CN114874297A - Mycobacterium tuberculosis proteome, screening method and application thereof - Google Patents
Mycobacterium tuberculosis proteome, screening method and application thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
Abstract
The invention discloses a mycobacterium tuberculosis proteome, a screening method and application thereof; the screening method of the mycobacterium tuberculosis proteome comprises the steps of hybridizing human T cell protein and a mycobacterium tuberculosis complete proteome chip, and selecting mycobacterium tuberculosis protein with positive sites to obtain the mycobacterium tuberculosis proteome; the mycobacterium tuberculosis proteome is used for preparing detection reagents related to the mycobacterium tuberculosis, preparing tuberculosis treatment reagents, researching the action mechanism of the mycobacterium tuberculosis and the like; the mycobacterium tuberculosis proteome provides potential targets for diagnosis and treatment of tuberculosis, particularly for research on pathogenesis of mycobacterium tuberculosis and development of tuberculosis vaccines.
Description
Technical Field
The invention relates to a mycobacterium tuberculosis proteome, a screening method and application thereof, belonging to the technical field of biology.
Background
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection is a chronic infectious wasting disease that has become a global problem threatening human health. The worldwide tuberculosis report of 2021 issued by WHO shows that in 2020, the number of people with latent tuberculosis infection in the world is nearly 20 hundred million, the number of new tuberculosis patients is 987 thousands, the incidence rate is 127/10 thousands, the estimated incidence number and incidence rate continuously show a descending trend, but the descending amplitude is slower than that of the current year. The influence of new coronary pneumonia epidemic situation is eliminated, tuberculosis is the first death cause of a single infection source and is the 13 th leading cause of death in the world. In recent years, the emergence of drug-resistant tuberculosis, especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), and the continued increase in drug-resistant rates of mycobacterium tuberculosis, has presented a greater challenge to anti-tuberculosis chemotherapy. In addition, the prevention effect of the conventional BCG on pulmonary tuberculosis of children and adults is unstable, and the immunological mechanism of action is not completely clear, which also causes the predicament of tuberculosis prevention. However, the occurrence, development and transformation of tuberculosis are related to factors such as the bacterial load of the body and the virulence thereof, and the immune function of the body plays a very important role. Therefore, the immune response and escape mechanism of the mycobacterium tuberculosis are fully understood, and the method has important theoretical guiding significance for clinical diagnosis and treatment of tuberculosis and research on novel tuberculosis vaccines and immunotherapy.
Mycobacterium tuberculosis is an intracellular pathogenic bacterium, mainly parasitizing macrophages, which evades the immune killing action of host cells through various strategies, thus slowly growing and surviving in the host for a long time. This occurs through molecular interactions between specific pathogen proteins and host immune cells. Molecular interactions can affect changes in molecular function, leading to pathogens altering their gene expression processes, regulating the transition of pathogens from a replicating (growth) state to a non-replicating (dormant) state, and triggering alternative mechanisms for energy production. Thus, these intermolecular interactions are critical for the survival of Mycobacterium tuberculosis in the host, and they can regulate the host's immune response to bacterial infection and acquire nutrients required for its growth. This suggests that proteins that interact with the host upon infection with mycobacterium tuberculosis may be potential targets for the design of novel antitubercular drugs and vaccines.
Disclosure of Invention
In order to overcome the defects of the prior art, the first object of the invention is to provide a mycobacterium tuberculosis proteome, which provides potential targets for the diagnosis and treatment of tuberculosis, particularly the pathogenesis of mycobacterium tuberculosis and the research of developing tuberculosis vaccines.
The second purpose of the invention is to provide a screening method of the mycobacterium tuberculosis proteome, which has objective results, high efficiency, reduced cost and certain superiority.
The third purpose of the invention is to provide an application of the mycobacterium tuberculosis proteome, which has good auxiliary development and application for preparing detection reagents and treatment reagents or researching the action mechanism of mycobacterium tuberculosis.
The first purpose of the invention can be achieved by adopting the following technical scheme: rv3768, Rv1848, Rv0028, Rv0301, Rv1022, Rv0095, Rv2523, Rv0546, Rv1778, Rv0100, Rv1724, Rv1875, Rv3735, RV3284, Rv0577, Rv3849, Rv2697, Rv2360, Rv0489, Rv3246, Rv3841, Rv3285, Rv0764, Rv2837, Rv2907, Rv3756, Rv3118, Rv3291, Rv2534, Rv0865, Rv 366, Rv3688, Rv3295, Rv3283, Rv 321404, Rv0036, Rv1719, Rv3855, Rv2731, Rv273, Rv 2325, Rv 06408180, Rv 329258, Rv 03649, Rv 033546, Rv 329258, Rv 323946, Rv 03649, Rv 323946, Rv 323970, Rv 3282, Rv3, Rv 3282, Rv 3779 Rv3, Rv379, Rv3, Rv379, Rv 3782, Rv379, Rv373, Rv379, Rv 3782, Rv379, Rv 3782 Rv379, Rv 3782, Rv379, Rv 3757, Rv379, Rv 3747, Rv379, rv2492, Rv2614, Rv1034, RV2514, Rv3322, Rv1112, Rv0952, Rv1919, Rv1636, Rv0350, Rv0119, Rv0283, Rv2996, Rv2862, Rv0984, Rv2680, Rv0351, Rv0310, MT3289, Rv0396, Rv3160, Rv1451, Rv0056, MT3268, Rv3715, Rv0511, Rv2478, Rv1413, Rv2473, Rv2540, Rv1957, Rv0772, Rv3309, Rv3301, Rv2841, Rv3799, Rv0684, Rv2114, MT, Rv2944, RV2026, Rv0045, MT 067, MT 3368, Rv 33287, Rv 3327, Rv319, Rv328, Rv379, rv3248, MT3269, Rv1080, Rv3002, Rv0191, MT2625, MT1196, Rv2909, MT3780, Rv1667, Rv1202, Rv0030, Rv0113, Rv0110, Rv2765, Rv1829, Rv1414, RV1416, Rv1685, Rv2310, Rv0801, Rv3678, MT1364, Rv0099, Rv3709, Rv2202, Rv1045, Rv1606, Rv2333, RV1331, Rv3838, Rv3406, MT3, Rv2818, Rv3039, Rv 3617672, Rv1816, Rv0518, Rv259, Rv2557, Rv 2591, Rv 8, Rv1, Rv3567, Rv 3537, Rv 3512437, Rv039, Rv 1722, Rv 1728, Rv039, Rv 989, Rv 982, Rv 989, Rv 982, Rv 988, Rv 989, Rv 982, Rv 989, Rv 988, Rv 982, Rv 989, Rv 982, Rv 988, Rv 982, Rv 989, Rv 988, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 982, Rv 989, Rv 988 Rv 989, Rv 989, Rv 982, Rv 989, Rv 988 Rv 989, Rv 988, Rv 989, Rv 982, Rv 988 Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, R, At least one of Rv2495c, Rv0585c, Rv1168c, Rv3835, Rv3341, MT1342, Rv2937, Rv1698, Rv3437, Rv1362c, Rv3024c and Rv 0155.
