Mycobacterium tuberculosis antigen composition and its application, Mycobacterium tuberculosis infection diagnosis kit

Abstract

The present invention provides a tubercle bacillus antigen composition and its application, and a tubercle bacillus infection diagnosis kit, and relates to the field of biotechnology. The tubercle bacillus antigen composition includes an antigen in the form of a mixture of polypeptide fragments, each of which independently contains a plurality of polypeptides, each of which independently originates from continuous amino acid residues in a target protein, and the sum of the sequences of all polypeptides covers the expected region of the target protein sequence. The target protein includes ESAT‑6 protein, CFP10 protein and Rv2003c partial protein. The tubercle bacillus antigen composition improves the sensitivity of diagnosis of tubercle bacillus infection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a tuberculosis bacillus antigen composition and application thereof, and a tuberculosis bacillus infection diagnosis kit. Background Art

[0002] Tuberculosis is a chronic respiratory infectious disease that seriously endangers human health. Its pathogen is Mycobacterium tuberculosis. Mycobacterium tuberculosis is mainly transmitted through the respiratory tract and can also enter the human body through the digestive tract. Mycobacterium tuberculosis has strong resistance to the outside world and can survive for more than 5 months in damp places. Therefore, sputum carrying bacteria is the main source of infection of Mycobacterium tuberculosis. Healthy people can inhale droplets sprayed by patients' coughs or sneezes, which can cause pulmonary Mycobacterium tuberculosis infection.

[0003] The most effective way to control the spread and development of tuberculosis is early detection and early treatment. Existing tuberculosis diagnosis can be divided into four categories according to the method: etiology, molecular biology, imaging and immunology. Among them, etiology and molecular biology are effective means of diagnosis, and imaging and immunology are supplemented as auxiliary diagnostic methods.

[0004] Etiology includes two methods: sputum smear and sputum culture. Sputum smear is to make the patient's sputum into a glass slide and use a microscope to directly look for Mycobacterium tuberculosis. This method is low-cost but extremely low in sensitivity, and can only detect about 20% of tuberculosis patients. Sputum culture uses selective culture medium to culture the patient's sputum to observe whether Mycobacterium tuberculosis grows. This method is more sensitive than sputum smear, but it is still only about 40%, and the culture time is as long as 4 weeks, so there are still a large number of tuberculosis patients who cannot be detected.

[0005] With the development of molecular biology, a nucleic acid detection method for tuberculosis has emerged. This method does not require the survival of tuberculosis bacteria in sputum, so the sensitivity is further improved to about 50% compared with sputum smears, and the culture time is greatly shortened. This sensitivity is already the limit of tuberculosis diagnosis. There are still nearly half of the patients who cannot be detected by diagnostic means, and some patients are not infected in the lungs (i.e., extrapulmonary tuberculosis), and do not produce sputum and therefore cannot be detected.

[0006] As an auxiliary diagnostic method, imaging requires that the tuberculosis lesions are relatively large in order to be observed by imaging equipment. At the same time, imaging alone cannot distinguish tuberculosis from other lung diseases such as pneumonia and lung cancer. Therefore, patients who cannot be diagnosed generally need the attending physician to make a diagnosis based on experience, comprehensive imaging, immunology, clinical manifestations and other evidence.

[0007] Since tuberculosis bacteria basically do not induce humoral immunity of the human immune system, the effect of conventional immunological antibody detection is poor. And tuberculosis bacteria almost never enter the blood circulation system, so immunological antigen detection is also ineffective. In recent years, the immunological detection of tuberculosis has mainly focused on the cellular immunity of the immune system, namely the interferon release assay (IGRA). The principle is to co-culture the antigens carried by tuberculosis bacteria with lymphocytes. The lymphocytes of tuberculosis patients will produce a strong secondary immune response due to the initial immunization, while the lymphocytes of healthy people will only trigger the primary immune response because they have not been exposed to the antigens carried by tuberculosis bacteria. Their immune strength is weaker than the secondary immunization and the activation time is longer than the secondary immunization.

[0008] After continuous technological development, the mainstream application of IGRA (Interferon-Gamma Release Assay) is to use two antigens, ESAT-6 and CFP-10, which are unique to tuberculosis bacteria, to detect the level of interferon-gamma released by lymphocytes after co-culture to determine the infection status of the sample. Its sensitivity and specificity can reach about 80%~85%. However, this performance level makes IGRA only exist as an auxiliary diagnosis, and its sensitivity and specificity need to be improved. Further improving the sensitivity can make its detection rate extremely high, and it can be used as an effective screening method to control the spread of tuberculosis and achieve early detection; because it has a higher sensitivity than molecular biological methods, further improving the specificity can make its diagnosis accuracy close to that of etiology and molecular biology methods, and it is expected to make a contribution to the gold standard for the diagnosis of tuberculosis.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The purpose of the present invention is to provide a Mycobacterium tuberculosis antigen composition to improve the sensitivity of Mycobacterium tuberculosis infection diagnosis.

