KR102157770B1 - Antigen peptides composition for diagnosing tuberculosis of domestic animal and use thereof - Google Patents

Abstract

The present invention relates to a method and kit for diagnosing tuberculosis in livestock using an antigenic peptide.
The method and kit for diagnosing tuberculosis in livestock using the antigenic peptide of the present invention have high specificity and sensitivity by measuring the cytokine of interferon (IFN)-γ according to T lymphocyte or NK cell activation. Excellent effect on

Description

Antigen peptides composition for diagnosing tuberculosis of domestic animal and use thereof for diagnosis of tuberculosis in livestock

The present invention relates to an antigenic peptide composition for diagnosis of tuberculosis in livestock, and a method and use thereof.

Tuberculosis causes serious public health and economic damage to humans and animals. There are major Mycobacterium genus bacteria that cause tuberculosis, including Mycobacterium turbiosis, Mycobacterium bovis, and Mycobacterium carneti. Among them, especially in cattle, Mycobacterium bovis is known as a direct causative agent of tuberculosis. As in human tuberculosis, tuberculosis in cattle is generally diagnosed by delayed hypersensitivity reaction, that is, by observing swelling and induration after inoculation of a tuberculosis-derived protein body. However, this diagnostic method has a great inconvenience in that it is observed after intradermal vaccination and 3 days, so studies on the development of a diagnostic method by measuring specific antibody values have been continued. The intradermal diagnosis method, which is used as an official field diagnosis method for tuberculosis in cattle, measures the degree of cellular immune response involving phagocytic cells and T cells among the immune responses to tuberculosis antigens in cattle. This cellular immune response increases as the tuberculosis lesion progresses in bovine, but decreases at the end of tuberculosis in bovine. On the other hand, the antibody response to the tuberculosis antigen in bovine does not show a response in the early stages of tuberculosis in bovine, but gradually increases as the tuberculosis lesion in bovine progresses. Therefore, measuring antibodies that specifically respond to bovine tuberculosis antigens at a time when the cellular immune response decreases is based on the negative diagnosis results of late bovine tuberculosis infected subjects, which can be seen in intradermal diagnosis using cellular immune responses. Since it can be diagnosed as positive by antibody detection, bovine tuberculosis-specific antibody diagnosis using humoral immune response is useful as a complementary diagnosis method with intradermal diagnosis using cellular immune response.

On the other hand, looking at what was applied as a serologic diagnosis of tuberculosis in cattle, there may be mentioned complement binding reaction, enzyme immunoassay, immunochromatography, and latex bead aggregation reaction. These serological diagnostic methods show many differences in specificity and sensitivity. In general, specificity is high, while sensitivity tends to be low. These results seem to be characteristic of the slow proliferation of Mycobacterium tuberculosis, and the immune response that occurs accordingly is primarily a cellular immune response followed by an antibody response, a secondary humoral immune response. Therefore, many researchers have been developing serological diagnostic methods such as enzyme immunoassay, immunochromatography, and latex bead aggregation using recombinant proteins and natural proteins to find specific antigens for tuberculosis, but have been commercialized so far. The number of examples used in this study is extremely limited, and it is difficult to use it largely as an official diagnosis method to replace intradermal diagnosis or nodule test.

The interferon gamma secretion test has been recently used to diagnose tuberculosis infection in livestock. Existing kits perform stimulation using PPD to activate T lymphocytes or NK cells, but PPD contains more than 200 antigens, some of which are also included in several non-TB mycobacteria, so it is non-specific for Mycobacterium tuberculosis and There is a problem in that the specificity is further lowered because a false-positive diagnosis result occurs depending on the concentration.

Therefore, there is a need in the art for a technology capable of sensitive and specific diagnosis of Mycobacterium tuberculosis in cattle and livestock. Under this background, the present inventors mixed several antigenic peptides derived from Early secreted antigenic target-6 (ESAT-6), Culture filtrate protein-10 (CFP-10), and Low Molecular Weight Protein Antigen 7 (TB10.4). The present invention was completed by developing a composition and a diagnostic method used as a stimulating antigen for diagnosis of tuberculosis infection.

