WO2017125007A1 - 用于诊断活动性结核的方法和试剂盒 - Google Patents

用于诊断活动性结核的方法和试剂盒 Download PDF

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WO2017125007A1
WO2017125007A1 PCT/CN2017/071462 CN2017071462W WO2017125007A1 WO 2017125007 A1 WO2017125007 A1 WO 2017125007A1 CN 2017071462 W CN2017071462 W CN 2017071462W WO 2017125007 A1 WO2017125007 A1 WO 2017125007A1
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sample
seq
difference
amino acid
acid sequence
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PCT/CN2017/071462
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French (fr)
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葛胜祥
刘永亮
陈梦媛
张军
夏宁邵
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厦门大学
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Priority to US16/071,639 priority Critical patent/US20190025301A1/en
Priority to JP2018537822A priority patent/JP2019508684A/ja
Priority to EP17741038.8A priority patent/EP3407071A4/en
Priority to AU2017209240A priority patent/AU2017209240A1/en
Priority to KR1020187023728A priority patent/KR20180104038A/ko
Publication of WO2017125007A1 publication Critical patent/WO2017125007A1/zh
Priority to HK19100852.6A priority patent/HK1258504A1/zh

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    • G—PHYSICS
    • 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
    • G01N33/53—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • 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
    • G—PHYSICS
    • 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
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • 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)
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention belongs to the fields of molecular biology, immunology and disease diagnosis.
  • the present invention relates to a method for diagnosing whether a subject has active tuberculosis, a method for determining a therapeutic effect of a therapy on active tuberculosis, and a candidate for screening for active tuberculosis The method of medicine.
  • the invention also relates to kits containing specific stimuli and reagents for detecting IL-6 levels.
  • Tuberculosis is a chronic infectious disease caused by M. tuberculosis infection. It is a very serious global health problem. There are about 9 million new tuberculosis patients in the world and 1.7 million deaths due to tuberculosis. In addition, it is estimated that approximately one-third of the world's population is latent to M. tuberculosis, a potential source of active tuberculosis.
  • the first priority is to discover and cure patients with infectious tuberculosis.
  • X-ray testing equipment has high requirements and low specificity. The rate of over-diagnosis, missed diagnosis and misdiagnosis is higher. High; in addition, X-ray examination can only find suspicious shadows in the lungs, and the purpose of detecting infectious patients is not achieved. Therefore, the ninth WHO Expert Committee Report on Tuberculosis in 1974 clearly pointed out that the method of actively detecting patients by group chest X-rays is not only In developed countries, it is not desirable in developing countries and must be abolished.
  • the sputum smear test can detect patients with infectious tuberculosis, with high specificity, simple equipment requirements, low cost and low technical requirements. However, there are also problems that are difficult to obtain, and many patients have dry cough and no sputum, or micro-bacteria are not easy to detect.
  • the widely used sputum smear detection sensitivity is between 34-80%. Although the detection sensitivity of sputum culture is higher than that of sputum smear, it takes a long time and has high requirements for diagnostic laboratories. In many countries with high TB burden, this method cannot be satisfied. Requirements for laboratory conditions. Immunoassay techniques may have great application value in the diagnosis of tuberculosis, especially after rapid diagnosis and bedside diagnosis.
  • the IFN- ⁇ release test (IGRAs) is a widely used TB immunodiagnostic method. It has been proved to have application value in the detection of Mycobacterium tuberculosis infection, and the detection effect has a significant advantage over the TST (tuberculin skin test).
  • TST tuberculin skin test
  • neither IGRAs nor TST methods can distinguish between latent tuberculosis infection (LTBI) and active tuberculosis; in high-TB burden countries, the application value of IGRAs is limited because of a high proportion of latent infections.
  • LTBI latent tuberculosis infection
  • active tuberculosis in high-TB burden countries, the application value of IGRAs is limited because of a high proportion of latent infections.
  • assisted diagnosis The current clinically used tuberculosis diagnosis still relies mainly on clinical symptoms, imaging diagnosis and pathogenic diagnosis. If it can accurately and quickly diagnose and perform appropriate chemotherapy in the early stage of tuberculosis, it will be of
  • RV0183 or "RV0183 protein” refers to a protein naturally occurring in Mycobacterium tuberculosis, belonging to monoacylglycerol lipase (MAGL) or hemolysis. Phospholipase (lysophospholipase).
  • the sequence of the RV0183 protein is well known in the art and can be found in various public databases (e.g., NCBI database accession number CP009480.1, AIR 12906.1, CCP 42909.1, NP_214697.2, or WP 003401112.1).
  • RV0183 when referring to the amino acid sequence of RV0183, the description is made with reference to the sequence shown in SEQ ID NO: 1.
  • M. tuberculosis includes both the standard strain H37Rv and a plurality of isolates, and there may be a difference between the amino acid sequences of the RV0183 proteins of the various isolates.
  • the RV0183 protein of different isolates of M. tuberculosis has extremely high amino acid sequence identity and has substantially the same biological function, although sequence differences may exist.
  • RV0183 includes not only the protein represented by SEQ ID NO: 1, but also the RV0183 protein of various M. tuberculosis isolates (for example, BAL64025.1, AOE34503.1, EGE52872.1). , or the RV0183 protein shown in CDM08372.1, and its natural or artificial variants.
  • the expression "amino acid residues 1-20 of the RV0183 protein” includes positions 1-20 of SEQ ID NO: 1. Amino acid residues, as well as corresponding fragments in the RV0183 protein of various M. tuberculosis isolates, and corresponding fragments in variants (natural or artificial) of the RV0183 protein.
  • the expression "corresponding segment” means a segment located at an equivalent position in the sequence to be compared when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.
  • RV0183 or "RV0183 protein” is not limited by any particular method of synthesizing a protein, and can be produced by conventional techniques known to those skilled in the art, such as DNA recombination techniques or chemical synthesis techniques.
  • PlcD or "PlcD protein” refers to a protein naturally occurring in Mycobacterium tuberculosis, belonging to the phospholipase C family.
  • the sequence of the PlcD protein is well known in the art, see, for example, Andersen ST, et al., Nature, 1998. 393 (6685): 537-44 (which is incorporated herein by reference), and NCBI database accession number CCP44521.1.
  • M. tuberculosis when referring to the amino acid sequence of PlcD, the description is made with reference to the sequence shown in SEQ ID NO: 3.
  • M. tuberculosis includes both the standard strain H37Rv and a plurality of isolates, and there may be a difference between the amino acid sequences of the PlcD proteins of the various isolates.
  • PlcD proteins of different isolates of M. tuberculosis have extremely high amino acid sequence identity and have substantially the same biological function, although sequence differences may exist.
  • the term "PlcD” includes not only the protein represented by SEQ ID NO: 3 but also the PlcD protein of various M. tuberculosis isolates, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the PlcD protein, it includes not only the sequence fragment of SEQ ID NO: 3, but also the corresponding sequence fragment in the PlcD protein of various M.
  • corresponding sequence fragment means a fragment located at an equivalent position in the sequence to be compared when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.
  • PlcD or "PlcD protein” is not limited by any particular method of synthesizing a protein, and can be produced by conventional techniques known to those skilled in the art, such as DNA recombination techniques or chemical synthesis techniques.
  • the expression "antigenic fragment of RV0183 or PlcD” refers to a fragment of an amino acid sequence (ie, a polypeptide) obtained by truncating the RV0183 or PlcD protein, which fragment has the same identity as the corresponding full length protein.
  • the amino acid sequence fragment shown by SEQ ID NOS: 5-25 in the invention is an antigenic fragment of RV0183.
  • the antigenic fragment is not limited by any particular method of synthesizing the polypeptide, and can be produced by conventional techniques known to those skilled in the art, such as DNA recombination techniques or chemical synthesis techniques.
  • RV0183, PlcD or an antigenic fragment thereof can be obtained by DNA recombination technology, for example, by using a cell-free expression system from a polynucleotide encoding these proteins or polypeptides
  • cell-free expression systems include, for example, Reticulocyte-based lysate-based expression system, wheat germ extract-based expression system, and E. coli extract-based expression system; or by using an in vivo expression system (eg, E. coli prokaryotic expression system, yeast eukaryotic expression system) Obtained from polynucleotides encoding these proteins or polypeptides.
  • RV0183, PlcD or an antigenic fragment thereof can be produced by chemical synthesis.
  • Methods for chemical total synthesis of proteins or polypeptides are well known in the art (see, for example, Raibaut L, et al., Top Curr Chem. 2015; 363: 103-54; Thapa P, et al. Molecules.
  • SPPS Solid Phase Peptide Synthesis
  • NCL Native Chemical Ligation
  • TAEC Transfer Active Ester Condensation
  • an antigenic fragment having an amino acid sequence as shown in SEQ ID NOS: 13, 14 and 19, respectively means an antigenic fragment having the amino acid sequence of SEQ ID NO: 13, A combination of an antigenic fragment having the amino acid sequence of SEQ ID NO: 14 and an antigenic fragment having the amino acid sequence of SEQ ID NO: 19. Other similar expressions have similar meanings.
  • the term "agent capable of detecting IL-6” refers to a substance that is capable of specifically binding to IL-6. Such materials are well known in the art or can be prepared by methods well known in the art, such as antibodies, targeting polypeptides or aptamers. In general, it is particularly preferred that such agents are capable of determining the level of IL-6 in a sample by immunological detection.
  • immunological assays is particularly advantageous because it exploits the specific interaction/binding affinity between antigen-antibodies. Thus, as long as the reagent retains reactivity with specific binding to IL-6, the reagent can be used to determine the level of IL-6 in the sample by immunological detection (ie, the reagent can be used to detect IL-6).
  • the reagent for detecting IL-6 may also be a nucleic acid aptamer that binds to IL-6, including but not limited to those described in PCT International Application WO2014159669 and "Gupta S, et al., J Biol Chem. 2014; 289(12): Nucleic acid aptamers in 8706-19", all of which are incorporated herein by reference.
  • the term "specifically binds" refers to a non-random binding reaction between two molecules (ie, a binding molecule and a target molecule), such as a reaction between an antibody and the antigen to which it is directed.
  • the binding affinity between the two molecules can be described by the K D value.
  • the K D value refers to the ratio of kd (the specific binding molecule-dissociation rate of the target molecule interaction; also known as koff) to ka (the association rate of the specific binding molecule-target molecule interaction; also known as kon)
  • the dissociation constant obtained is either kd/ka expressed as a molar concentration (M). The smaller the K D value, the tighter the binding of the two molecules and the higher the affinity.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D values may be determined by methods known in the art, e.g. BIACORE instrument measured using surface plasmon resonance technique (SPR).
  • immunological assay refers to an assay that utilizes specific interaction/binding affinity between antigen-antibodies, which is generally useful for detecting the presence of a particular antigen or antibody in a sample or Level.
  • immunological assays are well known to those skilled in the art and include, but are not limited to, ELISA assays, Elispot assays, Western blots, surface plasmon resonance assays, and the like.
  • ELISA assays ELISA assays
  • Elispot assays Elispot assays
  • Western blots Western blots
  • surface plasmon resonance assays and the like.
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain CL.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with high denaturation (referred to as complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy/light chain pair form an antibody binding site, respectively.
  • Amino acid to each region or domain Compatible with Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342 : The definition of 878-883.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • an "antigen-binding fragment" of an antibody refers to one or more portions of a full length antibody that retain the ability to bind to the same antigen (eg, IL-6) to which the antibody binds, and integrity.
  • Antibodies compete for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 Paul, W., ed., 2nd Ed., Raven Press, N. Y. (1989), which is incorporated herein by reference in its entirety for all purposes.
  • Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabody Diabody and a polypeptide comprising at least a portion of an antibody sufficient to confer specific ability to bind the antigen to the polypeptide.
  • CDR complementarity determining region
  • the term "Aptamer” refers to a single-stranded oligonucleotide capable of binding to a target protein of interest or other biological target molecule with high affinity and high specificity, which is foldable to form
  • a thermodynamically stable three-dimensional structure of Stem-Loop, Hairpin, Pseudoknot or G-tetramer for example by structural complementarity, base stacking force, van der Waals The force, hydrogen bonding or electrostatic interaction specifically binds to the target protein or other biological target molecule of interest.
  • the aptamer may be DNA or RNA, and may also contain a nucleic acid analog (eg, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a glycol nucleic acid (GNA), or a threose nucleic acid (TNA).
  • a nucleic acid analog eg, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a glycol nucleic acid (GNA), or a threose nucleic acid (TNA).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • targeting polypeptide refers to a polypeptide molecule that can specifically bind to a target protein of interest.
  • the targeting polypeptide may comprise a natural amino acid, a synthetic amino acid or an amino acid mimetic that functions in a manner similar to a naturally occurring amino acid.
  • Naturally occurring amino acids are those encoded by the genetic code and those amino acids that are later modified, for example, hydroxyproline, ⁇ -hydroxyglutamate, O-phosphoserine, phosphothreonine or phosphotyrosine.
  • polypeptide solution it binds the target protein can be used to target affinity dissociation equilibrium constant (i.e., K D value) Describe.
  • K D value affinity dissociation equilibrium constant
  • K D value is greater than about 10 -3 M
  • K D value is usually considered to represent a non-binding or non-specific binding.
  • a targeting polypeptide that specifically binds to the target protein can be obtained by methods known to those skilled in the art, such as by phage display technology or protein microarray technology.
  • the term "detectable label” refers to any composition detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. In the present invention, it is particularly preferred that such a marker can be applied to immunological detection (for example, an enzyme-linked immunoassay, a radioimmunoassay, a fluorescent immunoassay, a chemiluminescent immunoassay, etc.).
  • Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Red albumin (PE), Texas Red, rhodamine, quantum dot or cyanine dye derivatives (eg Cy7, Alexa 750), acridinium ester compounds, magnetic beads (eg, a heat-sensitive marker such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex, etc.) beads, and avidin modified for binding to the above markers (for example, streptavidin) Biotin.
  • enzymes e.
  • Patents that teach the use of such markers include, but are not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241 each incorporated herein by reference.
  • the markers encompassed in the present invention can be detected by methods known in the art.
  • the radioactive label can be detected using a photographic film or a scintillation counter
  • the fluorescent label can be detected using a photodetector to detect the emitted light.
  • Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and the calorimetric label is detected by simply visualizing the stained label.
  • the term "specific stimuli” refers to a PBMC capable of stimulating only active tuberculosis patients to produce IL-6, but not an inactive tuberculosis population (eg, latent tuberculosis, old age) PBMC from tuberculosis patients or non-tuberculosis patients (negative for IGRA) produces IL-6; or refers to substances that stimulate PBMC in active tuberculosis patients to produce significantly higher levels of IL-6 secretion than inactive Tuberculosis population (eg, latent tuberculosis, old tuberculosis, or non-tuberculosis (negative IGRA)).
  • Specific stimulators of the invention include RV0183, PlcD or antigenic fragments thereof.
  • the specific stimulator contains no or almost no endotoxin.
  • Methods for removing endotoxin from the above specific stimuli are well known in the art, such as ion exchange chromatography, affinity chromatography or extraction.
  • non-specific stimuli refers to a substance that is capable of activating most or all of the lymphocytes without being specifically restricted by TCR or BCR. After lymphocyte activation, a large amount of cytokines can be secreted (for example, IL-6).
  • cytokines for example, IL-6.
  • Substances useful as non-specific stimuli are well known in the art and include, but are not limited to, mitogens, phytohemagglutinin (PHA), Concanavalin A (ConA), Pokeweed mitogen (PWM), lipopolysaccharide ( LPS) or staphylococcal protein A (SPA).
  • the non-specific stimulator can be included in a culture medium (eg, a cell culture medium such as RPMI-1640 medium and DMEM medium).
  • the term "statistical analysis value” refers to a value obtained by statistically analyzing the detection results obtained by various detection methods.
  • Various statistical analysis methods are well known in the art (see, for example, PCT International Application WO2009064901) and include, but are not limited to, linear combinations of test results, linear regression models, logistic regression models, linear discriminant analysis (LDA) models, nearest neighbor models. Or microarray predictive analysis (PAM).
  • PAM microarray predictive analysis
  • the statistical analysis value is a value obtained by statistical analysis by a logistic regression model.
  • Logistic regression model is described in detail, for example, "Hu Chunyan. Combined detection of four tumor markers in ovarian cancer serum [D]. Guangzhou: Sun Yat-sen University, 2008: 1-39".
  • reference value refers to a value that reflects the condition of an inactive tuberculosis population.
  • reference values include, for example, IL in a sample of inactive tuberculosis (eg, latent tuberculosis, old tuberculosis, or non-tuberculosis (negative IGRA)) based on specific stimulation of the original stimulus (eg, latent tuberculosis, old tuberculosis, or non-tuberculosis) 6 level difference, a normal value or range of values determined; and statistical analysis of the values obtained from samples of inactive tuberculosis population (eg, the difference in IL-6 levels as described above) The resulting values (statistical analysis values).
  • ROC Receiver Operating Characteristic
  • PBMC peripheral blood mononuclear cell
  • T cells lymphocytes
  • B cells lymphocytes
  • NK cells monocytes
  • dendritic cells dendritic cells
  • peripheral blood leukocyte layer refers to a component formed by peripheral anticoagulation after natural sedimentation, centrifugation or density gradient centrifugation, mainly by white blood cells (including peripheral blood mononuclear cells) and Platelet composition. After anticoagulation, the upper layer of plasma, the lower layer of red blood cells, and a thin layer of white film between them, which accounts for about 1% of the total blood volume, are called the white film layer.
  • subject includes, but is not limited to, various animals, particularly mammals, such as humans.
  • anticoagulant refers to an agent or substance that is capable of preventing blood coagulation, such materials being well known in the art including, but not limited to, heparin, EDTA, oxalate (eg, sodium oxalate) , potassium oxalate, ammonium oxalate), sodium citrate (sodium citrate).
  • oxalate eg, sodium oxalate
  • potassium oxalate eg, potassium oxalate
  • ammonium oxalate sodium citrate
  • sodium citrate sodium citrate
  • the term "diluent” is preferably an electrolyte solution capable of maintaining cell osmotic pressure, and if necessary, the solution also has a function of maintaining physiological pH.
  • solutions are well known in the art and include, but are not limited to, Alsever's solution, Earle's Balanced Salt Solution (EBSS), Gey's Balanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS).
  • PBS phosphate buffered saline
  • DPBS Dunsen's phosphate buffered saline
  • RBSS Ringer's balanced salt solution
  • SBSS Simm's balanced salt solution
  • TRIS buffer TBS
  • TBS Tyrode's Balanced Salt Solution
  • the term "culture fluid” or “medium” refers to a nutrient that is capable of maintaining cellular activity.
  • the nutrients contain amino acids, vitamins, carbohydrates, inorganic salts and the like.
  • Such nutrients are well known in the art and include, but are not limited to, RPMI-1640 medium or DMEM medium.
  • the purpose of adding a culture solution or a medium to a sample from the subject is to maintain the activity of cells, particularly PBMC, in the sample during the stimulation of the original stimulus.
  • Methods for maintaining the activity of cells in blood components are well known in the art, and those skilled in the art can select according to actual needs.
  • a culture solution when the sample is whole blood, a culture solution may be added, for example, adding a suitable amount of glucose, sodium chloride, potassium chloride, etc. in a phosphate buffer or physiological saline; In some embodiments, the culture solution is added with a suitable amount of glucose and potassium chloride in a phosphate buffer.
  • a medium such as a cell culture medium, may be added, for example, suitable for maintaining blood cells, particularly PBMC. Active cell culture medium, such as RPMI-1640 medium or DMEM medium.
  • active tuberculosis means that a tuberculosis lesion is in an active phase, such as a sputum smear positive, or accompanied by associated symptoms such as hypothermia, cough, weight loss, fatigue, poor appetite, and the like.
  • tuberculosis includes tuberculosis and extrapulmonary tuberculosis, including, for example, lymphatic tuberculosis, tuberculous meningitis, tuberculous peritonitis, intestinal tuberculosis, renal tuberculosis, epididymal tuberculosis, female reproductive system tuberculosis (including fallopian tubes, uterus) Endometrial, ovarian tuberculosis) and bone and joint tuberculosis.
  • lymphatic tuberculosis tuberculous meningitis
  • tuberculous peritonitis include intestinal tuberculosis, renal tuberculosis, epididymal tuberculosis, female reproductive system tuberculosis (including fallopian tubes, uterus) Endometrial, ovarian tuberculosis) and bone and joint tuberculosis.
  • the inventors of the present application screened a large number of M. tuberculosis RD region antigens, and unexpectedly found that RV0183, PlcD or its antigenic fragment stimulates the production of large amounts of IL-6 in peripheral blood of active tuberculosis patients, thereby distinguishing between active tuberculosis and inactive tuberculosis (eg, latent tuberculosis, old tuberculosis, or non-tuberculosis) ).
  • active tuberculosis eg, latent tuberculosis, old tuberculosis, or non-tuberculosis
  • the IGRAs and TST methods commonly used in the field were incapable of distinguishing between latent tuberculosis infection (LTBI) and active tuberculosis. Based on this finding, the inventors have developed a new method for diagnosing active tuberculosis.
  • the invention provides a kit comprising one or more of RV0183, PlcD or an antigenic fragment thereof, and an agent capable of detecting IL-6.
  • the RV0183 has the amino acid sequence set forth in SEQ ID NO: 1; and/or the PlcD has the amino acid sequence set forth in SEQ ID NO: 3.
  • the kit comprises RV0183 and/or PlcD.
  • the kit comprises one or more antigenic fragments of RV0183.
  • the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-25.
  • the kit comprises an antigenic fragment having the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 19, respectively.
  • the kit further comprises a combination of the following antigenic fragments:
  • the kit comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 5-25, respectively.
  • the agent capable of detecting IL-6 is a substance capable of specifically binding to IL-6, such as an antibody, a targeting polypeptide or a nucleic acid aptamer.
  • the reagent also carries a detectable label.
