CN106589082B

CN106589082B - Screening and application of active tuberculosis diagnostic molecules

Info

Publication number: CN106589082B
Application number: CN201510680521.0A
Authority: CN
Inventors: 潘卫庆; 徐新东; 周方斌
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2015-10-19
Filing date: 2015-10-19
Publication date: 2021-02-12
Anticipated expiration: 2035-10-19
Also published as: CN106589082A

Abstract

The invention provides a screening and application of active tuberculosis diagnostic molecules. Specifically, the invention discloses high-throughput screening of important antigens of mycobacterium tuberculosis and application of the important antigens in active tuberculosis diagnosis. The inventor screens 92 positive antigens which can be recognized by the serum of a tuberculosis patient based on a GST fusion expression high-throughput functional protein screening technology, wherein 14 antigens present strong positive reactions. The active tuberculosis serological detection shows that the positive antigens have higher sensitivity and specificity when used for detecting the active tuberculosis. The antigen combination composed of the 6 proteins TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP6 is used for tuberculosis serological detection, and laboratory verification shows that the combination has high sensitivity and specificity for detecting mycobacterium tuberculosis and has application value in tuberculosis diagnosis and monitoring.

Description

Screening and application of active tuberculosis diagnostic molecules

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to screening and application of active tuberculosis diagnostic molecules.

Background

Tuberculosis (Tuberculosis) is an infectious disease caused by infection of the Mycobacterium Tuberculosis Complex, mainly respiratory infections. Because of the invention of the BCG vaccine (BCG) and the wide application of the drugs for effectively treating tuberculosis, tuberculosis is effectively controlled once.

Since the 90 s of the last century, the globalization of large-scale population mobility, the abuse of antibiotics, the relaxation of consciousness of people in preventing tuberculosis, the resurgence of tuberculosis and the heavy scaring of soil become important infectious diseases seriously threatening human health. International organizations such as WHO make a thousand-year development target plan for suppressing tuberculosis prevalence in the early century, and although the new morbidity and mortality of tuberculosis are reduced to a certain extent, the effect is not significant, tuberculosis is not completely eliminated, and the prevention and control situation of tuberculosis in the world is still severe. It is estimated that nearly 1/3 people worldwide are infected with Mycobacterium tuberculosis, about 2000 million tuberculosis patients, 860 million new cases per year, and at least 130 million people die of the disease. At present, the countries with the most serious tuberculosis burden are mainly concentrated in Asia and Africa, and the number of tuberculosis patients in 22 countries with high tuberculosis burden is the second world in China.

In fact, the problem of tuberculosis is becoming more complex due to the emergence of multi-drug resistant tuberculosis strains, widely drug resistant tuberculosis strains, etc., the spread of HIV/AIDS in the tuberculosis epidemic, and the reduced immunoprotection of the traditional tuberculosis vaccine BCG.

The lack of an effective diagnostic method for tuberculosis infection is an important reason why tuberculosis is difficult to eradicate. The traditional tuberculosis diagnosis method has many defects, the detection rate of sputum smear microscopy is low, the culture of the sputum tubercle bacillus wastes time and labor, the imaging examination as an auxiliary means cannot accurately identify tiny focuses, and the like. Tuberculosis diagnosis techniques developed in recent years include PCR amplification techniques, rapid sputum culture, and novel imaging, but these techniques are cost-limited in developing countries and are not suitable for large-scale monitoring and screening.

TST is the most widely used tuberculosis infection detection means, but has low sensitivity, is easily influenced by BCG (BCG) inoculation, has cross infection with other mycobacteria and the like. IGRAs diagnose tuberculosis infection by detecting gamma interferon release caused by specific antigen stimulation of mycobacterium tuberculosis in organisms. Compared with TST, IGRAs have higher specificity in areas where BCG is widely inoculated due to the use of RD (region of difference) region deletion gene-encoded antigens, such as CFP-10, ESAT-6, TB7.7 and the like, but the sensitivity is still to be improved, and more uncertain results appear particularly in immunosuppressed people. Furthermore, neither TST nor IGRAs are able to distinguish between active and latent tuberculosis, which makes their clinical application in areas with high prevalence of tuberculosis extremely limited.

Therefore, there is an urgent need in the art to develop more accurate and effective methods and tools for diagnosing tuberculosis infection.

Disclosure of Invention

The object of the present invention is to provide an accurate and effective method and tool for diagnosing tuberculosis infection.

In a first aspect of the invention, there is provided a combination (or set) of tuberculosis positive antigens, said combination of antigens comprising n active tuberculosis antigens, wherein n is a positive integer from 5 to 100, and said active tuberculosis antigens are selected from the group consisting of:

(a) a polypeptide having a sequence as set forth in any one of SEQ ID No. 1-92;

(b) a fusion protein of a polypeptide with a sequence shown in any one of SEQ ID NO. 1-92 and GST;

(c) a fusion polypeptide formed by fusion of 2-10 (preferably 3-7) polypeptides of (a);

(d) any combination of (a), (b), and (c) above.

In another preferred embodiment, n is 6 to 50, more preferably n is 6 to 20.

In another preferred embodiment, the antigen combination comprises an active tuberculosis antigen selected from the group consisting of:

(a1) a polypeptide with a sequence shown in SEQ ID No. 5, 6 or 8;

(b1) fusion protein of polypeptide with the sequence shown in SEQ ID No. 5, 6 or 8 and GST;

(c1) the polypeptide with the sequence shown in SEQ ID No. 5, 6 or 8 is fused with other different active tuberculosis antigens to form a fusion polypeptide.

In another preferred embodiment, the antigen combination comprises the amino acid sequences as set forth in SEQ ID NOs: 1. 2, 3, 4, 5 and 6 or their corresponding fusion proteins or fusion polypeptides.