[0011] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0012] In a first aspect, a Mycobacterium tuberculosis antigen composition is provided, wherein the Mycobacterium tuberculosis antigen composition comprises a mixture of ESAT-6 polypeptide fragments, a mixture of CFP10 polypeptide fragments, and a mixture of Rv2003c polypeptide fragments;

[0013] Each of the polypeptide fragment mixtures independently contains a plurality of polypeptides, the polypeptides of the same polypeptide fragment mixture are derived from continuous amino acid residues in the same target protein, and the sum of the sequences of all the polypeptides covers the expected region of the target protein sequence;

[0014] The target protein of the ESAT-6 polypeptide fragment mixture is the ESAT-6 protein, and the expected region is at least 80% of the full length of the ESAT-6 protein; the target protein of the CFP10 polypeptide fragment mixture is the CFP10 protein, and the expected region is at least 80% of the full length of the CFP10 protein; the target protein of the Rv2003c polypeptide fragment mixture is the Rv2003c protein, and the expected region is at least 80% of the 1st to (50th to 60th) positions of the Rv2003c protein.

[0015] In a second aspect, there is provided a use of the tubercle bacillus antigen composition of the first aspect in detecting tubercle bacillus or anti-tuberculosis bacillus antibodies for non-diagnostic and therapeutic purposes, or in preparing a kit for diagnosing tuberculosis infection.

[0016] In a third aspect, a Mycobacterium tuberculosis infection diagnostic kit according to the first aspect is provided, the kit comprising the Mycobacterium tuberculosis antigen composition according to the first aspect, and a substance for detecting a marker; the marker comprises IFN-γ.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention improves the tubercle bacillus antigen composition, uses polypeptide fragments derived from ESAT-6 protein, CFP10 protein and Rv2003c partial protein as antigens, and effectively improves the detection sensitivity without reducing the IGRA specificity in the IGRA experiment with IFN-γ as the detection marker. In the preferred scheme, the present invention also optimizes the detection marker of the IGRA experiment, and in the preferred marker combination, the sensitivity of IGRA is increased to nearly 95%, which helps to make IGRA the most effective tuberculosis screening method currently available. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 It is a scatter plot of IFN-γ concentration in the culture supernatant of Example 1;

[0021] Figure 2 The ROC curve diagram of the Mycobacterium tuberculosis infection diagnosis kit in Example 1;

[0022] Figure 3 It is a scatter plot of IL-2 concentration in the culture supernatant of Example 2;

[0023] Figure 4 The ROC curve diagram of the Mycobacterium tuberculosis infection diagnosis kit in Example 2;

[0024] Figure 5 It is a scatter plot of IP-10 concentration in the culture supernatant of Example 3;

[0025] Figure 6 The ROC curve diagram of the Mycobacterium tuberculosis infection diagnosis kit of Example 3;

[0026] Figure 7 It is a scatter plot of IL-6 concentration in the culture supernatant of Example 4;

[0027] Figure 8 The ROC curve diagram of the Mycobacterium tuberculosis infection diagnosis kit of Example 4;

[0028] Fig. 9 It is a scatter plot of TNF-α concentration in the culture supernatant of Example 5;

[0029] Fig.10 The ROC curve diagram of the Mycobacterium tuberculosis infection diagnostic kit in Example 5 is shown;

[0030] Fig.11 It is a scatter plot of IFN-γ concentration in the culture supernatant of Comparative Example 1;

[0031] Fig.12 The ROC curve of the Mycobacterium tuberculosis infection diagnosis kit of comparative example 1 is shown;

[0032] Fig.13 It is a scatter plot of IFN-γ concentration in the culture supernatant of Comparative Example 2;

[0033] Fig.14 The ROC curve of the Mycobacterium tuberculosis infection diagnosis kit of comparative example 2 is shown;

[0034] Fig.15 It is a scatter plot of IFN-γ concentration in the culture supernatant of Comparative Example 3;

[0035] Fig.16 The ROC curve of the tuberculosis infection diagnosis kit of comparative example 3 is shown;

[0036] Fig.17 It is a scatter plot of IFN-γ concentration in the culture supernatant of Comparative Example 4;

[0037] Fig.18 The ROC curve of the Mycobacterium tuberculosis infection diagnosis kit of comparative example 4 is shown;

[0038] Fig.19 It is a scatter plot of IFN-γ concentration in the culture supernatant of Comparative Example 5;

[0039] Fig. 20This is the ROC curve diagram of the Mycobacterium tuberculosis infection diagnostic kit of comparative example 5. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Definition of terms:

[0042] Herein, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual degree of importance between these entities or actions.

[0043] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0044] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0045] As used herein, the terms "each... is independently selected from" and "... are each independently selected from" and "... are independently selected from" are interchangeable and should be understood in a broad sense, meaning that in a class of objects, the specific features of each specific object do not affect each other, and in this class of objects, the specific features of any two objects may be the same or different.

[0046] Herein, when an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0047] As used herein, "and / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0048] In this article, tuberculosis refers to Mycobacterium tuberculosis. In this article, "tuberculosis bacillus", "tuberculosis", "Mycobacterium tuberculosis" and "tuberculosis bacteria" can be used interchangeably.

[0049] In this article, ESAT-6 refers to Early Secretory Antigen Target-6, which is a protein produced by Mycobacterium tuberculosis and is one of the key virulence factors of the bacterium. It is secreted in the early stages of Mycobacterium tuberculosis infection and can stimulate the body to produce a strong immune response, especially T cell immune response, and release high levels of IFN-γ.

