitis and its application {Protein for dianosing porcine proliferative enteritis and application

The present invention relates to a protein for diagnosing swine proliferative ileitis and its application. More specifically, the present invention relates to LI0147 ORF, which is expected to be the outer membrane protein of Lawsonia intracellularis in connection with the diagnosis of the causative agent of swine proliferative ileitis. It relates to a 95 amino acid sequence protein fragment of a high immunogenic site and to expressing this protein fragment and using it as an antigen to diagnose swine proliferative ileitis.

Swine proliferative ileitis is caused by Gram-negative intracellular parasites called Lawsonia intracellularis. Farm surveys in Europe, Asia, and North America have found that one-fourth to one-half of farms in pig farms are seriously infected, and more than 50% of farms in Korea are diagnosed with ileitis. Serological studies have shown that over 90% of pig farms in most countries have L. intracellularis. In particular, this disease is a disease that is increasing in the incidence of pigs in developed countries, which is relatively hygienic.

The spread of the disease is caused by vertical infection through feces of infected sows and horizontal infection in piglets through feces of infected pigs.

Proliferative ileitis is a major problem in the swine industry in Korea and around the world, and many pig farmers are suffering economic damage from swine proliferative ileitis, but the research on the causative organism is difficult to separate and culture. . In the United Kingdom and the United States, the successful study of pure culture of Lawsonia intracellularis, the causative agent of proliferative ileitis, has been actively conducted in the laboratory. As a result of this research, the genome information of Lawsonia intracellularis is registered in the NCBI Gene Bank. ACCESSION: NC008011).

In addition, in the conventional method of swine ileitis (causative bacterium: Lawsonia intracellularis), 1) diagnosis by clinical symptoms and histopathologic findings, 2) identification of causative bacteria, and 3) individual diagnosis by PCR are used. Has been.

However, the existing clinicopathological methods or methods by identification of causative bacteria should confirm whether the bacteria penetrate into the tissue by microscopic confirmation after tissue sections and staining. The condition is required and the time required for diagnosis is high.

The present invention solves the above problems, and the object of the present invention as devised by the necessity of the above is to provide a kit for diagnosing swine proliferative ileitis, which is a major disease occurring in pigs in a short time in a single reaction will be.

Another object of the present invention is to provide a method for diagnosing swine proliferative ileitis, which is a major disease occurring in pigs within a short time in one reaction.

In order to achieve the above object, the present invention provides a protein for diagnosing swine proliferative ileitis having the amino acid sequence of SEQ ID NO: 1.

The present invention also provides an antibody that specifically recognizes the protein of the present invention.

The present invention also provides a composition for diagnosing swine proliferative ileitis comprising the protein of the present invention.

In another aspect, the present invention provides a kit for diagnosing swine proliferative ileitis comprising the protein of the present invention.

In another aspect, the present invention (a) provides a biological sample; (b) binding to the protein of claim 1 of Lawsonia intracellularis when Lawsonia intracellularis is present in the biological sample Under conditions that allow for the formation of an antibody / antigen complex, the biological sample is reacted with the protein of claim 1; (c) detecting the presence of the complex to detect the presence of the antibody of Lawsonia intracellularis in the sample. Provided are methods for detecting antibodies or antigens against Lawsonia intracellularis in a biological sample, including detecting the presence.

In another aspect, the present invention provides a method for the treatment of swine proliferative ileitis comprising reacting an antibody according to the present invention to a sample comprising Lawsonia intracellularis and judging a sample showing a positive response to the antibody as swine proliferative ileitis. Provide diagnostic methods.

Hereinafter, the present invention will be described.

One exemplary embodiment of the present invention provides a method for efficiently diagnosing infection of Lawsonia intracellularis via LI0147 in pigs.

Another exemplary embodiment provides a recombinant protein having the amino acid sequence of SEQ ID NO: 1.

The amino acid sequence of SEQ ID NO: 1 is obtained by analyzing the antigenic and hydrophobic regions of the LI0147 ORF protein of Lawsonia intracellularis, and the sequence is different from each other. The amino acid sequence of SEQ ID NO: 1 is the 59th to 154th amino acids of LI0147 ORF A group consisting of sequences, it is an immunogenic antigen specific for Lawsonia intracellularis. SEQ ID NO: 1 may be useful for diagnosing swine proliferative ileitis as a specific immunogenic antigen for swine proliferative ileitis.