In another preferred embodiment, the antigen combination comprises the amino acid sequences as set forth in SEQ ID NOs: 1-14 or a corresponding fusion protein or fusion polypeptide thereof.

In another preferred embodiment, the antigen combination comprises the amino acid sequences as set forth in SEQ ID NOs: 5. 6, 8, 9, 10, 11, 12 and 14 or 1, 2, 3, 4, 5, 6, 7 or 8 polypeptides or their corresponding fusion proteins or fusion polypeptides.

In another preferred embodiment, the antigen combination comprises the amino acid sequences as set forth in SEQ ID NOs: 1 and 2 or a corresponding fusion protein or fusion polypeptide thereof.

In another preferred embodiment, the antigen combination has an active tuberculosis detection accuracy of greater than or equal to 60% (more preferably greater than or equal to 60%) and a sensitivity of greater than or equal to 85% (more preferably greater than or equal to 88%).

The invention also provides an active tuberculosis antigen selected from the group consisting of:

(a) a polypeptide having a sequence as set forth in any one of SEQ ID No. 1-92;

(c) a fusion polypeptide formed by fusing 2-10 (preferably 3-7) polypeptides in (a).

In a second aspect of the invention, there is provided the use of a positive antigen or a combination of tuberculosis positive antigens as described in the first aspect for the preparation of a reagent or kit for the detection of active tuberculosis.

In another preferred embodiment, the reagent comprises a serum detection reagent, a detection plate, or a chip.

In a third aspect of the invention, there is provided an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide encoding a positive antigen or combination of antigens according to the first aspect;

(b) a polynucleotide complementary to polynucleotide (a).

In another preferred embodiment, the polynucleotide is a polynucleotide encoding an amino acid sequence shown in any one of SEQ ID NOS: 1-14 (preferably, encoding SEQ ID NOS: 1-6).

In another preferred embodiment, the polynucleotide is a polynucleotide encoding an amino acid sequence shown in any one of

SEQ ID NOs

5, 6 and 8.

In a fourth aspect of the invention, there is provided a vector comprising the polynucleotide of the third aspect.

In a fifth aspect of the invention, there is provided a host cell comprising the vector of the fourth aspect, or having the polynucleotide of the third aspect chromosomally integrated therein.

In another preferred embodiment, the host cell is E.coli.

In a sixth aspect of the present invention, there is provided a method for preparing a positive antigen for Mycobacterium tuberculosis, comprising the steps of:

(a) culturing the host cell of the fifth aspect under conditions suitable for expression;

(b) isolating said positive antigen of Mycobacterium tuberculosis from the culture.

In another preferred embodiment, the amino acid sequence of the antigen is shown as any one of SEQ ID NO 1-14; preferably, the amino acid sequence of the antigen is shown in SEQ ID NO 1-6.

In another preferred embodiment, the amino acid sequence of the antigen is as shown in any one of

SEQ ID NOs

5, 6 and 8.

In a seventh aspect of the present invention, there is provided a kit for detecting active tuberculosis, the kit comprising: a container or carrier; and a positive antigen combination according to the first aspect of the invention located within or on the carrier.

In another preferred example, each active tuberculosis antigen in the positive antigen combination is respectively positioned in different containers, or respectively positioned on different carriers, or respectively positioned at different detection areas (or detection points) of the carriers.

In another preferred embodiment, the kit further comprises an enzyme binding solution, a reaction substrate and optionally instructions; preferably, the kit may further comprise a component selected from the group consisting of: a reaction stop solution, a sample diluent, and a washing solution.

In another preferred embodiment, the solid phase carrier is coated with a peptide having an amino acid sequence as shown in SEQ ID NO:1-6, and optionally a positive antigen selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 7-92 (in particular the positive antigen shown in SEQ ID NO. 8).

In another preferred embodiment, the kit or the reagent is used for detecting whether a sample to be detected is infected with mycobacterium tuberculosis.

In another preferred example, the sample to be tested is from a population in an epidemic area of Mycobacterium tuberculosis.

In an eighth aspect of the present invention, there is provided a reagent for detecting active tuberculosis, the reagent comprising: a solid support and a positive antigen combination according to the first aspect of the invention coated on the solid support.

In another preferred embodiment, the reagent is a protein chip.

In another preferred embodiment, the protein chip is used for detecting blood samples or serum samples.

In another preferred embodiment, each active tuberculosis antigen in the positive antigen combination is located on a different carrier or located in a different detection area (or detection point) of the carrier.

In a ninth aspect of the invention, there is provided a standard selected from the group consisting of:

(i) n active tuberculosis antigens, n is a positive integer of 5-100, and the active tuberculosis antigens are selected from the following group:

(a) a polypeptide having a sequence as set forth in any one of SEQ ID No. 1-92;

(d) any combination of (a), (b), and (c) above.

(ii) (ii) antigen-antibody complexes formed by the n active tuberculosis antigens in (i) and the corresponding antibodies, respectively.

In another preferred embodiment, n is 6 to 50, more preferably n is 6 to 20.

In another preferred embodiment, the antigen combination comprises an active tuberculosis antigen selected from the group consisting of: a polypeptide with the sequence shown in SEQ ID No. 5, 6 or 8.

In another preferred embodiment, each of said compounds comprises:

(1) a positive antigen with an amino acid sequence as shown in any one of SEQ ID NO 1-92 (preferably as shown in SEQ ID NO 1-14, more preferably as shown in SEQ ID NO 1-4, 5, 6 or 8); and

(2) an antibody that specifically binds to the positive antigen in (1);

and the antigen and antibody are bound together.

In a tenth aspect of the present invention, there is provided an antibody combination (or antibody set) comprising n antibodies, wherein the antibodies specifically bind to n active tuberculosis antigens respectively in the active tuberculosis antigen combination of the first aspect of the present invention, and n is a positive integer of 5 to 100.