[0050] In this article, CFP10 refers to Culture Filtrate Protein-10, a secretory protein of Mycobacterium tuberculosis encoded by the RD1 region gene Rv3874. It is a low molecular weight specific secretory protein and a target antigen for T cells that can induce delayed-type hypersensitivity reactions in the skin and stimulate peripheral blood mononuclear cells to produce specific IFN-γ.

[0051] Herein, Rv2003c refers to the protein encoded by the gene numbered Rv2003c in the genome of Mycobacterium tuberculosis, which belongs to the PE / PPE family. The PE / PPE family is a class of conserved proteins unique to Mycobacterium tuberculosis that play an important role in bacterial virulence, growth and host immune response.

[0052] Unless otherwise specified, the amino acid sequence positions herein are all counted from the N-terminus to the C-terminus.

[0053] In a first aspect, a Mycobacterium tuberculosis antigen composition is provided, wherein the Mycobacterium tuberculosis antigen composition comprises antigens in the form of a mixture of polypeptide fragments, wherein each mixture of polypeptide fragments is composed of polypeptides derived from the same target protein.

[0054] Each of the polypeptide fragment mixtures independently contains several polypeptides, each polypeptide independently originates from continuous amino acid residues in the target protein, and the sum of the sequences of all polypeptides covers the expected region of the target protein sequence. The expected region can be a continuous or discontinuous sequence in the full length of the target protein, preferably a continuous sequence.

[0055] The sum of the sequences of all the polypeptides covers the expected region of the target protein sequence, which means that in a mixture of polypeptide fragments, the sequence of each polypeptide can match a fragment in the expected region, and when the sequences of all the polypeptides are correctly arranged and spliced, the full picture of the expected region of the target protein can be completely restored. There may be repeated fragments in the sequences of any two polypeptides, and the repeated parts may be located at the nitrogen end and / or carbon end of the polypeptides. For example, one polypeptide is derived from the 1st to 20th positions of the target protein, another polypeptide is derived from the 15th to 30th positions of the target protein, another polypeptide is derived from the 25th to 40th positions of the target protein, another polypeptide is derived from the 35th to 48th positions of the target protein, and another polypeptide is derived from the 49th to 60th positions of the target protein... and after the sequences of all the polypeptides are correctly arranged and spliced, the spliced ​​sequence covers the entire length of the expected region of the target protein sequence. When the expected region is a discontinuous sequence, the spliced ​​sequence covers the entire length of the expected region of the target protein sequence after splicing.

[0056] In an optional embodiment, the expected region is a continuous region in the target protein, and each of the polypeptide fragment mixtures independently contains N polypeptides, where N is a positive integer.

[0057] According to the position of the first amino acid residue at the N-terminus of each polypeptide sequence in the expected region sequence, they are labeled as the 1st polypeptide, the 2nd polypeptide... the i-th polypeptide... to the N-th polypeptide from the N-terminus to the C-terminus; 1≤i≤N.

[0058] The first to the N-1th peptides conform to the following rules:

[0059] The length of each polypeptide fragment is independently 5 to 25 aa (for example, but not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 aa).

[0060] The sequence of the first polypeptide starts at the first position of the N-terminus of the expected region sequence, that is, the first polypeptide sequence is from the 1st to the 10th to 20th position of the expected region.

[0061] There are 0 to 10 aa repeating sequences at the C-terminus of the i-1 polypeptide and the N-terminus of the sequence of the i-th polypeptide, for example, but not limited to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aa repeats; 0 means no repeats, for example, the sequence of the first polypeptide is from the 1st to the 10th position of the expected region, and the sequence of the second polypeptide is from the 11th to the 15th position of the expected region. Other exemplary repeating modes are, for example: the sequence of the first polypeptide is from the 1st to the 10th position of the expected region, and the sequence of the second polypeptide is from the 10th to the 15th position of the expected region, then there is 1 aa repeat; the sequence of the first polypeptide is from the 1st to the 10th position of the expected region, and the sequence of the second polypeptide is from the 6th to the 15th position of the expected region, then there is 5 aa repeat; the sequence of the first polypeptide is from the 1st to the 15th position of the expected region, and the sequence of the second polypeptide is from the 6th to the 15th position of the expected region, then there is 10 aa repeat.

[0062] The Nth polypeptide (the polypeptide at the most C-terminal end of the sequence in the expected region) complies with the following rules:

[0063] The sequence of the Nth polypeptide starts from the expected region sequence site where any amino acid residue in the N-1th polypeptide is located, and ends at the last position of the expected region sequence, so as to meet the requirement that the sum of the sequences of all polypeptides covers the expected region of the target protein sequence.

[0064] In a specific embodiment: among the 1st to (N-1)th polypeptides, each polypeptide has a length of 15 aa, and there is a 5 aa repeating sequence at the C-terminus of the i-1th polypeptide and the N-terminus of the sequence of the i-th polypeptide.

[0065] This embodiment means that the first polypeptide, the second polypeptide...the Nth polypeptide are respectively located at positions 1 to 15, 11 to 25, 21 to 35, 31 to 45, 41 to 55, 51 to 65...in the expected region until the entire expected region is covered.

