CN111948387B

CN111948387B - Application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of tuberculosis vaccine

Info

Publication number: CN111948387B
Application number: CN201910409579.XA
Authority: CN
Inventors: 毕利军; 张泓泰; 王雅果; 张先恩; 朱国峰
Original assignee: Tibikon Biotechnology Guangdong Co ltd; Institute of Biophysics of CAS
Current assignee: Tibikon Biotechnology Guangdong Co ltd; Institute of Biophysics of CAS
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2023-09-12
Anticipated expiration: 2039-05-16
Also published as: CN111948387A

Abstract

The invention discloses application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of tuberculosis vaccines. The Rv1485 protein is a protein composed of an amino acid sequence shown as a sequence 2 in a sequence table. Experiments prove that the Rv1485 protein induces effective Th1 type humoral and cellular immune response, is beneficial to the control of tuberculosis, and can protect hosts from being affected by mycobacterium tuberculosis. The invention has important application value.

Description

Application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of tuberculosis vaccine

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of tuberculosis vaccines.

Background

Achieving the tuberculosis termination strategy of the world health organization by 2030 requires a new potential in various aspects of tuberculosis prevention and control, including the development of new vaccines, diagnostics and pharmaceuticals. BCG vaccine introduced nearly 100 years ago was of low potency but is still the only vaccine licensed, which increases resistance to antitubercular drugs and fatal synergy between tuberculosis and aids viruses, leading to the recurrence of this ancient disease, posing a global threat. Infectious pathogens are currently the leading cause of death in tuberculosis. Development of an improved vaccine may be the most effective strategy to achieve the elimination of tuberculosis. Since the intersection of this century, a great deal of investment has been made in vaccine research, and 12 candidate vaccines have been generated in the current pipeline at different clinical trial stages; however, the major bottleneck is the lack of antigen candidates that induce a protective immune response, a key determinant of vaccine efficacy. There is a need to develop whole system antigen screening studies to identify more extensive, more effective immunodominant protective antigens.

BCG is an attenuated live strain of Mycobacterium bovis, and has been widely used since 1921, and has been shown to prevent severe forms of tuberculosis in children, but is poorly and non-uniform in effect on adult tuberculosis and may lead to BCG disease transmitted in the immunodeficiency group. In the search for alternatives, the main immunization strategy being explored is to replace BCG with recombinant live BCG or genetically attenuated Mycobacterium Tuberculosis (MTB) vaccines (both with higher safety and protective efficacy), the limited immunity conferred by primary vaccines and "immunotherapy" (vaccines that can shorten active tuberculosis or potential tuberculosis infection (LTBI) treatment) methods using viral vectors or subunit vaccines, with the aim of enhancing and expanding the immune response initially induced by infants vaccinated with BCG (also rBCG or attenuated MTB vaccines) before contacting MTB by administering subunit vaccines (consisting of proteins, peptides or DNA). The protective effect of BCG begins to diminish during childhood or puberty. The protective efficacy of the antigen as a subunit is critical to the success of this immunization strategy. However, to date, only 11 antigens (PepA, PPE18, ag85A, ag85B, TB 10.4.4, PPE42, esxV, esxW, rv1813, ESAT-6 and Rv2660c, respectively) that induce a sufficiently protective immune response have been widely used. Three protein/adjuvant vaccines are currently tested in phase II and H56 is currently tested in phase IIa: IC31, combining Mycobacterium tuberculosis antigens Ag85B, ESAT and Rv2660c with IC31 adjuvant, M72/AS01E is currently undergoing phase IIb testing in HIV-negative adults infected with Mycobacterium tuberculosis in Kenya, south Africa and Zanya, and is a subunit vaccine comprising Mycobacterium tuberculosis antigens (32A and 39A) and adjuvant. Although the steady development of candidate vaccines through clinical trials is encouraging, the anti-tuberculosis protection provided by previously developed promising candidate vaccines (such as MVA 85A) was not significantly increased over vaccinated bcg (Refs) alone, which is an important reminder to the tuberculosis vaccine development community and challenges in developing effective tuberculosis vaccines.

Insufficient knowledge of the nuclear immune response is an additional challenge in developing effective vaccines, particularly because there are no known immunologically relevant protections or biomarkers for therapeutic efficacy. Bcg is reported to induce T helper cell 1 (Th 1) type responses, mainly involving IFN-gamma (REFs) produced by cd4+ T cells. In order to be effective as an enhancer for protecting tuberculosis, the ideal candidate antigen should induce a cellular immune response at least as strong as BCG, including cd4+ and cd8+ T cells, as well as non-conventional T cells such as γδ T cells and CD 1-restricted αβ T cells (Refs). IFN-gamma plays a key role in the control of tuberculosis in humans and mice. TB diagnostic kits based on IFN- γ release in response to stimulation by MTB antigen have been developed and are useful tools for screening for potent MTB antigens, which can be used as biomarkers for mycobacterium tuberculosis infection or as candidate promoters for further development into protein subunit vaccines.