In another preferred embodiment, the antibody is a monoclonal antibody that binds to a polypeptide having an amino acid sequence set forth in SEQ ID NO:1-14 (preferably as shown in SEQ ID NO:1-4, 5, 6 or 8).

In an eleventh aspect of the invention, there is provided a use of the antibody of the tenth aspect of the invention for:

(a) preparing a reagent or a kit for detecting active tuberculosis; and

(b) used as a positive antibody control for active tuberculosis.

In a twelfth aspect of the present invention, there is provided a method for high throughput screening of Mycobacterium tuberculosis antigens, comprising the steps of:

(1) carrying out fusion expression on candidate protein antigen containing a signal peptide or a transmembrane region and a marker protein GST;

(2) placing the fusion protein obtained in the step (1) in a hole of a multi-well plate chelated with GSH, thereby capturing the GST fusion protein;

(3) placing serum containing antibodies to mycobacterium tuberculosis into the wells of the multi-well plate of step (2);

(4) and (3) screening the holes forming the specific antigen-antibody compound, wherein the corresponding candidate protein is the positive antigen of the mycobacterium tuberculosis.

In another preferred embodiment, 5 to 600, preferably 20 to 500 GST fusion proteins are placed in the wells of a multi-well plate in step (2).

In another preferred example, the sample to be tested in step (3) is human serum.

In another preferred embodiment, the screening method in step (3) is ELISA.

In a thirteenth aspect of the present invention, there is provided a method for detecting whether a specimen is infected with mycobacterium tuberculosis, comprising the steps of:

(1) contacting the combination of positive antigens of mycobacterium tuberculosis with a sample to be detected;

(2) detecting whether the sample contains a specific antibody that binds to the positive antigen of any of the first aspect.

In another preferred embodiment, the amino acid sequence of the positive antigen is as shown in SEQ ID NO:1-92 (preferably as shown in any one of SEQ ID NOs: 1-14, more preferably as shown in any one of SEQ ID NOs: 1-4, 5, 6 or 8).

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of high throughput screening of Mycobacterium tuberculosis antigens.

Figure 2 shows the expression and purification results of TBGP 4. Wherein lanes are as follows: lane M: a molecular weight standard; lane 1 is disrupted cells; lane 2 is the supernatant of the disrupted cells; lane 3 broken pellet; lane 4 is purified protein.

FIG. 3 shows the purified recombinant protein of TBGP 1-6. Wherein lanes are as follows: m: a molecular weight standard; lanes 1-6 are TBGP1, 2, 3, 4, 5, and 6, respectively.

FIG. 4 shows the results of sensitivity of normal human and patient to TBGP1-66 antigens.

Detailed Description

The inventor of the invention has conducted extensive and intensive research, and based on a GST fusion expression high-throughput functional protein screening technology, 92 positive antigens capable of being recognized by the serum of a tuberculosis patient are screened for the first time, wherein 14 antigens present strong positive reactions, and a combination of 6 strongest positive antigens is used for mycobacterium tuberculosis serological detection, and the result shows that the antigen combination has high sensitivity and high specificity in detecting mycobacterium tuberculosis. The kit can be used for preparing a reagent or a kit for diagnosing and monitoring tuberculosis. The present invention has been completed based on this finding.

Definition of

As used herein, the terms "combination of antigens" and "antigen set" are used interchangeably to refer to a combination or set of two or more antigen combinations. In the combination or set, each individual antigen acts as an element of the set. It will be appreciated that in the antigen combination and antigen set described, the antigens may be independent of each other (e.g. located in a detection spot (or detection zone) of the detection plate or detection chip) or may be mixed together. The antigen combination of the invention is particularly suitable for detection reagents (including kits, chips and the like) for detecting active tuberculosis. Furthermore, the antigen combination of the present invention is also particularly suitable as a standard or positive control for a kit or a detection method for detecting tuberculosis (especially active tuberculosis).

As used herein, the terms "antibody combination" and "antibody set" are used interchangeably.

As used herein, the term "TBGP 1-6" refers to TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP 6.

Positive antigen

In the present invention, the terms "antigen" or "positive antigen" are used interchangeably and refer to a protein or polypeptide capable of specifically binding to an antibody against Mycobacterium tuberculosis. The term also includes polypeptide fragments that are immunogenic (e.g., any one of the polypeptides of SEQ ID No.:1-92 or immunogenic fragments thereof). The antigen is a substance which can stimulate an organism to generate (specific) immune response and can be combined with an immune response product antibody and sensitized lymphocytes in vitro to generate immune effect (specific reaction). The basic properties of an antigen are two, the ability to induce an immune response, i.e., immunogenicity, and the ability to react with the product of the immune response, i.e., antigenicity.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or protein in the natural state in the living cell is not isolated or purified, the same polynucleotide or protein is isolated or purified if it is separated from other substances coexisting in the natural state. By "isolated positive antigen" is meant that the antigen is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify positive antigens using conventional methods, such as standard protein purification techniques. Substantially pure protein antigens produce a single major band on non-reducing polyacrylamide gels. The purity of the antigen can be analyzed by amino acid sequence analysis.

The antigens of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the antigens of the invention may be glycosylated and non-glycosylated. The antigens of the invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of the antigen. As used herein, the terms "fragment," "derivative," and "analog" refer to an antigen that retains substantially the same biological function or activity as the native antigen of the invention. The antigenic fragment, derivative or analogue of the invention may be (i) an antigen in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) an antigen having a substituent group in one or more amino acid residues, or (iii) an antigen formed by fusion of the antigen to another compound, such as a compound that extends the half-life of the antigen, e.g. polyethylene glycol, or (iv) an additional amino acid sequence fused to the sequence of the antigen. Such fragments, derivatives and analogs are within the purview of those skilled in the art.