The amino acid of SEQ ID NO: 1 was selected by analyzing the antigenic and hydrophobic regions of the LI0147 ORF protein of Lawsonia intracellularis. Antigenicity and hydrophobicity analysis can be performed using a known method, preferably antigenicity is analyzed according to the Kolaskar & Tongaonkar Antigenicity method, hydrophobicity can be analyzed according to the Parker method. The amino acid sequence of SEQ ID NO: 1 may be synthesized using a known method, preferably an oligonucleotide synthesizer, or may be made into a recombinant protein using the same.

Another embodiment of the present invention provides a recombinant protein having the amino acid sequence of SEQ ID NO: 1. The recombinant protein has immunogenicity to Lawsonia intracellularis and can be used to specifically diagnose porcine proliferative ileitis.

In order to select Lawsonia antigen protein, the genome sequence of Lawsonia intracellularis was identified from NCBI GenBank data (NCBI GenBank data, ACCESSION: NC008011), and LI0147, which is expected to encode outer membrane protein, was selected. From the amino acid sequences of these genes, antigenicity and hydrophilicity were analyzed to select specific sequences capable of detecting Lawsonia intracellularis, which were cloned into E. coli expression vectors. In addition, the insoluble protein was successfully purified using a buffer containing 8M urea. The purified protein provides a method for screening pigs infected with Lawsonia intracellularis and uninfected pigs using direct enzyme immunoassay.

By immunogenic antigen or fragment herein is meant a fragment of a protein having one or more epitopes that can be recognized by an antibody against a protein of the invention.

In order to achieve another object of the present invention, the present invention provides a diagnostic agent for porcine proliferative ileitis comprising an antibody that specifically binds to the protein of the present invention or immunogenic fragments thereof.

The antibody of the present invention may be a polyclonal antibody, but is preferably a monoclonal antibody.

Polyclonal antibodies can be prepared by injecting a fragment of the protein of the invention as an immunogen into an external host according to conventional methods known to those skilled in the art. Outer hosts include mammals such as mice, rats, sheep and rabbits. Immunogens are injected intramuscularly, intraperitoneally or subcutaneously, and are usually administered with an adjuvant to increase antigenicity. Blood is collected periodically from the external host to collect serum that shows improved titers and specificity for the antigen or to separate and purify the antibody therefrom.

Monoclonal antibodies can be prepared by techniques for generating immortalized cell lines by fusions known to those skilled in the art (Koeher and Milstein 1975, Nature, 256: 495). The manufacturing method is briefly described as follows. First, 20 ug of pure protein is immunized to Balb / C mice or peptides are synthesized and combined with bovine serum albumin to immunize mice. Thereafter, antigen-producing lymphocytes isolated from mice are fused with myeloma in humans or mice to produce immortalized hybridomas, and only hybridoma cells that produce the desired monoclonal antibody using ELISA method. After selection and propagation, monoclonal antibodies are isolated and purified from the culture.

In the method of the present invention, the detecting step directly detects the presence of the protein or immunogenic fragment thereof by two-dimensional (2-D) electrophoresis from the blood solution of the liver of the individual, or by contacting the blood with the antibody of the present invention, the antigen antibody. Indirectly confirming the presence of a protein or protein fragment of the invention through a reaction.

Currently known immunoassay as an antigen antibody reaction, enzyme immunoassay (ELISA, Coated tube), antibody-bound magnetic particles are bound to a tube, and then antigen-tracer and refractory contaminants are reacted competitively to induce enzymatic reaction. Magnetic particle method, latex particle method using antibody bound latex particle.

Diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art, and typically include lyophilized antibodies, buffers, stabilizers, inactive proteins and the like. The antibody may be labeled by radionuclides, fluorescent sources, enzymes, and the like.

Monoclonal antibodies of the present invention can be used in a variety of immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), as well as various pig growth through the development of antibodies showing a higher specificity and sensitivity It can also be used to develop protein chips with ileitis detection spectrum.