Disclosure of Invention

The invention aims to prepare tuberculosis vaccines.

The invention firstly protects the application of Rv1485 protein in preparing tuberculosis detection reagent, tuberculosis vaccine or antitubercular drug.

The Rv1485 protein may be a 1) or a 2) or a 3):

a1 Protein composed of amino acid sequences shown in sequence 2 in a sequence table;

a2 A fusion protein obtained by connecting a label with the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 2 in the sequence table and has the same function as the protein in the a 1).

Wherein, the sequence 2 in the sequence table consists of 344 amino acid residues.

In order to facilitate purification of the protein of a 1), a tag as shown in Table 11 may be attached to the amino-terminal or carboxyl-terminal of the protein shown in sequence 2 in the sequence listing.

TABLE 11 sequence of tags

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein of the above a 3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in the a 3) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

The gene encoding the protein in the above a 3) can be obtained by deleting one or several amino acid residues from the gene encoding the protein, and/or performing one or several base pair missense mutation, and/or ligating the coding sequence of the tag shown in Table 11 at the 5 'end and/or 3' end thereof.

The invention also protects a tuberculosis detection reagent, which can comprise at least one of b 1) to b 3):

b1 The Rv1485 protein;

b2 A nucleic acid molecule encoding said Rv1485 protein;

b3 A recombinant protein expressed by a recombinant bacterium comprising a nucleic acid molecule encoding said Rv1485 protein.

In the tuberculosis detection reagent, b 1) or b 2) or b 3) may be an active ingredient.

The invention also protects a T-SPOT.TB kit or an X.DOT-TB kit containing the tuberculosis detection reagent.

The kit may also contain a capture antibody and/or a detection antibody. The capture antibody may be a mouse monoclonal antibody against human or animal IFN-gamma. The detection antibody may be another mouse monoclonal antibody against a different epitope of human or animal IFN-gamma. The animal can be mouse, rat, rabbit, sheep, horse.

Any of the above kits may further comprise a positive standard and/or a negative standard. The positive standard may be a tuberculosis non-specific stimulating antigen. The negative standard may be an antigen-free cell culture broth. The tuberculosis non-specific stimulatory antigen may be phytohemagglutinin, PHA.

Any of the above kits may further comprise a readable carrier described as follows: when the number of SFCs detected by ESAT6/CFP10 exceeds a certain standard (not less than 6 for the T-SPOT. TB kit and not less than 11 for the X.DOT-TB kit), the test reading is considered positive for MTB infection; otherwise the test reading is considered negative for MTB infection.

The invention also protects a tuberculosis vaccine, which can comprise at least one of b 1) to b 3):

b1 The Rv1485 protein;

b2 A nucleic acid molecule encoding said Rv1485 protein;

In the tuberculosis vaccine, b 1) or b 2) or b 3) may be an active ingredient.

The invention also protects a tuberculosis vaccine which can be obtained by modifying recombinant BCG containing the Rv1485 protein.

The invention also provides antitubercular medicaments, which may include antibodies that bind to the Rv1485 protein.

In the above antitubercular drug, the antibody may be an antibody prepared using the Rv1485 protein as an immunogen.

In the above antitubercular drug, the antibody may be A1) or A2).

A1 Polyclonal antibodies prepared by immunizing animals (such as mice, rats, rabbits, sheep, and humans) with the Rv1485 protein as an immunogen.

A2 The Rv1485 protein is used as an immunogen to immunize animals (such as mice, rats, rabbits, sheep and humans) and the hybridoma technology or the DNA recombination technology is adopted to prepare the monoclonal antibody. The monoclonal antibody may be a humanized monoclonal antibody.

In the antitubercular drug, the antibody may be an active ingredient.

The antitubercular drug may specifically consist of any of the antibodies described above.

Experiments prove that the Rv1485 protein induces effective Th1 type humoral and cellular immune response, is beneficial to the control of tuberculosis, and can protect hosts from being affected by mycobacterium tuberculosis. The invention has important application value.

Drawings

FIG. 1 shows the experimental strategy for identifying MTB antigen protein and verifying the protective effect of MTB antigen protein in a model of BALB/c mice infected by tubercle bacillus. (1) 1781 MTB proteins (1728H 37Rv and 53 CDC1551 proteins) were cloned and expressed using Gateway technology and then purified from inclusion bodies using denaturation and renaturation techniques; (2) protein antigenicity was detected by IFN-. Gamma.ELISPOT method: proteins that induced IFN-gamma release in the first round of screening (PBMC samples from 8 suspected tuberculosis patients) were subjected to two additional screens (screening of PBMC samples from 2:20 suspected tuberculosis patients; screening of PBMC samples from 3:20 active tuberculosis patients and PBMC samples from 20 healthy donors). (3) Immunizing BABL/c mice with 20 candidate antigens screened by the third round of screening, and then attacking with mycobacterium tuberculosis H37 Rv; the protective effect of candidate antigens was assessed by detection of lung bacterial burden and histopathological analysis by assessment of cytokines and IgG2a secreted by spleen cells: the IgG1 ratio was evaluated for host immune response, yielding Rv1705c and Rv1485 with best results.