[0066] It should be noted that the lengths of any two polypeptides in the mixture of polypeptide fragments of the same target protein can be the same or different, and the lengths of the repeated fragments of two polypeptides adjacent to any one group of sequences in the expected region can be the same or different from the lengths of the repeated fragments of two polypeptides adjacent to other groups of sequences. For example, in the mixture of polypeptide fragments of the same target protein, the first polypeptide, the second polypeptide...the Nth polypeptide are successively the 1st to 10th, 5th to 25th, 23rd to 35th, 30th to 50th...in the expected region until the entire expected region is covered.

[0067] The sequence separation rules of polypeptides in the polypeptide fragment mixture of different target proteins in the tuberculosis antigen composition can be the same or different. For example, in the polypeptide fragment mixture of a target protein, the first polypeptide, the second polypeptide...the Nth polypeptide are sequentially the 1st to 10th, the 5th to 25th, the 23rd to 35th, the 30th to 50th...of the expected region until the entire expected region is covered; in the polypeptide fragment mixture of another target protein, the first polypeptide, the second polypeptide...the Nth polypeptide are sequentially the 1st to 15th, the 8th to 20th, the 15th to 35th, the 30th to 45th, the 40th to 55th...of the expected region until the entire expected region is covered.

[0068] The Mycobacterium tuberculosis antigen composition provided in the first aspect comprises three polypeptide fragment mixtures, namely, an ESAT-6 polypeptide fragment mixture, a CFP10 polypeptide fragment mixture and an Rv2003c polypeptide fragment mixture.

[0069] The ESAT-6 polypeptide fragment mixture is a target protein with ESAT-6 protein, and the expected region is at least 80% of the full length of the ESAT-6 protein, for example, but not limited to at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99% or covering the full length of the ESAT-6 protein. In an optional embodiment, the expected region is the full length of the ESAT-6 protein.

[0070] In an optional embodiment, the amino acid sequence of the ESAT-6 protein is the sequence shown in SEQ ID NO.1, or a sequence that has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% or 99% homology to the sequence shown in SEQ ID NO.1.

[0071] The CFP10 polypeptide fragment mixture is a target protein of CFP10 protein, and the expected region is at least 80% of the full length of the CFP10 protein, for example, but not limited to at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99% or covering the full length of the CFP10 protein. In an optional embodiment, the expected region is the full length of the CFP10 protein.

[0072] In an optional embodiment, the amino acid sequence of the CFP10 protein is the sequence shown in SEQ ID NO.2, or a sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% or 99% homology to the sequence shown in SEQ ID NO.2.

[0073] The RV2003C polypeptide fragment mixture is a fragment with the RV2003C protein as the target, and the expected region is at least 80% of the 1st to (50th to 60th) positions of the RV2003C protein sequence (for example, but not limited to, the 1st to 50th, 1st to 51st, 1st to 52nd, 1st to 53rd, 1st to 54th, 1st to 55th, 1st to 56th, 1st to 57th, 1st to 58th, 1st to 59th or 1st to 60th positions of the RV2003C protein sequence), for example, but not limited to, at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99% or covering the 1st to (50th to 60th) positions of RV2003C. In an optional embodiment, the expected region is the 1st to 55th positions of RV2003C.

[0074] In an optional embodiment, the amino acid sequence of the RV2003C protein is the sequence shown in SEQ ID NO.3, or a sequence that has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% or 99% homology to the sequence shown in SEQ ID NO.3.

[0075] In an optional embodiment, in the Mycobacterium tuberculosis antigen composition, the expected region of the ESAT-6 polypeptide fragment mixture is the full length of the ESAT-6 protein; the expected region of the CFP10 polypeptide fragment mixture is the full length of the CFP10 protein; and the expected region of the Rv2003c polypeptide fragment mixture is positions 1 to 55 of the Rv2003c protein.

[0076] In an optional embodiment, the mass ratio of any two polypeptides in each of the polypeptide fragment mixtures is (1-5):(1-5), for example, it can be but not limited to 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:4, 3:5 or 4:5, optionally 1:1.

[0077] In an optional embodiment, the mass ratio of the ESAT-6 polypeptide fragment mixture, the CFP10 polypeptide fragment mixture and the Rv2003c polypeptide fragment mixture is (1~5):(1~5):(1~5), for example, it can be but not limited to 1:1:1, 1:1:5, 1:2:5, 1:3:5, 1:4:5, 1:5:5, 2:1:5, 2:2:5, 2:3:5, 2:4:5, 2:5:5, 3:1:5, 3:2:5, 3:3:5, 3:4:5, 3:5:5, 4:1:5, 4:2:5, 4:3:5, 4:4:5, 4:5:5, 5:1:5, 5:2:5, 5:3:5 or 5:4:5, optionally 1:1:1.

[0078] In a second aspect, there is provided a use of the Mycobacterium tuberculosis antigen composition described in the first aspect in detecting Mycobacterium tuberculosis or anti-Mercobacterium tuberculosis antibodies for non-diagnostic and therapeutic purposes, or in preparing a Mycobacterium tuberculosis infection diagnostic kit.