Further, Rv3768, Rv1848, Rv0028, Rv0301, Rv1022, Rv0095, Rv2523, Rv0546, Rv1778, Rv0100, Rv1724, Rv 1871871875, Rv3735, Rv3284, Rv0577, Rv3849, Rv2697, Rv2360, Rv0489, Rv3246, Rv3841, Rv3285, Rv0764, Rv2837, Rv2907, Rv3756, Rv3118, Rv3291, Rv2534, Rv0865, Rv 6, Rv3688, Rv3295, Rv3283, Rv0036, Rv039, Rv1719, Rv3855, Rv31, Rv 4, Rv 2325, 273254, Rv 32818181818181818181818120, Rv309, Rv 3025309, Rv 3290, Rv 323025309, Rv 3290, Rv 289, Rv 289, Rv 3290, Rv 3230819, 289, Rv 3290, Rv 373290, Rv379, Rv 37779, Rv379, 289, Rv 3230819, 289, Rv 3290, Rv 3230819, Rv 3290, Rv 37798, Rv 373230819, 30819, Rv 3290, Rv 3730819, Rv 3290, R95, 289, R95, Rv, Rv33 c, Rv1112, Rv0952, Rv1919c, Rv1636, Rv0350, Rv0119, Rv0283, Rv2996c, Rv28 c, Rv0984, Rv2680, Rv0351, Rv0310c, MT3289, Rv0396, Rv3160c, Rv1451, Rv0056, MT1560.1, MT3268, Rv3715c, Rv0271c, Rv0511, Rv2478c, Rv1413, Rv2473, Rv2540c, Rv1957, Rv0772, Rv3309 8687428, Rv 28937, Rv 9341 3652, Rv37 c, Rv0684, Rv 1714, MT 3624272, Rv 3844, Rv 3306, Rv 388749 c, Rv 25046, Rv 22466, Rv 22467, Rv 22464, Rv 22467, Rv 2235467, Rv 25046, Rv 22464, Rv 22467, Rv 25046, Rv 22464, Rv 22467, Rv 2504, Rv 22467, Rv 33464, Rv 33467, Rv 2502, Rv 33464, Rv 33467, Rv 3346, Rv 2502, Rv339, Rv 2504, Rv 33464, Rv 2502, Rv 2504, Rv 33464, Rv 3382, Rv3, Rv 2504, Rv3, Rv 2504, Rv2, Rv 2504, Rv 3305, Rv3, Rv2, Rv3, Rv 2504, Rv3, Rv 3382, rv2851c, Rv3099c, Rv1677, Rv32 c, RV1379, Rv1377c, Rv1347c, Rv0491, Rv2959c, Rv2913c, Rv2718c, Rv1671, Rv1019, Rv1219c, Rv2179c, Rv2185c, RV1949c, Rv2513, Rv2149c, Rv1683, Rv3521, Rv1003, Rv1419, Rv0726c, Rv0970, Rv0078, Rv0821c, Rv2689c, Rv2825c, Rv3442c, Rv2436, Rv3465, Rv3748, Rv0232, Rv 00477, Rv 6855, Rv c, Rv 68576, Rv c, Rv 6857, c, 6857, c, Rv 6857, c, 6857, c and Rv 6857, c patients with tuberculosis, no tuberculosis, No. 3, No. Rv c, No. 3, No. Rv 6859, No. Rv c, No. Rv 6859, No. 2, No. 3, No. Rv 6859, No. 3, No. 2, No. Rv 6859, No. Rv c, No. Rv 6859, No. 3, No. Rv c, No. Rv 6859, No. Rv c, No. Rv 6859, No. Rv c, No. Rv 6859, No. Rv c, No. Rv 6859, No. R;
rv1080, Rv3002, Rv0191, MT2625, MT1196, Rv2909, MT3780, Rv1667, Rv1202, Rv0030, Rv0113, Rv0110, Rv2765, Rv1829, Rv1414, RV1416, Rv1685, Rv2310, Rv0801, Rv3678, MT1364, Rv0099, Rv3709, Rv2202, Rv1045, Rv1606, Rv2333, RV1331, Rv3838, Rv3406, MT3103, Rv2818, Rv3039, Rv3672, Rv1816, Rv 1760518, Rv1159, Rv2557, Rv3791, Rv2358, Rv3567, Rv 5, Rv 14437, Rv1448, Rv 01926, Rv 35059, Rv 35048, Rv 2502, Rv 989, Rv 256226, Rv379, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 98, Rv1168c, Rv3835, Rv3341, MT1342, Rv2937, Rv1698, Rv3437, Rv1362c, Rv3024c and Rv0155 are specific proteins for non-tuberculosis patients.
Further, the mycobacterium tuberculosis proteome hybridizes positively with T cell proteins.
The second purpose of the invention can be achieved by adopting the following technical scheme: a screening method of mycobacterium tuberculosis proteome comprises the steps of hybridizing human T cell protein and a mycobacterium tuberculosis whole proteome chip, and selecting mycobacterium tuberculosis protein with positive sites to obtain the mycobacterium tuberculosis proteome.
Further, human T cell proteins include T cell proteins from patients with tuberculosis and T cell proteins from patients with non-tuberculosis.
Further, human T cell proteins were obtained by flow cytometry; flow cytometry detection of cell immunity type as CD3 + And (4) carrying out T lymphocyte percentage and sorting to obtain human T cell protein.
The third purpose of the invention can be achieved by adopting the following technical scheme: the application of a mycobacterium tuberculosis proteome is characterized in that the mycobacterium tuberculosis proteome is used for preparing a detection reagent related to mycobacterium tuberculosis; the reagent comprises mycobacterium tuberculosis proteome; or a DNA molecule encoding the mycobacterium tuberculosis proteome; or a recombinant protein produced by a recombinant bacterium containing a DNA molecule.
Further, the reagent is a detection reagent for mycobacterium tuberculosis or a detection reagent for tuberculosis immune function state.
Or, the use of a mycobacterium tuberculosis proteome for the preparation of a tuberculosis treatment agent.