FIG. 2 shows the performance of the IFN-. Gamma.release assay to evaluate the Mycobacterium tuberculosis proteins obtained and the recombinant CFP10 purified by commercial CFP10 (Rv 3874).

FIG. 3 is a comparison of IFN- γ release levels detected by the T.SPOT-TB kit and the X-DOT.TB kit.

FIG. 4 is a screen of 49 candidate antigens associated with a host immune response from 1781 Mycobacterium tuberculosis proteins by screening for IFN-gamma release from PBMC samples from suspected tuberculosis patients. A is the antigenic activity of 1781 MTB proteins assayed by the ELISPOT IFN- γ release assay, releasing IFN- γ from 8 PBMC samples (from suspected tuberculosis patients), one MTB protein per column; clove flower column: MTB protein stimulating IFN- γ release in PBMC samples; light blue column: no IFN-gamma released MTB protein was detected; of the 188 putative antigen proteins identified in the first round of screening, 49 stimulated IFN-gamma release in > 40% of the PBMC samples. B is a second round of screening, selection of 20 PBMC samples (from patients suspected of having tuberculosis).

FIG. 5 is a graph demonstrating the protective efficacy of 20 candidate Mycobacterium tuberculosis T cell antigens in a BALB/c mouse challenge model. A is the bacterial load in the lungs of mice vaccinated with 20 MTB candidate antigens 6 weeks after challenge with mycobacterium tuberculosis H37Rv (n=4 per group). Positive control: BCG, negative control: and (5) brine. BALB/c mice were inoculated subcutaneously with saline, BCG or one of 20 candidate MTB T cell antigens, then 5 x 10 after 6 weeks ⁵ CFU mycobacterium tuberculosis H37Rv intravenous infection. Mice were euthanized after an additional 8 weeks. Bacterial load in the lungs was assessed by scoring CFU. One-way analysis of variance (ANOVA) was used for the inter-group comparison. The data shown are log10 representations ± SEM. B is 6 weeks after infection of Mycobacterium tuberculosis H37RvRepresentative images of lungs from Rv1705c or Rv1485 vaccinated BALB/c mice. Lungs from BCG and PBS vaccinated mice served as controls. C. Pathological changes in the lungs of vaccinated mice. The following figures: representative images of hematoxylin-eosin stained sections of lung tissue from mice vaccinated with candidate MTB T cell antigen and control mice (scale bar: 200 μm). Upper left insert plate: acid-fast (AF) staining of lung tissue sections showed bacterial load; the arrow indicates AF-positive bacteria (scale bar, 50 μm).

FIG. 6 shows the immune response of Rv1705c or Rv1485 immunized mice. A is Rv1705c or Rv1485 specific IgG, igG1 and IgG2a antibody levels in immunized mice. Serum from mice immunized with Rv1705c or Rv1485 was collected three weeks after final immunization and specific IgG, igG1 and IgG2a antibodies were detected by ELISA. The results shown are average log10 endpoint titers (±sem), n=3. IgG2a: the proportion of IgG1 is shown in the lower right panel. B. Cytokines (IL-2, IL-6, TNF- α, IFN- α, IL-4, IL-10) were released from spleen cells isolated from Rv1705c or Rv1485 immunized mice. The mouse spleen cells were harvested from immunized mice four weeks after the last immunization. Cells were seeded into 24 well plates and incubated with either Rv1705c (10. Mu.g/mL) or Rv1485 (10. Mu.g/mL), PPD (purified protein derivative; 10. Mu.g/mL, positive control) or RPMI1640 medium (negative control) for 24h (IL-2 assay) or at 37℃for 72h (all other cytokines were detected). Cytokines were detected in cell supernatants using commercial IL-2, IL-6, TNF- α, IFN- α, IL-4 and IL-10ELISA kits. Results are shown as mean ± SEM. * **: p <0.05. The results presented are from two independent experiments.

FIG. 7 is a characterization study of the Rv1705c epitope.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The quantitative tests in the following examples were all set up in triplicate and the results averaged.

The Mycobacterium bovis BCG strain is a product of ATCC company, catalog number 35734. The Mycobacterium bovis BCG strain is hereinafter referred to as BCG.