As mentioned above, the protein of the present invention can be used as a diagnostic agent to detect the presence of Lawsonia intracellularis reactive antibodies in a biological sample to determine the presence of Lawsonia intracellularis infection. For example, the presence of antibodies reactive to the proteins of the invention can be detected using standard electrophoresis and immunodiagnostic techniques, including competition, direct response or immunoassay such as sandwich type assays. Such assays include, but are not limited to, Western blots; Coagulation test; Enzyme labeled and mediated immune assays such as ELISA; Biotin / avidin type assays; Radioimmunoassay; Immunoelectrophoresis; And immunoprecipitation. Such reactions generally involve identifying labels such as fluorescence, chemiluminescence, radioactive activity, enzymatic labels or dye molecules, or include other methods of detecting the formation of an antigen and the antibody or antibodies that react with it. .

Assays described above generally involve isolating unbound antibodies in a liquid phase from a solid phase support to which the antigen-antibody complexes are bound. Solid supports that can be used in the practice of the present invention include substrates such as nitrocellulose (eg, in the form of membranes or microtiter wells); Polyvinylchloride (eg, sheet or microtiter wells); Polystyrene latex (eg, beads or microtiter plates); Polyvinylidene fluoride; Diazotized paper; nylon membrane; And activated beads, magnetically reactive beads, and the like.

Typically, the solid support first reacts with a solid phase component (eg, a protein of one or more Lawsonia intracellularis) under suitable binding conditions to ensure that the component is sufficiently anchored to the support. Sometimes, the immobilization of the antigen to the support can be enhanced by binding the antigen to peptides with better binding properties. Suitable binding proteins include, but are not limited to, macromolecules such as serum albumin, including bovine serum albumin (BSA), kiolimpet hemocyanin, immunoglobulin molecules, tyroglobulins, ovalbumins, and other proteins known to those skilled in the art. Included. Other molecules that can be used to bind the antigen to the support include polysaccharides, polylactic acid, polyglycolic acid, polymerizable amino acids, amino acid copolymers, and the like. Such molecules and methods of binding such molecules to antigens are known to those skilled in the art. Brinkley, M.A. Bioconjugate Chem. (1992) 3: 2-13; Hashida et al., J. Appl. Biochem. (1984) 6: 56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30: 117-124.

After reacting the solid support with the solid phase component, any unfixed solid phase component is removed from the support by washing, and the support bound component is subsequently found to contain ligand residues (eg, antibodies to immobilized antigen) under suitable binding conditions. Contact with the expected biological sample. After washing to remove any unbound ligand, a secondary binder moiety is added under suitable binding conditions, where the secondary binder can optionally be bound to the bound ligand. The presence of secondary binders can then be detected by using techniques known in the art.

More particularly, ELISA methods are used wherein the wells of the microtiter plates can be coated with the required protein. Biological samples containing or expected to contain anti-protein immunoglobulin molecules are then added to the coated wells. After sufficient incubation to allow the antibody to bind to the immobilized antigen, the plate is washed to remove unbound residues and detectably labeled secondary binding molecules added. The secondary binding molecules are allowed to react with any captured sample antibody, the plates are washed, and the presence of secondary binding molecules is detected by using methods known in the art.

Thus, in one specific embodiment, the presence of the bound anti-antigen ligand from the biological sample can be readily detected by using secondary binders that include antibodies directed against the antibody ligand. Many anti-pig immunoglobulin (Ig) molecules that can be readily conjugated to enzyme labels detectable by using methods known to those skilled in the art such as horseradish peroxidase, alkaline phosphatase or urease are known in the art. It is. Appropriate enzyme substrates are then used to generate a detectable signal. In other related embodiments, competitive type ELISA techniques can be practiced using methods known to those skilled in the art.

The assay is performed in solution such that antibodies specific for the proteins of the invention form complexes under precipitation conditions. In one particular embodiment, the proteins of the invention can be bound to solid phase particles (eg, agarose beads, etc.) by using binding techniques known in the art, such as by direct chemical bonding or indirect bonding. have. The antigen coated particles are then contacted with biological samples that are expected to contain antibodies to the protein under suitable binding conditions. Crosslinking between bound antibodies leads to the formation of particle-antigen-antibody complex aggregates that can be precipitated and separated from the sample by using washing and / or centrifugation. The reaction mixture can be analyzed to determine the presence or absence of the antibody-antigen complex by using any of a number of standard methods, such as the immunodiagnostic method described above.