An object of the present invention (a) as an antigen peptide composition for diagnosis of tuberculosis in livestock, any one or more early secreted antigenic target-6 (ESAT-6) derived antigen peptide selected from the group consisting of SEQ ID NO: 1 to 3, SEQ ID NO: 4 Any one or more culture filtrate protein-10 (CFP-10) derived antigen peptides selected from the group consisting of 9 and any one or more Low Molecular Weight Protein Antigen 7 (TB10.4) selected from the group consisting of SEQ ID NOs: 10 to 20 Reacting the derived antigenic peptide with the sample; And

It is to provide a method for diagnosing tuberculosis in livestock comprising; (b) measuring whether interferon-γ is secreted.

Another object of the present invention is any one or more early secreted antigenic target-6 (ESAT-6) derived antigen peptides selected from the group consisting of SEQ ID NOs: 1 to 3, and any one or more selected from the group consisting of SEQ ID NOs: 4 to 9 A kit for diagnosis of tuberculosis in livestock comprising an antigen peptide derived from culture filtrate protein-10 (CFP-10) and any one or more Low Molecular Weight Protein Antigen 7 (TB10.4) derived antigen peptides selected from the group consisting of SEQ ID NOs: 10 to 20 Is to provide.

The present invention (a) as an antigen peptide composition for diagnosis of tuberculosis in livestock, any one or more early secreted antigenic target-6 (ESAT-6) derived antigen peptides selected from the group consisting of SEQ ID NOs: 1 to 3, SEQ ID NOs: 4 to 9 Any one or more culture filtrate protein-10 (CFP-10)-derived antigen peptides selected from the group consisting of and any one or more Low Molecular Weight Protein Antigen 7 (TB10.4)-derived antigens selected from the group consisting of SEQ ID NOs: 10 to 20 Reacting the peptide with the sample; And

It provides a method for diagnosing tuberculosis in livestock comprising; (b) measuring whether interferon-γ is secreted.

The method for diagnosing tuberculosis in livestock of the present invention has high specificity and sensitivity by measuring the cytokine of interferon (IFN)-γ in response to T lymphocyte or NK cell activation, and exhibits excellent effects in rapid and convenient diagnosis of tuberculosis in livestock.

In the present invention, the "diagnosis" is to determine whether a subject, such as a livestock, currently has a specific disease or disease, to determine the prognosis of a subject suffering from a specific disease or disease, or therametrics (Eg, monitoring the condition of the subject to provide information on treatment efficacy). For the purposes of the present invention, the diagnosis is to confirm the presence or absence of the disease or disorder described above.

The above livestock refers to wild animals domesticated or improved by humans such as horses, pigs, cows, chickens, ducks, goats or sheep. Preferably the livestock is cattle.

The method for diagnosing tuberculosis in livestock of the present invention includes (a) an antigen peptide composition for diagnosis of tuberculosis in livestock, and is an antigen peptide derived from one or more early secreted antigenic target-6 (ESAT-6) selected from the group consisting of SEQ ID NOs: 1 to 3 , Any one or more culture filtrate protein-10 (CFP-10) derived antigen peptides selected from the group consisting of SEQ ID NOs: 4 to 9 and any one or more Low Molecular Weight Protein Antigen 7 selected from the group consisting of SEQ ID NOs: 10 to 20 ( TB10.4)-derived antigenic peptide is reacted with the sample.

Samples according to the present invention include, but are not limited to, whole blood, serum, plasma, sputum, urine, pleural fluid, ascites fluid, cerebrospinal fluid, or brocho alveolar lavage. In one embodiment according to the present invention, it may be blood, in particular serum.