  • the agent determines the level of IL-6 in the sample by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • the agent comprises an antibody against IL-6 or an antigen binding fragment thereof. Further, in certain preferred embodiments, the agent measures the level of IL-6 by ELISA.
  • the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. Further, in certain preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.
  • the kit further comprises one or more devices or reagents selected from 1) to 5):
  • a blood collection device such as a pyrogen-free vacuum blood collection tube
  • an anticoagulant such as heparin
  • non-specific stimuli such as plant lectin or concanavalin A
  • Diluent such as phosphate buffer or saline.
  • the kit is for diagnosing active tuberculosis, determining the therapeutic effect of a therapy on active tuberculosis, or screening for a drug candidate capable of treating active tuberculosis.
  • the invention provides the use of a specific stimulator in a kit for the diagnosis of active tuberculosis; wherein the specific stimulator is selected from the group consisting of RV0183, PlcD or an antigenic fragment thereof One or more of them.
  • the RV0183 has the amino acid sequence set forth in SEQ ID NO: 1; and/or the PlcD has the amino acid sequence set forth in SEQ ID NO: 3.
  • the specific stimulator is selected from the group consisting of RV0183, PlcD, or a combination thereof.
  • the specific stimulator is selected from one or more antigenic fragments of RV0183.
  • the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-25.
  • the specific stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 13, 14, and 19, respectively.
  • the specific stimulator further comprises a combination of the following antigenic fragments:
  • the specific stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 5-25, respectively.
  • the kit includes an agent capable of detecting IL-6, such as an antibody, targeting polypeptide or nucleic acid aptamer capable of specifically binding to IL-6.
  • an agent capable of detecting IL-6 such as an antibody, targeting polypeptide or nucleic acid aptamer capable of specifically binding to IL-6.
  • the reagent also carries a detectable label.
  • the agent determines the level of IL-6 in the sample by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • the agent comprises an antibody against IL-6 or an antigen binding fragment thereof. Further, in certain preferred embodiments, the agent measures the level of IL-6 by ELISA.
  • the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. Further, in certain preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.
  • the kit further comprises one or more devices or reagents selected from 1) to 5):
  • a blood collection device such as a pyrogen-free vacuum blood collection tube
  • an anticoagulant such as heparin
  • non-specific stimuli such as plant lectin or concanavalin A
  • Diluent such as phosphate buffer or saline.
  • the kit diagnoses whether the subject has active tuberculosis by a method comprising the steps of:
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood white layer, and may optionally include other groups For example, an anticoagulant, a diluent, and the like.
  • PBMC peripheral blood mononuclear cells
  • the difference or statistical analysis obtained from the difference when the difference or statistical analysis obtained from the difference is greater than a reference value, indicating that the subject providing the sample has active tuberculosis; when the difference or by the difference When the value obtained by the statistical analysis value is not greater than the reference value, it indicates that the subject providing the sample does not have active tuberculosis.
  • step (3) the difference is statistically analyzed using a statistical model selected from the group consisting of linear combination, linear regression model, logistic regression model, and linear discriminant analysis (LDA) model. , nearest neighbor model or microarray predictive analysis (PAM). Further, in certain preferred embodiments, in step (3), the difference is statistically analyzed using a logistic regression model.
  • a statistical model selected from the group consisting of linear combination, linear regression model, logistic regression model, and linear discriminant analysis (LDA) model. , nearest neighbor model or microarray predictive analysis (PAM).
  • PAM microarray predictive analysis
  • step (1) at least two specific stimuli are used to stimulate one or more samples from the subject together or separately as a sample to be tested, wherein the specific The stimuli are each independently selected from the group consisting of RV0183, PlcD or antigenic fragments thereof. Further, in certain preferred embodiments, in step (1), at least two samples are separately stimulated as samples to be tested using RV0183 and PlcD. Or, in certain preferred embodiments, in step (1), at least one sample is co-stimulated using one or more antigenic fragments of RV0183 as a sample to be tested.
  • the specific stimulator in step (1), is placed in a culture medium, such as a cell culture medium, such as RPMI-1640 medium or DMEM medium, and then used to stimulate the source.
  • a culture medium such as a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • a sample of the subject is described to produce a sample to be tested.
  • step (1) further comprises stimulating at least one sample as a positive control sample using a non-specific stimuli.
  • the non-specific stimulator comprises a plant lectin or concanavalin A.
  • a medium eg, a cell culture medium such as RPMI-1640 medium or DMEM medium
  • a sample from the subject is incubated or diluted to produce a negative control sample.
  • the method further comprises one or more of the following steps: (a) obtaining a sample from the subject using a blood collection device; (b) using Anticoagulant treatment of blood collection device or from a sample of the subject; (c) treating the sample from the subject with a culture or culture medium; and, (d) diluting the sample from the subject with a dilution.
  • stimulating stimuli are used from the stimulating temperature at a temperature at which the cell (eg, PBMC) activity is high (eg, at 36-38 ° C, eg, at about 37 ° C) Sample of the subject.
  • a temperature at which the cell (eg, PBMC) activity is high eg, at 36-38 ° C, eg, at about 37 ° C
  • step (1) the sample from the subject is stimulated with the stimulator for not less than 12 h, such as 15-24 h, such as 20-24 h.
  • the invention provides the use of a specific stimulator in a kit for determining the therapeutic effect of a therapy on active tuberculosis; wherein the specific stimuli are selected from the group consisting of RV0183 Or one or more of PlcD or an antigenic fragment thereof.
  • the RV0183 has the amino acid sequence set forth in SEQ ID NO: 1; and/or the PlcD has the amino acid sequence set forth in SEQ ID NO: 3.
  • the specific stimulator is selected from the group consisting of RV0183, PlcD, or a combination thereof.
  • the specific stimulator is selected from one or more antigenic fragments of RV0183.
  • the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-25.
  • the specific stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 13, 14, and 19, respectively.
  • the specific stimulator further comprises a combination of the following antigenic fragments:
  • the specific stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 5-25, respectively.
  • the kit includes an agent capable of detecting IL-6, such as an antibody, targeting polypeptide or nucleic acid aptamer capable of specifically binding to IL-6.
  • an agent capable of detecting IL-6 such as an antibody, targeting polypeptide or nucleic acid aptamer capable of specifically binding to IL-6.
  • the reagent also carries a detectable label.
  • the agent determines the level of IL-6 in the sample by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • the agent comprises an antibody against IL-6 or an antigen binding fragment thereof. Further, in certain preferred embodiments, the agent measures the level of IL-6 by ELISA.
  • the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. Further, in certain preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.
  • the kit further comprises one or more devices or reagents selected from 1) to 5):
  • a blood collection device such as a pyrogen-free vacuum blood collection tube
  • an anticoagulant such as heparin
  • non-specific stimuli such as plant lectin or concanavalin A
  • a diluent such as phosphate buffer or physiological saline
  • the kit determines the therapeutic effect of a therapy on active tuberculosis by a method comprising the steps of:
  • said specific stimulating element is selected from one or more of RV0183, PlcD or an antigenic fragment thereof;
  • step (3) measuring the IL-6 level of each sample in the step (2) using a reagent capable of detecting IL-6, and calculating the difference of the IL-6 level of the sample to be tested and the negative control sample as the first difference ;
  • the heterosexual stimulus is selected from one or more of RV0183, PlcD or an antigenic fragment thereof;
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood white layer, and may optionally include other groups For example, an anticoagulant, a diluent, and the like.
  • PBMC peripheral blood mononuclear cells
  • the second difference when the second difference is greater than the first difference, or when the statistical analysis value of the second difference is greater than a statistical analysis value of the first difference, Indicating that the therapy is ineffective for the treatment of active tuberculosis; when the second difference is less than the first difference, or when the statistical analysis of the second difference is less than the statistical analysis of the first difference At the time, it indicates that the therapy is effective for the treatment of active tuberculosis.
  • the first difference and the second difference are statistically analyzed using a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM). Further, in certain preferred embodiments, in step (8), the first difference and the second difference are statistically analyzed using a logistic regression model.
  • a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM).
  • LDA Linear Discriminant Analysis
  • PAM microarray predictive analysis
  • steps (2) and (6) the pre-treatment sample and the post-treatment sample are subjected to the same treatment (eg, under the same conditions, using the same specific stimuli for treatment) ).
  • at least two specific stimuli are used to stimulate one or more samples from the subject together or separately as a sample to be tested, wherein the specific stimuli are each independently selected from the group consisting of RV0183, PlcD or antigenic fragments thereof.
  • at least two samples are separately stimulated as samples to be tested using RV0183, and PlcD.
  • at least one sample is co-stimulated using one or more antigenic fragments of RV0183 as a sample to be tested.
  • the subject is a mammal, such as a human.
  • the therapy comprises administering to the subject an anti-tuberculosis drug, such as isoniazid, Fuping, streptomycin, pyrazinamide, ethambutol or any combination thereof.
  • an anti-tuberculosis drug such as isoniazid, Fuping, streptomycin, pyrazinamide, ethambutol or any combination thereof.
  • the specific stimulator in steps (2) and (6), is placed in a medium, such as a cell culture medium, such as RPMI-1640 medium or DMEM medium, and then used A sample from the subject is stimulated to produce a sample to be tested.
  • a medium such as a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • steps (2) and (6) further comprise stimulating at least one sample as a positive control sample using a non-specific stimuli.
  • the non-specific stimulator comprises a plant lectin or concanavalin A.
  • a medium that does not contain the specific stimulating pro- and non-specific stimuli eg, a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • the sample from the subject is incubated or diluted to produce a negative control sample.
  • a pre-treatment sample from the subject is obtained using a blood collection device.
  • a post-treatment sample from the subject is obtained using a blood collection device.
  • the method prior to performing step (1), further comprises one or more of the following steps: (a) treating the blood collection device with an anticoagulant or from the subject a sample; (b) treating the sample from the subject with a culture or culture medium; and, (c) diluting the sample from the subject with a dilution.
  • the method prior to performing step (5), further comprises one or more of the following steps: (a) treating the blood collection device with an anticoagulant or from the subject a sample; (b) treating the sample from the subject with a culture or culture medium; and, (c) diluting the sample from the subject with a dilution.
  • the stimuli are used at a temperature at which the cells (eg, PBMC) are more active (eg, at 36-38 ° C, eg, at about 37 ° C) A sample from the subject is stimulated.
  • the sample from the subject is stimulated with the stimulator for not less than 12 h, such as 15-24 h, such as 20-24 h.
  • the invention provides the use of a specific stimulator in a kit for screening a drug candidate capable of treating active tuberculosis; wherein the specific stimuli are selected from the group consisting of RV0183, PlcD Or one or more of its antigenic fragments.
  • the RV0183 has the amino acid sequence set forth in SEQ ID NO: 1; and/or the PlcD has the amino acid sequence set forth in SEQ ID NO: 3.
  • the specific stimulator is selected from the group consisting of RV0183, PlcD, or a combination thereof.
  • the specific stimulator is selected from one or more antigenic fragments of RV0183.
  • the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-25.
  • the specific stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 13, 14, and 19, respectively.
  • the specific stimulator further comprises a combination of the following antigenic fragments:
  • the specific stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 5-25, respectively.
  • the kit includes an agent capable of detecting IL-6, such as an antibody, targeting polypeptide or nucleic acid aptamer capable of specifically binding to IL-6.
  • an agent capable of detecting IL-6 such as an antibody, targeting polypeptide or nucleic acid aptamer capable of specifically binding to IL-6.
  • the reagent also carries a detectable label.
  • the agent determines the level of IL-6 in the sample by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • the agent comprises an antibody against IL-6 or an antigen binding fragment thereof. Further, in certain preferred embodiments, the agent measures the level of IL-6 by ELISA.
  • the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. Further, in certain preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.
  • the kit further comprises one or more devices or reagents selected from 1) to 5):
  • a blood collection device such as a pyrogen-free vacuum blood collection tube
  • an anticoagulant such as heparin
  • non-specific stimuli such as plant lectin or concanavalin A
  • Diluent such as phosphate buffer or saline.
  • kit screens for a drug candidate capable of treating active tuberculosis by a method comprising the steps of:
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood white layer, and may optionally include other groups For example, an anticoagulant, a diluent, and the like.
  • PBMC peripheral blood mononuclear cells
  • the second difference when the second difference is greater than the first difference, or when the statistical analysis value of the second difference is greater than a statistical analysis value of the first difference, Indicating that the screened drug is ineffective for treating active tuberculosis; when the second difference is less than the first difference, or when the statistical analysis value of the second difference is less than the statistical analysis of the first difference At the time of the value, it indicates that the drug being screened is effective for the treatment of active tuberculosis.
  • the first difference and the second difference are statistically analyzed using a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM). Further, in certain preferred embodiments, in step (8), the first difference and the second difference are statistically analyzed using a logistic regression model.
  • a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM).
  • LDA Linear Discriminant Analysis
  • PAM microarray predictive analysis
  • steps (2) and (6) the pre-treatment sample and the post-treatment sample are subjected to the same treatment (eg, under the same conditions, using the same specific stimuli for treatment) ).
  • at least two specific stimuli are used to separately or co-stimulate one or more samples from the animal as test samples, wherein The specific stimuli are each independently selected from RV0183, PlcD or antigenic fragments thereof.
  • at least two samples are separately stimulated as samples to be tested using RV0183, and PlcD.
  • at least one sample is co-stimulated using one or more antigenic fragments of RV0183 as a sample to be tested.
  • the model animal is a non-human mammal, such as a mouse, guinea pig, rabbit, or non-human primate (eg, cynomolgus or macaque).
  • non-human mammal such as a mouse, guinea pig, rabbit, or non-human primate (eg, cynomolgus or macaque).
  • the subject is a mammal, such as a human.
  • the specific stimulator in steps (2) and (6), is placed in a medium, such as a cell culture medium, such as RPMI-1640 medium or DMEM medium, and then used A sample from the animal is stimulated to produce a sample to be tested.
  • a medium such as a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • steps (2) and (6) further comprise stimulating at least one sample as a positive control sample using a non-specific stimuli.
  • the non-specific stimulator comprises a plant lectin or concanavalin A.
  • a medium that does not contain the specific stimulating pro- and non-specific stimuli eg, a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • the sample from the animal is incubated or diluted to produce a negative control sample.
  • a pre-treatment sample from the animal is obtained using a blood collection device.
  • a post-treatment sample from the animal is obtained using a blood collection device.
  • the method prior to performing step (1), further comprises one or more of the following steps: (a) treating the blood collection device or the sample from the animal with an anticoagulant; (b) treating the sample from the animal with a culture medium or a medium; and, (c) diluting the sample from the animal with a diluent.
  • the method prior to performing step (5), further comprises one or more of the following steps: (a) treating the blood collection device or the sample from the animal with an anticoagulant; (b) treating the sample from the animal with a culture medium or a medium; and, (c) diluting the sample from the animal with a diluent.
  • the stimuli are used at a temperature at which the cells (eg, PBMC) are more active (eg, at 36-38 ° C, eg, at about 37 ° C) Samples from the animal are stimulated.
  • the sample from the animal is stimulated with the stimulator for not less than 12 h, such as 15-24 h, such as 20-24 h.
  • the invention provides a method for diagnosing whether a subject has active tuberculosis, comprising the steps of:
  • step (3) measuring the IL-6 level of each sample in the step (2), and calculating the difference of the IL-6 level of the sample to be tested and the negative control sample;
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood white layer, and may optionally include other groups For example, an anticoagulant, a diluent, and the like.
  • PBMC peripheral blood mononuclear cells
  • the table when the difference or the statistical analysis value obtained from the difference is greater than a reference value, the table The subject providing the sample has active tuberculosis; when the difference or the statistical analysis value obtained from the difference is not greater than the reference value, it indicates that the subject providing the sample does not have active tuberculosis.
  • the difference in step (4), is statistically analyzed using a statistical model selected from the group consisting of linear combination, linear regression model, logistic regression model, and linear discriminant analysis (LDA) model. , nearest neighbor model or microarray predictive analysis (PAM). Further, in certain preferred embodiments, in step (4), the difference is statistically analyzed using a logistic regression model.
  • a statistical model selected from the group consisting of linear combination, linear regression model, logistic regression model, and linear discriminant analysis (LDA) model. , nearest neighbor model or microarray predictive analysis (PAM).
  • PAM microarray predictive analysis
  • the RV0183 has the amino acid sequence set forth in SEQ ID NO: 1; and/or the PlcD has the amino acid sequence set forth in SEQ ID NO: 3.
  • the antigenic fragment is an antigenic fragment of RV0183. Further, in certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-25.
  • the subject is a mammal, such as a human.
  • step (2) at least two specific stimuli are used to stimulate one or more samples from the subject as a test sample, wherein the specific The stimuli are each independently selected from the group consisting of RV0183, PlcD or antigenic fragments thereof.
  • step (2) at least two samples are separately stimulated as samples to be tested using RV0183, and PlcD. Or, in certain preferred embodiments, in step (2), at least one sample is co-stimulated using one or more antigenic fragments as a sample to be tested. Further, in certain preferred embodiments, in step (2), at least one sample is co-stimulated as a sample to be tested using a combination of the following antigenic fragments:
  • the specific stimulator in step (2), is placed in a culture medium, such as a cell culture medium, such as RPMI-1640 medium or DMEM medium, and then used to stimulate the source.
  • a culture medium such as a cell culture medium, such as RPMI-1640 medium or DMEM medium.
  • Subject The sample is used to produce a sample to be tested.
  • a medium eg, a cell culture medium such as RPMI-1640 medium or DMEM medium
  • a sample from the subject is incubated or diluted to produce a negative control sample.
  • the level of IL-6 in the sample is determined by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • an antibody against IL-6 or an antigen binding fragment thereof is used to detect the level of IL-6, for example, by ELISA.
  • the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. Further, in certain preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.
  • step (2) further comprises stimulating at least one sample as a positive control sample using a non-specific stimuli.
  • the non-specific stimulator comprises a plant lectin or concanavalin A.
  • step (1) prior to step (1), one or more of the following steps are further included: (a) obtaining a sample from the subject; (b) adding an anticoagulant to the sample For example, heparin; (c) obtaining PBMC or PBMC-containing blood components (for example, peripheral blood leukocyte layer) from the sample; (d) adding a culture solution or a medium to the sample; (e) diluting the sample.
  • an anticoagulant for example, heparin
  • PBMC or PBMC-containing blood components for example, peripheral blood leukocyte layer
  • stimulating stimulus is used from the said stimulating temperature at a temperature at which the cell (eg, PBMC) activity is high (eg, at 36-38 ° C, eg, at about 37 ° C) Sample of the subject.
  • a temperature at which the cell (eg, PBMC) activity is high eg, at 36-38 ° C, eg, at about 37 ° C
  • step (2) the sample from the subject is stimulated with the stimulator for not less than 12 h, such as 15-24 h, such as 20-24 h.
  • the present invention provides a method for determining the therapeutic effect of a therapy on active tuberculosis, comprising the steps of:
  • the heterosexual stimulus is selected from one or more of RV0183, PlcD or an antigenic fragment thereof;
  • step (3) measuring the IL-6 level of each sample in the step (2), and calculating the difference of the IL-6 level of the sample to be tested and the negative control sample as the first difference;
  • said specific stimulating element is selected from one or more of RV0183, PlcD or an antigenic fragment thereof;
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood white layer, and may optionally include other groups For example, an anticoagulant, a diluent, and the like.
  • PBMC peripheral blood mononuclear cells
  • the second difference when the second difference is greater than the first difference, or when the statistical analysis value of the second difference is greater than a statistical analysis value of the first difference, Indicating that the therapy is ineffective for the treatment of active tuberculosis; when the second difference is less than the first difference, or when the statistical analysis of the second difference is less than the statistical analysis of the first difference At the time, it indicates that the therapy is effective for the treatment of active tuberculosis.
  • the first difference and the second difference are statistically analyzed using a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM). Further, in certain preferred embodiments, in step (8), the first difference and the second difference are statistically analyzed using a logistic regression model.
  • a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM).
  • LDA Linear Discriminant Analysis
  • PAM microarray predictive analysis
  • the RV0183 has the amino acid sequence set forth in SEQ ID NO: 1; and/or the PlcD has the amino acid sequence set forth in SEQ ID NO: 3.
  • the antigenic fragment is an antigenic fragment of RV0183. Further, in certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-25.
  • the subject is a mammal, such as a human.
  • the therapy comprises administering to the subject an anti-tuberculosis drug, such as isoniazid, rifampicin, streptomycin, pyrazinamide, ethambutol, or any combination thereof.
  • an anti-tuberculosis drug such as isoniazid, rifampicin, streptomycin, pyrazinamide, ethambutol, or any combination thereof.
  • steps (2) and (6) the pre-treatment sample and the post-treatment sample are subjected to the same treatment (eg, under the same conditions, using the same specific stimuli for treatment) ).
  • steps (2) and (6) at least two specific stimuli are used to stimulate one or more samples from the subject together or separately as a sample to be tested, wherein the specific stimuli are each independently selected from the group consisting of RV0183, PlcD or antigenic fragments thereof.
  • steps (2) and (6) at least two samples are separately stimulated as samples to be tested using RV0183, and PlcD. Or, in certain preferred embodiments, in steps (2) and (6), at least one sample is co-stimulated using one or more antigenic fragments as a sample to be tested. Further, in certain preferred embodiments, in steps (2) and (6), at least one sample is co-stimulated as a sample to be tested using a combination of the following antigenic fragments:
  • the specific stimulator in steps (2) and (6), is placed in a medium, such as a cell culture medium, such as RPMI-1640 medium or DMEM medium, and then used A sample from the subject is stimulated to produce a sample to be tested.
  • a medium such as a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • a medium that does not contain the specific stimulating pro- and non-specific stimuli eg, a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • a sample from the subject is incubated or diluted to produce a negative control sample.
  • the level of IL-6 in the sample is determined by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • an antibody against IL-6 or an antigen binding fragment thereof is used to detect the level of IL-6, for example, by ELISA.