In another embodiment, an immunoaffinity matrix is provided wherein a group of polyclonal antibodies from a biological sample expected to contain an antibody against a desired peptide of the invention is immobilized on a substrate. In this regard, initial affinity purification of the sample can be performed by using immobilized antigen.

Many methods are known in the art for immobilizing immunoglobulins (in gratuitous or specific fragments) at high yields and good retention levels of antibody binding activity. Although not limited to any particular method, immobilized Protein A or Protein G can be used to immobilize immunoglobulins.

Thus, when immunoglobulin molecules are immobilized to provide an immunoaffinity matrix, the protein fragments are contacted with the bound antibody under suitable binding conditions. After any nonspecifically bound antigen has been washed out of the immunoaffinity support, the presence of the bound antigen can be measured by assaying the label using methods known in the art.

In addition, the antibodies to be raised against the proteins of the present invention may be used in the assays described above to detect the presence of antibodies to the proteins in a given sample. Such assays are basically performed as described above and are known to those skilled in the art.

As can be seen through the present invention, the protein fragment of the present invention can be used to efficiently diagnose the presence of Lawsonia intracellularis infection in pigs, can be used to efficiently detect the presence of Lawsonia intracellularis antibody, pig proliferative ileitis It can be used for diagnosis.

1 is a diagram showing the antigenicity (Kolaskar & Tongaonkar Antigenicity method) (FIG. 1A) and hydrophilicity analysis (Parker method) (FIG. 1B) of LI0147.
2 is a diagram showing the results of purification after expression of the lawsonia antigen in E. coli.
Figure 3 is a diagram showing the results of direct enzyme immunoassay (direct ELISA) of recombinant Lawsonia-His fusion protein.

The following examples are intended to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

Example  One. Lawsonia antigen And hydrophilicity analysis

The genome sequence of Lawsonia intracellularis was identified from NCBI GenBank data (NCBI80: 11) to select Lawsonia antigen protein. Among them, LI0147, an ORF expected to encode outer membrane protein, was selected. The amino acid sequence was identified based on the nucleotide sequence of LI0147. From this information, the antigenicity was analyzed using Kolaskar & Tongaonkar Antigenicity method, and the hydrophilicity was analyzed using Parker method. The results are shown in Fig. According to Fig. 1, the 59th to 154th amino acid portions showed high hydrophilicity and antigenicity, and this portion was selected as the antigen of the present invention.

Example  2. lawsonia antigen  Preparation of expression vector

(A) Preparation of synthetic lawsonia antigen gene

The base sequence of LI0147, the antigenic protein of lawsonia, was taken from the NCBI GenBank data (NCBI GenBank data, ACCESSION: NC008011), and oligonucleotides of the synthesized gene were synthesized in Bionics, Korea.

name The sequence (5 '- > 3') Other LI0147F GAATTCATTCTAGGTACTATTACTGGT (SEQ ID NO: 3) EcoR I LI0147R GTCGACATTGAAGATACTCGAGCCG (SEQ ID NO: 4) Sal I

Table 1 shows information on oligonucleotides used to prepare lawsonia antigens.

Nucleotide sequence Amino acid sequence 5-attctaggt actattactg gtggagctgt tggtggtgtt cttggtagtt taatcggtgg aggatcagga agaatattat caactgttgt tggtgctgga gcaggggctg ttgctggaaa cattgctgaa agaaaaatta caacgcaaca aggtctcgaa atagaagtga aacttgataa tggtcaaatt atttctatag ttcaaggtgc tgatcaatct tttagtcctg gcgaacgtgt tcgagtacta cgtggaagtg atggttcggc tcgagtatct tcaata-3 (SEQ ID NO: 2) 1-ILGTITGGAVGGVLGSLIGGGSGRILSTVVGAGAGAVAGNIAERKITTQQGLEIEVKLDNGQIISIVQGADQSFSPGERVRVLRGSDGSARVSSI-95 (SEQ ID NO: 1)

Table 2 relates to the nucleotide sequence and amino acid sequence encoding the Lawsonia antigen amplified by the PCR method.