[0079] In a third aspect, a Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the Mycobacterium tuberculosis antigen composition of the first aspect, and a substance for detecting a marker, wherein the marker comprises IFN-γ.

[0080] In an optional embodiment, the marker further includes one or more of IL-2, IP-10, IL-6 and TNF-α.

[0081] In an optional embodiment, the markers include IFN-γ and IL-2.

[0082] In an alternative embodiment, the markers include IFN-γ and TNF-α.

[0083] In an optional embodiment, the working concentration of the Mycobacterium tuberculosis antigen composition is 1-10 μg / ml, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg / ml.

[0084] The substances used to detect markers include, but are not limited to, one or more of detection reagents, kits, antibody chips, antibody probes, and detection equipment. More specifically, the substances used to detect markers include, but are not limited to, reagents used to achieve immunofluorescence staining detection, flow sorting detection, immunoblotting detection, immunohistochemistry detection, ELISA detection, immunochromatography detection, or immunomagnetic bead detection; exemplary reagents used in the above methods include, but are not limited to, one or more of tracer markers, solid phase carriers, buffer reagents, salts, primary antibodies, secondary antibodies, colorimetric substrates, blocking solutions, washing solutions, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality control products, and markers.

[0085] In an optional embodiment, the tuberculosis infection diagnosis kit comprises using the tuberculosis antigen composition described in the first aspect to fully contact a sample, and then detecting the level of the marker released by the sample, wherein the sample contains lymphocytes. When the marker level exceeds a threshold value, it is determined that the sample is derived from a subject infected with tuberculosis.

[0086] In an optional implementation, the threshold is a cut-off value obtained through a ROC curve.

[0087] In an optional embodiment, the Mycobacterium tuberculosis infection diagnosis kit further comprises a sample separation reagent and / or a cell culture reagent.

[0088] In an alternative embodiment, the sample includes a cell mixture containing lymphocytes or blood containing lymphocytes.

[0089] In an optional embodiment, the sample includes whole blood, and during the detection, it is necessary to simultaneously detect the marker levels in the plasma or serum separated from the sample co-cultured with the Mycobacterium tuberculosis antigen composition and the marker levels in the plasma or serum not co-cultured with the Mycobacterium tuberculosis antigen composition to deduct background interference.

[0090] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0091] In the following examples and comparative examples:

[0092] The ESAT-6 polypeptide fragment mixture is synthesized according to the following rules according to the full length of the ESAT-6 protein sequence: positions 1 to 15, positions 11 to 25, positions 21 to 35, positions 31 to 45, positions 41 to 55, positions 51 to 65 ... until the entire protein sequence is covered. The amino acid sequence of the ESAT-6 protein is shown in SEQ ID NO.1.

[0093] SEQ ID NO.1:

[0094] MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA, 95aa.

[0095] The CFP10 polypeptide fragment mixture is synthesized according to the following rules according to the full length of the CFP10 protein sequence: positions 1 to 15, positions 11 to 25, positions 21 to 35, positions 31 to 45, positions 41 to 55, positions 51 to 65... until the entire protein sequence is covered. The amino acid sequence of the CFP10 protein is shown in SEQ ID NO.2.

[0096] SEQ ID NO.2:

[0097] MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF, 100aa.

[0098] The Rv2003c full-length polypeptide fragment mixture is synthesized according to the following rules according to the full length of the Rv2003c protein sequence: positions 1 to 15, positions 11 to 25, positions 21 to 35, positions 31 to 45, positions 41 to 55, positions 51 to 65 ... until the entire protein sequence is covered. The amino acid sequence of the Rv2003c protein is shown in SEQ ID NO.3.

[0099] SEQ ID NO.3:

[0100] MVKRSRATRLSPSIWSGWESPQCRSIRARLLLPRGRSRPPNADCCWNQLAVTPDTRMPASSAAGRDAAAYDAWYDSPTGRPILATEVAALRPLIEVFAQPRLEIGVGTGRFADLLGVRFGLDPSRDALMFARRRGVLVANAVGEAV PFVSRHFGAVLMAFTLCFVTDPAAIFRETRRLLADGGGLVIGFLPRGTPWADLYALRAARGQPGYRDARFYTAAELEQLLADSGFRVIARRCTLHQPPGLARYDIEAAHDGIQAGAGFVAISAVDQAHEPKDDHPLESE, 285aa.

[0101] The Rv2003c (1-55) polypeptide fragment mixture is synthesized according to the following rules based on the 1-55 positions of the Rv2003c protein sequence: 1-15, 11-25, 21-35, 31-45 and 41-55. The amino acid sequence of the Rv2003c protein is shown in SEQ ID NO.3.

[0102] All recombinant proteins and peptides were diluted to 10 μg / ml using cell culture medium and then mixed in equal proportions to ensure that the concentration of Mycobacterium tuberculosis antigen after mixing was 10 μg / ml.

[0103] The method for detecting samples using the kits of the following embodiments and comparative examples is as follows:

[0104] 1. To avoid background interference caused by cytokines in plasma, PBMCs were extracted from whole blood for all samples.

[0105] 2. Dilute the cells to 5×10 6 The suspension of 5 μg / ml was mixed with tuberculosis antigen in equal proportions, that is, the working concentration of tuberculosis antigen was 5 μg / ml.