The antigenic peptide composition according to the present invention is an early secreted antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10), or low molecular weight known as an antigen expressed in mycobacterium bovis . It is an antigenic peptide that has excellent binding ability to Protein Antigen 7 (TB10.4). In particular, an antigenic peptide sequence that is highly reactive to tuberculosis was selected from the entire protein sequence.

That is, the antigen peptide composition for diagnosing tuberculosis in livestock according to the present invention is suitable for diagnosing tuberculosis in cows, especially cows infected with microbacterium bovis , among livestock.

The antigen peptide composition according to the present invention is preferably an antigen peptide derived from early secreted antigenic target-6 (ESAT-6) of SEQ ID NOs: 1 to 3, and an antigen peptide derived from culture filtrate protein-10 (CFP-10) of SEQ ID NOs: 4 to 9 And it may include an antigen peptide derived from Low Molecular Weight Protein Antigen 7 (TB10.4) of SEQ ID NOs: 10 to 20. The reaction between the antigen peptide composition and the sample is an antigen-antibody complex reaction.

The method for diagnosing tuberculosis in livestock of the present invention includes the step of (b) measuring whether interferon-γ is secreted.

In the diagnosis of tuberculosis in livestock by the interferon-γ secretion test (measurement) according to the present invention, the presence or absence of infection is diagnosed by measuring interferon gamma secreted by reacting T lymphocytes or NK cells sensitized to Mycobacterium tuberculosis to a tuberculosis antigen. The greatest advantage of the interferon-γ secretion test according to the present invention is that it uses a Mycobacterium tuberculosis-specific antigen, so it is not affected by the majority of non-TB mycobacterial infections, so the specificity and sensitivity of the diagnosis of tuberculosis infection are high.

In particular, in livestock, TB10.4, and the case of attempting to diagnose tuberculosis infection using an antigenic peptide composition using an antigenic peptide combination of ESAT-6, CFP-10, and TB10.4 is hardly confirmed. In the present invention, ESAT- 6, by confirming the excellent specificity and sensitivity of the antigenic peptide composition of CFP-10 and TB10.4, the present invention was completed.

Detection by antigen-antibody complex reaction can be performed using a known method, but is not limited thereto, but immunoprecipitation assays including Radial Immunodiffusion, immunoelectrophoresis or reverse current electrophoresis , RIA (Radioimmunoassay), or ELISA (Enzyme Linked Immunosorbent Assay). Preferably, it may be ELISA (Enzyme Linked Immunosorbent Assay).

In this detection process, the control group in the present application is a normal control group in which there is no clinical tuberculosis symptom and no active lesion is found, that is, a healthy animal or a sample derived from a healthy animal that shows negative in all of the healthy animal tuberculosis test.

When the secretion of interferon-γ is measured and the secretion is increased above a certain level, it is determined that livestock is infected with tuberculosis.

The present invention is any one or more early secreted antigenic target-6 (ESAT-6) derived antigen peptide selected from the group consisting of SEQ ID NOs: 1 to 3, any one or more culture filtrate protein- selected from the group consisting of SEQ ID NOs: 4 to 9 It provides a kit for diagnosis of tuberculosis in livestock comprising an antigen peptide derived from 10 (CFP-10) and any one or more Low Molecular Weight Protein Antigen 7 (TB10.4) derived antigen peptides selected from the group consisting of SEQ ID NOs: 10 to 20.

The kit for diagnosing tuberculosis in livestock according to the present invention has high specificity and sensitivity by measuring the cytokine of interferon (IFN)-γ in response to T lymphocyte or NK cell activation, and exhibits excellent effects in rapid and convenient diagnosis of tuberculosis in livestock. . .

The present invention is preferably an antigen peptide derived from Early secreted antigenic target-6 (ESAT-6) of SEQ ID NOs: 1 to 3, an antigen peptide derived from Culture filtrate protein-10 (CFP-10) of SEQ ID NOs: 4 to 9, and SEQ ID NO: 10 to It provides a kit for diagnosis of tuberculosis in livestock containing an antigenic peptide derived from Low Molecular Weight Protein Antigen 7 (TB10.4) of 20.