Further, the tuberculosis treating agent includes a polyclonal antibody obtained with a mycobacterium tuberculosis proteome as an immunogen and/or a monoclonal antibody recognizing an antigen protein of the mycobacterium tuberculosis proteome.
Or, an application of the mycobacterium tuberculosis proteome, and the mycobacterium tuberculosis proteome is used for preparing the vaccine.
Or, an application of the mycobacterium tuberculosis proteome, the mycobacterium tuberculosis proteome is used for researching the action mechanism of the mycobacterium tuberculosis.
Compared with the prior art, the invention has the beneficial effects that:
1. the mycobacterium tuberculosis proteome provides potential targets for diagnosis and treatment of tuberculosis, particularly research on pathogenesis of mycobacterium tuberculosis and development of tuberculosis vaccines;
2. the screening method of the mycobacterium tuberculosis proteome is obtained through the interaction of protein-protein functions, has objective screening result and high efficiency, reduces the cost and has certain superiority;
3. the mycobacterium tuberculosis proteome of the invention has wide application, can analyze the function of Mtb as a system and identify the mode and the attribute of the system; the kit is used for preparing a detection reagent and a treatment reagent or researching the action mechanism of the mycobacterium tuberculosis, and has good auxiliary development and application.
Drawings
FIG. 1 shows the result of SDS-PAGE detection in HV group;
FIG. 2 shows SDS-PAGE detection of TB group;
FIG. 3 is a TB group chip scan view;
FIG. 4 is a view of a HV block chip scan;
FIG. 5 shows the results of SDS-PAGE detection of Jurkat group;
FIG. 6 shows the result of Western Blot detection;
FIGS. 7-8 show the Dot Blot detection results;
FIG. 9 is a Jurkat group chip scan view;
FIG. 10 is a Venn diagram.
Detailed Description
The invention will be further described with reference to the accompanying drawings and the detailed description below:
a method for screening a mycobacterium tuberculosis proteome comprising
T cell protein obtaining step: human T cell proteins were obtained by flow cytometry; flow cytometry detection of cell immunity type as CD3 + The percentage of T lymphocytes is sorted to obtain human T cell protein;
and (3) hybridizing: respectively hybridizing the T cell protein of the tuberculosis patient and the T cell protein of the non-tuberculosis patient with the full-protein group chip of the mycobacterium tuberculosis, and selecting the mycobacterium tuberculosis protein with positive sites to obtain the mycobacterium tuberculosis protein group.
The mycobacterium tuberculosis proteome comprises common proteins of non-tuberculosis patients and specific proteins of the non-tuberculosis patients;
common proteins of patients with non-tuberculosis and tuberculosis comprise Rv3768, Rv1848, Rv0028, Rv0301, Rv1022, Rv0095, Rv2523, Rv0546, Rv1778, Rv0100, Rv1724, Rv1875, Rv3735, RV3284, Rv0577, Rv3849, Rv2697, Rv2360, Rv0489, Rv3246, Rv3841, Rv3285, Rv0764, Rv2837, Rv2907, Rv3756, Rv3118, Rv3291, Rv2534, Rv0865, Rv1876, Rv3688, Rv3295, Rv3283, RV1404, Rv0036, Rv1719, Rv3855, Rv2731, Rv2324, Rv 2325, Rv 0640649, Rv 323546, Rv 036427, Rv 0358, Rv3246, Rv 03649, Rv 328180, Rv 6427, Rv329, Rv 0320, Rv 6427, Rv 3282, Rv 6427, Rv329, Rv 6427, Rv 289, Rv 6420, Rv 178, Rv 6420, Rv 1746, Rv 649, Rv 6420, Rv 649, Rv 6420, Rv 649, Rv179, Rv 649, Rv 6408 Rv 649, Rv 6408 Rv 649, Rv379, Rv 649, Rv 6408 Rv 649, Rv379, Rv 649, Rv 6420, Rv 6408 Rv 649, Rv 6420, Rv 649, Rv379, Rv 649, Rv379, Rv 649, Rv379, Rv 649, Rv379, Rv 649, Rv379, Rv 649, Rv 64No. Rv 649, Rv379, Rv 649, Rv379, Rv 649, Rv379, Rv 64043, Rv 649, Rv379, Rv 649, Rv379, Rv 64043, Rv379, R, Rv3677, Rv1463, Rv0258, Rv2492, Rv2614, Rv1034, RV2514, Rv3322, Rv1112, Rv0952, Rv1919, Rv1636, Rv0350, Rv0119, Rv0283, Rv2996, Rv2862, Rv0984, Rv2680, Rv0351, Rv0310, MT3289, Rv0396, Rv3160, Rv1451, Rv0056, MT3268, Rv3715, Rv0271, Rv0511, Rv2478, Rv 1410043, Rv2473, Rv2540, Rv1957, Rv0772, Rv3309, Rv3301, Rv2841, Rv3799, Rv0684, Rv2114, MT, Rv 062427, Rv 28677, Rv 289, Rv 2529, Rv 2509, Rv329, Rv 32059, Rv379, Rv319, Rv379, Rv319, Rv379, Rv 3726, Rv379, Rv 3781, Rv379, Rv 3781, Rv379, Rv 3781, Rv379, Rv 3726, Rv379, Rv 3726, Rv 3781, Rv379, Rv 3781, Rv319, Rv 3781, Rv379, Rv 3726, Rv 3781, Rv 3726, Rv 3781, Rv 3726, Rv379, Rv 3726, Rv379, Rv 3781, Rv 3781, Rv379, Rv 3781, Rv 3781, Rv379, Rv379, Rv379, Rv 3781, Rv379, Rv 3781, Rv379, Rv 3781 No. 3, Rv 3781 No. 3, Rv379, Rv 3781 No. 3, Rv 3781, Rv379, Rv 3781 No. 3, Rv No. 3, Rv 3781 No., At least one of Rv3676, Rv3717, Rv2711, Rv3248c and MT 3269;
the specific protein of patients with non-tuberculosis patients comprises Rv1080, Rv3002, Rv0191, MT2625, MT1196, Rv2909, MT3780, Rv1667, Rv1202, Rv0030, Rv0113, Rv0110, Rv2765, Rv1829, Rv1414, RV1416, Rv1685, Rv2310, Rv0801, Rv3678, MT1364, Rv0099, Rv3709, Rv2202, Rv1045, Rv1606, Rv 2332333, RV1331, Rv3838, Rv3406, MT3103, Rv2818, Rv3039, Rv3672, Rv 0516, Rv0518, Rv1159, Rv2557, Rv 91, Rv0518, Rv 353567, Rv 37037, Rv 14437, Rv1448, Rv 0335729, Rv 1728, Rv 2502, Rv 089, Rv 988, Rv 989, Rv 35089, Rv 1728, Rv 35088, Rv 35729, Rv359, Rv 1728, Rv 35729, Rv359, Rv 35729, Rv 982, Rv 1728, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 988, Rv 989, Rv 988, Rv 982, Rv 1728, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 729, Rv 982, Rv 988, Rv 982, Rv 729, Rv 989, Rv 988, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 982, Rv 988, Rv 989, Rv 982, Rv 989, Rv 988 Rv 989 Rv 982 Rv 989, Rv 982 Rv 729 Rv 989, Rv 988 Rv 989, Rv 989 Rv 988 Rv 989, Rv 988 Rv 729, Rv 988 Rv 989, Rv 988, Rv 989, Rv 988 Rv 982, Rv 989, Rv 988, Rv 982, Rv 988 Rv 982, Rv 989, Rv 988, Rv 989, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 989, R, At least one of RV1395, RV0072, RV1766, RV1439c, RV1536, RV2495c, RV0585c, RV1168c, RV3835, RV3341, MT1342, RV2937, RV1698, RV3437, RV1362c, RV3024c, and RV 0155.