  • the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. Further, in certain preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.
  • steps (2) and (6) further comprise stimulating at least one sample as a positive control sample using a non-specific stimuli.
  • the non-specific stimulator comprises a plant lectin or concanavalin A.
  • step (1) prior to step (1), one or more of the following steps are further included: (a) adding an anticoagulant, such as heparin, to the pre-treatment sample; (b) from before treatment The sample is obtained from PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer); (c) adding a culture solution or a medium to the pre-treatment sample; and, (d) diluting the pre-treatment sample.
  • an anticoagulant such as heparin
  • step (5) prior to step (5), one or more of the following steps are further included: (a) adding an anticoagulant, such as heparin, to the post-treatment sample; (b) after treatment The sample is obtained from PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer); (c) a culture solution or a medium is added to the post-treatment sample; and, (d) the post-treatment sample is diluted.
  • an anticoagulant such as heparin
  • the stimuli are used at a temperature at which the cells (eg, PBMC) are more active (eg, at 36-38 ° C, eg, at about 37 ° C) A sample from the subject is stimulated.
  • the sample from the subject is stimulated with the stimulator for not less than 12 h, such as 15-24 h, such as 20-24 h.
  • the invention provides a method for screening for a drug candidate capable of treating active tuberculosis comprising the steps of:
  • step (3) measuring the IL-6 level of each sample in the step (2), and calculating the difference of the IL-6 level of the sample to be tested and the negative control sample as the first difference;
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood white layer, and may optionally include other groups For example, an anticoagulant, a diluent, and the like.
  • PBMC peripheral blood mononuclear cells
  • the second difference when the second difference is greater than the first difference, or when the statistical analysis value of the second difference is greater than a statistical analysis value of the first difference, Indicating that the screened drug is ineffective for treating active tuberculosis; when the second difference is less than the first difference, or when the statistical analysis value of the second difference is less than the statistical analysis of the first difference At the time of the value, it indicates that the drug being screened is effective for the treatment of active tuberculosis.
  • the first difference and the second difference are statistically analyzed using a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM). Further, in certain preferred embodiments, in step (8), the first difference and the second difference are statistically analyzed using a logistic regression model.
  • a statistical model selected from the group consisting of: linear combination, linear regression model, logistic regression model, Linear Discriminant Analysis (LDA) model, nearest neighbor model, or microarray predictive analysis (PAM).
  • LDA Linear Discriminant Analysis
  • PAM microarray predictive analysis
  • the RV0183 has the amino acid sequence set forth in SEQ ID NO: 1; and/or the PlcD has the amino acid sequence set forth in SEQ ID NO: 3.
  • the antigenic fragment is an antigenic fragment of RV0183. Further, in certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-25.
  • the model animal is a non-human mammal, such as a mouse, guinea pig, rabbit, or non-human primate (eg, cynomolgus or macaque).
  • non-human mammal such as a mouse, guinea pig, rabbit, or non-human primate (eg, cynomolgus or macaque).
  • the subject is a mammal, such as a human.
  • steps (2) and (6) the pre-treatment sample and the post-treatment sample are subjected to the same treatment (eg, under the same conditions, using the same specific stimuli for treatment) ).
  • steps (2) and (6) at least two specific stimuli are used to stimulate one or more samples from the animal together or separately as a sample to be tested, wherein The specific stimuli are each independently selected from RV0183, PlcD or antigenic fragments thereof.
  • steps (2) and (6) at least two samples are separately stimulated as samples to be tested using RV0183, and PlcD. Or, in certain preferred embodiments, in steps (2) and (6), at least one sample is co-stimulated using one or more antigenic fragments as a sample to be tested. Further, in certain preferred embodiments, in steps (2) and (6), at least one sample is co-stimulated as a sample to be tested using a combination of the following antigenic fragments:
  • the specific stimulator in steps (2) and (6), is placed in a medium, such as a cell culture medium, such as RPMI-1640 medium or DMEM medium, and then used A sample from the animal is stimulated to produce a sample to be tested.
  • a medium such as a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • a medium that does not contain the specific stimulating pro- and non-specific stimuli eg, a cell culture medium, such as RPMI-1640 medium or DMEM medium
  • the sample from the animal is incubated or diluted to produce a negative control sample.
  • the level of IL-6 in the sample is determined by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • an antibody against IL-6 or an antigen binding fragment thereof is used to detect the level of IL-6, for example, by ELISA.
  • the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. Further, in certain preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.
  • steps (2) and (6) further comprise stimulating at least one sample as a positive control sample using a non-specific stimuli.
  • the non-specific stimulator comprises a plant lectin or concanavalin A.
  • step (1) prior to step (1), one or more of the following steps are further included: (a) adding an anticoagulant, such as heparin, to the pre-treatment sample; (b) from before treatment The sample is obtained from PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer); (c) adding a culture solution or a medium to the pre-treatment sample; and, (d) diluting the pre-treatment sample.
  • an anticoagulant such as heparin
  • step (5) prior to step (5), one or more of the following steps are further included: (a) adding an anticoagulant, such as heparin, to the post-treatment sample; (b) after treatment The sample is obtained from PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer); (c) a culture solution or a medium is added to the post-treatment sample; and, (d) the post-treatment sample is diluted.
  • an anticoagulant such as heparin
  • the stimuli are used at a temperature at which the cells (eg, PBMC) are more active (eg, at 36-38 ° C, eg, at about 37 ° C) Samples from the animal are stimulated.
  • the sample from the animal is stimulated with the stimulator for not less than 12 h, such as 15-24 h, such as 20-24 h.
  • the invention provides a polypeptide library comprising:
  • polypeptide library further comprises a combination of the following polypeptides:
  • the polypeptide library comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 5-25, respectively.
  • the polypeptide library is capable of inducing a sample to produce IL-6; wherein the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood single Nuclear cells (PBMC), or peripheral blood leukocytes, and optionally other components, such as anticoagulants, diluents, and the like.
  • PBMC peripheral blood mononuclear cells
  • whole blood eg, anticoagulated whole blood
  • PBMC peripheral blood single Nuclear cells
  • leukocytes peripheral blood leukocytes
  • other components such as anticoagulants, diluents, and the like.
  • the polypeptide library is used to diagnose active tuberculosis, to determine the therapeutic effect of a therapy on active tuberculosis or to screen for a drug candidate capable of treating active tuberculosis.
  • a specific stimulator (RV0183, PlcD or an antigenic fragment thereof) can stimulate a large amount of IL-6 in peripheral blood of active tuberculosis patients, thereby distinguishing between active tuberculosis and inactive tuberculosis population.
  • active tuberculosis e.g, latent tuberculosis, old tuberculosis, or non-tuberculosis
  • non-tuberculosis eg, latent tuberculosis, old tuberculosis, or non-tuberculosis
  • stimulation of samples from the subject is accomplished by collecting whole blood and adding a specific stimulator, and then culturing under appropriate conditions; experimental conditions, technical capabilities of the person , equipment and environmental requirements are not high; compared with traditional tuberculosis diagnostic methods (such as bacterial culture, sputum coating, X-ray detection, etc.), the detection of active TB patients has higher sensitivity and specificity, and Shorten the time required for diagnosis; the cost is not high and the range of use is wide.
  • tuberculosis diagnostic methods such as bacterial culture, sputum coating, X-ray detection, etc.
  • Figure 1 shows the results of SDS-PAGE analysis showing recombinant antigen RV0183 purified in two steps by Ni-NTA and DEAE columns after efficient expression in E. coli ER2566.
  • lane 1 represents the protein molecular weight standard
  • lane 2 represents the protein RV0183 after two-step purification.
  • Figure 2 shows the results of SDS-PAGE analysis showing the recombinant antigen Plcd which was highly expressed in E. coli ER2566 and purified by Ni-NTA and renatured by inclusion bodies.
  • lane M represents the protein molecular weight standard
  • lane 1 represents the denatured inclusion body
  • lane 2 represents the protein Plcd after affinity column chromatography
  • lane 4 represents the dialyzed reticular protein Plcd.
  • Figure 3 shows the results of analysis of IL-6 levels in whole blood of a subject using recombinant antigen RV0183. After 1 ⁇ g of recombinant antigen RV0183 stimulated 500 ⁇ l of whole blood for 20 ⁇ 2 h, the plasma samples were diluted 2-fold. The results showed that IL-6 secretion levels in active tuberculosis patients were significantly higher than those in latent infections. (Note: *** means p ⁇ 0.001)
  • FIG 4 shows the results of analysis of IL-6 levels in whole blood of a subject using recombinant antigen RV0183.
  • aTB active TB patients
  • LTBI latent M. tuberculosis
  • IGRA-physical examination non-infected individuals
  • Figure 5 shows the results of analysis of IL-6 levels in whole blood of a subject using recombinant antigen RV0183. After 2 ⁇ g of recombinant antigen RV0183 stimulated 1 ml of whole blood for 20 ⁇ 2 h, the plasma samples were diluted 5 times. The results showed that the level of IL-6 secretion in clinically active tuberculosis patients was significantly higher than that in non-tuberculous mycobacteria. (Note: *** means p ⁇ 0.001)
  • Figure 6 shows the results of analysis of IL-6 levels in whole blood of a subject using antigenic fragments of RV0183.
  • the results showed that 21 antigenic fragments of RV0183 can stimulate the increase of IL-6 secretion in whole blood of active tuberculosis patients in vitro, while in healthy controls, there is basically no IL-6 response, green to red indicates response. The strength increases.
  • Figures 7A-7B show the results of IL-6 level analysis of the whole blood of a subject, respectively, using recombinant antigen RV0183 and recombinant antigen PlcD.
  • recombinant antigen RV0183 and 2 ⁇ g of recombinant antigen Plcd were stimulated for 1 ⁇ 2 hours of whole blood for 20 ⁇ 2 h, the plasma samples were diluted 5 times.
  • the results showed that the levels of IL-6 secretion in active tuberculosis patients after recombinant antigen RV0183 and recombinant antigen PlcD were significantly higher than those in latent and non-infected populations, among which aTB represents active tuberculosis and HC represents healthy controls.
  • Figure 8 shows the results of ROC analysis of the combined detection of recombinant antigen RV0183 and recombinant antigen Plcd. The results showed that the combined detection of recombinant antigen RV0183 and recombinant antigen Plcd can improve the sensitivity of detecting active tuberculosis patients. Degree and specificity.
  • Figure 9 shows the results of analysis of IL-6 levels in whole blood of a subject using the RV0183 polypeptide library.
  • Twenty-one peptide libraries consisting of antigenic fragments of RV0183 stimulated whole blood samples of clinical tuberculosis patients, old tuberculosis individuals, and non-tuberculous lung disease cases in vitro. The results showed that IL-6 secretion levels were significantly higher in active tuberculosis patients than in inactive tuberculosis specimens. (Note: ** means p ⁇ 0.01, *** means p ⁇ 0.001)
  • Nucleotide sequence of recombinant protein RV0183 (SEQ ID NO: 2)
  • Nucleotide sequence of recombinant protein Plcd (SEQ ID NO: 4)
  • RV0183 polypeptide library peptide sequence (P1-P21) (SEQ ID NOs: 5-25)
  • RV0183-p1 MTTTRTERNFAGIGDVRIVY (SEQ ID NO: 5)
  • RV0183-p2 GDVRIVYDVWTPDTAPQAVV (SEQ ID NO: 6)
  • RV0183-p3 TAPQAVVVLAHGLGEHARRY (SEQ ID NO: 7)
  • RV0183-p4 GEHARRYDHVAQRLGAAGLV (SEQ ID NO: 8)
  • RV0183-p5 LGAAGLVTYALDHRGHGRSG (SEQ ID NO: 9)
  • RV0183-p6 RGHGRSGGKRVLVRDISEYT (SEQ ID NO: 10)
  • RV0183-p7 RDISEYTADFDTLVGIATRE (SEQ ID NO: 11)
  • RV0183-p8 VGIATREYPGCKRIVLGHSM (SEQ ID NO: 12)
  • RV0183-p9 IVLGHSMGGGIVFAYGVERP (SEQ ID NO: 13)
  • RV0183-p10 AYGVERPDNYDLMVLSAPAV (SEQ ID NO: 14)
  • RV0183-p11 VLSAPAVAAQDLVSPVVAVA (SEQ ID NO: 15)
  • RV0183-p12 SPVVAVAAKLLGVVVPGLPV (SEQ ID NO: 16)
  • RV0183-p13 VVPGLPVQELDFTAISRDPE (SEQ ID NO: 17)
  • RV0183-p14 AISRDEVEVVQAYNTDPLVHH (SEQ ID NO: 18)
  • RV0183-p15 TDPLVHHGRVPAGIGRALLQ (SEQ ID NO: 19)
  • RV0183-p16 IGRALLQVGETMPRRAPALT (SEQ ID NO: 20)
  • RV0183-p17 RRAPALTAPLLVLHGTDDRL (SEQ ID NO: 21)
  • RV0183-p18 HGTDDRLIPIEGSRRLVECV (SEQ ID NO: 22)
  • RV0183-p19 RRLVECVGSADVQLKEYPGL (SEQ ID NO: 23)
  • RV0183-p20 LKEYPGLYHEVFNEPERNQV (SEQ ID NO: 24)
  • RV0183-p21 EPERNQVLDDVVAWLTERL (SEQ ID NO: 25)
  • plcD-1-F TTCAACCATCGCCGCCTCTACCA (SEQ ID NO: 26)
  • plcD-1-R CCATCGCCGCCTCTACCAGT (SEQ ID NO: 27)
  • plcD-2-F GGATCCATGGATGCCGGCGTCAG (SEQ ID NO: 28)
  • plcD-2-R AAGCTTTTAGCACGGACCGCTCG (SEQ ID NO: 29)
  • the target gene fragment was artificially synthesized.
  • the BamHI restriction site was added to the 5' end of the target gene fragment, and the EcoRI restriction site was added to the 3' end.
  • the vector was pMD18T, and the ends were digested. Site of the RV0183 plasmid.
  • RV0183 plasmid was digested with BamHI/EcoRI.
  • the digested product was recovered by agarose gel electrophoresis.
  • the pTO-T7 vector was digested with BamHI/EcoRI (for information, see Luo Wenxin, Zhang Jun, Yang Haijie et al. Construction and preliminary application of a prokaryotic expression vector with enhancer, Journal of Bioengineering, 2000, 16(5) :578-581) was ligated with the restriction enzyme encoding RV0183 to obtain the expression vector pTO-T7-RV0183 containing the RV0183 gene fragment.
  • the constructed pTO-T7-RV0183 expression vector was transformed into E. coli ER2566 (laboratory preservation) and plated in solid LB medium (LB medium containing kana (final concentration 100 ⁇ g/ml)).
  • Solid LB medium LB medium containing kana (final concentration 100 ⁇ g/ml)
  • static culture at 37 ° C until the single colony is clearly identifiable.
  • a single colony was picked into liquid LB medium (containing 100 ⁇ g/ml kanamycin), and cultured at 37 ° C, 180 rpm for 8 hours with shaking.
  • the bacterial solution was then transferred to a 500 ml liquid LB medium (containing 100 ug/ml kanamycin) in a culture flask, and shake cultured at 37 ° C, 180 rpm.
  • a 500 ml liquid LB medium containing 100 ug/ml kanamycin
  • IPTG was added to a final concentration of 0.2 mM/L and induction culture was continued for 4 hours at 37 ° C, 180 rpm.
  • the culture was then centrifuged at 5000 rpm for 10 min, and the cells were collected.
  • the recombinant protein RV0183 Since the recombinant protein RV0183 is used to stimulate whole blood, it is necessary to remove endotoxin from the protein product. Methods for purifying recombinant protein disease from bacterial proteins to remove endotoxin are known to those skilled in the art. In the present embodiment, the following exemplary methods are used.
  • the collected cells were suspended in a buffer of 50 mM TB8.0, placed in an ice bath and disrupted by ultrasonication, and then centrifuged at 12000 rpm for 10 min to collect inclusion bodies.
  • Ni-NTA column The supernatant of the target protein was expressed and purified using a self-assembled Ni-NTA column (media manufacturer: Qiagen). Briefly, the sample was loaded onto a Ni-NTA column, washed with 0.2% sodium deoxycholate, 50 mM TB 8.0 to remove a portion of the endotoxin, then washed with 200 ml of 50 mM TB 8.0 to remove sodium deoxycholate; The target protein was eluted by deliquoring 100 ml (150 mM imidazole, 50 mM TB8.0).
  • DEAE column purification The above eluted protein was further purified using a DEAE column.
  • the upper column buffer was 50 mM TB 8.0 and the eluent was 400 mM NaCl, 50 mM TB 8.0.
  • the eluted protein was dialyzed to 50 mM TB 8.0 for storage.
  • the buffer used was prepared by using water for injection, and the experimental containers used were dry-baked at 200 ° C for more than 2 hours.
  • the recombinant protein RV0183 purified in two steps was detected by SDS-PAGE, and the results are shown in Fig. 1.
  • the results showed that the recombinant protein RV0183 had a molecular weight of about 30 KD, and after purification by the above two steps, the purity of the recombinant protein RV0183 was over 95%.
  • the endotoxin in the purified recombinant protein RV0183 was detected using a guanidine reagent. The results showed that the endotoxin content of the recombinant protein RV0183 was less than 100 EU/mg after the above two steps of purification.
  • the nested PCR method was used to obtain the target fragment by two rounds of PCR using M. tuberculosis H37Rv genomic DNA as a template. .
  • the components were mixed in a 0.5 ml Ependorf tube according to the following table to establish a 20 ⁇ l volume PCR reaction system.
  • Amplification procedure pre-denaturation at 95 ° C for 10 min, heat-transition, transfer to 95 ° C for 1 min, 55 ° C for 1 min, 72 ° C for 3 min, 30 cycles, 72 ° C extension for 10 minutes.
  • the components were mixed in a 0.5 ml Ependorf tube as follows to form a 50 ⁇ l PCR reaction system.
  • Amplification procedure pre-denaturation at 95 ° C for 10 min, denaturation at 95 ° C for 1 min, annealing at 55 ° C for 1 min, extension at 72 ° C for 3 min, 30 cycles, and extension at 72 ° C for 10 min.
  • the amplified product was identified and recovered by 1% agarose gel electrophoresis.
  • the following components were mixed in a 0.5 mL Ependorf tube, and reacted at 16 ° C for 12 hours to obtain a pMD18-T cloning vector to which a gene fragment of interest was ligated.
  • the recovered product was identified by 1% agarose electrophoresis, and it was confirmed that the recovery was successful.
  • the pMD18-T vector containing the gene fragment of interest was digested, and the cloned target fragment was recovered, and a part of the cloning vector was taken for sequencing.
  • the pMD-18T digested target gene fragment was ligated with the digested PTO-T7 vector.
  • the ligation system was 1 ⁇ l of PTO-T7 vector, 6 ⁇ l of the target gene fragment, 1 ⁇ l of 10 ⁇ T4 DNA Ligase Buffer, 1 ⁇ l of T4 DNA ligase, and the total reaction volume was 10 ⁇ l. .
  • the connection time is 2h.
  • the procedure was the same as the above-mentioned ligation step, using ER2566 competent cells as a transformation target, and after transformation, ER2566 was coated on LB resistant medium containing kanamycin.
  • a small amount of expression identifies a strain with the correct expression of the target protein. After incubation at 37 ° C for 4 h, 1 mmol/L IPTG was added, and induction was carried out for 4 h at 37 ° C, and the cells were collected by centrifugation at 9000 g for 6 min. After the cells were purged, the precipitate was collected by centrifugation, and resuspended in TB 8.0 buffer by adding 5 mL per 200 mL of the bacterial solution. The suspension was resuspended by ultrasonication, and the treatment time was calculated for 3 minutes per 500 mL of bacterial solution.
  • the sample was collected by centrifugation at 12000 g for 10 min to collect the crushed precipitate.
  • the crushed product precipitate was repeatedly purged 3 times with TB 8.0 solution, and each time it was resuspended, it was shaken for 15 min in a 37 ° C incubator.
  • the pellet was resuspended in 15 mL of TB 8.0 solution containing 6 M urea, and then thoroughly blown. Most of the precipitate was blown up and centrifuged at 12000 g for 10 min to collect the supernatant into the next purification stage.
  • Ni-NTA column The supernatant of the target protein was expressed and purified using a self-assembled Ni-NTA column (media manufacturer: Qiagen). The sample was loaded onto a Ni-NTA column, washed with 0.2% sodium deoxycholate, 50 mM TB8.0 + 6 M urea to remove part of the endotoxin, and then washed with 200 ml of 50 mM TB8.0 + 6 M urea to remove sodium deoxycholate; The protein of interest was eluted with 100 ml of eluent (150 mM imidazole, 50 mM TB 8.0, 6 M urea).
  • the inclusion body Since the plcD protein belongs to the inclusion body expression, the inclusion body is denatured and dissolved in 6M urea. After affinity chromatography, the denaturing agent is gradually removed by gradient dialysis. Dithiothreitol (DTT) is added during the dialysis to prevent errors. Disulfide bonds help the protein form correctly folded and remain soluble.
  • DTT Dithiothreitol
  • Triton X-114 was added to the protein solution at a final concentration of 1% (w/v), and the two phases were mixed at 4 ° C and shaken for 15 min.
  • the protein solution containing Triton X-114 was placed in a 37 ° C water bath for 10 min, and the system was cloudy, and oil droplets appeared in the solution. Centrifuge at 12000 g for 10 min at 25 ° C and carefully remove the aqueous phase. Repeat the above steps once to collect the aqueous phase.
  • the treated protein solution was assayed for endotoxin content using a sputum reagent gel method.
  • the sample was dispensed into 9 endotoxin-free 2 ml EP tubes, 500 ⁇ l of peripheral blood in each EP tube; 15 ⁇ l of culture medium was added to the EP tube (culture solution formula: 133.33 mg/ml D-glucose and 166.67 were added to 30 ⁇ l PBS).