The present invention is an antigen peptide derived from Early secreted antigenic target-6 (ESAT-6) of SEQ ID NOs: 1 to 3, an antigen peptide derived from Culture filtrate protein-10 (CFP-10) of SEQ ID NOs: 4 to 9, and SEQ ID NOs: 10 to 20 Antigen peptide derived from Low Molecular Weight Protein Antigen 7 (TB10.4); And it provides a kit for diagnosis of tuberculosis in livestock comprising a detection antibody that specifically binds to interferon-γ.

The tuberculosis diagnostic kit may perform an antigen-antibody reaction with a sample including the above antigenic peptide in one tube. Then, a kit related to Radial Immunodiffusion, immunoprecipitation assay including immunoelectrophoresis or reverse current electrophoresis, Radioimmunoassay (RIA) or ELISA (Enzyme Linked Immunosorbent Assay) capable of measuring interferon-γ secretion It may further include a device.

In addition, the tuberculosis diagnostic kit includes an adsorbed support and a gastric antigen peptide, and the detection antibody specifically binds to interferon-γ, and includes a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radioactive material; Or it provides a kit that is labeled with a material containing colored particles including colloidal gold particles or colored latex particles.

The support is a solid support and includes, for example, microplates, microarrays, chips, glass, beads or particles, or membranes, but is not limited thereto.

The method and kit for diagnosing tuberculosis in livestock of the present invention have high specificity and sensitivity by measuring the cytokine of interferon (IFN)-γ in response to T lymphocyte or NK cell activation, and have excellent effects in rapid and convenient diagnosis of tuberculosis in livestock. Show.

1 is a result of measuring interferon-γ in a cow infected with tuberculosis using the antigen peptide composition according to the present invention and showing its OD value.
2 is a result of measuring interferon-γ in a cow infected with tuberculosis using the antigen peptide composition according to the present invention and showing its OD value.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Preparation of tuberculosis antigen peptide

Early secreted antigenic target-6 (ESAT-6), Culture filtrate protein-10 (CFP-10), and Low Molecular Weight Protein Antigen 7 (TB10.4) are known as antigens expressed in mycobacterium bovis . Based on the presence, antigenic peptide sequences having good reactivity against tuberculosis were selected from the entire protein sequences of ESAT-6, CFP-10 and TB10.4.

The sequences selected for ESAT-6, CFP-10 and TB10.4 are shown in Tables 1 to 3, respectively.

ESAT-6 (EsxA): early secreted antigenic target-6 MTEQQWNFAGIEAAASAI (SEQ ID NO: 1) AGIEAAASAIQGNVTSIHSL (SEQ ID NO: 2) ATATELNNALQNLARTISEA (SEQ ID NO: 3)

CFP-10: culture filtrate protein-10 LAQEAGNFERISGDLKTQI (SEQ ID NO: 4) RISGDLKTQIDQVESTAGSL (SEQ ID NO: 5) SLQGQWRGAAGTAAQAAVVR (SEQ ID NO: 6) RFQEAANKQKQELDEISTNI (SEQ ID NO: 7) KQELDEISTNIRQAGVQYSR (SEQ ID NO: 8) IRQAGVQYSRADEEQQQAL (SEQ ID NO: 9)

TB10.4 (EsxH): Low Molecular Weight Protein Antigen 7 EsxH MSQIMYNYPAMLGHAGDMA (SEQ ID NO: 10) AMLGHAGDMAGYAGTLQSL (SEQ ID NO: 11) AGYAGTLQSLGAEIA (SEQ ID NO: 12) TLQSLGAEIAVEQAA (SEQ ID NO: 13) GAEIAVEQAALQSAW (SEQ ID NO: 14) VEQAALQSAWQGDTG (SEQ ID NO: 15) LQSAWQGDTGITYQAWQAQW (SEQ ID NO: 16) ITYQAWQAQWNQAMEDLVRA (SEQ ID NO: 17) NQAMEDLVRAYHAMSSTHEA (SEQ ID NO: 18) YHAMSSTHEANTMAMMAR (SEQ ID NO: 19) EANTMAMMARDTAEAAKWGG (SEQ ID NO: 20)

Based on the selected sequence, it was commissioned to Genscript to synthesize and purify Fabtide.