The application of the mycobacterium tuberculosis proteome comprises the following steps:
the mycobacterium tuberculosis proteome is used for preparing a detection reagent for mycobacterium tuberculosis or a detection reagent for the tuberculosis immune function state; the reagent comprises mycobacterium tuberculosis proteome; or a DNA molecule encoding the mycobacterium tuberculosis proteome; or a recombinant protein produced by a recombinant bacterium containing a DNA molecule;
the mycobacterium tuberculosis proteome is used for preparing tuberculosis treatment reagents; the therapeutic agent comprises a polyclonal antibody obtained by taking a mycobacterium tuberculosis proteome as an immunogen and/or a monoclonal antibody recognizing an antigen protein of the mycobacterium tuberculosis proteome;
the mycobacterium tuberculosis proteome is used for researching the action mechanism of the mycobacterium tuberculosis, such as an immune escape mechanism which the mycobacterium tuberculosis may have.
Example 1:
the study subjects were divided into two groups according to known circumstances:
(1) non-tuberculosis patient group (HV, 39 patients): the group had no tuberculosis and symptoms and signs of related mycosis, and had negative TST test (except for early allergy; immune system interference: application of immunosuppressants such as glucocorticoid or malnutrition, measles, pertussis, etc.), and immune hypofunction: severe tuberculosis, various critical patients, immune deficiency of lymphocyte system, etc.);
(2) tuberculosis patient group (TB, 32 patients): tuberculosis patients who were initially diagnosed as tuberculosis and were not treated; the patients with pulmonary tuberculosis are confirmed to be diagnosed through clinical, laboratory and imaging examination according to the standard of pulmonary tuberculosis diagnosis (WS 288-2017). The patient with primary diagnosis of pulmonary tuberculosis refers to a patient who is found for the first time, does not receive any anti-tuberculosis drug treatment or is treated by irregular and unreasonable anti-tuberculosis treatment chemotherapy after the pulmonary tuberculosis is found, but the treatment course is less than 1 month; a recurrent tuberculosis patient refers to a patient who has failed the initial treatment or healed but has recurred again (a patient who has received irregular, unreasonable chemotherapy for more than 1 month after removal of the diagnosed tuberculosis).
Obtaining of T cell protein:
1. isolation of Peripheral Blood Mononuclear Cells (PBMC):
(1) 5mL of collected fresh anticoagulation blood (heparin anticoagulant) is transferred into a 15mL centrifuge tube;
(2) adding PBS with the same amount into each centrifugal tube, and uniformly mixing;
(3) another new 15mL centrifuge tube is taken, and 5mL of lymphocyte separation solution is added;
(4) slowly adding the diluted blood into a centrifugal tube containing lymphocyte separation liquid;
(5)2000rpm, room temperature, centrifuging for 30 min;
(6) sucking the second layer of haze cells (PBMC) into a 15mL centrifuge tube;
(7) adding a proper amount of PBS, washing PBMC, centrifuging at 1000rpm at room temperature for 10 min;
(8) discarding the supernatant, and repeating the step (7);
(9) the supernatant was discarded, PBMCs were resuspended in appropriate amounts of PBS and counted.
2. Flow cytometry detection and sorting of T cells:
(1) transferring the PBMC into a 1.5mL EP tube, adding a fluorescent dye Alexa Fluor647Mouse Anti-Human CD3(5 μ L Alexa antibody/100 μ L cell suspension) and a negative isotype control antibody respectively, and incubating for 20min at room temperature in a dark place;
(2) adding a proper amount of PBS, mixing uniformly, centrifuging at 1000rpm and 4 ℃ for 5 min;
(3) discarding the supernatant, adding 1mL PBS to gently blow and wash, removing the unbound antibody, centrifuging at 1000rpm and 4 ℃ for 5min, and repeating for 2 times;
(4) discarding the supernatant, adding 1mL of PBS to resuspend PBMC marked by the fluorescent antibody, and detecting on a machine;
(5) the flow cytometer (BD, FACS Aria III) was adjusted to work optimally, and the negative isotype control group of the fluorescently labeled antibody was added, followed by the experimental group of fluorescently labeled antibody. Detecting CD3 as cell immune type + T lymphocyte percentage and sorting.
3. T cell protein extraction and detection:
(1) each 1 × 10 6 Cell lysis by adding 100. mu.L to each cellOscillating for 5-10s, standing on ice for 10min, and repeating for 3 times; 12000g, centrifuging for 30min at 4 ℃, and obtaining supernatant which is T cell protein solution;
(2) the protein concentration of the HV group measured by the BCA method is 141 ng/mu L, the protein concentration of the TB group is 494 ng/mu L, and all sample concentrations meet the requirements of subsequent experiments;
(3) protein bands were detected after SDS-PAGE and Coomassie blue staining, and all samples were total protein as shown in FIGS. 1 and 2.
Example 2:
screening for mycobacterium tuberculosis proteins that interact with human T cell proteins:
MtbProt TM the mycobacterium tuberculosis proteome chip has 4262 mycobacterium tuberculosis recombinant proteins which comprise 3829 proteins coded by mycobacterium tuberculosis standard strain H37Rv genes and 433 proteins coded by mycobacterium tuberculosis pathogenic bacteria CDC1551 genes, the integral coverage rate is up to 91 percent, and the chip is the first mycobacterium tuberculosis proteome chip in the world at present.