Oligonucleotides amplified by the primers by PCR method were prepared to contain restriction sites for cloning into the expression vector pET28a (+). When using the expression vector pET28a (+) the forward oligonucleotide contains an EcoRI restriction site and the reverse oligonucleotide contains a SalI restriction site.

When PCR was performed using the primers LI0147F and LI0147R, a total of 288 bp (96 amino acids, 15 kDa) LI0147-His gene that could be introduced into pET28a (+) was synthesized (Table 2).

PCR reaction (50ul volume) was set as follows.

Oligonucleotides Sense primer and Anti-Sense Primer (Table 1) were used for each dNTP (dATP, dCTP, dGTP and dTTP), and 20 pmol of DNA polymerase (1U) and 1X buffer, respectively. The reaction was incubated at 95 ° C. for 5 minutes, amplified by circulating 15 seconds at 95 ° C. for 15 seconds, 60 seconds at 60 ° C., and 60 seconds at 72 ° C., and left at 72 ° C. for 10 minutes. PCR-products were purified using Qiagen PCR spin column.

 (B) Cloning of PCR Products

PCR products amplified for introduction into pET28a (+) (Novagen, USA) as mentioned above were digested and gel-separated with restriction endonucleases EcoRI + SalI (New England Biolab, USA) pET28a (+).

The binding product is used to transform DH5-alpa (Invitrogen, USA) receptor cells. The transformed cells were cultured in LB medium (DIFCO, USA) to which 50 μg / ml of kanamycin was added. DNAs for minipreps were obtained by incubating overnight from colony medium and digested with ECoRI + SalI to select the desired clones.

Example  3. Lawsonia  Induction of Growth and Expression of Escherichia Coli Strains with Antigen Plasmids

Spawns of each E. coli clone were incubated overnight in a shaking orbital incubator at 37 ° C. in 50 ml of sterile LB to which 50 μg / ml of kanamycin was added. 50 ml of inoculum was transferred from the seed to a flask containing 0.5 liters of sterile LB added with 50 μg / ml of kanamycin, incubated at 37 ° C. until they were medium-proliferated, and with 0.2 mM isopropylthiogalactoside (IPTG). Induced by incubating at 37 ℃ for 8 hours. After the induction phase, cells were pelleted in centrifuge and harvested according to standard procedures. Pelletized cells were sonicated with Lysis-Equilibration buffer / Wash (LEW, 50 mM NaH2PO4, 300 mM NaCl, pH to 8.0) and analyzed by 12% SDS-PAGE. Store at −70 ° C. until next step (FIG. 2).

Example  4. Lawsonia  Production of antigens

Frozen cells obtained from Example 3 were prepared according to each polyhistidine-tagged protein purification standard procedure. Each standard procedure is as follows.

Lawsonia-His fusion protein was resuspended by addition of Lysis-Equilibration buffer / Wash (LEW, 50 mM NaH 2 PO 4, 300 mM NaCl, pH to 8.0) and the cells were disrupted by ultrasonication (Ultrasonication). Insoluble lysates were prepared by centrifugation of cell-lysate (10,000 rpm, 30 minutes), and this protein was dissolved in Denaturing Solublilization buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 8 M Urea, pH to 8.0), and then 0.45 um. It was filtered through an injection filter (Syringe filter, Sartorius). Antigen proteins were purified by nickel affinity chromatography and insoluble cell lysates were loaded into Ni-NTA His Bind Resin (Novagen) column equilibrated with Denaturing Solublilization buffer. After washing the column with 3 vol volume denaturing solublilization buffer, Lawsonia-His protein was eluted with a buffer solution of Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 8M Urea, 250 mM Imidazole, pH 8.0). . The eluted fragments were analyzed on 12% SDS-PAGE. Purified fusion protein was buffered overnight in PBS buffer, and refrigerated by passing through a 0.2um filter.

Example  5. Recombination Lawsonia - His  Direct enzyme immunoassay of fusion proteins ( direct ELISA Through)

ELISA analysis was performed using the fusion protein antigen of the present invention prepared in Example 3.