Example 2. Confirmation of detection of Mycobacterium tuberculosis by antigen peptides in livestock infected with tuberculosis

1 ml of bovine blood was collected, 10 μg/ml and 15 μg/ml of the antigen peptides of SEQ ID NOs: 1 to 20 were added and mixed well, and then reacted at 37° C. for 20 hours (available from 16 to 24 hours). . At the same time, mitogen, which is used as a control for cell activation, and PPD A (Avium), which is a control to confirm infection with bovis, were reacted together. Thereafter, the cultured tube was centrifuged at 3000 g for 15 minutes to collect plasma, and the collected plasma was measured for interferon gamma generated using ELISA (OD value measurement using a 450 nm filter) method.

The results of the experiment on cattle infected with tuberculosis are shown in Table 4 and Fig. 1, respectively.

Table 4 and FIG. 1 show the experimental results for cow 1 infected with tuberculosis.

As shown in Table 4 and Fig. 1, the experimental results using the antigenic peptide of the present invention also showed a high OD value for IFN-γ. From the above results, it was confirmed that the antigen peptide composition according to the present invention has reactivity against tuberculosis infection in cattle and can be used as a kit suitable for diagnosing positive and negative.

Example 3. Confirmation of detection of Mycobacterium tuberculosis by antigen peptides in livestock infected with tuberculosis

Kits used for diagnosing tuberculosis in cattle are known as Ruminant IFN-γ kit (ID.vet), TVperon kit (Bionote), and Bovi-check kit (Chungang vaccine). In comparison with these kits, the results of detection of Mycobacterium tuberculosis of the above synthesized antigenic peptide sequences according to the present invention were compared. 1 ml of bovine blood was collected, 10 μg/ml and 15 μg/ml of the antigen peptides of SEQ ID NOs: 1 to 20 were added and mixed well, and then reacted at 37° C. for 20 hours (available from 16 to 24 hours). . At the same time, mitogen, which is used as a control for cell activation, and PPD A (Avium), which is a control to confirm infection with bovis, were reacted together. At this time, PPD A and PPD B were added to the blood according to the instructions of each kit, mixed well, and then reacted at 37°C for 20 hours (available from 16 to 24 hours). Thereafter, the cultured tube was centrifuged at 3000 g for 15 minutes to collect plasma, and the collected plasma was measured for interferon gamma generated using ELISA (OD value measurement using a 450 nm filter) method.

Table 5 and Figure 2 shows the experimental results for cow 2 infected with tuberculosis.

As shown in Table 5 and 2, the OD value was higher in the experimental results using the antigen peptide of the present invention than the OD value of interferon gamma for PPD B of the kit, respectively. In particular, in the case of tuberculosis-infected cow 2, it was confirmed that antigenic peptides were more reactive than conventional kits, and could be used as a kit suitable for diagnosing positive and negative tuberculosis infection.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