[0106] 3. Incubate at 37°C, 100% humidity, 5% CO2 for 16-24 hours.

[0107] 4. The Mycobacterium tuberculosis infection diagnostic kits of Examples 1 to 9 and Comparative Examples 1 to 4 were used to detect the concentration of markers corresponding to the culture supernatant, and then the results were statistically analyzed.

[0108] A total of 42 confirmed TB cases (ATB / positive cases) and 37 healthy persons with no history of TB contact (HC / negative cases) were tested.

[0109] Example 1

[0110] Provided is a Mycobacterium tuberculosis infection diagnostic kit, comprising a Mycobacterium tuberculosis antigen composition and a double-antibody sandwich ELISA detection reagent for detecting markers.

[0111] The tuberculosis antigen composition is a mixture of ESAT-6 polypeptide fragments, a mixture of CFP10 polypeptide fragments and a mixture of Rv2003c (1-55) polypeptide fragments.

[0112] The marker is IFN-γ.

[0113] The test results are shown in Table 1, Table 2, Figure 1 and Figure 2 As shown, the AUC of the ROC curve is 0.8488, the detection sensitivity of the kit is 88.10%, the specificity is 83.78%, the total compliance rate is 86.08%, and the Kappa value is 0.7199.

[0114] Table 1 IFN-γ concentration in the supernatant of each sample cell in Example 1 (pg / mL)

[0115]

[0116] Table 2 Four-grid table of test results of Example 1

[0117]

[0118] Example 2

[0119] A Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting a marker, wherein the marker is IL-2.

[0120] The test results are shown in Table 3, Table 4, Figure 3 and Figure 4 As shown, the AUC of the ROC curve is 0.7703, the detection sensitivity of the kit is 57.14%, the specificity is 94.59%, the total compliance rate is 74.68%, and the Kappa value is 0.5044.

[0121] Table 3 IL-2 concentration in the supernatant of each sample cell in Example 2 (pg / mL)

[0122]

[0123] Table 4 Four-grid table of test results of Example 2

[0124]

[0125] Example 3

[0126] A Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting a marker, wherein the marker is IP-10.

[0127] The test results are shown in Table 5, Table 6, Figure 5 and Figure 6 As shown, the AUC of the ROC curve is 0.8127, the detection sensitivity of the kit is 88.10%, the specificity is 67.57%, the total compliance rate is 78.48%, and the Kappa value is 0.5630.

[0128] Table 5 IP-10 concentration in the cell supernatant of each sample in Example 3 (pg / mL)

[0129]

[0130] Table 6 Four-grid table of test results of Example 3

[0131]

[0132] Example 4

[0133] Provided is a Mycobacterium tuberculosis infection diagnostic kit, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting a marker, wherein the marker is IL-6.

[0134] The test results are shown in Table 7, Table 8, Figure 7 and Figure 8 As shown, the AUC of the ROC curve is 0.5630, the detection sensitivity of the kit is 45.24%, the specificity is 83.78%, the total compliance rate is 63.29%, and the Kappa value is 0.2825.

[0135] Table 7 IL-6 concentration in the supernatant of each sample cell in Example 4 (pg / mL)

[0136]

[0137] Table 8 Four-grid table of test results of Example 4

[0138]

[0139] Example 5

[0140] A Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting a marker, wherein the marker is TNF-α.

[0141] The test results are shown in Table 9, Table 10, Fig. 9 and Fig.10 As shown, the AUC of the ROC curve is 0.7223, the detection sensitivity of the kit is 64.29%, the specificity is 89.19%, the total compliance rate is 75.95%, and the Kappa value is 0.5255.

[0142] Table 9 TNF-α concentration in the supernatant of each sample cell in Example 5 (pg / mL)

[0143]

[0144] Table 10 Four-grid table of test results of Example 5

[0145]

[0146] Example 6

[0147] A Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting markers, wherein the markers are IFN-γ and IL-2.

[0148] The test results are shown in Table 11. The test kit has a sensitivity of 95.24%, a specificity of 83.78%, an overall compliance rate of 89.87%, and a Kappa value of 0.7953.

[0149] Table 11 Four-grid table of test results of Example 6

[0150]

[0151] Example 7

[0152] A Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting markers, wherein the markers are IFN-γ and IP-10.

[0153] The test results are shown in Table 12. The test kit has a sensitivity of 90.48%, a specificity of 67.57%, an overall compliance rate of 79.75%, and a Kappa value of 0.5880.

[0154] Table 12 Four-grid table of test results of Example 7

[0155]

[0156] Example 8

[0157] A Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting markers, wherein the markers are IFN-γ and IL-6.

[0158] The test results are shown in Table 13. The test kit has a sensitivity of 92.86%, a specificity of 78.38%, an overall compliance rate of 86.08%, and a Kappa value of 0.7181.

[0159] Table 13 Four-grid table of test results of Example 8

[0160]

[0161] Example 9

[0162] A Mycobacterium tuberculosis infection diagnostic kit is provided, comprising the same Mycobacterium tuberculosis antigen composition as in Example 1 and a double antibody sandwich ELISA detection reagent for detecting markers, wherein the markers are IFN-γ and TNF-α.