1. Sample marking and detection:
(1) labeling the sample with Cy3 fluorescein according to the instructions of the kit CyDye Protein LabellingCY3 MONO 5-PACK (GE, PA 23001);
(2) detecting the fluorescence labeling effect of the samples by using Western blot and Dot blot, wherein all the samples are qualified; the sample signal is in positive correlation with the sample gradient, the minimum detection limit is 0.5ng, and the detection result meets the requirements of subsequent experiments;
(3) and (3) sealing: taking out the protein chip from a refrigerator at-80 deg.C, rewarming at 4 deg.C, adding 300 μ L of sealing solution into each well, placing in a side-swinging shaking table, and sealing at 4 deg.C for 3 hr;
(4) sample incubation: removing the blocking solution, quickly adding the prepared sample incubation solution (final concentration is 1 μ g/mL) at 200 μ L/hole, placing on a side-shaking table, and incubating overnight at 4 ℃ (from this step, taking care to avoid light);
(5) cleaning: placing the chip in a horizontal shaking table, cleaning with cleaning solution at room temperature for 3 times and 5 min/time, and cleaning with ultrapure water at room temperature for 2 times and 5 min/time;
(6) and (3) drying: absorbing residual water from the edge of the chip by using absorbent paper without contacting the surface of the chip;
(7) scanning: scanning the dried chip according to the operation specification and the use instruction of the scanner, if the chip is not scanned in time, storing the chip in a light-proof slide glass box at-20 ℃, and completing the scanning within three days;
the hybridization results are shown in FIGS. 3 and 4, and in the enlarged view of FIG. 4, the reference numeral 1 is a positive spot, and the reference numeral 2 is a positive control spot (Cy 3-BSA). Except for the positive control points, the chips after the T cell proteins of the non-tuberculosis patients and the tuberculosis patients are hybridized with the mycobacterium tuberculosis protein have positive sites, which indicates that the corresponding interaction proteins are screened;
(8) data extraction: the image obtained by scanning is analyzed by GenePix Pro v6.0 software to obtain original data, then the normalized signal-to-noise ratio (SNR) value of all proteins on the chip is taken as a calculation object, the positive level of the site is judged, and the mycobacterium tuberculosis proteome is screened out by taking the SNR (signal-to-noise ratio) more than or equal to 1.5 as a threshold value.
By studying protein-protein function interactions, Mtb can be used as a system to analyze its function and to identify patterns and attributes of the system, which can further distinguish the specific interaction of mycobacterium tuberculosis proteins between acquired and innate immunity. Experimental studies have found and identified some protein-protein interactions (PPIs) between Mtb and humans. Yeast two-hybrid (Y2H) system, affinity pull-down (AP), and Mass Spectrometry (MS) are the two main methods for finding these interactions. However, Y2H has a sensitivity of only 20%, and AP/MS can detect proteins in the complex, but when multiple proteins are purified simultaneously, it is not possible to distinguish between direct and indirect interactions between the proteins. Furthermore, these methods are not only time consuming and costly, especially when high throughput modes are employed. In recent years, based on homology, mutual ordering, interaction domains and structures between gene sequences, various computational methods have been developed to predict PPI between host and pathogen. However, most of these methods have yet to be rigorously validated. Therefore, the accuracy of the computational method to predict host-pathogen PPI is largely unknown.
The embodiment combines the flow cytometry and the protein chip technology, the screening process has certain technical difficulty, and the protein-protein interaction is objectively analyzed in a high-flux mode under natural conditions, so that compared with the original method, the method not only saves time and reduces cost, but also has certain superiority.
Carrying out isogeny analysis on the screened mycobacterium tuberculosis proteins and drawing a Venn diagram;
rv3768, Rv1848, Rv0028, Rv0301, Rv1022, Rv00 0095c, Rv2523c, Rv0546c, Rv17 1778c, Rv0100, Rv1724, Rv1875, Rv3735, RV3284, Rv 0568577, Rv3849, Rv 2668597, Rv2360c, Rv0489, Rv3246c, Rv3841, Rv3285, Rv0764c, Rv 68537 c, Rv 6858, Rv 6855, Rv 68537 c, Rv 6853, Rv 68537 c, Rv 6855, Rv 6853, Rv 68537 c, Rv 6858, Rv 6853, Rv c, Rv 6858 68537 c, Rv 6858, Rv 6853 No. c, No. 6853 No. c, No. 6858, No. 6855, No. c, No. 5, No. 5 No. 4, No. 4 No. 5 No. 2 No. 4, No. 2 No. 4, No. 4, No. 4, No. 4, No. 4 No. 2 No. 4, No. 2 No. 4,6858, No. 4,6858,6858,6858,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,6854,, Rv1657, Rv1794, Rv1142, Rv3198, Rv1896, Rv3279, Rv1531, Rv2461, Rv3154, Rv1015, Rv3007, Rv2511, Rv3677, Rv1463, Rv0258, Rv2492, Rv2614, Rv1034, RV2514, Rv3322, Rv1112, Rv0952, Rv1919, Rv1636, Rv0350, Rv0119, Rv0283, Rv2996, Rv2862, Rv0984, Rv2680, Rv0351, Rv0310, MT 89, Rv0396, Rv3160, Rv1451, Rv0056, MT3268, Rv3715, Rv0271, Rv1, Rv 063, Rv 0628063, Rv 281417, Rv 2846, Rv319, Rv 289, Rv319, Rv1379, Rv 289, Rv319, Rv3309, Rv 289, Rv1379, Rv 289, Rv 989, Rv 289, Rv1379, Rv 989, Rv1379, Rv 289, Rv 289, Rv1379, Rv 289, Rv1379, Rv 289, Rv1379, Rv 289, Rv 289, Rv1379, Rv 289, Rv 289, Rv1379, Rv 289, Rv 289, Rv1379, Rv 289, Rv1379, Rv1379, Rv 289, Rv1379, Rv 1378, Rv1379, Rv 289, Rv1379, Rv 289, Rv1379, Rv 289, Rv1379, Rv2499c, Rv3213C, Rv0956, Rv0429c, Rv1718, Rv0534C, Rv3609c, Rv0041, Rv1056, Rv1264, Rv2293c, Rv3899C, Rv3676, Rv3717, Rv2711, Rv3248c and MT 3269;
rv1080, Rv3002, Rv0191, MT2625, MT1196, Rv2909, MT3780, Rv1667, Rv1202, Rv0030, Rv0113, Rv0110, Rv2765, Rv1829, Rv1414, RV1416, Rv1685, Rv2310, Rv0801, Rv3678, MT1364, Rv0099, Rv3709, Rv2202, Rv1045, Rv1606, Rv 2331025 3, RV1331, Rv3838, Rv3406, MT3103, Rv2818, Rv3039, Rv3672, Rv1816, Rv0518, Rv039, Rv2557, Rv3791, Rv 8, Rv1, Rv 353567, Rv 351247, Rv039, Rv 35098, Rv 1722, Rv 059, Rv 989, Rv088, Rv 989, Rv 35088, Rv 1728, Rv 989, Rv358, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 982, Rv 988, Rv 982, Rv 989, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 988 Rv 989, Rv 982, Rv 988 Rv 982, Rv 988 Rv 982, Rv 988 Rv 989, Rv 989, Rv 988 Rv, Rv 989, Rv 988 Rv, Rv 988 Rv, Rv 982, Rv 989, Rv 988 Rv, Rv 982, Rv 988, Rv 989, Rv 982, Rv 989, Rv 982, Rv 988 Rv, Rv 988, Rv 988, Rv 982, Rv 988, rv1932, Rv2535C, Rv1395, Rv0072, Rv1766, Rv1439c, Rv1536, Rv2495c, Rv0585c, Rv1168c, Rv3835, Rv3341, MT1342, Rv2937, Rv1698, Rv3437, Rv1362c, Rv 302384 3024c and Rv 0155.