  • mM KCl wherein the PBS formula is: Na 2 HPO4 ⁇ 12H 2 O 2.9 g, KH 2 PO4 0.24 g, NaCl 8 g, KCl 0.2 g, super pure water to 1 L.
  • the N tube contains no stimulating antigen
  • Ta contains 1 ⁇ g of stimulating antigen RV0183
  • P tube contains PHA 20 ug.
  • peripheral blood was added to the EP tube, the mixture was inverted and mixed, and cultured in an incubator at 37 ° C for 20-24 hours; plasma samples were collected at 5000 rpm to measure the cytokine content in the plasma.
  • the collected plasma samples were subjected to cytokine quantitative assay using a commercial Milliplex kit (Merck Millipore, St. Charles, Missouri, USA) (Cat. No. HCYTMAG-60K-PX38, HCP2MAG-62K-PX23, HCP3MAG-63K-PX11).
  • the detection indicators include 69 kinds of EGF (see Table 2), and the detection platform is Luminex 200 (the lotion, standard, control, and magnetic beads in the following steps are all from the kit).
  • the detection steps include: 1) adding 200 ⁇ l of the washing solution to each well of the 96-well assay plate, shaking at room temperature for 10 min, removing the washing solution; 2) adding 25 ⁇ l of the standard product and the control substance to the corresponding well, and repeating the double well; 3) Add 25 ⁇ l of assay buffer to the sample well; 4) Add 25 ⁇ l of plasma matrix per well to the standard wells, control wells, and blank control wells (matrix refers to one sample dilution in the kit); 5) add 25 ⁇ l per well Plasma sample; 6) add 25 ⁇ l magnetic beads per well at 2-8 ° C overnight; 7) shift In addition to the liquid in the well, wash the solution in 200 ⁇ l/well, wash twice; 8) add 25 ⁇ l of detection antibody per well, incubate for 1 h at room temperature; 9) add 25 ⁇ l of Steptavidin-Phycoerythrin per well, react at room temperature for 30 min; 10) remove the liquid in the well.
  • test results are shown in Table 3.
  • aTB active tuberculosis
  • LTBI latent infection
  • Inpatients with tuberculosis were active tuberculosis patients, and IGRA-positive specimens with no clinical symptoms were latent infections).
  • Active tuberculosis patients all of them are clinically diagnosed active tuberculosis patients, 79 cases; 2) M. tuberculosis latent infection population and healthy people: 37 medical workers participated in the pair, 14 cases of medical care Tuberculosis IGRAs test results were positive but no clinical tuberculosis symptoms and no history of tuberculosis, identified as latent infection; another 23 cases of IGRAs A health care worker with a negative test result is judged to be a healthy person without M. tuberculosis infection.
  • the IL-6 ELISA test kit (Xiamen Wantai Bohai Bio) was used to detect IL-6 levels in plasma.
  • Active tuberculosis patients 207 clinically diagnosed active tuberculosis patients;
  • Each subject collects 3 ml of peripheral blood, and the whole blood is dispensed into three 2 ml EP tubes, and each EP tube is dispensed with 1 ml; three EP tubes are labeled as N, Ta, and P, respectively.
  • 30 ⁇ l of the culture solution was added to the tube (formulation is the same as in Example 4); wherein the culture solution added to the N tube contained no antigen, and 30 ⁇ l of the culture solution added to the Ta tube contained 2 ⁇ g of the above recombinant antigen.
  • RV0183, 30 ⁇ l of the culture solution added to the P tube contains 40 ⁇ g of PHA;
  • the EP tube was placed in a centrifuge, centrifuged at 5000 rpm for 10 min, and plasma samples were collected for IL-6 secretion level detection;
  • the IL-6 ELISA test kit (Xiamen Wantai Bohai Bio) was used to detect IL-6 levels in plasma.
  • the IL-6 detection method was the same as that described in Example 4.
  • the EP tube was placed in a centrifuge, centrifuged at 5000 rpm for 10 min, and plasma samples were collected for IL-6 secretion level detection;
  • the IL-6 ELISA test kit (Xiamen Wantai Bohai Bio) was used to detect IL-6 levels in plasma.
  • the IL-6 detection method was the same as that described in Example 4.
  • Example 7 Screening for active tuberculosis patients using a combination of RV0183 and PlcD
  • aTB (IGRA+) represents a clinically confirmed active tuberculosis patient with a positive TB-IGRA test
  • aTB (IGRA-) represents a clinically confirmed active tuberculosis patient with a negative TB-IGRA test
  • HC (IGRA+) represents a healthy person with a positive TB-IGRA test
  • HC (IGRA-) represents a healthy person with a negative TB-IGRA test.
  • Each subject collects 4 ml of peripheral blood, and the whole blood is dispensed into 4 2 ml EP tubes, and each EP tube is dispensed with 1 ml; 4 EP tubes are labeled as N, Ta1, Ta2, and P, respectively.
  • Add 30 ⁇ l of the culture solution to the EP tube (formulation is the same as in Example 4); wherein the culture solution added to the N tube contains no antigen, 30 ⁇ l of the culture solution added to the Ta1 tube contains 2 ⁇ g of the above recombinant antigen RV0183, and 30 ⁇ l of the culture solution added to the Ta2 contains 2 ⁇ g of recombinant antigen Plcd, 30 ⁇ l of culture medium added to the P tube contains 40 ⁇ g of PHA;
  • the EP tube was placed in a centrifuge, centrifuged at 5000 rpm for 10 min, and plasma samples were collected for IL-6 secretion level detection;
  • the IL-6 ELISA test kit (Xiamen Wantai Bohai Bio) was used to detect IL-6 levels in plasma.
  • the IL-6 detection method was the same as that described in Example 4.
  • Example 8 Value of other antigens of Mycobacterium tuberculosis for screening and screening of active tuberculosis
  • the EP tube was placed in a centrifuge, centrifuged at 5000 rpm for 10 min, and plasma samples were collected for IL-6 secretion level detection;
  • the IL-6 ELISA test kit (Xiamen Wantai Bohai Bio) was used to detect IL-6 levels in plasma.
  • the IL-6 detection method was the same as that described in Example 4.
  • the test results are shown in Table 4.
  • the levels of IL-6 in the samples stimulated by ESAT-6, CFP-10, RV1009, RV1884c, RV2389c, RV2450c, RV3097c, and RV3542 were not found in active tuberculosis (aTB) and latent infection (LTBI). Significant changes, that is, the above antigens cannot be used to distinguish between active tuberculosis and latent infections.
  • Example 9 Screening of active tuberculosis patients in different types of lung disease patients using the RV0183 polypeptide library
  • Active tuberculosis cases 101 cases of clinically diagnosed active tuberculosis patients;

Abstract

一种用于诊断受试者是否患有活动性结核的方法，一种用于判断一种疗法对活动性结核的治疗效果的方法，一种用于筛选能够治疗活动性结核的候选药物的方法，以及一种含有特异性刺激原和检测IL-6水平的试剂的试剂盒，所述方法和试剂盒属于分子生物学、免疫学以及疾病诊断领域。

Description

用于诊断活动性结核的方法和试剂盒 技术领域
本发明属于分子生物学、免疫学以及疾病诊断领域。具体而言，本发明涉及，用于诊断受试者是否患有活动性结核的方法，用于判断一种疗法对活动性结核的治疗效果的方法，以及用于筛选能够治疗活动性结核的候选药物的方法。本发明还涉及含有特异性刺激原和检测IL-6水平的试剂的试剂盒。
背景技术
结核病(Tuberculosis，TB)是由结核分枝杆菌感染引起的慢性传染病，是一种非常严重的全球性健康问题，全球每年新增结核病人约有900万，因结核病死亡的人数高达170万。此外，据估计全球约有1/3人口为结核分枝杆菌潜伏感染者，这类人群为潜在的活动性结核病人来源。
控制结核病，首要是发现并治愈传染性结核病病人。X射线应用于临床检测后，为发现结核病病人提供了一种简便、易行、快速、敏感性高的方法；然而，X射线检测设备要求高，特异性低，过诊、漏诊、误诊率较高；此外X射线检测只能发现肺部可疑阴影，达不到检测出传染性病人的目的，因此1974年WHO第9次结核病专家委员会报告明确指出采取团体胸部X射线主动发现病人的方法，不仅在发达国家，在发展中国家都是不可取的，必须废止。
痰涂片检测可以检出传染性肺结核病病人，特异性高，设备要求简单，费用低，技术要求不高。但也存在取痰不易的问题，不少患者干咳无痰，或微量排菌不易检出。广泛应用的痰涂片检测灵敏度在34-80％之间，虽然痰培养检测灵敏度高于痰涂片，但是耗时长，对诊断实验室的要求高，在很多高结核负担国家无法满足该方法对实验室条件的要求。免疫检测技术在结核诊断上可能有很大的应用价值，尤其在实现快速诊断和床边诊断以后。
IFN-γ释放实验(IGRAs)是一种广泛应用的TB免疫诊断方法，在结核分支杆菌感染检测上已经被证明具有应用价值，检测效果比TST(tuberculin skin test)有显著的优势。但是，IGRAs和TST方法均不能区分结核潜伏性感染(LTBI)和活动性结核；在高结核负担国家因有很高比例潜伏感染人群，IGRAs的应用价值受到限制，其仍然只能作 为一种辅助诊断手段。目前临床普遍采用的结核诊断仍然主要依赖临床症状、影像学诊断和病原学诊断。如果能够在结核发病早期准确快速确诊并进行适当的化疗，对减轻结核病人的危害及结核传染的控制将有重要价值。
因此，本领域需要简单、方便、快速且具有高准确性和特异性的活动性结核诊断方法。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“RV0183”或“RV0183蛋白”是指，天然存在于结核分枝杆菌(Mycobacterium tuberculosis)中的一种蛋白，属于单酰基甘油脂肪酶(monoacylglycerol lipase，MAGL)或溶血磷脂酶(lysophospholipase)。RV0183蛋白的序列是本领域公知的，并且可参见各种公共数据库(例如NCBI数据库登录号CP009480.1，AIR12906.1，CCP42909.1，NP_214697.2，或WP 003401112.1)。
在本发明中，当提及RV0183的氨基酸序列时，参照SEQ ID NO：1所示的序列来进行描述。然而，本领域技术人员理解，结核分枝杆菌既包括标准株H37Rv，也可包括多种分离株，并且各种分离株的RV0183蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是结核分枝杆菌的不同分离株的RV0183蛋白在氨基酸序列上具有极高的同一性，并且具有实质上相同的生物学功能。同时，本领域技术人员理解，在RV0183蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“RV0183”不仅包括SEQ ID NO：1所示的蛋白，而且应包括各种结核分枝杆菌分离株的RV0183蛋白(例如BAL64025.1，AOE34503.1，EGE52872.1，或CDM08372.1所示的RV0183蛋白)，以及其天然或人工的变体。并且，当描述RV0183蛋白的序列片段时，其不仅包括SEQ ID NO：1的序列片段，还包括各种结核分枝杆菌分离株的RV0183蛋白中的相应序列片段，以及SEQ ID NO：1的天然或人工变体中的相应序列片段。因此，表述“RV0183蛋白的第1-20位氨基酸残基”包括，SEQ ID NO：1的第1-20位 氨基酸残基，以及各种结核分枝杆菌分离株的RV0183蛋白中的相应片段，以及RV0183蛋白的变体(天然或人工)中的相应片段。在本发明中，表述“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
在本发明中，术语“RV0183”或“RV0183蛋白”不受任何特定的合成蛋白的方法限制，可通过本领域技术人员已知的常规技术产生，例如DNA重组技术或化学合成技术。
如本文中所使用的，术语“PlcD”或“PlcD蛋白”是指，天然存在于结核分枝杆菌(Mycobacterium tuberculosis)中的一种蛋白，属于磷脂酶C(phospholipase C)家族。PlcD蛋白的序列是本领域公知的，参见例如Andersen ST，et al.，Nature，1998.393(6685)：537-44(其通过引用并入本文)，以及NCBI数据库登录号CCP44521.1。
在本发明中，当提及PlcD的氨基酸序列时，参照SEQ ID NO：3所示的序列来进行描述。然而，本领域技术人员理解，结核分枝杆菌既包括标准株H37Rv，也可包括多种分离株，并且各种分离株的PlcD蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是结核分枝杆菌的不同分离株的PlcD蛋白在氨基酸序列上具有极高的同一性，并且具有实质上相同的生物学功能。同时，本领域技术人员理解，在PlcD蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PlcD”不仅包括SEQ ID NO：3所示的蛋白，而且应包括各种结核分枝杆菌分离株的PlcD蛋白，以及其天然或人工的变体。并且，当描述PlcD蛋白的序列片段时，其不仅包括SEQ ID NO：3的序列片段，还包括各种结核分枝杆菌分离株的PlcD蛋白中的相应序列片段，以及SEQ ID NO：3的天然或人工变体中的相应序列片段。在本发明中，表述“相应序列片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
在本发明中，术语“PlcD”或“PlcD蛋白”不受任何特定的合成蛋白的方法限制，可通过本领域技术人员已知的常规技术产生，例如DNA重组技术或化学合成技术。
如本文中所使用的，表述“RV0183或PlcD的抗原性片段”是指，RV0183或PlcD蛋白经过截短后得到的氨基酸序列片段(即，多肽)，该片段具有与相应的全长蛋白相同的生物学活性，即，可刺激活动性结核患者的外周血产生IL-6，从而区分活动性结核和非活动性结核人群(例如，潜伏性结核感染者、陈旧性结核患者或非结核感染者)。例如本 发明中SEQ ID NO：5-25所示的氨基酸序列片段即为RV0183的抗原性片段。在本发明中，该抗原性片段不受任何特定的合成多肽的方法限制，可通过本领域技术人员已知的常规技术产生，例如DNA重组技术或化学合成技术。
在本发明中，RV0183、PlcD或其抗原性片段(即，多肽)可以通过DNA重组技术获得，例如通过使用无细胞表达系统从编码这些蛋白或多肽的多核苷酸获得(无细胞表达系统包括例如基于网织红细胞裂解物的表达系统、基于麦胚提取物的表达系统以及基于大肠杆菌提取物的表达系统)；或通过使用体内表达系统(例如，大肠杆菌原核表达系统、酵母真核表达系统)从编码这些蛋白或多肽的多核苷酸获得。作为另外一种选择，RV0183、PlcD或其抗原性片段(即，多肽)可以通过化学合成产生。蛋白或多肽化学全合成的方法在本领域内是熟知的(参见，例如，Raibaut L，et al.，Top Curr Chem.2015；363：103-54；Thapa P，et al.Molecules.2014；19(9)：14461-83；Dawson PE，et al.，Science，1994；266(5186)：776-9；和Wang P，et al.，Tetrahedron Lett，1998，39(47)：88711-14；其通过引用并入本文)，并且包括但不限于：固相肽合成技术(Solid Phase Peptide Synthesis，SPPS)或液相分段合成技术(例如，天然化学连接法(Native Chemical Ligation，NCL)、叠氮法(Azide method)、转移活化酯法(Transfer Active Ester Condensation，TAEC))。
如本文中所使用的，表述“分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段”是指，具有SEQ ID NO：13所示的氨基酸序列的抗原性片段、具有SEQ ID NO：14所示的氨基酸序列的抗原性片段、和具有SEQ ID NO：19所示的氨基酸序列的抗原性片段的组合。其他类似的表述具有类似的含义。
如本文中所使用的，术语“能够检测IL-6的试剂”是指，能够和IL-6特异性结合的物质。这类物质是本领域所公知的，或者可以利用本领域公知的方法制备得到，例如为抗体、靶向多肽或核酸适体等。通常而言，特别优选的是，此类试剂能够通过免疫学检测来确定样品中IL-6的水平。免疫学检测的使用是特别有利的，因为其利用了抗原-抗体之间的特异性相互作用/结合亲和力。因此，只要试剂保留了与IL-6特异性结合的反应性，那么该试剂即可以通过免疫学检测来确定样品中IL-6的水平(也即，该试剂即可用作能够检测IL-6的试剂)。保留与IL-6特异性结合的反应性的各种试剂是本领域技术人员容易想到并且可容易获得的，其包括但不限于，抗IL-6的抗体或其抗原结合片段，例如抗IL-6的多克隆抗体或单克隆抗体，例如IgG抗体或IgM抗体。作为另一种选择，所述能够 检测IL-6的试剂也可以为结合IL-6的核酸适体，其包括但不限于描述于PCT国际申请WO2014159669中以及“Gupta S，et al.，J Biol Chem.2014；289(12)：8706-19”中的核酸适体，其全部通过引用并入本文。
如本文中所使用的，术语“特异性结合”是指，两分子(即结合分子与靶分子)之间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。两分子之间的结合亲和力可用KD值描述。KD值是指由kd(特定的结合分子-靶分子相互作用的解离速率；亦称为koff)与ka(特定结合分子-靶分子相互作用的缔合速率；亦称为kon)之比得到的解离常数，或者指表示为摩尔浓度(M)的kd/ka。KD值越小，两分子结合越紧密，亲和力越高。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。KD值可通过本领域熟知的方法确定，例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
如本文中所使用的，术语“免疫学检测”是指，利用抗原-抗体之间的特异性相互作用/结合亲和力来进行的测定，其一般可用于检测特定抗原或者抗体在样品中的存在或水平。此类免疫学测定是本领域技术人员公知的，包括但不限于，ELISA检测，Elispot检测，Western印迹，表面等离子共振法等。关于免疫学测定的详细描述，可参见例如，Fundamental Immunology，Ch.7Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分 配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，抗体的“抗原结合片段”是指，全长抗体的一个或多个部分，所述部分保持结合抗体所结合的相同抗原(例如，IL-6)的能力，与完整抗体竞争对抗原的特异性结合。通常参见，Fundamental Immunology，Ch.7Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab′、F(ab′)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性结合抗原能力的抗体的至少一部分。
如本文中所使用的，术语“核酸适体(Aptamer)”是指，能够高亲和性和高特异性地结合目的靶蛋白或其它生物靶分子的单链寡核苷酸，其可折叠形成例如茎环(Stem-Loop)、发夹(Hairpin)、假结(Pseudoknot)或G-四聚体(G-tetramer)的热力学稳定的三维空间结构，通过例如结构互补、碱基堆积力、范德华力、氢键或静电作用与目的靶蛋白或其它生物靶分子特异性结合。核酸适体可以为DNA或RNA，也可以包含核酸类似物(例如锁核酸(LNA)、肽核酸(PNA)、二醇核酸(GNA)或苏糖核酸(TNA))。获得结合特定靶蛋白的核酸适体的方法是本领域公知的，例如SELEX(Systematic evolution of ligands by exponential enrichment)筛选技术。
如本文中所使用的，术语“靶向多肽”是指，可以特异性结合目的靶蛋白的多肽分子。在本发明中，该靶向多肽可包含天然氨基酸、合成的氨基酸或采用与天然存在的氨基酸类似的方式起作用的氨基酸模拟物(mimetics)。天然存在的氨基酸为通过遗传密码来编码的那些以及后来修饰的那些氨基酸，例如，羟基脯氨酸、γ-羟基谷氨酸盐、O-磷酸丝氨酸、磷酸苏氨酸或磷酸酪氨酸。在本发明中，可基于亲和力来确定靶向多肽与其目的靶蛋白之间的“特异性”，该亲和力可用靶向多肽与其所结合的目的靶蛋白的解离平衡常数(即，KD值)进行描述。KD值越低，靶向多肽与其所结合的目的靶蛋白之间的结合强度 越强。在本领域中通常已知，大于约10-3M的KD值通常被认为表示非结合或非特异性结合。取决于具体的目的靶蛋白，可以通过本领域技术人员已知的方法获得特异性结合该靶蛋白的靶向多肽，例如通过噬菌体展示技术或蛋白质微阵列技术进行筛选。