[0163] The test results are shown in Table 14. The test kit has a sensitivity of 90.48%, a specificity of 83.78%, an overall compliance rate of 87.34%, and a Kappa value of 0.7450.

[0164] Table 14 Four-grid table of test results of Example 9

[0165]

[0166] Comparative Example 1

[0167] Provided is a Mycobacterium tuberculosis infection diagnostic kit, comprising a Mycobacterium tuberculosis antigen composition and a double-antibody sandwich ELISA detection reagent for detecting markers.

[0168] The tuberculosis antigen composition is a mixture of ESAT-6 polypeptide fragments and a mixture of CFP10 polypeptide fragments.

[0169] The marker is IFN-γ.

[0170] The test results are shown in Table 15, Table 16, Fig.11 and Fig.12 As shown, the AUC of the ROC curve is 0.7979, the detection sensitivity of the kit is 80.95%, the specificity is 83.78%, the total compliance rate is 82.28%, and the Kappa value is 0.6453.

[0171] Table 15 IFN-γ concentration in the supernatant of each sample cell in Comparative Example 1 (pg / mL)

[0172]

[0173] Table 16 Four-grid table of test results of comparative example 1

[0174]

[0175] Comparative Example 2

[0176] Provided is a Mycobacterium tuberculosis infection diagnostic kit, comprising a Mycobacterium tuberculosis antigen composition and a double-antibody sandwich ELISA detection reagent for detecting markers.

[0177] The tuberculosis antigen composition is a recombinant protein containing ESAT-6 fragment, CFP10 fragment and Rv2003c fragments 1 to 55, and the structure is as follows:

[0178] ESAT6-linker-CFP10-linker-Rv2003c Rv2003c (1~55)-linker-His Tag.

[0179] The recombinant protein uses an Escherichia coli prokaryotic expression system, the expression vector is pET-32a, and the expression strain is BL21 (DE3).

[0180] The marker is IFN-γ.

[0181] The test results are shown in Table 17, Table 18, Fig.13 and Fig.14 As shown, the AUC of the ROC curve is 0.8008, the detection sensitivity of the kit is 85.71%, the specificity is 75.68%, the total compliance rate is 81.01%, and the Kappa value is 0.6169.

[0182] Table 17 IFN-γ concentration in the supernatant of each sample cell in Comparative Example 2 (pg / mL)

[0183]

[0184] Table 18 Test results of comparative example 2 Four-grid table Four-grid table

[0185]

[0186] Comparative Example 3

[0187] Provided is a Mycobacterium tuberculosis infection diagnostic kit, comprising a Mycobacterium tuberculosis antigen composition and a double-antibody sandwich ELISA detection reagent for detecting markers.

[0188] The tuberculosis antigen composition is a mixture of ESAT-6 polypeptide fragments and a mixture of Rv2003c (1-55) polypeptide fragments.

[0189] The marker is IFN-γ.

[0190] The test results are shown in Table 19, Table 20, Fig.15 and Fig.16 As shown, the AUC of the ROC curve is 0.7597, the detection sensitivity of the kit is 73.81%, the specificity is 83.78%, the total compliance rate is 78.48%, and the Kappa value is 0.5713.

[0191] Table 19 IFN-γ concentration in the supernatant of each sample cell in Comparative Example 3 (pg / mL)

[0192]

[0193] Table 20 Test results of comparative example 3 Four-grid table Four-grid table

[0194]

[0195] Comparative Example 4

[0196] Provided is a Mycobacterium tuberculosis infection diagnostic kit, comprising a Mycobacterium tuberculosis antigen composition and a double-antibody sandwich ELISA detection reagent for detecting markers.

[0197] The tuberculosis antigen composition is a mixture of CFP10 polypeptide fragments and a mixture of Rv2003c (1-55) polypeptide fragments.

[0198] The marker is IFN-γ.

[0199] The test results are shown in Table 21, Table 22, Fig.17 and Fig.18 As shown, the AUC of the ROC curve is 0.7597, the detection sensitivity of the kit is 66.67%, the specificity is 89.19%, the total compliance rate is 77.22%, and the Kappa value is 0.5497.

[0200] Table 21 IFN-γ concentration in the supernatant of each sample cell in Comparative Example 4 (pg / mL)

[0201]

[0202] Table 22 Test results of comparative example 4 Four-grid table Four-grid table

[0203]

[0204] Comparative Example 5

[0205] Provided is a Mycobacterium tuberculosis infection diagnostic kit, comprising a Mycobacterium tuberculosis antigen composition and a double-antibody sandwich ELISA detection reagent for detecting markers.

[0206] The tuberculosis bacillus antigen composition is a mixture of ESAT-6 polypeptide fragments, a mixture of CFP10 polypeptide fragments and a mixture of Rv2003c full-length polypeptide fragments.

[0207] The marker is IFN-γ.

[0208] The test results are shown in Table 23, Table 24, Fig.19 and Fig. 20 As shown, the AUC of the ROC curve is 0.7979, the detection sensitivity of the kit is 88.10%, the specificity is 75.68%, the total compliance rate is 82.28%, and the Kappa value is 0.6418.