In the present invention, the term "antigen" also includes variants having the same function. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the antigen such as acetylation, carboxylation, glycosylation, in vivo or in vitro. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

Taking the active positive antigen of Mycobacterium tuberculosis of the present invention as an example, typical positive antigens of Mycobacterium tuberculosis include

(a) A polypeptide with an amino acid sequence as shown in any one of SEQ ID NO 1-92;

(b) 1-92, and one or more amino acid residues are substituted, deleted or added to form derivative polypeptides with the same or similar immunogenicity;

(c) and SEQ ID NO:1-92 (preferably 95%, more preferably 98%, most preferably 99%) of an immunogenic polypeptide; or

(d) Active fragments of polypeptides (a) to (c) having immunogenicity.

Antigen coding sequences

The polynucleotide encoding the antigen of the present invention may be in the form of DNA or in the form of RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The term "polynucleotide encoding an antigen of the present invention" may include a polynucleotide encoding the antigen, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention.

The nucleotide sequence of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, especially open reading frame sequences, and the extracted genome of a standard strain of Mycobacterium tuberculosis can be used as a template to amplify to obtain the relevant sequences. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence encoding the antigen of the invention (or a fragment or derivative thereof) can be obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the antigen sequences of the invention by chemical synthesis. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.

Antigen preparation method

The preparation method of the antigen of the invention comprises the following steps:

(1) transforming or transducing a suitable host cell with a polynucleotide encoding a positive antigen of the invention (or a variant thereof), or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the antigen of the invention from the culture medium or the cells.

In the present invention, the polynucleotide sequence may be inserted into a recombinant expression vector. Methods well known to those skilled in the art can be used to construct expression vectors containing antigen-encoding DNA sequences and appropriate transcription/translation control signals. Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the antigenic protein of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The antigenic protein may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the antigenic protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Antigen-antibody complexes

As used herein, the term "antigen-antibody complex" refers to a complex formed by the reversible binding of an antibody to an antigen via secondary bonds such as electrostatic forces, hydrogen bonds, van der waals forces, etc., which are capable of activating complement, participating in immune regulation, mediating type III hypersensitivity, etc.

The present invention provides an antigen-antibody complex, said complex comprising: (i) a positive antigen with an amino acid sequence shown in SEQ ID NO. 1-92 (preferably with an amino acid sequence shown in SEQ ID NO. 1-14, more preferably with an amino acid sequence shown in SEQ ID NO. 1-6); and (ii) an antibody that specifically binds to the positive antigen in (i); and the antigen and antibody are bound together.

The invention also provides the use of said antigen-antibody complex for:

(a) preparing a reagent or a kit for detecting mycobacterium tuberculosis; and (b) as a positive control for the detection of Mycobacterium tuberculosis.

Antigen screening method

The invention relates to a method for screening high-throughput protein antigen based on glutathione-S-transferase (GST) fusion protein expression, which comprises the following steps:

(1) the method for predicting the secretion and transmembrane protein of the mycobacterium tuberculosis by using bioinformatics method, in a preferred embodiment of the invention, the database is preferably protein sequence of NCBI, and the prediction software is preferably SignalP 3.0 (in a preferred embodiment, the protein sequence of NCBI is a protein sequence of NCBI, and the protein sequence of NCBI is a protein sequence of the NCBI, and the protein sequence of the NCBI ishttp:// www.cbs.dtu.dk/services/SignalP/) And TMHMM 2.0(http://www.cbs.dtu.dk/services/ TMHMM-2.0/)；

(2) Amplifying target genes corresponding to each protein, cloning the target genes to an expression vector, preferably a GST expression vector, and inducing thalli to express the antibody-GST fusion protein after converting escherichia coli;

(3) placing the fusion protein in a hole of a multi-well plate chelated with GSH, and capturing the GST fusion protein;

(4) and (3) placing the serum containing the mycobacterium tuberculosis antibody in a porous plate, carrying out antigen-antibody detection, screening holes for forming a specific antigen-antibody compound, wherein the corresponding candidate protein is the positive antigen of the mycobacterium tuberculosis. High throughput screening can be achieved by simultaneously placing 5-1000 (preferably 10-500, more preferably 20-100) GST fusion proteins in the wells of a multiwell plate.

Reagent kit

The invention also provides a kit for detecting the mycobacterium tuberculosis. Generally, the kit of the invention comprises the following components: a container or carrier; and a positive antigen according to the first aspect of the invention located within the container or on the carrier.

In another preferred embodiment, the kit further comprises an enzyme binding solution, a reaction substrate and optionally instructions. Preferably, the kit may further comprise a component selected from the group consisting of: a reaction stop solution, a sample diluent, and a washing solution.

A preferred kit for detecting Mycobacterium tuberculosis comprises: the kit comprises a container, and a coating solution, a horseradish peroxidase-labeled mouse anti-human IgG (H + L) antibody, a substrate solution TMB, a diluted concentrated sulfuric acid reaction stop solution and a sample diluent which are positioned in the container. Preferably, blank controls, positive and negative controls are included and are used to monitor compliance of the test procedure.

Vaccine and pharmaceutical composition

The invention also provides a vaccine and an antigen pharmaceutical composition. The vaccine and antigen pharmaceutical compositions of the present invention comprise an effective amount of at least one protein antigen of the present invention (i.e., a tuberculosis positive antigen) and a pharmaceutically acceptable adjuvant and/or carrier. Wherein, in the vaccine composition of the present invention, one or more protein antigens may be contained, preferably the content of the protein antigens is 0.01 to 99 wt%, more preferably 0.1 to 90 wt%, based on the total weight of the composition.