如本文中所使用的，术语“可检测的标记”是指，可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何组合物。在本发明中，特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的，包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，
Figure PCTCN2017071462-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。本发明中涵盖的标记物可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。
如本文中所使用的，术语“特异性刺激原”是指，能够仅刺激活动性结核患者的PBMC产生IL-6，而不能刺激非活动性结核人群(例如，潜伏性结核感染者、陈旧性结核患者或非结核感染者(IGRA检测阴性))的PBMC产生IL-6的物质；或者是指这样的物质，其刺激活动性结核患者的PBMC产生的IL-6分泌量显著高于非活动性结核人群(例如，潜伏性结核感染者、陈旧性结核患者或非结核感染者(IGRA检测阴性))。本发明的特异性刺激原包括RV0183、PlcD或其抗原性片段。在本发明的优选实施方案中，所述特异性刺激原不含有或几乎不含有内毒素。去除上述特异性刺激原中的内毒素的方法为本领域所公知，例如离子交换层析法、亲和层析法或萃取法。
如本文中所使用的，术语“非特异性刺激源”是指，能够激活多数或全部淋巴细胞，而不受TCR或BCR特异性限制的物质。淋巴细胞活化后，可大量分泌细胞因子(例如， IL-6)。可用作非特异性刺激源的物质是本领域熟知的，包括但不限于有丝分裂原，植物血凝素(PHA)、刀豆蛋白A(ConA)、商陆丝裂原(PWM)、脂多糖(LPS)或葡萄球菌蛋白A(SPA)。在某些实施方案中，所述非特异性刺激原可包含在培养基(例如细胞培养基，例如RPMI-1640培养基和DMEM培养基)中。
如本文中所使用的，术语“统计分析值”是指，对通过各种检测方法得到的检测结果进行统计分析后得到的值。各种统计分析方法是本领域公知的(参见，例如PCT国际申请WO2009064901)，并且包括但不限于检测结果的线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)。通常而言，特别优选的是，所述统计分析值为通过Logistic回归模型进行统计分析得到的值。Logistic回归模型具体描述于例如“胡春艳.四种肿瘤标志物在卵巢癌血清中的联合检测[D].广州：中山大学，2008：1-39”。
如本文中所使用的，术语“参考值”(也称为最佳诊断界值)是指能够反映非活动性结核人群的状况的值。在本发明中，参考值包括例如，根据特异性刺激原刺激前后非活动性结核人群(例如，潜伏性结核感染者、陈旧性结核患者或非结核感染者(IGRA检测阴性)样品中的IL-6水平的差值，所确定的一个正常数值或数值范围；以及，对从非活动性结核人群的样品获得的检测值(例如，前文所述的IL-6水平的差值)进行统计分析而得到的值(统计分析值)。确定最佳诊断界值的方法是本领域熟知的，包括但不限于受试者工作特征曲线分析(Receiver Operating Characteristic(ROC)curve analysis)，其详细描述于例如“Habibzadeh F，et al.，Biochem Med(Zagreb).2016；26(3)：297-307”和“陈卫中等.ROC曲线中最佳工作点的选择[J].中国卫生统计，2006，23：157-158”。
如本文中所使用的，术语“外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)”是指，外周血中具有单个细胞核的细胞的总称，包括但不限于淋巴细胞(T细胞、B细胞、NK细胞)、单核细胞或树突状细胞。从外周血中获得PBMC的方法是本领域熟知的，包括但不限于Ficoll分层液法或Percoll分层液法。
如本文中所使用的，术语“外周血白膜层”是指，外周抗凝血经过自然沉降、离心或密度梯度离心后形成的一个组分，主要由白细胞(包括外周血单个核细胞)和血小板组成。抗凝血经过离心以后会形成上层的血浆、下层的红细胞以及二者之间薄薄的一层白色膜状物，约占血液总体积的1％，被称为白膜层。
如本文中所使用的，术语“受试者”包括但不限于各种动物，特别是哺乳动物，例如人。
如本文中所使用的，术语“抗凝剂”是指，能够阻止血液凝固的试剂或物质，这类物质是本领域熟知的，包括但不限于肝素、EDTA、草酸盐(例如，草酸钠、草酸钾、草酸铵)、枸橼酸钠(柠檬酸钠)。
在本发明中，术语“稀释液”优选为能够维持细胞渗透压的电解质溶液，必要时该溶液也具备保持生理pH值的作用。这类溶液是本领域熟知的，包括但不限于阿氏液(Alsever′s solution)、Earle′s平衡盐溶液(EBSS)、Gey′s平衡盐溶液(GBSS)、Hanks′平衡盐溶液(HBSS)、磷酸盐缓冲液(PBS)、杜氏磷酸盐缓冲液(DPBS)、Puck′s平衡盐溶液、Ringer′s平衡盐溶液(RBSS)、Simm′s平衡盐溶液(SBSS)、TRIS缓冲液(TBS)、Tyrode′s平衡盐溶液(TBSS)、生理盐水或林格氏液(Ringer′s Solution)。
如本文中所使用的，术语“培养液”或“培养基”是指，能够维持细胞活性的养料。通常所述养料含有氨基酸、维生素、碳水化合物、无机盐等。这类养料是本领域熟知的，包括但不限于RPMI-1640培养基或DMEM培养基。在本发明中，在来自所述受试者的样品中加入培养液或培养基的目的是为了在刺激原刺激的过程中维持样品中的细胞特别是PBMC的活性。维持血液成分中细胞的活性的方法为本领域所公知，本领域技术人员可以根据实际需要进行选择。在某些实施方案中，当样品为全血时，可以加入培养液，所述培养液例如为在磷酸盐缓冲液或生理盐水中添加适量的葡萄糖、氯化钠和氯化钾等；在某些实施方案中，所述培养液为在磷酸盐缓冲液中添加适量的葡萄糖和氯化钾。在某些实施方案中，当样品为外周血单个核细胞(PBMC)、外周血白膜层或其它含有PBMC的血液成分时，可以加入培养基，例如细胞培养基，例如适合维持血细胞特别是PBMC活性的细胞培养基，例如RPMI-1640培养基或DMEM培养基。
如本文中所使用的，术语“活动性结核”是指，结核病灶处于活动期，例如痰涂片阳性，或者同时伴有相关症状，如低热、咳嗽、消瘦、乏力、食欲差等。在本发明中，结核包括肺结核和肺外结核，所述肺外结核例如包括淋巴结核、结核性脑膜炎、结核性腹膜炎、肠结核、肾结核、附睾结核、女性生殖系统结核(包括输卵管、子宫内膜、卵巢结核)和骨关节结核等。
本申请的发明人筛选了大量结核分枝杆菌RD区抗原，出人意料地发现，RV0183、 PlcD或其抗原性片段可刺激活动性结核患者的外周血产生大量IL-6，从而区分活动性结核和非活动性结核人群(例如，潜伏性结核感染者、陈旧性结核患者或非结核感染者)。而在本申请之前，本领域中常用的IGRAs和TST方法均不能区分潜伏性结核感染(LTBI)和活动性结核。基于这一发现，本发明人开发了新的诊断活动性结核的方法。
因此，在一个方面，本发明提供了一种试剂盒，其包括RV0183、PlcD或其抗原性片段中的一种或数种，和能够检测IL-6的试剂。
在某些优选的实施方案中，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列。
在某些优选的实施方案中，所述试剂盒包含RV0183和/或PlcD。
在某些优选的实施方案中，所述试剂盒包含一种或多种RV0183的抗原性片段。进一步，在某些优选的实施方案中，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25。
在某些优选的实施方案中，所述试剂盒包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段。
任选地，所述试剂盒还包括下列抗原性片段的组合：
1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段，或
4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述试剂盒包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述能够检测IL-6的试剂为能够和IL-6特异性结合的物质，例如抗体、靶向多肽或核酸适体。
任选地，所述试剂还带有可检测的标记。
在某些优选的实施方案中，所述试剂通过免疫学检测来测定所述样品中IL-6的水平。进一步，在某些优选的实施方案中，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
在某些优选的实施方案中，所述试剂包括抗IL-6的抗体或其抗原结合片段。进一步，在某些优选的实施方案中，所述试剂通过ELISA来测定IL-6的水平。
在某些优选的实施方案中，所述抗IL-6的抗体为单克隆抗体或多克隆抗体。进一步，在某些优选的实施方案中，所述抗IL-6的抗体为IgG抗体或IgM抗体。
在某些优选的实施方案中，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
1)采血装置，例如无热原真空采血管；
2)抗凝剂，例如肝素；
3)培养液或培养基；
4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
5)稀释液，例如磷酸盐缓冲液或生理盐水。
在某些优选的实施方案中，所述试剂盒用于诊断活动性结核、判断一种疗法对活动性结核的治疗效果或筛选能够治疗活动性结核的候选药物。
在另一个方面，本发明提供了特异性刺激原在制备试剂盒中的用途，所述试剂盒用于诊断活动性结核；其中，所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种。
在某些优选的实施方案中，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列。
在某些优选的实施方案中，所述特异性刺激原选自RV0183、PlcD或其组合。
在某些优选的实施方案中，所述特异性刺激原选自一种或多种RV0183的抗原性片段。进一步，在某些优选的实施方案中，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25。
在某些优选的实施方案中，所述特异性刺激原包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段。
任选地，所述特异性刺激原还包括下列抗原性片段的组合：
1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段， 或
4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述特异性刺激原包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述试剂盒包括能够检测IL-6的试剂，例如能够和IL-6特异性结合的抗体、靶向多肽或核酸适体。
任选地，所述试剂还带有可检测的标记。
在某些优选的实施方案中，所述试剂通过免疫学检测来测定所述样品中IL-6的水平。进一步，在某些优选的实施方案中，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
在某些优选的实施方案中，所述试剂包括抗IL-6的抗体或其抗原结合片段。进一步，在某些优选的实施方案中，所述试剂通过ELISA来测定IL-6的水平。
在某些优选的实施方案中，所述抗IL-6的抗体为单克隆抗体或多克隆抗体。进一步，在某些优选的实施方案中，所述抗IL-6的抗体为IgG抗体或IgM抗体。
在某些优选的实施方案中，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
1)采血装置，例如无热原真空采血管；
2)抗凝剂，例如肝素；
3)培养液或培养基；
4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
5)稀释液，例如磷酸盐缓冲液或生理盐水。
在某些优选的实施方案中，所述试剂盒通过包括下述步骤的方法来诊断所述受试者是否患有活动性结核：
(1)使用特异性刺激原刺激来自所述受试者的至少一份样品作为待测样品，同时将来自所述受试者的未经刺激的样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(2)使用能够检测IL-6的试剂测定步骤(1)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值；和
(3)将所述差值与参考值进行比较，或对所述差值进行统计学分析以获得统计分析值，并将该统计分析值与参考值进行比较，并判断所述受试者是否患有活动性结核；
其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层，并且可任选地包含其他组分，例如抗凝剂、稀释液等。
在某些优选的实施方案中，当该差值或由该差值得到的统计分析值大于参考值时，表明提供该样品的受试者患有活动性结核；当该差值或由该差值得到的统计分析值不大于参考值时，表明提供该样品的受试者未患有活动性结核。
在某些优选的实施方案中，在步骤(3)中，利用选自下列的统计模型对所述差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)。进一步，在某些优选的实施方案中，在步骤(3)中，使用Logistic回归模型对所述差值进行统计分析。
在某些优选的实施方案中，在步骤(1)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段。进一步，在某些优选的实施方案中，在步骤(1)中，使用RV0183和PlcD分别刺激至少两份样品作为待测样品。或，在某些优选的实施方案中，在步骤(1)中，使用一种或多种RV0183的抗原性片段共同刺激至少一份样品作为待测样品。
在某些优选的实施方案中，在步骤(1)中，将所述特异性刺激原置于培养基，例如细胞培养基，例如RPMI-1640培养基或DMEM培养基，然后用于刺激来自所述受试者的样品，以产生待测样品。
在某些优选的实施方案中，步骤(1)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品。进一步，在某些优选的实施方案中，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A。
在某些优选的实施方案中，在步骤(1)中，用不含有所述特异性刺激原和非特异性刺激原的培养基(例如细胞培养基，例如RPMI-1640培养基或DMEM培养基)温育或稀释来自所述受试者的样品，以产生阴性对照样品。
在某些优选的实施方案中，在步骤(1)之前，所述方法还包括下列步骤中的一项或多项：(a)使用采血装置从所述受试者获得样品；(b)使用抗凝剂处理采血装置或来自所 述受试者的样品；(c)使用培养液或培养基处理来自所述受试者的样品；和，(d)使用稀释液稀释来自所述受试者的样品。
在某些优选的实施方案中，在步骤(1)中，在细胞(例如，PBMC)活性较高的温度下(例如在36-38℃，例如在37℃左右)使用刺激原刺激来自所述受试者的样品。
在某些优选的实施方案中，在步骤(1)中，用刺激原刺激来自所述受试者的样品不少于12h，例如15-24h，例如20-24h。
在另一个方面，本发明提供了特异性刺激原在制备试剂盒中的用途，所述试剂盒用于判断一种疗法对活动性结核的治疗效果；其中，所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种。
在某些优选的实施方案中，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列。
在某些优选的实施方案中，所述特异性刺激原选自RV0183、PlcD或其组合。
在某些优选的实施方案中，所述特异性刺激原选自一种或多种RV0183的抗原性片段。进一步，在某些优选的实施方案中，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25。
在某些优选的实施方案中，所述特异性刺激原包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段。
任选地，所述特异性刺激原还包括下列抗原性片段的组合：
1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段，或
4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述特异性刺激原包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述试剂盒包括能够检测IL-6的试剂，例如能够和IL-6特异性结合的抗体、靶向多肽或核酸适体。
任选地，所述试剂还带有可检测的标记。
在某些优选的实施方案中，所述试剂通过免疫学检测来测定所述样品中IL-6的水平。进一步，在某些优选的实施方案中，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
在某些优选的实施方案中，所述试剂包括抗IL-6的抗体或其抗原结合片段。进一步，在某些优选的实施方案中，所述试剂通过ELISA来测定IL-6的水平。
在某些优选的实施方案中，所述抗IL-6的抗体为单克隆抗体或多克隆抗体。进一步，在某些优选的实施方案中，所述抗IL-6的抗体为IgG抗体或IgM抗体。
在某些优选的实施方案中，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
1)采血装置，例如无热原真空采血管；
2)抗凝剂，例如肝素；
3)培养液或培养基；
4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
5)稀释液，例如磷酸盐缓冲液或生理盐水；
在某些优选的实施方案中，所述试剂盒通过包括下述步骤的方法来判断一种疗法对活动性结核的治疗效果：
(1)在对受试者进行所述疗法之前，获得来自所述受试者的至少两份样品，作为治疗前样品；
(2)使用特异性刺激原刺激来自所述受试者的至少一份治疗前样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(3)使用能够检测IL-6的试剂测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
(4)对所述受试者进行所述疗法；
(5)在对所述受试者进行所述疗法之后，获得来自所述受试者的至少两份样品，作为治疗后样品；
(6)使用特异性刺激原刺激来自所述受试者的至少一份治疗后样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特 异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
(8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层，并且可任选地包含其他组分，例如抗凝剂、稀释液等。
在某些优选的实施方案中，当所述第二差值大于所述第一差值时，或当所述第二差值的统计分析值大于所述第一差值的统计分析值时，表明所述疗法对活动性结核的治疗无效；当所述第二差值小于所述第一差值时，或当所述第二差值的统计分析值小于所述第一差值的统计分析值时，表明所述疗法对活动性结核的治疗有效。
在某些优选的实施方案中，在步骤(8)中，利用选自下列的统计模型对所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)。进一步，在某些优选的实施方案中，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析。
在某些优选的实施方案中，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理(例如，在相同的条件下，使用相同的特异性刺激原进行处理)。在某些优选的实施方案中，在步骤(2)和(6)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段。进一步，在某些优选的实施方案中，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品。或，在某些优选的实施方案中，在步骤(2)和(6)中，使用一种或多种RV0183的抗原性片段共同刺激至少一份样品作为待测样品。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在某些优选的实施方案中，所述疗法包括对受试者施用抗结核药物，例如异烟肼、利 福平、链霉素、吡嗪酰胺、乙胺丁醇或其任何组合。
在某些优选的实施方案中，在步骤(2)和(6)中，将所述特异性刺激原置于培养基，例如细胞培养基，例如RPMI-1640培养基或DMEM培养基，然后用于刺激来自所述受试者的样品，以产生待测样品。
在某些优选的实施方案中，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品。进一步，在某些优选的实施方案中，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A。
在某些优选的实施方案中，在步骤(2)和(6)中，用不含有所述特异性刺激原和非特异性刺激原的培养基(例如细胞培养基，例如RPMI-1640培养基或DMEM培养基)温育或稀释来自所述受试者的样品，以产生阴性对照样品。
在某些优选的实施方案中，在步骤(1)中，使用采血装置获得来自所述受试者的治疗前样品。
在某些优选的实施方案中，在步骤(5)中，使用采血装置获得来自所述受试者的治疗后样品。
在某些优选的实施方案中，在进行步骤(1)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述受试者的样品；(b)使用培养液或培养基处理来自所述受试者的样品；和，(c)使用稀释液稀释来自所述受试者的样品。
在某些优选的实施方案中，在进行步骤(5)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述受试者的样品；(b)使用培养液或培养基处理来自所述受试者的样品；和，(c)使用稀释液稀释来自所述受试者的样品。
在某些优选的实施方案中，在步骤(2)和(6)中，在细胞(例如，PBMC)活性较高的温度下(例如在36-38℃，例如在37℃左右)使用刺激原刺激来自所述受试者的样品。
在某些优选的实施方案中，在步骤(2)和(6)中，用刺激原刺激来自所述受试者的样品不少于12h，例如15-24h，例如20-24h。
在另一个方面，本发明提供了特异性刺激原在制备试剂盒中的用途，所述试剂盒用于筛选能够治疗活动性结核的候选药物；其中，所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种。
在某些优选的实施方案中，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列。
在某些优选的实施方案中，所述特异性刺激原选自RV0183、PlcD或其组合。
在某些优选的实施方案中，所述特异性刺激原选自一种或多种RV0183的抗原性片段。进一步，在某些优选的实施方案中，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25。
在某些优选的实施方案中，所述特异性刺激原包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段。
任选地，所述特异性刺激原还包括下列抗原性片段的组合：
1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段，或
4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述特异性刺激原包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述试剂盒包括能够检测IL-6的试剂，例如能够和IL-6特异性结合的抗体、靶向多肽或核酸适体。
任选地，所述试剂还带有可检测的标记。
在某些优选的实施方案中，所述试剂通过免疫学检测来测定所述样品中IL-6的水平。进一步，在某些优选的实施方案中，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
在某些优选的实施方案中，所述试剂包括抗IL-6的抗体或其抗原结合片段。进一步，在某些优选的实施方案中，所述试剂通过ELISA来测定IL-6的水平。
在某些优选的实施方案中，所述抗IL-6的抗体为单克隆抗体或多克隆抗体。进一步，在某些优选的实施方案中，所述抗IL-6的抗体为IgG抗体或IgM抗体。
在某些优选的实施方案中，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
1)采血装置，例如无热原真空采血管；
2)抗凝剂，例如肝素；
3)培养液或培养基；
4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
5)稀释液，例如磷酸盐缓冲液或生理盐水。
在某些优选的实施方案中，所述试剂盒通过包括下述步骤的方法来筛选能够治疗活动性结核的候选药物：
(1)在给模型动物施用候选药物之前，获得来自所述动物的至少两份样品，作为治疗前样品；
(2)使用特异性刺激原刺激来自所述动物的至少一份治疗前样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(3)使用检测IL-6的试剂测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
(4)给所述动物施用候选药物；
(5)在给所述动物施用所述候选药物之后，获得来自所述动物的至少两份样品，作为治疗后样品；
(6)使用特异性刺激原刺激来自所述动物的至少一份治疗后样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
(8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层，并且可任选地包含其他组分，例如抗凝剂、稀释液等。
在某些优选的实施方案中，当所述第二差值大于所述第一差值时，或当所述第二差值的统计分析值大于所述第一差值的统计分析值时，表明被筛选的药物对治疗活动性结核无效；当所述第二差值小于所述第一差值时，或当所述第二差值的统计分析值小于所述第一差值的统计分析值时，表明被筛选的药物对活动性结核的治疗有效。
在某些优选的实施方案中，在步骤(8)中，利用选自下列的统计模型对所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)。进一步，在某些优选的实施方案中，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析。
在某些优选的实施方案中，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理(例如，在相同的条件下，使用相同的特异性刺激原进行处理)。在某些优选的实施方案中，在步骤(2)和(6)中，使用至少两种特异性刺激原分别或共同刺激一份或多份来自所述动物的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段。进一步，在某些优选的实施方案中，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品。或，在某些优选的实施方案中，在步骤(2)和(6)中，使用一种或多种RV0183的抗原性片段共同刺激至少一份样品作为待测样品。
在某些优选的实施方案中，所述模型动物是非人哺乳动物，例如小鼠、豚鼠、兔或非人灵长类动物(例如食蟹猴或猕猴)。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在某些优选的实施方案中，在步骤(2)和(6)中，将所述特异性刺激原置于培养基，例如细胞培养基，例如RPMI-1640培养基或DMEM培养基，然后用于刺激来自所述动物的样品，以产生待测样品。
在某些优选的实施方案中，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品。进一步，在某些优选的实施方案中，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A。
在某些优选的实施方案中，在步骤(2)和(6)中，用不含有所述特异性刺激原和非特异性刺激原的培养基(例如细胞培养基，例如RPMI-1640培养基或DMEM培养基)温育或稀释来自所述动物的样品，以产生阴性对照样品。
在某些优选的实施方案中，在步骤(1)中，使用采血装置获得来自所述动物的治疗前样品。
在某些优选的实施方案中，在步骤(5)中，使用采血装置获得来自所述动物的治疗后样品。
在某些优选的实施方案中，在进行步骤(1)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述动物的样品；(b)使用培养液或培养基处理来自所述动物的样品；和，(c)使用稀释液稀释来自所述动物的样品。