Example 3:
as shown by the results of example 2, which respectively screen 2 groups of subjects for proteins shared by non-tuberculous patients and tuberculosis patients and proteins specific to non-tuberculous patients, it is suggested that the expression of these proteins may be selectively inhibited to exert different regulatory effects during the invasion of Mtb into host cells, and thus it is presumed that the proteins are related to the immune escape mechanism of Mtb, so that the Mycobacterium tuberculosis proteome is useful for studying the action mechanism of Mycobacterium tuberculosis; especially, according to the condition that the mycobacterium tuberculosis proteome is divided into two groups of common proteins of non-tuberculosis patients and specific proteins of the non-tuberculosis patients, the immune escape mechanism of Mtb can be further researched.
Example 4:
based on MtbProt TM Mycobacterium tuberculosis proteome chip screens for proteins of Mycobacterium tuberculosis that interact with proteins of the Jurkat cell line:
to verify the results of example 2, the Jurkat cell line was selected as a control in this example. The Jurkat cell line belongs to an acute T cell leukemia cell line, is a suspension cell, has high growth speed, and the cells gather into a grape bunch shape in the best culture state, are round and bright and are similar to fish eggs.
1. Resurgence of Jurkat cells:
(1) taking out the frozen Jurkat cells from the liquid nitrogen tank, quickly putting the frozen tube into water at 37 ℃, and continuously and gently shaking until the cells are completely dissolved;
(2) transferring the cells into a 15mL centrifuge tube, adding 2mL RPMI-1640 culture solution, centrifuging at 1000rpm for 5min, and discarding the supernatant;
(3) resuspend the cells in appropriate amounts of complete medium (RPMI-1640+10 v/v% fetal bovine serum);
(4)25cm 2 5mL of the pre-warmed complete medium was added to the cell culture flask, and then 1mL of the resuspended cells was added and gently mixed.
(5)5v/v％CO 2 And cultured at 37 ℃.
2. Jurkat cell subculture:
(1) transferring the cells in the culture bottle into a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, and removing the supernatant;
(2) resuspend the cells in appropriate amounts of complete medium (RPMI-1640+10 v/v% fetal bovine serum);
(3)25cm 2 adding 5mL of preheated complete culture medium into the cell culture bottle, then adding 1mL of resuspended cells, and gently mixing;
(4)5％CO 2 culturing at 37 ℃;
the growth of the cells which are just recovered is slow, about 3-4 days passes through one generation, after two generations, 2-3 days passes through one generation, the cell morphology is observed under an inverted microscope after each passage, and subsequent experiments can be carried out when the cells pass through proper generations.
3. Jurkat cell protein extraction and detection:
(1) each 1 × 10 6 Adding 100 μ L cell lysate into each cell, oscillating for 5-10s, standing on ice for 10min, and repeating for 3 times; 12000g, centrifuging for 30min at 4 ℃, and obtaining supernatant which is Jurkat cell protein solution;
(2) the protein concentration measured by the BCA method is 3492 ng/mu L, and the concentration of all samples meets the requirements of subsequent experiments;
(3) protein bands were detected after SDS-PAGE, stained with Coomassie Brilliant blue, and all samples were total protein as shown in FIG. 5;
4. selecting a mycobacterium tuberculosis protein that interacts with a Jurkat cell line protein:
(1) sample labeling and detection
1) According to the kit CyDye Protein LabellingCY3 MONO 5-PACK (GE, PA 23)
001) The instructions of (1), labeling the sample with Cy3 fluorescein;
2) detecting the fluorescent marking effect of the sample by using Western blot and Dot blot, wherein FIG. 6 shows the detection result of Western blot (including the result of HV group and TB group), and all samples are marked to be qualified; FIGS. 7 to 8 show the Dot Blot detection results (including the results of HV group and TB group), where the sample signal is positively correlated with the sample gradient, the minimum detection limit is 0.5ng, and the detection results satisfy the requirements of the subsequent experiments;
(2) and (3) sealing: taking out the protein chip from a refrigerator at-80 deg.C, rewarming at 4 deg.C, adding 300 μ L of sealing solution into each well, placing in a side-swinging shaking table, and sealing at 4 deg.C for 3 hr;
(3) sample incubation: removing the blocking solution, quickly adding the prepared sample incubation solution (final concentration is 1 μ g/ml) at 200 μ l/well, placing on a side-shaking table, and incubating overnight at 4 ℃ (from this step, taking care to avoid light);
(4) cleaning: placing the chip in a horizontal shaking table, cleaning with cleaning solution at room temperature for 3 times and 5 min/time, and cleaning with ultrapure water at room temperature for 2 times and 5 min/time;
(5) and (3) drying: absorbing residual water from the edge of the chip by using absorbent paper without contacting the surface of the chip;
(6) scanning: scanning the dried chip according to the operation specification and the use instruction of the scanner, if the chip is not scanned in time, storing the chip in a light-proof slide glass box at-20 ℃, and completing the scanning within three days;
(7) data extraction: the scanned image is analyzed by GenePix Pro v6.0 software to obtain original data, then the normalized signal-to-noise ratio (SNR) values of all proteins on the chip are taken as calculation objects, the positive level of the site is judged, and potential positive proteins are screened out by taking SNR (signal-to-noise ratio) more than or equal to 1.5 as a threshold value.