In another preferred embodiment, the protein antigen is selected from the group consisting of:

(a) a polypeptide having a sequence as set forth in any one of SEQ ID No. 1-92;

(b) 1-92 with other carrier protein (such as GST);

(d) any combination of (a), (b), and (c) above.

It will be appreciated that other elements such as optional linker peptides, tag sequences, signal peptides, secretion peptides, etc. may be included in the fusion proteins and fusion polypeptides.

In the present invention, commonly used vaccine adjuvants can be classified into 4 types: inorganic adjuvants such as aluminum hydroxide, alum, etc.; organic adjuvants, microorganisms and their products but excluding mycobacteria (mycobacterium tuberculosis, bacillus calmette-guerin), brevibacterium, bordetella pertussis, endotoxins, bacterial extracts (muramyl dipeptide), etc.; synthetic adjuvants such as artificially synthesized double-stranded polynucleotides (double-stranded polyadenylic acid, uridylic acid), levamisole, isoprinosine, and the like; oil agents, such as Freund's adjuvant, peanut oil emulsion adjuvant, mineral oil, vegetable oil, etc. Commonly used carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the administration mode, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. An effective amount of an antigen of the invention can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). It is usually administered at a dose of about 0.00001mg to 50mg/kg of animal body weight (preferably 0.0001mg to 10mg/kg of animal body weight) per day, and satisfactory control effects can be obtained. The formulated antigenic pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal.

The invention also provides a method for detecting whether a sample is infected with the mycobacterium tuberculosis, which comprises the following steps:

the positive antigen of mycobacterium tuberculosis is contacted with a sample to be detected, and whether the sample contains a specific antibody combined with any positive antigen of the invention is detected. In a preferred embodiment of the present invention, the amino acid sequence of the positive antigen is as shown in SEQ ID NO:1-92 (preferably SEQ ID NO:1-14, more preferably SEQ ID NO: 1-6).

The main advantages of the invention are:

1. the antigen sensitivity and specificity are high, for example, the sensitivity of a single antigen of TBGP1 reaches 30 percent, and the specificity of TBGP1-6 is higher than 90 percent;

2. the detection method and the detection kit can be accurately and effectively applied to diagnosis of TB patients, particularly detection of active TB;

3. the detection method and the detection kit are simple and convenient to operate, and the antigen can be read by a universal ELISA method.

4. The detection result is stable and the repeatability is high.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1

High throughput screening of important antigens of mycobacterium tuberculosis

1. Prediction of mycobacterium tuberculosis secretion and transmembrane proteins

Downloading a mycobacterium tuberculosis genome database from the NCBI, along with the corresponding protein sequences; the protein sequence was predicted by introducing it into SignalP 3.0(http:// www.cbs.dtu.dk/services/SignalP /) which is a signal peptide on-line prediction software.

The protein sequence was introduced into transmembrane structure online prediction software (http:// www.cbs.dtu.dk/services/TMHMM /), and prediction was performed.

Based on the prediction analysis, the present inventors selected over 600 proteins, which contained the sequence of the signal peptide protein, and a sequence having a transmembrane structure.

2. Cloning of genes

Designing primers by a conventional method, amplifying a target gene by PCR, recovering a target fragment, carrying out enzyme digestion, cloning the target fragment into a conventional commercially available expression vector pGEX-4T-1, transforming commercially available Escherichia coli Top10, and sequencing a positive clone to confirm that the sequence is correct.

3. Inducible expression of recombinant proteins

Extracting the plasmid by using a conventional method, and transforming the recombinant pGEX-4T-1 recombinant plasmid into Escherichia coli B121(DE 3); selecting a monoclonal, inoculating a 96-hole bacterium deep-hole culture plate, and standing overnight at 37 ℃ at 250 rpm; inoculating 1% of a 96-well deep-well bacterial culture plate containing 1ml of 2 XYT (containing 100. mu.g/ml Amp) liquid medium per well, and culturing at 37 ℃ and 250rpm for about three hours until OD600 becomes 0.4-1.0; adding IPTG to the final concentration of 1mmol/L, inducing the target protein expression at 37 ℃ and 250 rpm; inducing expression for 6hr, and stopping culture; collecting thallus, and freezing at-80 deg.C.

4. Solubilization of Inclusion bodies

Repeatedly freezing and thawing the thallus for 2 times; adding 300 mu l B-PER (adding DNase, RNase, PMSF and the like) to crack the thallus, blowing and uniformly mixing, and transferring to an EP tube; centrifuging at 13,000rpm at room temperature for 5 min; the supernatant is removed to a new Ep tube and is frozen and preserved for standby, and the precipitate is the inclusion body; 1% Triton-X100 PBS was prepared as a washing solution, and the inclusion bodies were washed at 300. mu.l/tube. Blowing with a gun, and mixing for 10-20min on a rotary mixer; centrifuging at 13,000rpm at room temperature for 5min, and discarding the supernatant; repeating the step 5, centrifuging and discarding the supernatant; dissolving the inclusion body by using an inclusion body dissolving solution; and (4) uniformly stirring, shaking at room temperature overnight, and fully dissolving the inclusion body.