The recombinant Lawsonia-His fusion protein antigen was diluted to 10 µg / ml using PBS, and coated with two peptides at a concentration of 100 µl / well in each well of a 96 well plate (Maxisorp, Nunc, Denmark).

The plate was stored at 4 ° C. for one day, washed three times with PBST (1X PBS + 0.1% Tween-20), then blocked at room temperature for 30 minutes with PBS with 5% skim milk powder, and with PBST. Washed three times.

As a porcine sample for diagnosis, 10 rats infected with Lawsonia intracellularis and 10 serum infected were used. The serum was diluted 1: 100 with PBS and reacted at room temperature at a concentration of 100 μl / well for 2 hours, and washed 5 times with PBST. HRP-conjugated goat anti-pig IgG (Serotec, UK) diluted 1: 500 was used as a secondary antibody. The secondary antibody was also reacted at room temperature for 1 hour and then washed 7 times with PBST. The detection of the bound secondary antibody was reacted with a TMB substrate (BioFX, SurModics, USA) for 10 minutes at room temperature, 5N HCl was administered as well / 20 and the reaction was measured at 450nm after completion of the reaction to observe the reactivity.

Based on the above results, it can be confirmed that the diagnosis of Lawsonia intracellularis infection can be effectively performed in porcine serum through the diagnosis method using the Lawsonia-His fusion protein presented in the present invention (FIG. 3).

<110> Konkuk University Industrial Cooperation Corp. <120> Protein for dianosing porcine proliferative enteritis and          application <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 95 <212> PRT <213> Lawsonia intracellularis <400> 1 Ile Leu Gly Thr Ile Thr Gly Gly Ala Val Gly Gly Val Leu Gly Ser   1 5 10 15 Leu Ile Gly Gly Gly Ser Gly Arg Ile Leu Ser Thr Val Val Gly Ala              20 25 30 Gly Ala Gly Ala Val Ala Gly Asn Ile Ala Glu Arg Lys Ile Thr Thr          35 40 45 Gln Gln Gly Leu Glu Ile Glu Val Lys Leu Asp Asn Gly Gln Ile Ile      50 55 60 Ser Ile Val Gln Gly Ala Asp Gln Ser Phe Ser Pro Gly Glu Arg Val  65 70 75 80 Arg Val Leu Arg Gly Ser Asp Gly Ser Ala Arg Val Ser Ser Ile                  85 90 95 <210> 2 <211> 285 <212> DNA <213> Lawsonia intracellularis <400> 2 attctaggta ctattactgg tggagctgtt ggtggtgttc ttggtagttt aatcggtgga 60 ggatcaggaa gaatattatc aactgttgtt ggtgctggag caggggctgt tgctggaaac 120 attgctgaaa gaaaaattac aacgcaacaa ggtctcgaaa tagaagtgaa acttgataat 180 ggtcaaatta tttctatagt tcaaggtgct gatcaatctt ttagtcctgg cgaacgtgtt 240 cgagtactac gtggaagtga tggttcggct cgagtatctt caata 285 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gaattcattc taggtactat tactggt 27 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtcgacattg aagatactcg agccg 25

Claims (7)

An antigen protein fragment for diagnosing swine proliferative ileitis having the amino acid sequence set forth in SEQ ID NO: 1. An antibody that specifically recognizes the protein fragment of claim 1. Pigment proliferative ileitis diagnostic composition comprising the protein fragment of claim 1. Pig diagnostic proliferative ileitis comprising the protein fragment of claim 1. Pig diagnostic proliferative ileitis comprising the antibody of claim 2. (a) providing a biological sample;
(b) in the biological sample, when Lawsonia intracellularis is present, it binds to the protein fragment of Claim 1 of Lawsonia intracellularis to form an antibody / antigen complex. Reacting the biological sample with the protein of claim 1;
(c) detecting an antibody of Lawsonia intracellularis in a biological sample, including detecting the presence of the complex to detect the presence of an antibody of Lawsonia intracellularis in the sample. How to detect it. A method for diagnosing swine proliferative ileitis comprising reacting an antibody according to claim 2 with a sample comprising Lawsonia intracellulum and judging a sample showing a positive response to the antibody as swine proliferative ileitis.

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