在某些优选的实施方案中，在进行步骤(5)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述动物的样品；(b)使用培养液或培养基处理来自所述动物的样品；和，(c)使用稀释液稀释来自所述动物的样品。
在某些优选的实施方案中，在步骤(2)和(6)中，在细胞(例如，PBMC)活性较高的温度下(例如在36-38℃，例如在37℃左右)使用刺激原刺激来自所述动物的样品。
在某些优选的实施方案中，在步骤(2)和(6)中，用刺激原刺激来自所述动物的样品不少于12h，例如15-24h，例如20-24h。
在另一个方面，本发明提供了一种用于诊断受试者是否患有活动性结核的方法，其包括下述步骤：
(1)提供来自所述受试者的至少两份样品；
(2)使用特异性刺激原刺激至少一份样品作为待测样品，并将未经刺激的样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种；
(3)测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值；和
(4)将所述差值与参考值进行比较，或对所述差值进行统计学分析以获得统计分析值，并将该统计分析值与参考值进行比较，并判断所述受试者是否患有活动性结核；
其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层，并且可任选地包含其他组分，例如抗凝剂、稀释液等。
在某些优选的实施方案中，当该差值或由该差值得到的统计分析值大于参考值时，表 明提供该样品的受试者患有活动性结核；当该差值或由该差值得到的统计分析值不大于参考值时，表明提供该样品的受试者未患有活动性结核。
在某些优选的实施方案中，在步骤(4)中，利用选自下列的统计模型对所述差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)。进一步，在某些优选的实施方案中，在步骤(4)中，使用Logistic回归模型对所述差值进行统计分析。
在某些优选的实施方案中，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列。
在某些优选的实施方案中，所述抗原性片段为RV0183的抗原性片段。进一步，在某些优选的实施方案中，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在某些优选的实施方案中，在步骤(2)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段。
在某些优选的实施方案中，在步骤(2)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品。或，在某些优选的实施方案中，在步骤(2)中，使用一种或多种抗原性片段共同刺激至少一份样品作为待测样品。进一步，在某些优选的实施方案中，在步骤(2)中，使用下列抗原性片段的组合共同刺激至少一份样品作为待测样品：
1)分别具有如SEQ ID NO：13、14、19所示的氨基酸序列的抗原性片段，
2)分别具有如SEQ ID NO：5、11、13-14、19、22所示的氨基酸序列的抗原性片段，
3)分别具有如SEQ ID NO：7-8、11-14、19所示的氨基酸序列的抗原性片段，
4)分别具有如SEQ ID NO：5-7、11-14、19、22、24所示的氨基酸序列的抗原性片段，
5)分别具有如SEQ ID NO：5、8-10、12-15、19、22-25所示的氨基酸序列的抗原性片段；或
6)分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，在步骤(2)中，将所述特异性刺激原置于培养基，例如细胞培养基，例如RPMI-1640培养基或DMEM培养基，然后用于刺激来自所述受试者 的样品，以产生待测样品。
在某些优选的实施方案中，在步骤(2)中，用不含有所述特异性刺激原和非特异性刺激原的培养基(例如细胞培养基，例如RPMI-1640培养基或DMEM培养基)温育或稀释来自所述受试者的样品，以产生阴性对照样品。
在某些优选的实施方案中，在步骤(3)中，通过免疫学检测来测定所述样品中IL-6的水平。进一步，在某些优选的实施方案中，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
在某些优选的实施方案中，在步骤(3)中，使用抗IL-6的抗体或其抗原结合片段来检测IL-6的水平，例如通过ELISA来进行测定。
在某些优选的实施方案中，所述抗IL-6的抗体为单克隆抗体或多克隆抗体。进一步，在某些优选的实施方案中，所述抗IL-6的抗体为IgG抗体或IgM抗体。
在某些优选的实施方案中，步骤(2)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品。进一步，在某些优选的实施方案中，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A。
在某些优选的实施方案中，在步骤(1)之前，还包括下列步骤中的一项或多项：(a)从所述受试者获得样品；(b)向样品中加入抗凝剂，例如肝素；(c)从样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(d)向样品中加入培养液或培养基；(e)稀释样品。
在某些优选的实施方案中，在步骤(2)中，在细胞(例如，PBMC)活性较高的温度下(例如在36-38℃，例如在37℃左右)使用刺激原刺激来自所述受试者的样品。
在某些优选的实施方案中，在步骤(2)中，用刺激原刺激来自所述受试者的样品不少于12h，例如15-24h，例如20-24h。
在另一个方面，本发明提供了一种用于判断一种疗法对活动性结核的治疗效果的方法，其包括下述步骤：
(1)在对受试者进行所述疗法之前，获得来自所述受试者的至少两份样品，作为治疗前样品；
(2)使用特异性刺激原刺激来自所述受试者的至少一份治疗前样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特 异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(3)测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
(4)对所述受试者进行所述疗法；
(5)在对所述受试者进行所述疗法之后，获得来自所述受试者的至少两份样品，作为治疗后样品；
(6)使用特异性刺激原刺激来自所述受试者的至少一份治疗后样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
(8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层，并且可任选地包含其他组分，例如抗凝剂、稀释液等。
在某些优选的实施方案中，当所述第二差值大于所述第一差值时，或当所述第二差值的统计分析值大于所述第一差值的统计分析值时，表明所述疗法对活动性结核的治疗无效；当所述第二差值小于所述第一差值时，或当所述第二差值的统计分析值小于所述第一差值的统计分析值时，表明所述疗法对活动性结核的治疗有效。
在某些优选的实施方案中，在步骤(8)中，利用选自下列的统计模型对所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)。进一步，在某些优选的实施方案中，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析。
在某些优选的实施方案中，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列。
在某些优选的实施方案中，所述抗原性片段为RV0183的抗原性片段。进一步，在某些优选的实施方案中，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在某些优选的实施方案中，所述疗法包括对受试者施用抗结核药物，例如异烟肼、利福平、链霉素、吡嗪酰胺、乙胺丁醇或其任何组合。
在某些优选的实施方案中，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理(例如，在相同的条件下，使用相同的特异性刺激原进行处理)。在某些优选的实施方案中，在步骤(2)和(6)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段。
在某些优选的实施方案中，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品。或，在某些优选的实施方案中，在步骤(2)和(6)中，使用一种或多种抗原性片段共同刺激至少一份样品作为待测样品。进一步，在某些优选的实施方案中，在步骤(2)和(6)中，使用下列抗原性片段的组合共同刺激至少一份样品作为待测样品：
1)分别具有如SEQ ID NO：13、14、19所示的氨基酸序列的抗原性片段，
2)分别具有如SEQ ID NO：5、11、13-14、19、22所示的氨基酸序列的抗原性片段，
3)分别具有如SEQ ID NO：7-8、11-14、19所示的氨基酸序列的抗原性片段，
4)分别具有如SEQ ID NO：5-7、11-14、19、22、24所示的氨基酸序列的抗原性片段，
5)分别具有如SEQ ID NO：5、8-10、12-15、19、22-25所示的氨基酸序列的抗原性片段；或
6)分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，在步骤(2)和(6)中，将所述特异性刺激原置于培养基，例如细胞培养基，例如RPMI-1640培养基或DMEM培养基，然后用于刺激来自所述受试者的样品，以产生待测样品。
在某些优选的实施方案中，在步骤(2)和(6)中，用不含有所述特异性刺激原和非特异性刺激原的培养基(例如细胞培养基，例如RPMI-1640培养基或DMEM培养基) 温育或稀释来自所述受试者的样品，以产生阴性对照样品。
在某些优选的实施方案中，在步骤(3)中，通过免疫学检测来测定所述样品中IL-6的水平。进一步，在某些优选的实施方案中，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
在某些优选的实施方案中，在步骤(3)中，使用抗IL-6的抗体或其抗原结合片段来检测IL-6的水平，例如通过ELISA来进行测定。
在某些优选的实施方案中，所述抗IL-6的抗体为单克隆抗体或多克隆抗体。进一步，在某些优选的实施方案中，所述抗IL-6的抗体为IgG抗体或IgM抗体。
在某些优选的实施方案中，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品。进一步，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A。
在某些优选的实施方案中，在步骤(1)之前，还包括下列步骤中的一项或多项：(a)向治疗前样品中加入抗凝剂，例如肝素；(b)从治疗前样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗前样品中加入培养液或培养基；和，(d)稀释治疗前样品。
在某些优选的实施方案中，在步骤(5)之前，还包括下列步骤中的一项或多项：(a)向治疗后样品中加入抗凝剂，例如肝素；(b)从治疗后样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗后样品中加入培养液或培养基；和，(d)稀释治疗后样品。
在某些优选的实施方案中，在步骤(2)和(6)中，在细胞(例如，PBMC)活性较高的温度下(例如在36-38℃，例如在37℃左右)使用刺激原刺激来自所述受试者的样品。
在某些优选的实施方案中，在步骤(2)和(6)中，用刺激原刺激来自所述受试者的样品不少于12h，例如15-24h，例如20-24h。
在另一个方面，本发明提供了一种用于筛选能够治疗活动性结核的候选药物的方法，其包括下述步骤：
(1)在给模型动物施用候选药物之前，获得来自所述动物的至少两份样品，作为治疗前样品；
(2)使用特异性刺激原刺激来自所述动物的至少一份治疗前样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(3)测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
(4)给所述动物施用候选药物；
(5)在给所述动物施用所述候选药物之后，获得来自所述动物的至少两份样品，作为治疗后样品；
(6)使用特异性刺激原刺激来自所述动物的至少一份治疗后样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
(7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
(8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层，并且可任选地包含其他组分，例如抗凝剂、稀释液等。
在某些优选的实施方案中，当所述第二差值大于所述第一差值时，或当所述第二差值的统计分析值大于所述第一差值的统计分析值时，表明被筛选的药物对治疗活动性结核无效；当所述第二差值小于所述第一差值时，或当所述第二差值的统计分析值小于所述第一差值的统计分析值时，表明被筛选的药物对活动性结核的治疗有效。
在某些优选的实施方案中，在步骤(8)中，利用选自下列的统计模型对所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)。进一步，在某些优选的实施方案中，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析。
在某些优选的实施方案中，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列。
在某些优选的实施方案中，所述抗原性片段为RV0183的抗原性片段。进一步，在某些优选的实施方案中，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25。
在某些优选的实施方案中，所述模型动物是非人哺乳动物，例如小鼠、豚鼠、兔或非人灵长类动物(例如食蟹猴或猕猴)。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在某些优选的实施方案中，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理(例如，在相同的条件下，使用相同的特异性刺激原进行处理)。在某些优选的实施方案中，在步骤(2)和(6)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述动物的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段。
在某些优选的实施方案中，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品。或，在某些优选的实施方案中，在步骤(2)和(6)中，使用一种或多种抗原性片段共同刺激至少一份样品作为待测样品。进一步，在某些优选的实施方案中，在步骤(2)和(6)中，使用下列抗原性片段的组合共同刺激至少一份样品作为待测样品：
1)分别具有如SEQ ID NO：13、14、19所示的氨基酸序列的抗原性片段，
2)分别具有如SEQ ID NO：5、11、13-14、19、22所示的氨基酸序列的抗原性片段，
3)分别具有如SEQ ID NO：7-8、11-14、19所示的氨基酸序列的抗原性片段，
4)分别具有如SEQ ID NO：5-7、11-14、19、22、24所示的氨基酸序列的抗原性片段，
5)分别具有如SEQ ID NO：5、8-10、12-15、19、22-25所示的氨基酸序列的抗原性片段；或
6)分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，在步骤(2)和(6)中，将所述特异性刺激原置于培养基，例如细胞培养基，例如RPMI-1640培养基或DMEM培养基，然后用于刺激来自所述动物的样品，以产生待测样品。
在某些优选的实施方案中，在步骤(2)和(6)中，用不含有所述特异性刺激原和非特异性刺激原的培养基(例如细胞培养基，例如RPMI-1640培养基或DMEM培养基)温育或稀释来自所述动物的样品，以产生阴性对照样品。
在某些优选的实施方案中，在步骤(3)中，通过免疫学检测来测定所述样品中IL-6的水平。进一步，在某些优选的实施方案中，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
在某些优选的实施方案中，在步骤(3)中，使用抗IL-6的抗体或其抗原结合片段来检测IL-6的水平，例如通过ELISA来进行测定。
在某些优选的实施方案中，所述抗IL-6的抗体为单克隆抗体或多克隆抗体。进一步，在某些优选的实施方案中，所述抗IL-6的抗体为IgG抗体或IgM抗体。
在某些优选的实施方案中，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品。进一步，在某些优选的实施方案中，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A。
在某些优选的实施方案中，在步骤(1)之前，还包括下列步骤中的一项或多项：(a)向治疗前样品中加入抗凝剂，例如肝素；(b)从治疗前样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗前样品中加入培养液或培养基；和，(d)稀释治疗前样品。
在某些优选的实施方案中，在步骤(5)之前，还包括下列步骤中的一项或多项：(a)向治疗后样品中加入抗凝剂，例如肝素；(b)从治疗后样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗后样品中加入培养液或培养基；和，(d)稀释治疗后样品。
在某些优选的实施方案中，在步骤(2)和(6)中，在细胞(例如，PBMC)活性较高的温度下(例如在36-38℃，例如在37℃左右)使用刺激原刺激来自所述动物的样品。
在某些优选的实施方案中，在步骤(2)和(6)中，用刺激原刺激来自所述动物的样品不少于12h，例如15-24h，例如20-24h。
在另一个方面，本发明提供了一种多肽库，其包括：
第一肽，其具有如SEQ ID NO：13所示的氨基酸序列；
第二肽，其具有如SEQ ID NO：14所示的氨基酸序列；和
第三肽，其具有如SEQ ID NO：19所示的氨基酸序列。
任选地，所述多肽库还包括下列多肽的组合：
1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的多肽，
2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的多肽，
3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的多肽，或
4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的多肽。
在某些优选的实施方案中，所述多肽库包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段。
在某些优选的实施方案中，所述多肽库能够诱导样品产生IL-6；其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层，并且可任选地包含其他组分，例如抗凝剂、稀释液等。
在某些优选的实施方案中，所述多肽库用于诊断活动性结核、判断一种疗法对活动性结核的治疗效果或筛选能够治疗活动性结核的候选药物。
发明的有益效果
本发明通过大量实验和反复摸索发现了特异性刺激原(RV0183、PlcD或其抗原性片段)可刺激活动性结核患者的外周血产生大量IL-6，从而区分活动性结核和非活动性结核人群(例如，潜伏性结核感染者、陈旧性结核患者或非结核感染者)，由此建立了一种简单、方便、快速且具有高准确性和特异性的活动性结核诊断方法。
在某些优选的实施方案中，通过采集全血，并添加特异性刺激原，然后在适当条件下培养，即可完成对来自所述受试者样品的刺激；对实验条件、人员的技术能力、设备和环境的要求不高；与传统的结核诊断手段(如菌培、痰涂、X光检测等)相比，对活动性结核病人的检出具有较高的灵敏度和特异性，而且大大缩短了诊断所需的时间；成本不高，使用范围较广。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了SDS-PAGE分析的结果，其显示大肠杆菌(E.coil)ER2566中高效表达后经Ni-NTA和DEAE柱两步纯化的重组抗原RV0183。其中，泳道1代表蛋白质分子量标准，泳道2代表经两步纯化后的蛋白RV0183。
图2显示了SDS-PAGE分析的结果，其显示大肠杆菌(E.coil)ER2566中高效表达后经Ni-NTA纯化和包涵体复性后的重组抗原Plcd。其中，泳道M代表蛋白质分子量标准，泳道1代表变性后包涵体，泳道2代表经亲和柱层析后的蛋白Plcd，泳道4代表透析复性后的蛋白Plcd。
图3显示了使用重组抗原RV0183刺激受试者全血的IL-6水平分析结果。1μg重组抗原RV0183刺激500μl全血20±2h后，血浆标本2倍稀释。结果显示活动性结核病人IL-6分泌水平显著高于潜伏感染人群。(注：＊＊＊表示p＜0.001)
图4显示了使用重组抗原RV0183刺激受试者全血的IL-6水平分析结果。2μg重组抗原RV0183刺激1ml全血20±2h后，血浆标本5倍稀释。结果显示，活动性结核病人(aTB)的IL-6分泌水平显著高于结核分枝杆菌潜伏感染人群(LTBI(IGRA+体检))和非感染人群(IGRA-体检)。(注：＊＊＊表示p＜0.001)
图5显示了使用重组抗原RV0183刺激受试者全血的IL-6水平分析结果。2μg重组抗原RV0183刺激1ml全血20±2h后，血浆标本5倍稀释。结果显示，临床活动性结核病人IL-6分泌水平显著高于非结核分枝杆菌感染者。(注：＊＊＊表示p＜0.001)
图6显示了使用RV0183的抗原性片段刺激受试者全血的IL-6水平分析结果。结果显示，RV0183的21条抗原性片段，均能在体外刺激活动性结核病人全血中IL-6分泌水平的升高，而在健康对照中，基本无IL-6应答，绿色到红色表示反应强度增加。
图7A-7B分别显示了使用重组抗原RV0183和重组抗原PlcD分别刺激受试者全血的IL-6水平分析结果。2μg重组抗原RV0183和2μg重组抗原Plcd分别刺激1ml全血20±2h后，血浆标本5倍稀释。结果显示，重组抗原RV0183和重组抗原PlcD分别刺激后活动性结核病人IL-6分泌水平均显著高于潜伏感染人群和非感染人群，其中aTB代表活动性结核，HC代表健康对照。
图8显示了重组抗原RV0183和重组抗原Plcd联合检测的ROC分析结果。结果显示，重组抗原RV0183和重组抗原Plcd的联合检测能够提高检测活动性结核患者的灵敏 度和特异性。
图9显示了使用RV0183多肽库刺激受试者全血的IL-6水平分析结果。21条RV0183的抗原性片段组成的多肽库共同体外刺激临床结核病人、陈旧性结核个体、非结核肺病病例的全血标本。结果显示，在活动性结核病人中，IL-6分泌水平显著高于非活动性结核标本。(注：＊＊表示p＜0.01，＊＊＊表示p＜0.001)
序列信息
本发明涉及的序列信息提供于下面的表1中。
表1：序列信息
Figure PCTCN2017071462-appb-000002
重组蛋白RV0183的氨基酸序列(SEQ ID NO：1)
Figure PCTCN2017071462-appb-000003
重组蛋白RV0183的核苷酸序列(SEQ ID NO：2)
Figure PCTCN2017071462-appb-000004
Figure PCTCN2017071462-appb-000005
重组蛋白Plcd的氨基酸序列(SEQ ID NO：3)
Figure PCTCN2017071462-appb-000006
重组蛋白Plcd的核苷酸序列(SEQ ID NO：4)
Figure PCTCN2017071462-appb-000007
Figure PCTCN2017071462-appb-000008
RV0183多肽库肽段序列(P1-P21)(SEQ ID NO：5～25)
RV0183-p1：MTTTRTERNFAGIGDVRIVY(SEQ ID NO：5)
RV0183-p2：GDVRIVYDVWTPDTAPQAVV(SEQ ID NO：6)
RV0183-p3：TAPQAVVVLAHGLGEHARRY(SEQ ID NO：7)
RV0183-p4：GEHARRYDHVAQRLGAAGLV(SEQ ID NO：8)
RV0183-p5：LGAAGLVTYALDHRGHGRSG(SEQ ID NO：9)
RV0183-p6：RGHGRSGGKRVLVRDISEYT(SEQ ID NO：10)
RV0183-p7：RDISEYTADFDTLVGIATRE(SEQ ID NO：11)
RV0183-p8：VGIATREYPGCKRIVLGHSM(SEQ ID NO：12)
RV0183-p9：IVLGHSMGGGIVFAYGVERP(SEQ ID NO：13)
RV0183-p10：AYGVERPDNYDLMVLSAPAV(SEQ ID NO：14)
RV0183-p11：VLSAPAVAAQDLVSPVVAVA(SEQ ID NO：15)
RV0183-p12：SPVVAVAAKLLGVVVPGLPV(SEQ ID NO：16)
RV0183-p13：VVPGLPVQELDFTAISRDPE(SEQ ID NO：17)
RV0183-p14：AISRDPEVVQAYNTDPLVHH(SEQ ID NO：18)
RV0183-p15：TDPLVHHGRVPAGIGRALLQ(SEQ ID NO：19)
RV0183-p16：IGRALLQVGETMPRRAPALT(SEQ ID NO：20)
RV0183-p17：RRAPALTAPLLVLHGTDDRL(SEQ ID NO：21)
RV0183-p18：HGTDDRLIPIEGSRRLVECV(SEQ ID NO：22)
RV0183-p19：RRLVECVGSADVQLKEYPGL(SEQ ID NO：23)
RV0183-p20：LKEYPGLYHEVFNEPERNQV(SEQ ID NO：24)
RV0183-p21：EPERNQVLDDVVAWLTERL(SEQ ID NO：25)
引物(5’-3’)(SEQ ID NO：26～29)
plcD-1-F：TTCAACCATCGCCGCCTCTACCA(SEQ ID NO：26)
plcD-1-R：CCATCGCCGCCTCTACCAGT(SEQ ID NO：27)
plcD-2-F：GGATCCATGGATGCCGGCGTCAG(SEQ ID NO：28)
plcD-2-R：AAGCTTTTAGCACGGACCGCTCG(SEQ ID NO：29)
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法(例如，分子生物学实验方法和免疫检测法)。参见，例如，Sambrook等人，Molecular Cloning：A Laboratory Manual，第2版，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(1989)；和Ausubel等人，Current Protocols in Molecular Biology，Greene Publishing Associates(1992)，其全部通过引用合并入本文。按照制造商的说明书进行酶促反应和纯化技术，如在本领域内通常使用的或如本文描述的。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.重组蛋白RV0183的克隆、表达和去内毒素纯化
1.包含重组蛋白RV0183的编码序列的表达载体的构建
1.1.1目的基因片段的获得
目的基因片段为人工合成，为了能够方便的引入表达载体，目的基因片段合成时5’端添加BamHI酶切位点，3’端添加EcoRI酶切位点，载体为pMD18T，得到两端含有酶切位点的RV0183质粒。
酶切：使用BamHI/EcoRI双酶切人工构建的RV0183质粒。琼脂糖凝胶电泳回收酶切产物。
1.1.2目的基因片段的融合和表达载体的构建
使用BamHI/EcoRI双酶切的pTO-T7载体(载体信息参见罗文新，张军，杨海杰等，一种带增强子的原核高效表达载体的构建及初步应用，生物工程学报，2000，16(5)：578-581)和编码RV0183的酶切产物连接，获得含有RV0183基因片段的表达载体pTO-T7-RV0183。
2.重组蛋白RV0183的表达与去内毒素纯化
将构建好的pTO-T7-RV0183表达载体转化入大肠杆菌ER2566(实验室保存)，将其涂布于含卡那霉素(Kan，终浓度100μg/ml)的固体LB培养基(LB培养基成分：10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，下同)上，并在37℃下静置培养至单菌落清晰可辨。挑取单菌落至液体LB培养基(含100μg/ml卡那霉素)中，在37℃，180rpm下震荡培养8小时。然后将菌液转接至500ml液体LB培养基(含100ug/ml卡那霉素)的培养瓶中，在37℃，180rpm下震荡培养。当培养瓶中液体的OD600达0.6-0.8时，加入IPTG至0.2mM/L的终浓度并在37℃，180rpm下继续诱导培养4小时。然后5000rpm离心培养物10min，收集菌体。