5. Renaturation of inclusion bodies

Centrifuging the dissolved solution at 13,000rpm at room temperature for 5min, and collecting the supernatant; adding 50 mul of solution obtained by denaturation and 1ml of renaturation solution into each well of a 96-well deep-well culture plate; mixing, standing at room temperature, and shaking overnight; centrifuging and collecting the supernatant;

6. serum adsorption

Transforming pGEX-4T-1 plasmid into commercial Escherichia coli Bl21(DE3), preparing 100ml of GST expression bacterial liquid and Bl21(DE3) bacterial liquid respectively, centrifugally collecting thalli, resuspending the thalli by using 10ml of PBS, and collecting supernatant for later use after ultrasonic crushing; mixing 1ml of column volume GSH filler, 3ml of GST expression supernatant and 9ml of PBS, mixing for 1h at room temperature, centrifuging at 1000rpm, collecting precipitate, and washing with 10ml of PBS for five times, wherein GST protein is bound on the filler; resuspend GST/Filler with 10ml PBS, split 1.9ml per tube; adding 100 μ l patient serum into subpackage tube, and mixing at room temperature for 5 hr to adsorb antibody against GST in serum; centrifuge at 1000rpm for 5min, remove the filler, collect the supernatant, at which point the serum dilution is 1: 20; adding 3ml of Bl21(DE3) lysis supernatant to 2ml of GST-adsorbed serum, and mixing at room temperature for 5h, wherein the step is to adsorb antibacterial protein antibody, and the serum dilution is 1: 50; centrifuging at 13000rpm for 10min, and subpackaging 500 μ l per tube for freezing for use.

7. Screening for seropositive clones

7.1 adding 20 mul renaturation protein liquid and 80 mul PBS in each hole in a GSH-micropore plate, incubating overnight at 4 ℃; meanwhile, GST expression bacterial liquid is used as a negative control, PBS is used as a blank control, and after positive protein is screened, the clone is used as a positive control.

The microplate layout is shown in table 1 below:

TABLE 1

S1

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

S13

S14

S15

S16

S17

S18

S19

S20

S21

S22

S23

S24

S25

S26

S27

S28

S29

S30

S31

S32

S33

S34

S35

S36

S37

S38

S39

S40

S41

S42

S43

S44

S45

B

-

+

Where "B" represents a blank control, "-" represents a negative control, and "+" represents a positive control.

7.2PBST washing five times; sealing for 2h at room temperature with 200 μ l of 5% milk powder/PBST; PBST washing five times;

7.3 adsorbed serum according to 1:20 diluted with 5% milk powder, final serum dilution was 1:1000, parts by weight; 100 μ l/well, binding at 37 ℃ for 1 h; PBST washing five times;

7.4 according to the ratio of 1:20,000, diluting the HRP enzyme-labeled mouse anti-human IgG (H + L) antibody, 100 μ L/hole, and combining for 1H at 37 ℃; PBST wash 5 times;

7.5 adding 100. mu.l of commercial Super Signal ELISA Femto for detection, and reading the fluorescence intensity value at 425 nm;

7.6 calculating the ratio R of the fluorescence intensity of the sample and the negative control, and judging whether the protein is positive protein according to the size of R. If R is more than or equal to 2, the test result is judged to be positive. The formula for R is as follows:

the results of using ten active tuberculosis patient sera from 409 mycobacterium tuberculosis secreted and transmembrane proteins are shown in fig. 1, and 92 positive clones (with response to at least one patient serum) were screened in total, of which 14 strong positive clones (R is greater than or equal to 10 or with response to more than two patient sera) were TBGP1, TBGP2, TBGP3, TBGP4, TBGP5, TBGP6, TBGP7, TBGP8, TBGP9, TBGP10, TBGP11, TBGP12, TBGP13 and TBGP14 proteins.

The 92 proteins are respectively named as TBGPn, wherein n is a positive integer from 1 to 92, namely TBGP1 to TBGP92, and the sequences of the 92 proteins are respectively shown as SEQ ID No. 1 to 92.

Example 2

Serological value assessment of Strong Positive clone diagnostic sensitivity

1. Selecting 14 strong positive clones determined in the example 1, selecting 42 samples of the serum of active tuberculosis patients and 22 samples of healthy serum, and verifying the 14 strong positive antigens;

2. respectively coating 14 antigens into a polystyrene micropore plate, and standing overnight at 4 ℃;

3. 200ul of 5% milk powder/PBST, and sealing for 2h at room temperature; PBST wash 3 times;

4. adding adsorbed serum at a ratio of 1:50, 100 ul/well, binding at 37 deg.C for 1h, and washing with PBST for 5 times;

5. diluting HRP enzyme-labeled mouse anti-human IgG (H + L) antibody according to a ratio of 1:10000, binding at 37 ℃ for 1H at 100 ul/hole, and washing with PBST for 5 times;

6. 100ul of chromogenic substrate was added for detection, and the OD was read at 450nm to calculate sensitivity and specificity.

7. Data processing

And if the OD value of the to-be-detected hole is larger than or equal to the cutoff value (the negative control plus 3 times of the standard deviation of the negative control), the to-be-detected hole is positive. When the ratio of the OD value of the hole to be detected to the cutoff value is more than 1, the hole to be detected is positive, and the ratio is more than 2, the hole to be detected is strong positive; negative results were obtained when the ratio was less than 1.

8. Results

Specific results are shown in Table 2

TABLE 2 evaluation of positive antigen serological response values

The results show that the sensitivity of 14 antigens is between 9.5% and 33.3%, and the specificity is higher than 80%. Wherein the highest value of the two antigens TBGP1 and TBGP2 is 33.3 percent. Sensitivity of TBGP6 and TBGP8 were both up to about 26% with specificity of 95-100%.

In addition, a single antigen cannot satisfy the diagnostic test of tuberculosis, and therefore, multiple antigens need to be used in combination.

Statistical calculations show that the combination of 6 antigens TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP6 can be significantly improved in sensitivity to about 71.4% without significant reduction in specificity (86.4%), which the inventors will further study in the following examples.

Example 3 expression and purification of 6 antigens such as TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP6

Construction of the expression vector pET28 (a): the TBGP gene was cleaved from the recombinant pGEX-4T-1 and ligated into the conventional commercially available pET28(a) vector by Kan⁺Plate screening positiveAnd (4) cloning.