5. As a result:
(1) the extracted Jurkat cell protein and Mycobacterium tuberculosis proteome chip hybridization results are shown in FIG. 9, except for the positive control point, the positive site appears on the chip after the Jurkat cell protein and Mycobacterium tuberculosis protein are hybridized, which indicates that the corresponding interaction protein is screened.
(2) The invention screens 93 mycobacterium tuberculosis proteins which interact with Jurkat cell proteins, and the proteins are respectively as follows: rv3855, Rv0937, Rv3768, Rv1848, Rv0028, Rv0301, Rv1022, Rv0025, Rv0095, Rv2523, Rv0546, Rv2293, Rv3609, Rv1778, Rv0100, Rv1724, Rv1875, Rv3735, Rv3284, Rv2847, Rv0577, Rv1264, Rv3849, Rv2697, Rv2360, Rv0489, Rv3246, Rv3676, Rv3841, Rv3285, Rv0764, Rv2837, Rv2907, Rv1718, Rv3756, Rv328, Rv3291, Rv2534, Rv0865, Rv1876, Rv3688, Rv3295, Rv 3231159, Rv 323783, Rv 32039, Rv3246, Rv 3222423, Rv329, Rv 3222448, Rv 322243, Rv 322249, Rv 32039, Rv 322249, Rv329, Rv 322249, Rv 322243, Rv 32039, Rv329, Rv 3264, Rv 322249, Rv329, Rv 32639, Rv329, Rv 32039, Rv 3264, Rv329, Rv 322249, Rv329, Rv 322568, Rv329, Rv 3264, Rv329, Rv 322568, Rv329, Rv 322568, Rv329, Rv 322568, Rv329, Rv 3264, Rv329, Rv 322568, Rv329, Rv 32639, Rv329, Rv 32639, Rv329, Rv 3264, Rv329, Rv 322568, Rv329, Rv 3264, Rv329, Rv 32639, Rv329, Rv328, Rv329, Rv 3264, Rv 32639, Rv 3264, Rv329, Rv 3264, Rv 32639, Rv329, Rv 3264, Rv329, Rv 3264, Rv329, Rv3295, Rv 3264, Rv329, Rv 3264, Rv 322568, Rv3295, Rv 3264, Rv 322568, Rv329, Rv 3264, Rv329, Rv 3264, Rv329, Rv 3264, Rv328, Rv 3264, Rv3295, Rv 3264, Rv 32639, Rv 3264, Rv 322568, Rv 3264, Rv329, Rv 3264, Rv 32.
The potential positive tuberculosis proteins screened from T cells of healthy people and tuberculosis patients were subjected to heterozygosity analysis and Venn diagram drawing, and as shown in FIG. 10, the 93 Mycobacterium tuberculosis proteins were all shared by non-tuberculosis patients, T cells of tuberculosis patients and Jurkat cells. Furthermore, the number of Mtb proteins interacting with T cells in tuberculosis patients was much greater than the Mtb proteins interacting with Jurkat cells, indicating that the cell line could not replace primary cells in some way; 137 specific proteins of patients with non-tuberculosis indicate that during the process of Mtb invading host cells, host protein expression may be selectively inhibited to exert different regulation effects
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (10)
1. A mycobacterium tuberculosis protein group, which is characterized by comprising Rv3768, Rv1848, Rv0028, Rv0301, Rv1022, Rv0095, Rv2523, Rv0546, Rv1778, Rv0100, Rv1724, Rv1875, Rv3735, RV3284, Rv0577, Rv3849, Rv2697, Rv2360, Rv0489, Rv3246, Rv3841, Rv3285, Rv0764, Rv2837, Rv2907, Rv3756, Rv3118, Rv3291, Rv2534, Rv0865, Rv1876, Rv3688, Rv3295, Rv3283, RV1404, Rv0036, Rv1719, Rv3855, Rv2731, Rv 062324, Rv 062325, Rv 3015, Rv 323546, Rv 03649, Rv 0358, Rv3246, Rv 0358, Rv359, Rv 649, Rv 64043, Rv 3239793, Rv 323946, Rv 3282, Rv 649, Rv 6427, Rv 3282, Rv 6427, Rv No. 5, Rv No. 5, Rv No. 5, Rv No. 3, Rv No. 5, Rv No. 3, Rv No. 5, Rv No. 3, Rv No. 3, Rv No. 3, Rv No. 3, Rv No. 3, Rv No. 3, Rv No. Rv 1722, Rv No. 4, Rv2511, Rv3677, Rv1463, Rv0258, Rv2492, Rv2614, Rv1034, RV2514, Rv3322, Rv1112, Rv0952, Rv1919, Rv1636, Rv0350, Rv0119, Rv0283, Rv2996, Rv2862, Rv0984, Rv2680, Rv0351, Rv0310, MT3289, Rv0396, Rv3160, Rv1451, Rv0056, MT, 3268, Rv3715, Rv0271, Rv2478, Rv1413, Rv2473, Rv2540, Rv1957, Rv0772, Rv3309, Rv3301, Rv2841, Rv3799, Rv0684, Rv064, Rv 06282423, Rv 2529, Rv 2509, Rv 2502, Rv379, Rv 3781 Rv379, Rv 3781 Rv379, Rv 3781 Rv, Rv379, Rv379, Rv 3781 Rv379, Rv, Rv3899, Rv3676, Rv3717, Rv2711, Rv3248, MT3269, Rv1080, Rv3002, Rv0191, MT2625, MT1196, Rv2909, MT3780, Rv1667, Rv1202, Rv0030, Rv0113, Rv0110, Rv2765, Rv1829, Rv1414, RV 6, Rv1685, Rv2310, Rv0801, Rv3678, MT1364, Rv0099, Rv3709, Rv2202, Rv1045, Rv1606, Rv 2332332333, RV1331, Rv3838, Rv3406, MT3103, Rv2818, Rv3039, Rv 361816, Rv1816, Rv 1818, Rv 35039, Rv 35089, Rv 35098, Rv 0335057, Rv 35729, Rv358, Rv 35729, Rv379, Rv 988, Rv 982, Rv 988, Rv 982, Rv 988, Rv 982, Rv 2508, Rv 989, Rv 988, Rv 989, Rv 2508, Rv 982, Rv 988, Rv 2508, Rv 989, Rv 2508, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988 Rv 989, Rv 989 Rv 988, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, Rv 988, Rv 989, At least one of Rv0072, Rv1766, Rv1439c, Rv1536, Rv2495c, Rv0585c, Rv1168c, Rv3835, Rv3341, MT1342, Rv2937, Rv1698, Rv3437, Rv1362c, Rv3024c and Rv 0155.