由于重组蛋白RV0183用于刺激全血，因此需要去除蛋白产物中的内毒素。从菌体蛋白中纯化重组蛋白病去除内毒素的方法是本领域技术人员已知的。在本实施例中，使用下列示例性方法。
将收集的菌体用50mM TB8.0的缓冲液悬浮，置于冰浴中并通过超声波进行破碎，然后以12000rpm离心10min，收集包涵体。
Ni-NTA柱纯化：目的蛋白上清表达，利用自组装的Ni-NTA柱(介质厂家：Qiagen)进行纯化。简言之，将样品加载至Ni-NTA柱，用0.2％脱氧胆酸钠，50mMTB8.0洗涤，以去除部分内毒素，然后用200ml 50mMTB8.0洗涤，以去除脱氧胆酸钠；最后用洗脱液100ml(150mM咪唑，50mMTB8.0)洗脱目的蛋白。
DEAE柱纯化：上述洗脱蛋白，继续使用DEAE柱纯化。上柱缓冲液为50mMTB8.0，洗脱液为400mMNaCl，50mMTB8.0。洗脱蛋白透析至50mMTB8.0保存。
在上述蛋白纯化过程中，所使用的缓冲液均由注射用水配制，所用实验容器均经200℃高温干烤2小时以上。
利用SDS-PAGE检测经两步纯化的重组蛋白RV0183，结果示于图1中。结果显示，重组蛋白RV0183分子量约为30KD，并且经过上述两步纯化后，重组蛋白RV0183的纯度达到95％以上。
使用鲎试剂检测经纯化的重组蛋白RV0183中的内毒素。结果显示，经上述两步纯化，重组蛋白RV0183的内毒素含量低于100EU/mg。
实施例2.重组蛋白Plcd的克隆、表达和去内毒素纯化
2.1包含重组蛋白PlcD的编码序列的表达载体的构建
PCR扩增获得目的基因片段
由于结核分枝杆菌基因组片段长，GC碱基含量高，直接扩增目的片段比较困难，因此采用巢式PCR方法，通过两轮PCR，以结核分枝杆菌H37Rv基因组DNA为模板扩增获得目的片段。
2.1.1PCR引物的设计
根据NCBI中记录的结核分枝杆菌H37Rv菌种的PlcD蛋白对应的基因序列，使用软件对引物进行评估和筛选，并添加相应的酶切位点。引物设计结果如下：
表2：引物序列(SEQ ID NO：26-29)
Figure PCTCN2017071462-appb-000009
2.1.2第一轮PCR扩增反应
按下表在0.5ml Ependorf管中混匀各个成分，建立20μl体积PCR反应体系。
plcD蛋白编码基因第一轮PCR反应体系
Figure PCTCN2017071462-appb-000010
扩增程序：95℃预变性10min，热启动后转入95℃变性1min，55℃退火1min，72℃延伸3min，进行30个循环，72℃延伸10分钟。
2.1.3第二轮PCR扩增反应
将第一轮PCR产物作为模板，按下表在0.5ml Ependorf管中混匀各成分，组成50μlPCR反应体系。
plcD蛋白编码基因第二轮PCR反应体系
Figure PCTCN2017071462-appb-000011
扩增程序：95℃预变性10min，95℃变性1min，55℃退火1min，72℃延伸3min，进行30个循环后，72℃延伸10min。扩增产物用1％琼脂糖凝胶电泳鉴定并回收。
2.1.4克隆载体的构建
在0.5mL Ependorf管中加入如下成分混合，16℃条件下反应12小时，获得连接有目的基因片段的pMD18-T克隆载体。
Figure PCTCN2017071462-appb-000012
2.1.5克隆载体连接产物转化
取40μL感受态DH5α细胞和10μL克隆载体连接产物混合，冰浴吸附15min。42℃热休克90s，迅速冰浴终止反应。加入200μL无抗生素的LB培养基，37℃振荡培养45min。取200μL培养液涂含有氨苄青霉素的固体培养基，37℃培养12h。
2.1.6克隆载体质粒回收
挑取单克隆菌落接种于5mL加有氨苄青霉素的液体LB培养基，37℃振荡培养过夜。取1.5mL菌液于1.5mL Eppendorf管中，12000g离心1min，弃上清。加入250μL溶液I，振荡使沉淀完全悬浮。加入250μL溶液II，轻轻翻转几下，使菌体裂解。加入350μL溶液III，充分混匀，置冰浴15min。12000rpm离心10min。吸上清转移到平衡好的质粒回收吸附柱上，室温静置5min，后续回收操作同胶回收步骤。
回收产物进行1％琼脂糖电泳鉴定，确认回收成功。对得到含目的基因片段的pMD18-T载体进行酶切，回收克隆后的目的片段，并取部分克隆载体送测序。
2.1.7PlcD表达克隆构建
pMD-18T酶切的目的基因片段与酶切处理的PTO-T7载体连接，连接体系为PTO-T7载体1μl，目的基因片段6μl，10×T4DNA Ligase Buffer 1μl，T4DNA连接酶1μl，反应总体积10μl。连接时间为2h。
2.2表达克隆质粒的转化与蛋白表达纯化
2.2.1表达载体连接物转化
步骤同上述连接步骤，使用ER2566感受态细胞作为转化对象，转化后将ER2566涂布在含卡那霉素的LB抗性培养基上。
2.2.2Plcd蛋白表达及纯化
目的蛋白的提取
活化在小量表达鉴定有正确目标蛋白表达的菌株。37℃条件培养4h后加入1mmol/L IPTG，保持37℃诱导4h，以9000g离心6min收集菌体。菌体吹洗后重新离心收集沉淀，按每200mL菌液加入5mL的比例加入TB 8.0缓冲液重悬。重悬菌液进行超声破碎处理，处理时间按每500mL菌液3min计算。超声后样品以12000g离心10min收集破碎后的沉淀。破碎产物沉淀用TB 8.0溶液反复吹洗3次，每次重悬后在37℃温箱中震荡15min。沉淀用15mL含6M尿素的TB 8.0溶液重悬后充分吹打，将大部分沉淀吹起后12000g离心10min收集上清进入下一步纯化阶段。
镍离子柱亲和层析
Ni-NTA柱纯化：目的蛋白上清表达，利用自组装的Ni-NTA柱(介质厂家：Qiagen)进行纯化。将样品加载至Ni-NTA柱，用0.2％脱氧胆酸钠，50mMTB8.0+6M尿素洗涤，以去除部分内毒素，然后用200ml 50mMTB8.0+6M尿素洗涤，以去除脱氧胆酸钠；最后用洗脱液100ml(150mM咪唑，50mMTB8.0，6M尿素)洗脱目的蛋白。
蛋白质的复性
由于plcD蛋白属于包涵体表达，本实验使用6M尿素使包涵体变性溶解，亲和层析纯化后采用梯度透析的方法逐步去除变性剂，透析过程中加入二硫苏糖醇(DTT)防止形成错误二硫键，帮助蛋白形成正确折叠，保持可溶状态。透析步骤见下表，结果示于图2。
plcD蛋白质透析复性操作步骤
Figure PCTCN2017071462-appb-000013
内毒素的去除和检测
根据Shigui Liu等人的方法(Liu SG，Tobias R.Removal of endotoxin from  recombinant protein preparations[J].Clinical Biochem，1997，30：455-463)，本实验采用非离子型去垢剂Triton X-114萃取对重组蛋白进行内毒素去除。
向蛋白溶液中加入终浓度为1％(w/v)的Triton X-114，在4℃条件下混匀两相，震荡15min。将含Triton X-114的蛋白溶液置于37℃水浴10min，可见体系浑浊，溶液中有油滴出现。25℃ 12000g离心10min，小心移出水相。反复以上步骤一次，收集水相。处理过后的蛋白溶液采用鲎试剂凝胶法检测内毒素含量。
实施例3.RV0183特异性T细胞反应标志物的筛选
3.1刺激样品的收集和处理
3.1.1体外刺激样品收集
用肝素抗凝管采集4例IGRAs检测阳性的住院活动性结核病人外周血5ml，采集4例IGRAs检测阳性的无临床症状潜伏感染者外周血5ml。
3.1.2样品刺激处理和收集
样品分装至9个无内毒素2ml EP管中，每个EP管中500μl外周血；EP管已分别添加15μl培养液(培养液配方：30μl PBS中添加133.33mg/ml的D-葡萄糖和166.67mM的KCl；其中PBS配方为：Na2HPO4·12H2O 2.9g，KH2PO4 0.24g，NaCl 8g，KCl 0.2g，补超纯水至1L。)，编号分别为N、Ta、P。其中N管不含刺激抗原，Ta中含1μg刺激抗原RV0183，P管中含PHA 20ug。
外周血加入EP管后，颠倒混匀，至于37℃恒温箱培养20-24h；5000rpm收集血浆样品，检测血浆中细胞因子含量。
3.2血浆中细胞因子含量检测
收集的血浆样品使用商品化的Milliplex试剂盒(Merck Millipore，St.Charles，Missouri，USA)(货号：HCYTMAG-60K-PX38，HCP2MAG-62K-PX23，HCP3MAG-63K-PX11)进行细胞因子定量检测，检测指标包括EGF等69种(见表2)，检测平台为Luminex 200(以下步骤中的洗液、标准品、质控品、磁珠均来自试剂盒中)。
检测步骤包括：1)在96孔检测板上每孔加200μl洗液，室温震荡10min，移除洗液；2)在相应的孔中添加25μl标准品和质控品，双孔重复；3)添加25μl assay buffer到样品孔；4)在标准品孔、质控品孔和空白对照孔中，每孔加25μl血浆基质(基质指试剂盒中一种样品稀释液)；5)每孔加25μl血浆样品；6)每孔加25μl磁珠2-8℃过夜；7)移 除孔内液体，200μl/孔洗液，洗两遍；8)每孔加检测抗体25μl，室温孵育1h；9)每孔加25μl Steptavidin-Phycoerythrin，室温反应30min；10)移除孔内液体，洗板两次；11)每孔加150μl鞘液，震荡混匀；11)在Luminex 200平台上读板；12)将每个样品Ta管的实际检测值减去N管的实际检测值而得到的差值作为最终结果(即，细胞因子实际增加值，以Ta-N表示)。(方法为双抗体夹心法)
检测结果见表3。分析细胞因子在活动性结核病人和潜伏感染人群中应答情况的不同，筛选出IL-6能够有效区分结核感染者是处于活动性结核(aTB)状态还是潜伏感染(LTBI)状态(有临床症状的住院结核病人为活动性结核病人，IGRA检测阳性无临床症状的标本为潜伏感染者)。
表3
Figure PCTCN2017071462-appb-000014
Figure PCTCN2017071462-appb-000015
实施例4.在结核感染人群中利用RV0183筛选活动性结核病人
4.1队列标本信息
4.1.1活动性结核病人：均为临床确诊活动性结核病人，79例；2)结核分枝杆菌潜伏感染人群和健康人群：共37例医护工作者参与到对列中来，其中14例医护结核IGRAs实验结果阳性但是无临床结核症状且无结核病史，判定为潜伏感染者；另外23例IGRAs 检测结果阴性的医护工作者判定为无结核分枝杆菌感染的健康者。
4.2样本刺激培养体系
4.2.1全血样本分装到三个2ml EP管中，每管分装500μl，3个EP管分别标记为N、Ta、P；N管中为15μl培养液(配方同实施例4)，不含抗原，Ta管中的15μl培养液含1μg上述重组抗原RV0183，P管中的15μl培养液含20μg PHA。
4.2.2全血分装后轻轻颠倒混匀，至于37℃恒温箱中温育20±2h。
4.2.3培养完成后，5000rpm离心10min收集血浆，检测IL-6水平。
4.3血浆IL-6水平检测
IL-6ELISA检测试剂盒(厦门万泰沧海生物)用来检测血浆中IL-6水平。
1)血浆标本处理，用20％新生牛血清(NBS)2倍稀释；2)加样温育：96孔IL-6检测板上，每孔加处理后的血浆100μl，37℃反应40min，洗板五次，甩干检测板；3)加酶温育：每孔加酶标二抗100μl，37℃恒温箱反应40min，洗板5次，甩干检测板；4)显色：加显色液反应10min，50μl终止液终止反应；5)测定：酶标仪波长与450nm处检测吸光值；6)将每个样品Ta管的实际检测值减去N管的实际检测值而得到的差值作为最终结果(即，细胞因子实际增加值，以Ta-N表示)。
检测结果见图3，1μg重组抗原刺激500μl外周血细胞，在结核分枝杆菌潜伏人群和活动性结核病人中IL-6的分泌水平有显著的差别。
实施例5.利用RV0183在普通人群中筛选活动性结核病人
5.1队列标本信息
5.1.1活动性结核病人：临床确诊活动性结核病人207例；
5.1.2门诊体检者及医护人员：综合科门诊体检者标本65例，其中结核IGRAs阳性标本且无临床症状者为结核分枝杆菌潜伏感染者；其中结核IGRAs阴性标本为非结核分枝杆菌感染者。医护人员82例，其中结核IGRAs阳性标本且无临床症状者为结核分枝杆菌潜伏感染者；其中结核IGRAs阴性标本为非结核分枝杆菌感染者。
5.2样本采集和样本刺激培养体系
5.2.1每位受检者采集3ml外周血，全血分装到3个2ml EP管中，每个EP管中分装1ml；3个EP管分别标为N、Ta、P，每个EP管中添加30μl培养液(配方同实施例4)；其中N管中添加的培养液不含抗原，Ta管中添加的30μl培养液含2μg上述重组抗原 RV0183，P管中添加的30μl培养液含40μg PHA；
5.2.2全血分装后，轻轻颠倒混匀，置于37℃恒温箱中静置培养20±2h；
5.2.3培养结束后，将EP管置于离心机，5000rpm离心10min，收集血浆标本，进行IL-6分泌水平检测；
5.3血浆中IL-6分泌水平检测
IL-6ELISA检测试剂盒(厦门万泰沧海生物)用来检测血浆中IL-6水平。
IL-6检测方法与实施例4中描述的方法相同。
检测结果见图4，RV0183刺激外周血细胞后，IL-6的分泌水平在活动性结核病人和结核分枝杆菌潜伏人群中有显著的差别；同时在活动性结核病人与非分枝结核杆菌感染者中IL-6的水平同样存在显著差别，结果见图5。上述结果表明，RV0183可用于活动性结核病人的检测。
实施例6.利用RV0183的抗原性片段筛选活动性结核病人
6.1队列标本信息
同实施例5。
6.2样本采集和样本刺激培养体系
6.2.1收集临床活动性结核标本，每例标本采集全血15ml，将全血分装至22个EP管中每管500μl全血，其中一个EP管为阴性对照管(N)，另外21支EP管编号为P1、P2、P3…P21，在P1管中添加1μg肽段RV0183-p1，P2管中添加1μg肽段RV0183-p2，依次类推，在P21管中添加1μg肽段RV0183-p21。
6.2.2全血分装后，轻轻颠倒混匀，置于37℃恒温箱中静置培养20±2h；
6.2.3培养结束后，将EP管置于离心机，5000rpm离心10min，收集血浆标本，进行IL-6分泌水平检测；
6.3血浆中IL-6分泌水平检测
IL-6ELISA检测试剂盒(厦门万泰沧海生物)用来检测血浆中IL-6水平。
IL-6检测方法与实施例4中描述的方法相同。
检测结果见图6，RV0183的抗原性片段(p1-p21)刺激的活动性结核病人全血中IL-6分泌水平出现不同程度的升高，而在健康对照中，基本无IL-6应答，结果说明上述抗原性片段可用于诊断活动性结核。
实施例7.利用RV0183和PlcD联合检测筛选活动性结核病人
7.1队列标本信息
aTB(IGRA+)代表TB-IGRA检测阳性的临床确诊活动性结核病人，aTB(IGRA-)代表TB-IGRA检测阴性的临床确诊活动性结核病人。HC(IGRA+)代表TB-IGRA检测阳性的健康体检者，HC(IGRA-)代表TB-IGRA检测阴性的健康体检者。其中aTB(IGRA+)标本55例，aTB(IGRA-)标本9例，HC(IGRA+)标本13例，HC(IGRA-)标本52例。
7.2样本采集和样本刺激培养体系
7.2.1每位受检者采集4ml外周血，全血分装到4个2mlEP管中，每个EP管中分装1ml；4个EP管分别标为N、Ta1、Ta2、P，每个EP管中添加30μl培养液(配方同实施例4)；其中N管中添加的培养液不含抗原，Ta1管中添加的30μl培养液含2μg上述重组抗原RV0183，Ta2中添加的30μl培养液含2μg重组抗原Plcd，P管中添加的30μl培养液含40μg PHA；
7.2.2全血分装后，轻轻颠倒混匀，置于37℃恒温箱中静置培养20±2h；
7.2.3培养结束后，将EP管置于离心机，5000rpm离心10min，收集血浆标本，进行IL-6分泌水平检测；
7.3血浆中IL-6分泌水平检测
IL-6ELISA检测试剂盒(厦门万泰沧海生物)用来检测血浆中IL-6水平。
IL-6检测方法与实施例4中描述的方法相同。
检测结果见图7A-7B，使用RV0183和Plcd分别刺激受试者全血后得到的IL-6实际变化水平在活动性结核病人筛查中具有互补效果。进一步用ROC分析来检验单独使用RV0183或PlcD以及两者联合检测在活动性结核诊断方面的性能。ROC曲线显示，RV0183和PlcD联合检测可以提高检测的灵敏度和特异性，结果见图8。
由上述结果可知，RV0183和Plcd刺激导致的IL-6实际变化水平可以单独或者联合用于活动性结核病人检测，具有较高的灵敏度和特异性。
实施例8.结核分枝杆菌的其它抗原用于活动性结核筛选诊断的价值探讨
8.1队列标本信息
收集临床诊断确诊活动性结核病人5例，无临床症状的潜伏感染者5例采集全血标本。
8.2样本采集和样本刺激培养体系
8.2.1采集全血标本10ml，分装至9只EP管中，每管1ml全血。其中1只EP管为阴性对照管(N)。其它8只EP管中分别添加2μg表3所列出的7种抗原中的一种；
8.2.2全血分装后，轻轻颠倒混匀，置于37℃恒温箱中静置培养20±2h；
8.2.3培养结束后，将EP管置于离心机，5000rpm离心10min，收集血浆标本，进行IL-6分泌水平检测；
8.3血浆中IL-6分泌水平检测
IL-6ELISA检测试剂盒(厦门万泰沧海生物)用来检测血浆中IL-6水平。
IL-6检测方法与实施例4中描述的方法相同。
检测结果见表4，ESAT-6、CFP-10、RV1009、RV1884c、RV2389c、RV2450c、RV3097c、RV3542这些抗原刺激后样品中IL-6水平在活动性结核(aTB)和潜伏感染(LTBI)中没有显著性变化，也即，上述抗原不能用于区分活动性结核和潜伏感染。
表4
Figure PCTCN2017071462-appb-000016
实施例9.利用RV0183多肽库在不同类型肺病患者标本中筛选活动性结核患者
9.1队列标本信息
9.1.1活动性结核病例：临床确诊活动性结核患者101例；
9.1.2陈旧性结核病例：无临床症状陈旧性结核患者19例；
9.1.3临床非结核肺病病例：其中IGRA检测阳性病例为结核分枝杆菌潜伏性感染者；其中IGRA检测阴性病例为非结核分枝杆菌感染者。
9.2样本采集和样本刺激培养体系
9.2.1样本采集：样本采集处理同实施例5。
9.2.2样本刺激培养：使用含有21条RV0183抗原性片段(SEQ ID NO：5-25)的多肽库作为刺激源标记(Ta)，刺激全血。将多肽库中的每种多肽配置成1μg/mL溶液，将各上述溶液等量混合后，取30μL进行试验。其它操作同实施例5。
9.3IL-6水平检测
同实施例5。
检测结果见图9，RV0183多肽库刺激全血标本后，活动性结核患者IL-6水平显著高于陈旧性肺结核和其它肺病患者，表明该多肽库可用于筛选活动性结核病人。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (8)

  1. 一种试剂盒，其包括RV0183、PlcD或其抗原性片段中的一种或数种，和
    能够检测IL-6的试剂；
    优选地，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述试剂盒包含RV0183和/或PlcD；
    优选地，所述试剂盒包含一种或多种RV0183的抗原性片段；更优选地，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25；
    优选地，所述试剂盒包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段；任选地，所述试剂盒还包括下列抗原性片段的组合：
    1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
    2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
    3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段，或
    4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段；
    优选地，所述试剂盒包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，所述能够检测IL-6的试剂为能够和IL-6特异性结合的物质，例如抗体、靶向多肽或核酸适体；任选地，所述试剂还带有可检测的标记；
    优选地，所述试剂通过免疫学检测来测定所述样品中IL-6的水平；更优选地，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法；
    优选地，所述试剂包括抗IL-6的抗体或其抗原结合片段；更优选地，所述试剂通过ELISA来测定IL-6的水平；
    优选地，所述抗IL-6的抗体为单克隆抗体或多克隆抗体；
    优选地，所述抗IL-6的抗体为IgG抗体或IgM抗体；
    优选地，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
    1)采血装置，例如无热原真空采血管；
    2)抗凝剂，例如肝素；
    3)培养液或培养基；
    4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
    5)稀释液，例如磷酸盐缓冲液或生理盐水；
    优选地，所述试剂盒用于诊断活动性结核、判断一种疗法对活动性结核的治疗效果或筛选能够治疗活动性结核的候选药物。
  2. 特异性刺激原在制备试剂盒中的用途，所述试剂盒用于诊断活动性结核；其中，所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种；
    优选地，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述特异性刺激原选自RV0183、PlcD或其组合；
    优选地，所述特异性刺激原选自一种或多种RV0183的抗原性片段；更优选地，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25；
    优选地，所述特异性刺激原包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段；任选地，所述特异性刺激原还包括下列抗原性片段的组合：
    1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
    2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
    3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段，或
    4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段；
    优选地，所述特异性刺激原包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，所述试剂盒包括能够检测IL-6的试剂，例如能够和IL-6特异性结合的抗体、靶向多肽或核酸适体；任选地，所述试剂还带有可检测的标记；
    优选地，所述试剂通过免疫学检测来测定所述样品中IL-6的水平；更优选地，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法；
    优选地，所述试剂包括抗IL-6的抗体或其抗原结合片段；更优选地，所述试剂通过 ELISA来测定IL-6的水平；
    优选地，所述抗IL-6的抗体为单克隆抗体或多克隆抗体；
    优选地，所述抗IL-6的抗体为IgG抗体或IgM抗体；
    优选地，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
    1)采血装置，例如无热原真空采血管；
    2)抗凝剂，例如肝素；
    3)培养液或培养基；
    4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
    5)稀释液，例如磷酸盐缓冲液或生理盐水；
    优选地，所述试剂盒通过包括下述步骤的方法来诊断所述受试者是否患有活动性结核：
    (1)使用特异性刺激原刺激来自所述受试者的至少一份样品作为待测样品，同时将来自所述受试者的未经刺激的样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (2)使用能够检测IL-6的试剂测定步骤(1)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值；和
    (3)将所述差值与参考值进行比较，或对所述差值进行统计学分析以获得统计分析值，并将该统计分析值与参考值进行比较，并判断所述受试者是否患有活动性结核；
    其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层；
    优选地，在步骤(3)中，利用选自下列的统计模型对所述差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)；更优选地，在步骤(3)中，使用Logistic回归模型对所述差值进行统计分析；
    优选地，在步骤(1)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段；更优选地，在步骤(1)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品；或，在步骤(1)中，使用一种或多种RV0183的抗原性片段共同刺激至少一份样品作为待测样品；
    优选地，步骤(1)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品；更优选地，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A；
    优选地，在步骤(1)之前，所述方法还包括下列步骤中的一项或多项：(a)使用采血装置从所述受试者获得样品；(b)使用抗凝剂处理采血装置或来自所述受试者的样品；(c)使用培养液或培养基处理来自所述受试者的样品；和，(d)使用稀释液稀释来自所述受试者的样品。
  3. 特异性刺激原在制备试剂盒中的用途，所述试剂盒用于判断一种疗法对活动性结核的治疗效果；其中，所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种；
    优选地，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述特异性刺激原选自RV0183、PlcD或其组合；
    优选地，所述特异性刺激原选自一种或多种RV0183的抗原性片段；更优选地，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25；
    优选地，所述特异性刺激原包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段；任选地，所述特异性刺激原还包括下列抗原性片段的组合：
    1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
    2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
    3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段，或
    4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段；
    优选地，所述特异性刺激原包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，所述试剂盒包括能够检测IL-6的试剂，例如能够和IL-6特异性结合的抗体、靶向多肽或核酸适体；任选地，所述试剂还带有可检测的标记；
    优选地，所述试剂通过免疫学检测来测定所述样品中IL-6的水平；更优选地，所述 免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法；
    优选地，所述试剂包括抗IL-6的抗体或其抗原结合片段；更优选地，所述试剂通过ELISA来测定IL-6的水平；
    优选地，所述抗IL-6的抗体为单克隆抗体或多克隆抗体；
    优选地，所述抗IL-6的抗体为IgG抗体或IgM抗体；
    优选地，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
    1)采血装置，例如无热原真空采血管；
    2)抗凝剂，例如肝素；
    3)培养液或培养基；