2. Expression of the protein: transforming B121(DE3) expression strain with recombinant pET28(a) vector, picking positive clone to prepare seed liquid, inoculating to triangle flask according to 1%

In a bottle, the temperature is 37 ℃, the rpm is 250, and the time is 3 hours; adding IPTG to induce expression, wherein the final concentration of IPTG is 1mM, the temperature is 37 ℃, the rpm is 250, and the time is 5 h;

3. bacterial cell lysis: collecting bacteria, and freeze thawing the bacteria twice; cracking the bacteria after twice freeze thawing with 10mlB-PER (adding DNase, RNase, PMSF and the like), transferring into a beaker, and slowly stirring for 30 min; ultrasonic: 10s ultrasonic treatment, 10s interval, 500W, 10 min.

4. Collecting a supernatant: centrifuging at 15000rpm for 10min, and collecting supernatant;

5. washing of inclusion bodies: centrifuging at 15000rpm for 10min, and collecting precipitate; the pellet was resuspended in 10ml inclusion body washes: scattering the precipitate with a gun, transferring into a beaker, performing ultrasonic treatment for 1-2min to thoroughly scatter the precipitate, and slowly stirring for 30 min; the formula of the cleaning solution is as follows: 0,.5M Urea, 0.1M NaH₂PO₄，0.01M Tris-HCl，0.5％Triton 100,PH＝8.0.

6. Solubilization of Inclusion bodies: the pellet was solubilized with 100ml of inclusion body lysis solution: scattering the precipitate with a gun, transferring into a beaker, performing ultrasonic treatment for 1-2min to thoroughly scatter the precipitate, and slowly stirring for 30 min; the formula of the dissolving solution is as follows: 8M Urea, 0.1M NaH₂PO₄，0.01M Tris-HCl，PH＝8.0.

And 7, Ni column purification: balancing the Ni column with a dissolving solution; mixing the protein solution and the protein solution according to the volume of 1ml column corresponding to 10ml, and incubating at 200rpm for 30 min; loading the mixed solution into a column, and collecting the liquid after the column; washing with 10 times of column volume of washing solution, and collecting the washing solution (Ni column washing solution formula: 0.1M NaH)₂PO₄20mM NaCl, 70mM imidazole, pH 8.0); elution with 10 column volumes of eluent: collecting eluent, 1 ml/tube (Ni column eluent formula: 0.1M NaH)₂PO₄20mM Nacl, 500mM imidazole, pH 8.0); SDS-PAGE detects protein purity in each collection tube.

8. Protein renaturation: dialyzing the purified protein solution in renaturation solution according to the volume of 1:10, carrying out renaturation at 4 ℃, and replacing fresh renaturation solution every 5 hours for 3 times; dialyzing the renatured protein in PBS containing 10% of glycerol according to the volume of 1:10, and changing fresh dialysate every 5h for 3 times at 4 ℃; SDS-PAGE detection; the concentration tube concentrates the protein solution to about 1ml, and the protein concentration is measured.

As shown in FIG. 2, lane 1 shows disrupted cells, using TBGP4 as an example; lane 2 is the supernatant of the disrupted cells; lane 3 broken pellet; lane 4 is purified protein. Thus, it was shown that TBGP4 was expressed as inclusion bodies, and a high-purity recombinant protein was obtained by protein purification, dialysis and renaturation.

As shown in FIG. 3, lanes 1-6 are purified recombinant proteins of TBGP1-6 (i.e., TBGP1, TBGP2, TBGP3, TBGP4, TBGP5, and TBGP6), respectively.

Example 4

Further study of antigen combinatorial sensitivity

Based on the serum detection of the invention, the combination of 6 antigens, TBGP1-6 (i.e. TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP6) has the highest sensitivity, and therefore, the present example further studies the sensitivity of the antigen combination.

96 and 49 parts of each of human serum samples of patients and normal persons were collected from the pulmonarily department hospital of Shanghai, and ELISA serum test was performed for further antigen sensitivity study.

The results of TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP66 antigens are shown in fig. 4, and the sensitivity and specificity of the antigen combination reach 67.7% and 91.8%, respectively.

Example 5

Preparing tuberculosis enzyme-linked immunodiagnosis reagent kit using antigen combination containing 6 antigens of TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP6 as detection antigens

1. Antigen-coated solid phase carrier: antigen combinations were coated in polystyrene reaction wells. Coating liquid: 1.5g of Na2CO₃And NaHCO₃Dissolved in 1000ml of distilled water.

2. Preparing an enzyme binding solution: murine anti-human IgG (H + L) antibody (Promega) labeled with HRP enzyme was used.

3. Preparing a substrate solution of the enzyme:

TMB liquid A: each ml of 0.005% sodium acetate-citric acid buffer solution contains 3.2. mu.L of 1.5% H₂O₂Storing at 4 ℃ in dark; TMB liquid: adding 0.08g of TMB into 40ml of DMSO (dimethyl sulfoxide), dissolving, adding 60ml of methanol, uniformly mixing, adding 100ml of 0.005% sodium acetate-citric acid buffer solution, oscillating for 1 hour in the dark, standing for 3 hours at room temperature, and storing in the dark at 4 ℃; before use, the solution A and the solution B are mixed in equal volume for use.

4. Preparing an enzyme reaction stopping solution: in 600ml ddH₂And slowly dripping 100ml of concentrated sulfuric acid into the O, continuously stirring, and metering to 900 ml.

5. Sample diluent: 5% milk powder/PBST.

6. Washing liquid: PBST.

The prepared kit is called TBGP-ELISA kit for short and is used in the subsequent examples.

Example 6

Detection method and operation program

1. Sample dilution

Diluting the serum to be detected by sample diluent according to the ratio of 1: 50.

2. Sample application reaction

Sample test wells 100. mu.l of diluted serum samples were added to each well and 2 replicates of each sample were tested. Setting 2 multiple wells for positive, negative and blank control, adding 100 μ l each of positive and negative control into the well, and adding only the diluent for blank control. Incubate at 37 ℃ for 60 minutes in the dark.