2. The mycobacterium tuberculosis proteome of claim 1, wherein the Rv3768, Rv1848, Rv0028, Rv0301, Rv1022, Rv0095, Rv2523, Rv0546, Rv1778, Rv0100, Rv1724, Rv1875, Rv3735, Rv3284, Rv0577, Rv3849, Rv2697, Rv2360, Rv0489, Rv3246, Rv3841, Rv3285, Rv 3264, Rv2837, Rv2907, Rv3756, Rv3118, Rv3291, Rv2534, Rv0865, Rv 1871876, Rv3688, Rv3295, Rv3283, Rv1404, Rv0036, Rv1719, Rv3855, Rv 3831, Rv 062324, Rv 320642, Rv 320342, Rv 32039, Rv329, Rv 32039, Rv 323546, Rv 32039, Rv 32359, Rv 32039, Rv 25649, Rv 2564039, Rv 2564032, Rv 32039, Rv 32359, Rv 258180, Rv 25649, Rv 2564032, Rv 2564039, Rv 258180, Rv 25649, Rv 2564043, Rv 258180, Rv 25649, Rv 2564043, Rv 25649, Rv253, Rv 25649, Rv259, Rv 25649, Rv253, Rv 25649, Rv253, Rv 25649, Rv259, Rv 25649, Rv253, Rv 25649, Rv253, Rv259, Rv 25649, Rv253, Rv 25649, Rv253, Rv259, Rv 25649, Rv253, Rv259, Rv 25649, Rv259, Rv 25649, Rv259, Rv 25649, Rv259, Rv 25649, Rv 2564043, Rv253, Rv 25649, Rv253, Rv 25649, Rv259, Rv 2548, Rv 25649, Rv253, Rv259, Rv 25649, Rv259, Rv 2564048, Rv259, Rv 2548, Rv253, Rv259, Rv253, Rv 25649, Rv259, Rv 2548, Rv259, Rv 25649, Rv259, Rv 2548, Rv253, Rv259, Rv 2548, Rv, Rv2461, Rv3154, Rv1015, Rv3007, Rv2511, Rv3677, Rv1463, Rv0258, Rv2492, Rv2614, Rv1034, RV2514, Rv3322, Rv1112, Rv0952, Rv1919, Rv1636, Rv0350, Rv0119, Rv0283, Rv2996, Rv2862, Rv0984, Rv2680, Rv0351, Rv0310, MT3289, Rv0396, Rv3160, Rv1451, Rv 0316, MT3268, Rv3715, Rv0511, Rv 05178, Rv1413, Rv2473, Rv2540, Rv 7, Rv 72, Rv3309, Rv3301, Rv2841, Rv 28059, Rv 289, Rv339, Rv 289, Rv 989, Rv 2029, Rv 289, Rv 2029, Rv 289, Rv 2029, Rv 289, Rv 2029, Rv 289, Rv 2029, Rv 289, Rv 2029, Rv 289, Rv 2029, Rv 89, Rv 2029, Rv 89, Rv 89, Rv 89, Rv 89, Rv 89, Rv 89, Rv, At least one of Rv0041, Rv1056, Rv1264, Rv2293c, Rv3899C, Rv3676, Rv3717, Rv2711, Rv3248c and MT3269 is a common protein for non-tuberculosis patients and tuberculosis patients;
rv1080, Rv3002, Rv0191, MT2625, MT1196, Rv2909, MT3780, Rv1667, Rv1202, Rv0030, Rv0113, Rv0110, Rv2765, Rv1829, Rv1414, RV1416, Rv1685, Rv2310, Rv0801, Rv3678, MT1364, Rv0099, Rv3709, Rv2202, Rv1606 2333, RV1, Rv3838, Rv3406, MT3103, Rv2818, Rv3039, Rv3672, Rv1816, Rv 1760518, Rv1159, Rv2557, Rv3791, Rv2358, Rv3567, Rv 5, Rv 14437, Rv1448, Rv 01926, Rv 35048, Rv039, Rv 35729, Rv379, Rv 089, Rv379, Rv 1722, Rv379, Rv 352352358, Rv 989, Rv 35729, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 982, Rv 989, Rv 982, Rv 989, Rv 988, Rv 989, Rv 982, Rv 989, Rv 98, Rv0585c, Rv1168c, Rv3835, Rv3341, MT1342, Rv2937, Rv1698, Rv3437, Rv1362c, Rv3024c and Rv0155 are specific proteins for non-tuberculosis patients.
3. The method for screening a mycobacterium tuberculosis proteome according to claim 1, comprising hybridizing human T-cell proteins and a mycobacterium tuberculosis whole proteome chip, and selecting a positive site of the mycobacterium tuberculosis protein to obtain the mycobacterium tuberculosis proteome.
4. The method for screening a mycobacterium tuberculosis proteome according to claim 3, wherein the human T cell proteins include T cell proteins of patients with tuberculosis and T cell proteins of patients without tuberculosis.
5. The method for screening a mycobacterium tuberculosis proteome of claim 3, wherein the human T cell protein is obtained by flow cytometry; the cytoimmunity type detected by the flow cytometry is CD3 + And (4) carrying out T lymphocyte percentage and sorting to obtain human T cell protein.
6. Use of the mycobacterium tuberculosis proteome of claim 1, wherein the mycobacterium tuberculosis proteome is used for preparing detection reagents related to mycobacterium tuberculosis; the reagent comprises a mycobacterium tuberculosis proteome; or a DNA molecule encoding the mycobacterium tuberculosis proteome; or a recombinant protein produced by a recombinant bacterium containing said DNA molecule.
7. The use of the mycobacterium tuberculosis proteome of claim 6, wherein the reagent is a detection reagent for mycobacterium tuberculosis or a detection reagent for the immune function status of tuberculosis.
8. Use of the mycobacterium tuberculosis proteome of claim 1, wherein the mycobacterium tuberculosis proteome is used for preparing a tuberculosis treatment agent.
9. The use of the mycobacterium tuberculosis proteome according to claim 8, wherein the tuberculosis treatment agent comprises a polyclonal antibody obtained with the mycobacterium tuberculosis proteome as an immunogen and/or a monoclonal antibody recognizing an antigen protein of the mycobacterium tuberculosis proteome.
10. Use of the mycobacterium tuberculosis proteome of claim 1, wherein the mycobacterium tuberculosis proteome is used for studying the action mechanism of mycobacterium tuberculosis.
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