    4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
    5)稀释液，例如磷酸盐缓冲液或生理盐水；
    优选地，所述试剂盒通过包括下述步骤的方法来判断一种疗法对活动性结核的治疗效果：
    (1)在对受试者进行所述疗法之前，获得来自所述受试者的至少两份样品，作为治疗前样品；
    (2)使用特异性刺激原刺激来自所述受试者的至少一份治疗前样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (3)使用能够检测IL-6的试剂测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
    (4)对所述受试者进行所述疗法；
    (5)在对所述受试者进行所述疗法之后，获得来自所述受试者的至少两份样品，作为治疗后样品；
    (6)使用特异性刺激原刺激来自所述受试者的至少一份治疗后样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
    (8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行 统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
    其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层；
    优选地，在步骤(8)中，利用选自下列的统计模型对所述所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)；更优选地，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析；
    优选地，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理；优选地，在步骤(2)和(6)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段；更优选地，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品；或，在步骤(2)和(6)中，使用一种或多种RV0183的抗原性片段共同刺激至少一份样品作为待测样品；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，所述疗法包括对受试者施用抗结核药物，例如异烟肼、利福平、链霉素、吡嗪酰胺、乙胺丁醇或其任何组合；
    优选地，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品；更优选地，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A；
    优选地，在步骤(1)中，使用采血装置获得来自所述受试者的治疗前样品；
    优选地，在步骤(5)中，使用采血装置获得来自所述受试者的治疗后样品；
    优选地，在进行步骤(1)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述受试者的样品；(b)使用培养液或培养基处理来自所述受试者的样品；和，(c)使用稀释液稀释来自所述受试者的样品；
    优选地，在进行步骤(5)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述受试者的样品；(b)使用培养液或培养基处理来自所述受试者的样品；和，(c)使用稀释液稀释来自所述受试者的样品。
  4. 特异性刺激原在制备试剂盒中的用途，所述试剂盒用于筛选能够治疗活动性结核的候选药物；其中，所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种；
    优选地，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述特异性刺激原选自RV0183、PlcD或其组合；
    优选地，所述特异性刺激原选自一种或多种RV0183的抗原性片段；更优选地，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25；
    优选地，所述特异性刺激原包含分别具有如SEQ ID NO：13、14和19所示的氨基酸序列的抗原性片段；任选地，所述特异性刺激原还包括下列抗原性片段的组合：
    1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的抗原性片段，
    2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的抗原性片段，
    3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的抗原性片段，或
    4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的抗原性片段；
    优选地，所述特异性刺激原包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，所述试剂盒包括能够检测IL-6的试剂，例如能够和IL-6特异性结合的抗体、靶向多肽或核酸适体；任选地，所述试剂还带有可检测的标记；
    优选地，所述试剂通过免疫学检测来测定所述样品中IL-6的水平；更优选地，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法；
    优选地，所述试剂包括抗IL-6的抗体或其抗原结合片段；更优选地，所述试剂通过ELISA来测定IL-6的水平；
    优选地，所述抗IL-6的抗体为单克隆抗体或多克隆抗体；
    优选地，所述抗IL-6的抗体为IgG抗体或IgM抗体；
    优选地，所述试剂盒还包含一种或多种选自1)-5)的装置或试剂：
    1)采血装置，例如无热原真空采血管；
    2)抗凝剂，例如肝素；
    3)培养液或培养基；
    4)非特异性刺激原，例如植物凝集素或刀豆球蛋白A；
    5)稀释液，例如磷酸盐缓冲液或生理盐水；
    优选地，所述试剂盒通过包括下述步骤的方法来筛选能够治疗活动性结核的候选药物：
    (1)在给模型动物施用候选药物之前，获得来自所述动物的至少两份样品，作为治疗前样品；
    (2)使用特异性刺激原刺激来自所述动物的至少一份治疗前样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (3)使用检测IL-6的试剂测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
    (4)给所述动物施用候选药物；
    (5)在给所述动物施用所述候选药物之后，获得来自所述动物的至少两份样品，作为治疗后样品；
    (6)使用特异性刺激原刺激来自所述动物的至少一份治疗后样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
    (8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
    其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层；
    优选地，在步骤(8)中，利用选自下列的统计模型对所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、 最近邻模型或微阵列预测分析(PAM)；更优选地，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析。
    优选地，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理；优选地，在步骤(2)和(6)中，使用至少两种特异性刺激原分别或共同刺激一份或多份来自所述动物的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段；更优选地，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品；或，在步骤(2)和(6)中，使用一种或多种RV0183的抗原性片段共同刺激至少一份样品作为待测样品；
    优选地，所述模型动物是非人哺乳动物，例如小鼠、豚鼠、兔或非人灵长类动物(例如食蟹猴或猕猴)；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品；更优选地，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A；
    优选地，在步骤(1)中，使用采血装置获得来自所述动物的治疗前样品；
    优选地，在步骤(5)中，使用采血装置获得来自所述动物的治疗后样品；
    优选地，在进行步骤(1)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述动物的样品；(b)使用培养液或培养基处理来自所述动物的样品；和，(c)使用稀释液稀释来自所述动物的样品；
    优选地，在进行步骤(5)之前，所述方法还包括下列步骤中的一项或多项：(a)使用抗凝剂处理采血装置或来自所述动物的样品；(b)使用培养液或培养基处理来自所述动物的样品；和，(c)使用稀释液稀释来自所述动物的样品。
  5. 用于诊断受试者是否患有活动性结核的方法，其包括下述步骤：
    (1)提供来自所述受试者的至少两份样品；
    (2)使用特异性刺激原刺激至少一份样品作为待测样品，并将未经刺激的样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或多种；
    (3)测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值；和
    (4)将所述差值与参考值进行比较，或对所述差值进行统计学分析以获得统计分析值，并将该统计分析值与参考值进行比较，并判断所述受试者是否患有活动性结核；
    其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层；
    优选地，在步骤(4)中，利用选自下列的统计模型对所述差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)；更优选地，在步骤(4)中，使用Logistic回归模型对所述差值进行统计分析；
    优选地，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述抗原性片段为RV0183的抗原性片段；更优选地，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，在步骤(2)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段；
    优选地，在步骤(2)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品；或，在步骤(2)中，使用一种或多种抗原性片段共同刺激至少一份样品作为待测样品；进一步优选地，在步骤(2)中，使用下列抗原性片段的组合共同刺激至少一份样品作为待测样品：
    1)分别具有如SEQ ID NO：13、14、19所示的氨基酸序列的抗原性片段，
    2)分别具有如SEQ ID NO：5、11、13-14、19、22所示的氨基酸序列的抗原性片段，
    3)分别具有如SEQ ID NO：7-8、11-14、19所示的氨基酸序列的抗原性片段，
    4)分别具有如SEQ ID NO：5-7、11-14、19、22、24所示的氨基酸序列的抗原性片段，
    5)分别具有如SEQ ID NO：5、8-10、12-15、19、22-25所示的氨基酸序列的抗原性片段；或
    6)分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，在步骤(3)中，通过免疫学检测来测定所述样品中IL-6的水平；进一步优选地，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法；
    优选地，在步骤(3)中，使用抗IL-6的抗体或其抗原结合片段来检测IL-6的水平，例如通过ELISA来进行测定；
    优选地，所述抗IL-6的抗体为单克隆抗体或多克隆抗体；
    优选地，所述抗IL-6的抗体为IgG抗体或IgM抗体；
    优选地，步骤(2)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品；更优选地，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A；
    优选地，在步骤(1)之前，还包括下列步骤中的一项或多项：(a)从所述受试者获得样品；(b)向样品中加入抗凝剂，例如肝素；(c)从样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(d)向样品中加入培养液或培养基；(e)稀释样品。
  6. 用于判断一种疗法对活动性结核的治疗效果的方法，其包括下述步骤：
    (1)在对受试者进行所述疗法之前，获得来自所述受试者的至少两份样品，作为治疗前样品；
    (2)使用特异性刺激原刺激来自所述受试者的至少一份治疗前样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (3)测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
    (4)对所述受试者进行所述疗法；
    (5)在对所述受试者进行所述疗法之后，获得来自所述受试者的至少两份样品，作为治疗后样品；
    (6)使用特异性刺激原刺激来自所述受试者的至少一份治疗后样品作为待测样品，同时将来自所述受试者的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述 待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
    (8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
    其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层；
    优选地，在步骤(8)中，利用选自下列的统计模型对所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)；更优选地，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析；
    优选地，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述抗原性片段为RV0183的抗原性片段；更优选地，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，所述疗法包括对受试者施用抗结核药物，例如异烟肼、利福平、链霉素、吡嗪酰胺、乙胺丁醇或其任何组合；
    优选地，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理；优选地，在步骤(2)和(6)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段；
    优选地，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品；或，在步骤(2)和(6)中，使用一种或多种抗原性片段共同刺激至少一份样品作为待测样品；进一步优选地，在步骤(2)和(6)中，使用下列抗原性片段的组合共同刺激至少一份样品作为待测样品：
    1)分别具有如SEQ ID NO：13、14、19所示的氨基酸序列的抗原性片段，
    2)分别具有如SEQ ID NO：5、11、13-14、19、22所示的氨基酸序列的抗原性片段，
    3)分别具有如SEQ ID NO：7-8、11-14、19所示的氨基酸序列的抗原性片段，
    4)分别具有如SEQ ID NO：5-7、11-14、19、22、24所示的氨基酸序列的抗原性片段，
    5)分别具有如SEQ ID NO：5、8-10、12-15、19、22-25所示的氨基酸序列的抗原性片段；或
    6)分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，在步骤(3)中，通过免疫学检测来测定所述样品中IL-6的水平；进一步优选地，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法；
    优选地，在步骤(3)中，使用抗IL-6的抗体或其抗原结合片段来检测IL-6的水平，例如通过ELISA来进行测定；
    优选地，所述抗IL-6的抗体为单克隆抗体或多克隆抗体；
    优选地，所述抗IL-6的抗体为IgG抗体或IgM抗体；
    优选地，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品；更优选地，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A；
    优选地，在步骤(1)之前，还包括下列步骤中的一项或多项：(a)向治疗前样品中加入抗凝剂，例如肝素；(b)从治疗前样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗前样品中加入培养液或培养基；和，(d)稀释治疗前样品；
    优选地，在步骤(5)之前，还包括下列步骤中的一项或多项：(a)向治疗后样品中加入抗凝剂，例如肝素；(b)从治疗后样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗后样品中加入培养液或培养基；和，(d)稀释治疗后样品。
  7. 用于筛选能够治疗活动性结核的候选药物的方法，其包括下述步骤：
    (1)在给模型动物施用候选药物之前，获得来自所述动物的至少两份样品，作为治疗前样品；
    (2)使用特异性刺激原刺激来自所述动物的至少一份治疗前样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗前样品作为阴性对照样品，其中所述特异性 刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (3)测定步骤(2)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第一差值；
    (4)给所述动物施用候选药物；
    (5)在给所述动物施用所述候选药物之后，获得来自所述动物的至少两份样品，作为治疗后样品；
    (6)使用特异性刺激原刺激来自所述动物的至少一份治疗后样品作为待测样品，同时将来自所述动物的未经刺激的至少一份治疗后样品作为阴性对照样品，其中所述特异性刺激原选自RV0183、PlcD或其抗原性片段中的一种或数种；
    (7)使用能够检测IL-6的试剂测定步骤(6)中各个样品的IL-6水平，并计算所述待测样品与阴性对照样品的IL-6水平的差值，作为第二差值；和
    (8)将所述第二差值与第一差值进行比较，或对所述第一差值和第二差值分别进行统计学分析以获得第一差值的统计分析值和第二差值的统计分析值，并将所述第二差值的统计分析值与所述第一差值的统计分析值进行比较，并判断所述疗法对活动性结核的治疗是否有效；
    其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层；
    优选地，在步骤(8)中，利用选自下列的统计模型对所述第一差值和第二差值进行统计分析：线性组合、线性回归模型、Logistic回归模型、线性判别分析(LDA)模型、最近邻模型或微阵列预测分析(PAM)；更优选地，在步骤(8)中，使用Logistic回归模型对所述第一差值和第二差值进行统计分析；
    优选地，所述RV0183具有如SEQ ID NO：1所示的氨基酸序列；和/或，所述PlcD具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述抗原性片段为RV0183的抗原性片段；更优选地，所述抗原性片段具有选自下列的氨基酸序列：SEQ ID NO：5-25；
    优选地，所述模型动物是非人哺乳动物，例如小鼠、豚鼠、兔或非人灵长类动物(例如食蟹猴或猕猴)；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，在步骤(2)和(6)中，对治疗前样品和治疗后样品进行相同的处理；优选 地，在步骤(2)和(6)中，使用至少两种特异性刺激原共同或分别刺激一份或多份来自所述受试者的样品作为待测样品，其中所述特异性刺激原各自独立地选自RV0183、PlcD或其抗原性片段；
    优选地，在步骤(2)和(6)中，使用RV0183、和PlcD分别刺激至少两份样品作为待测样品；或，在步骤(2)和(6)中，使用一种或多种抗原性片段共同刺激至少一份样品作为待测样品；进一步优选地，在步骤(2)和(6)中，使用下列抗原性片段的组合共同刺激至少一份样品作为待测样品：
    1)分别具有如SEQ ID NO：13、14、19所示的氨基酸序列的抗原性片段，
    2)分别具有如SEQ ID NO：5、11、13-14、19、22所示的氨基酸序列的抗原性片段，
    3)分别具有如SEQ ID NO：7-8、11-14、19所示的氨基酸序列的抗原性片段，
    4)分别具有如SEQ ID NO：5-7、11-14、19、22、24所示的氨基酸序列的抗原性片段，
    5)分别具有如SEQ ID NO：5、8-10、12-15、19、22-25所示的氨基酸序列的抗原性片段；或
    6)分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，在步骤(3)中，通过免疫学检测来测定所述样品中IL-6的水平；进一步优选地，所述免疫学检测选自ELISA检测、Elispot检测、Western印迹或表面等离子共振法；
    优选地，在步骤(3)中，使用抗IL-6的抗体或其抗原结合片段来检测IL-6的水平，例如通过ELISA来进行测定；
    优选地，所述抗IL-6的抗体为单克隆抗体或多克隆抗体；
    优选地，所述抗IL-6的抗体为IgG抗体或IgM抗体；
    优选地，步骤(2)和(6)还包括使用非特异性刺激原刺激至少一份样品作为阳性对照样品；更优选地，所述非特异性刺激原包括植物凝集素或刀豆球蛋白A；
    优选地，在步骤(1)之前，还包括下列步骤中的一项或多项：(a)向治疗前样品中加入抗凝剂，例如肝素；(b)从治疗前样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗前样品中加入培养液或培养基；和，(d)稀释治疗前样品；
    优选地，在步骤(5)之前，还包括下列步骤中的一项或多项：(a)向治疗后样品中加入抗凝剂，例如肝素；(b)从治疗后样品中获取PBMC或含有PBMC的血液成分(例如，外周血白膜层)；(c)向治疗后样品中加入培养液或培养基；和，(d)稀释治疗后样品。
  8. 多肽库，其包括：
    第一肽，其具有如SEQ ID NO：13所示的氨基酸序列；
    第二肽，其具有如SEQ ID NO：14所示的氨基酸序列；和
    第三肽，其具有如SEQ ID NO：19所示的氨基酸序列；
    任选地，所述多肽库还包括下列多肽的组合：
    1)分别具有如SEQ ID NO：5、11、22所示的氨基酸序列的多肽，
    2)分别具有如SEQ ID NO：7-8、11-12所示的氨基酸序列的多肽，
    3)分别具有如SEQ ID NO：5-7、11-12、22、24所示的氨基酸序列的多肽，或
    4)分别具有如SEQ ID NO：5、8-10、12、15、22-25所示的氨基酸序列的多肽；
    优选地，所述多肽库包含分别具有如SEQ ID NO：5-25所示的氨基酸序列的抗原性片段；
    优选地，所述多肽库能够诱导样品产生IL-6；其中，所述样品包含外周血单个核细胞(PBMC)，例如全血(例如抗凝全血)、外周血单个核细胞(PBMC)、或外周血白膜层；
    优选地，所述多肽库用于诊断活动性结核、判断一种疗法对活动性结核的治疗效果或筛选能够治疗活动性结核的候选药物。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110021367A1 (en) * 2009-07-27 2011-01-27 Gopal Balsubramanian Diagnosis and monitoring of mycobacterium tuberculosis infection
CN104017806A (zh) * 2014-05-08 2014-09-03 复旦大学 microRNA及其在制备活动性结核病检测试剂中的应用
CN104897893A (zh) * 2015-06-10 2015-09-09 复旦大学附属华山医院 一种基于结核特异性il-31检测的诊断结核分枝杆菌感染的试剂盒

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102004155B (zh) * 2010-02-12 2013-09-18 复旦大学附属华山医院 一种检测结核分枝杆菌感染的试剂、方法及应用
CN103122033B (zh) * 2011-11-21 2015-08-19 厦门大学 一种嵌合重组抗原及其用途
EP2687848A1 (en) * 2012-07-16 2014-01-22 Lionex GmbH Status of tuberculosis infection in an individual
CN103604933A (zh) * 2013-11-27 2014-02-26 华中科技大学同济医学院附属同济医院 基于抗原特异性TNF-α-ELISA检测活动性结核病的试剂盒及其应用
CN104020297B (zh) * 2014-06-10 2016-12-07 上海交通大学医学院 用于结核分枝杆菌感染检测及临床治疗效果监测的试剂盒及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110021367A1 (en) * 2009-07-27 2011-01-27 Gopal Balsubramanian Diagnosis and monitoring of mycobacterium tuberculosis infection
CN104017806A (zh) * 2014-05-08 2014-09-03 复旦大学 microRNA及其在制备活动性结核病检测试剂中的应用
CN104897893A (zh) * 2015-06-10 2015-09-09 复旦大学附属华山医院 一种基于结核特异性il-31检测的诊断结核分枝杆菌感染的试剂盒

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIA, HAO: "Jiéhé fēn zhī gǎn jūn Rv0183 dànbái duì fèipào jù shì xìbāo (RAW264.7) Shēngwù xué xiàoyìng de yánjiū = Study on the biological effects of Mycobacterium tuberculosis Rv0183 protein on alveolar macrophages (RAW264.7) ", MYCOBACTERIUM TUBERCULOSIS RV0183 PROTEIN ON THE BIOLOGY OF ALVEOLAR MACROPHAGES (RAW264.7) MASTER THESIS, 31 December 2010 (2010-12-31), pages 3, XP009513157 *
See also references of EP3407071A4 *
YANG, YONGHUI ET AL.: "Expression Levels of IL -6, IL -37 and TNF-a in Active Pulmonary Tuberculosis and Their Correlation", LABELED IMMUNOASSAYS AND CLINICAL MEDICINE, vol. 22, no. 12, 31 December 2015 (2015-12-31), pages 1239, XP009512090, ISSN: 1006-1703 *

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