3. Washing machine

And (4) spin-drying liquid in the holes, filling each hole with a washing solution, standing for 2 minutes, spin-drying, repeatedly washing for 4 times, and finally drying by beating.

4. Adding enzyme

Adding 100 μ L of HRP enzyme-labeled mouse anti-human IgG (H + L) antibody to each well, incubating at 37 ℃ in the dark for 1 hour, washing as above, and patting dry;

5. color development

Adding 100 mul of enzyme substrate color development liquid into each hole, and incubating for 10 minutes at 37 ℃ in a dark place; adding 50 mul of stop solution, and immediately detecting by using an enzyme-linked immunosorbent assay;

6. reading number

The blank was zeroed and the OD read at 450 nm.

7. Data processing

And if the OD value of the to-be-detected hole is larger than or equal to the negative control plus 3 times of the standard deviation of the negative control, the to-be-detected hole is positive. When the OD value of the negative control is lower than 0.05, the value is calculated as 0.05.

Example 7 kit sensitivity and specificity

The sensitivity and specificity of a commercial Mycobacterium tuberculosis antibody (IgG) detection Kit (available from Shanghai Rongsheng biological and pharmaceutical industries, Ltd., TAID-Kit for short) and the multiple antigen combination (TBGP-ELISA) prepared in example 5 were evaluated using a serum sample of a clinically confirmed active tuberculosis patient.

The serum samples were 288 active tuberculosis patients and 96 normal human sera collected from the pulmonaceae hospital in Shanghai.

The results are shown in Table 3.

TABLE 3

Among 288 samples of active tuberculosis patients, the TBGP-ELISA has 191 positive reactions and the sensitivity is 66.3 percent, which is consistent with the previous result, the TAID-Kit has 91 positive reactions and the sensitivity is 31.6 percent, and the difference between the two is obvious (P is less than 0.0001); among 96 normal human samples, TBGP-ELISA has 10 false positive reactions with specificity of 89.6%, TAID-Kit has 9 false positive reactions with specificity of 90.6%, and the two have no significant difference (P is 1.000); on the overall accuracy, the TBGP-ELISA accuracy is 72.1%, the TAID-Kit accuracy is 46.4%, and the difference between the TBGP-ELISA accuracy and the TAID-Kit accuracy is significant (P is less than 0.0001).

Discussion of the related Art

The serological diagnosis technology is fast and simple to operate and is applied to diagnosis of various diseases. The key point is to search tuberculosis antigens with high sensitivity and high specificity. The existing serological diagnostic antigens for tuberculosis are different in performance, and the sensitivity is between 0.09% and 59.7%. Serological diagnosis is not recommended by international organizations such as WHO and the like at present because of insufficient sensitivity and specificity. Therefore, the search for tuberculosis antigens with high sensitivity and high specificity is the key point for improving the diagnosis efficiency, which is also advocated by the WHO.

In view of the potential important functions and application values of mycobacterium tuberculosis secretory protein, membrane protein, RD protein, latency-related protein and the like, a high-throughput functional protein research method is needed to perform comprehensive and systematic analysis on the functions of mycobacterium tuberculosis in the aspects of tuberculosis diagnosis, immunity, host interaction and the like. The established GST fusion protein high-throughput screening technology is utilized to carry out serological screening on the established GST fusion protein library, a plurality of potential tuberculosis diagnosis antigen biomarkers are screened out, and a multi-antigen combination which can be used for active tuberculosis diagnosis is finally obtained through further serological value diagnosis evaluation.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. The tuberculosis positive antigen combination is characterized in that the antigen combination is a combination of antigens with amino acid sequences shown as SEQ ID NO: 1. 2, 3, 4, 5 and 6 or a fusion protein or fusion polypeptide corresponding thereto.

2. Use of a combination of tuberculosis-positive antigens as defined in claim 1 for the preparation of a reagent or kit for the detection of active tuberculosis.

3. An isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide encoding the positive antigen or combination of antigens of claim 1;

(b) a polynucleotide complementary to polynucleotide (a).

4. A vector comprising the polynucleotide of claim 3.

5. A host cell comprising the vector of claim 4, or having the polynucleotide of claim 3 integrated into its chromosome.

6. A method for preparing a positive antigen for mycobacterium tuberculosis comprising the steps of:

(a) culturing the host cell of claim 5 under conditions suitable for expression;

7. A kit for detecting active tuberculosis, the kit comprising: a container or carrier; and a positive antigen combination of claim 1 in or on the container.

8. A reagent for detecting active tuberculosis, the reagent comprising: a solid support and a positive antigen combination according to claim 1 coated on the solid support.

9. A standard, wherein the standard is selected from the group consisting of:

(i) the active tuberculosis antigen is an active tuberculosis antigen with amino acid sequences respectively shown as SEQ ID NO: 1. a combination of 6 polypeptides represented by 2, 3, 4, 5 and 6;

(ii) (ii) an antigen-antibody complex formed by the active tuberculosis antigen in (i) and the corresponding antibody.

10. An antibody combination, which comprises n antibodies, wherein the antibodies specifically bind to the active tuberculosis antigens in the active tuberculosis antigen combination of claim 1, respectively, and n is 6.

11. Use of the antibody combination according to claim 10 for:

(a) preparing a reagent or a kit for detecting active tuberculosis; and

(b) used as a positive antibody control for active tuberculosis.

12. A method for high-throughput screening of Mycobacterium tuberculosis antigens, comprising the steps of:

(4) screening the wells for the formation of specific antigen-antibody complexes, the corresponding candidate protein being the positive antigen of Mycobacterium tuberculosis as defined in claim 1.