<110> PIgenomics CO.,LTD <120> Antigen peptides composition for diagnosing tuberculosis of domestic animal and use thereof <130> Antigen <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: ESAT-6 (EsxA) <400> 1 Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser 1 5 10 15 Ala Ile <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: ESAT-6 (EsxA) <400> 2 Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val Thr Ser 1 5 10 15 Ile His Ser Leu 20 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: ESAT-6 (EsxA) <400> 3 Ala Thr Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr 1 5 10 15 Ile Ser Glu Ala 20 <210> 4 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: CFP-10 <400> 4 Leu Ala Gln Glu Ala Gly Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys 1 5 10 15 Thr Gln Ile <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: CFP-10 <400> 5 Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu Ser Thr 1 5 10 15 Ala Gly Ser Leu 20 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: CFP-10 <400> 6 Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr Ala Ala Gln Ala 1 5 10 15 Ala Val Val Arg 20 <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: CFP-10 <400> 7 Arg Phe Gln Glu Ala Ala Asn Lys Gln Lys Gln Glu Leu Asp Glu Ile 1 5 10 15 Ser Thr Asn Ile 20 <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: CFP-10 <400> 8 Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly Val 1 5 10 15 Gln Tyr Ser Arg 20 <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: CFP-10 <400> 9 Ile Arg Gln Ala Gly Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln 1 5 10 15 Gln Ala Leu <210> 10 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 10 Met Ser Gln Ile Met Tyr Asn Tyr Pro Ala Met Leu Gly His Ala Gly 1 5 10 15 Asp Met Ala <210> 11 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 11 Ala Met Leu Gly His Ala Gly Asp Met Ala Gly Tyr Ala Gly Thr Leu 1 5 10 15 Gln Ser Leu <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 12 Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala Glu Ile Ala 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 13 Thr Leu Gln Ser Leu Gly Ala Glu Ile Ala Val Glu Gln Ala Ala 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 14 Gly Ala Glu Ile Ala Val Glu Gln Ala Ala Leu Gln Ser Ala Trp 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 15 Val Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly 1 5 10 15 <210> 16 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 16 Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly Ile Thr Tyr Gln Ala Trp 1 5 10 15 Gln Ala Gln Trp 20 <210> 17 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 17 Ile Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala Met Glu Asp 1 5 10 15 Leu Val Arg Ala 20 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 18 Asn Gln Ala Met Glu Asp Leu Val Arg Ala Tyr His Ala Met Ser Ser 1 5 10 15 Thr His Glu Ala 20 <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 19 Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr Met Ala Met Met 1 5 10 15 Ala Arg <210> 20 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Angtigen Peptide: TB10.4 (EsxH) <400> 20 Glu Ala Asn Thr Met Ala Met Met Ala Arg Asp Thr Ala Glu Ala Ala 1 5 10 15 Lys Trp Gly Gly 20

Claims (7)

(a) Antigen peptide composition for diagnosis of bovine tuberculosis, derived from early secreted antigenic target-6 (ESAT-6) of SEQ ID NOs: 1 to 3, and culture filtrate protein-10 (CFP-10) of SEQ ID NOs: 4 to 9 An antigenic peptide composition consisting of an antigenic peptide and an antigenic peptide derived from Low Molecular Weight Protein Antigen 7 (TB10.4) of SEQ ID NOs: 10 to 20 was prepared from whole blood, serum, plasma, sputum, urine, pleural fluid, ascites fluid, cerebrospinal fluid and bronchoalveolar lavage fluid ( brocho alveolar lavage) reacting with a sample selected from the group consisting of; And
(b) measuring whether interferon-γ is secreted or not; tuberculosis diagnosis method in cattle comprising.
delete delete The method of claim 1, wherein the measurement of the secretion of interferon-γ in step (b) is an immunoprecipitation assay including Radial Immunodiffusion, immunoelectrophoresis or reverse current electrophoresis, Radioimmunoassay (RIA), and ELISA ( Enzyme Linked Immunosorbent Assay) by any one selected from the group consisting of tuberculosis diagnosis method in cattle.
delete Early secreted antigenic target-6 (ESAT-6) derived antigen peptide of SEQ ID NOs: 1 to 3, culture filtrate protein-10 (CFP-10) derived antigen peptide of SEQ ID NOs: 4 to 9, and low molecular weight of SEQ ID NO: 10 to 20 A kit for diagnosing bovine tuberculosis consisting of an antigen peptide derived from Protein Antigen 7 (TB10.4).
The kit for diagnosing bovine tuberculosis according to claim 6, further comprising a detection antibody that specifically binds to interferon-γ.

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