[0209] Table 23 IFN-γ concentration in the supernatant of each sample cell in Comparative Example 5 (pg / mL)

[0210]

[0211] Table 24 Test results of comparative example 5 Four-grid table Four-grid table

[0212]

[0213] The sensitivity, specificity, total coincidence rate and Kappa value of Examples 1 to 9 and Comparative Examples 1 to 5 are summarized in Table 25.

[0214] Table 25 Sensitivity, specificity, total coincidence rate and Kappa value of Examples 1 to 9 and Comparative Examples 1 to 5

[0215]

[0216] In summary, the experiment found that the combination of ESAT-6, CFP10, and Rv2003c (1~55) proteins in the form of recombinant proteins can improve sensitivity, but at the same time the specificity decreases significantly (Comparative Example 2); the antigen composition composed of ESAT-6 polypeptide fragment mixture, CFP10 polypeptide fragment mixture and Rv2003c full-length polypeptide fragment mixture has a better sensitivity improvement effect, but at the same time the specificity decreases significantly (Comparative Example 5); the combination of ESAT-6 polypeptide fragment mixture and CFP10 polypeptide fragment mixture cannot improve sensitivity (Comparative Example 1), and the sensitivity of ESAT-6 polypeptide fragment mixture and CFP10 polypeptide fragment mixture combined with Rv2003c (1~55) polypeptide fragment mixture respectively decreases significantly (Comparative Examples 3 and 4). Only the combination of ESAT-6 polypeptide fragment mixture, CFP10 polypeptide fragment mixture and Rv2003c (1~55) polypeptide fragment mixture can achieve a significant improvement in sensitivity while maintaining specificity.

[0217] The results of Examples 2 to 5 show that when the ESAT-6 polypeptide fragment mixture, the CFP10 polypeptide fragment mixture and the Rv2003c (1 to 55) polypeptide fragment mixture are used in combination as antigens, the sensitivity of IL-2, IL-6 and TNF-α as detection markers alone is reduced, but the specificity of IL-2 and TNF-α as detection markers alone can be improved; using IP-10 alone as a detection marker can improve the detection sensitivity but reduce the specificity.

[0218] The results of Examples 6 to 9 show that when the ESAT-6 polypeptide fragment mixture, the CFP10 polypeptide fragment mixture and the Rv2003c (1-55) polypeptide fragment mixture are used in combination as antigens, the sensitivity of the combined use of IFN-γ and IP-10 as a detection marker is not significantly improved, but the specificity is reduced (Example 7); the combined use of IFN-γ and TNF-α as a detection marker can improve the sensitivity while ensuring that the specificity does not decrease, but the effect is not as good as the combination of IFN-γ and IL-2 (Examples 6 and 9); the combination of IFN-γ and IL-6 improves the sensitivity while slightly reducing the specificity (Example 8); at the same time, since TNF-α exists in large quantities in the blood of tumor patients, it is possible that tumor patients and tuberculosis patients cannot be distinguished, so the most preferred embodiment is the combination of IFN-γ and IL-2 (Example 6).

[0219] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Mycobacterium tuberculosis antigen composition, characterized in that: It is composed of a mixture of ESAT-6 polypeptide fragments, a mixture of CFP10 polypeptide fragments, and a mixture of Rv2003c polypeptide fragments; The ESAT-6 polypeptide fragment mixture is synthesized according to the following rules according to the full length of the ESAT-6 protein sequence: positions 1 to 15, positions 11 to 25, positions 21 to 35, positions 31 to 45, positions 41 to 55, positions 51 to 65 ... until the entire protein sequence is covered, and the amino acid sequence of the ESAT-6 protein is shown in SEQ ID NO.1; The CFP10 polypeptide fragment mixture is synthesized according to the following rules according to the full length of the CFP10 protein sequence: positions 1 to 15, positions 11 to 25, positions 21 to 35, positions 31 to 45, positions 41 to 55, positions 51 to 65 ... until the entire protein sequence is covered. The amino acid sequence of the CFP10 protein is shown in SEQ ID NO. 2; The Rv2003c polypeptide fragment mixture is synthesized according to the following rules according to positions 1 to 55 of the Rv2003c protein sequence: positions 1 to 15, positions 11 to 25, positions 21 to 35, positions 31 to 45 and positions 41 to 55. The amino acid sequence of the Rv2003c protein is shown in SEQ ID NO. 3; The polypeptides in the tuberculosis antigen composition are mixed in equal proportions.

2. Use of the tubercle bacillus tuberculosis antigen composition according to claim 1 in detecting tubercle bacillus tuberculosis or anti-tuberculosis bacillus antibodies for non-diagnostic and therapeutic purposes or in preparing a kit for diagnosing tuberculosis infection.

3. A Mycobacterium tuberculosis infection diagnostic kit, characterized in that: The method comprises the tuberculosis antigen composition according to claim 1, and a substance for detecting a marker; the marker comprises IFN-γ.

4. The Mycobacterium tuberculosis infection diagnostic kit according to claim 3, characterized in that: The markers also include one or more of IL-2, IP-10, IL-6 and TNF-α.

5. The Mycobacterium tuberculosis infection diagnostic kit according to claim 4, characterized in that: The markers include IFN-γ and IL-2; or, the markers include IFN-γ and TNF-α.

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