Mycobacterium tuberculosis H37Rv strain is provided by Beijing thoracic hospital affiliated to university of capital medical science. Mycobacterium tuberculosis H37Rv strain hereinafter referred to as Mycobacterium tuberculosis.

Coli Rosetta (DE 3) is a product of Beijing full gold biotechnology Co. LB liquid medium is a product of the company oxoid, UK. The X.DOT-TB kit is a product of Guangdong body Bikang Biotechnology Co.

The T-SPOT.TB kit is a product of Oxford Immunotec, inc. of England. Both the IFN-gamma monoclonal capture antibody and the IFN-gamma detection antibody are components of the T-SPOT.TB kit.

Mycobacterium tuberculosis medium: an aqueous solution containing 0.5% (v/v) glycerol, 0.05% (v/v) Tween-80 and 10% (v/v) liquid Middlebrook7H 9 medium. Liquid Middlebrook7H 9 medium is a product of BD company.

MedieBurk7H10 medium: middlebrook7H10 medium containing 10% (v/v) OADC. Middlebrook7H10 medium is a product of BD company.

In the examples below, statistical analysis was performed using a graphic pad prism (version 6) and Excel. Data are expressed as ± SEM. Determining the statistical significance of the comparison data between groups using one-way analysis of variance (ANOVA), P <0.05; * P <0.01; * P <0.001. The significance of the inter-sample differences was analyzed using paired t-test, with statistical treatment at P <0.05.

IFN-y release assays in the examples below were performed using either the X.DOT-TB kit or the T-SPOT.TB kit.

The procedure for detection using the T-SPOT. TB kit was as follows:

(1) Blood samples were taken and PBMC were collected using Ficoll-Paque density gradient centrifugation.

(2) A96-well plate was used, 100. Mu.L of IFN-. Gamma.monoclonal capture antibody was added to each well, and coated overnight at 4 ℃.

(3) After the completion of step (2), the 96-well plate was taken, the liquid phase was discarded, washed twice (1 min each time) with PBS buffer at pH7.4 and 0.01M, and then the plate was dried.

(4) After completion of step (3), 200. Mu.L of PBS buffer, pH7.4, containing 2% (v/v) BSA, 0.01M was added to each well of the 96-well plate, and incubated at 37℃for 1 hour.

(5) After the step (4) is completed, taking the 96-well plate, discarding the liquid phase, adding RPMI1640 culture solution, and washing once.

(6) Taking the 96-well plate finished in the step (5), and adding 50 mu L of an aqueous solution of the mycobacterium tuberculosis protein (the concentration is 200 pmol) into each detection well; 50. Mu.L of an aqueous solution (200 pmol) of the fusion protein CFP10-ESAT6 was added to each control well; add 50. Mu.L of serum-free medium to each negative control well; mu.L of phytohemagglutinin solution (phytohemagglutinin dissolved in AIM V) was added to each positive control well ^TM Medium serum-free Medium was obtained at a concentration of 200 pmol).

(7) After completion of step (6), 100. Mu.L of PBMC (about 2.5X10) collected in step (1) was added to each well ⁶ Individual lymphocytes).

(8) After step (7) is completed, the 96-well plate is placed in an incubator at 37 ℃ with 5% CO ₂ Culturing for 18-20h.

(9) After the step (8) is completed, taking the 96-well plate, discarding the supernatant, adding 200 mu L of precooled ice water, firstly placing at-20 ℃ for 5min (aiming at cell lysis), and then placing at 4 ℃ for 5min (aiming at cell lysis).

(10) After completion of step (9), the 96-well plate was taken, the supernatant was discarded, washed 7 times with washing solution (200. Mu.L of washing solution each time, 1min each time), and patted dry.

(11) After completion of step (10), 100. Mu.L of IFN-gamma detection antibody dilution (made up of 999 parts by volume of pH7.4, 0.01M PBS buffer and 1 part by volume of IFN-gamma detection antibody) was added to each well of the 96-well plate, and incubated at 37℃for 1 hour.

(12) After the completion of step (11), the 96-well plate was taken, the supernatant was discarded, washed 5 times with washing solution (200. Mu.L of washing solution was added each time, and washing was performed for 1min each time), and the plate was dried.

(13) After completion of step (12), 100. Mu.L of a dilution of the alkaline phosphatase-labeled streptavidin (prepared by mixing 999 parts by volume of pH7.4, 0.01M PBS buffer and 1 part by volume of alkaline phosphatase-labeled streptavidin) was added to each well, and incubated at 37℃for 30min.

(13) After completion of step (12), the 96-well plate was taken, the supernatant was discarded, washed 5 times with washing solution (200. Mu.L of washing solution was added each time, and washing was performed for 1min each time), and the plate was dried.

(14) After the step (13) is completed, 100 mu L of BCIP/NBT substrate solution is added into each well of the 96-well plate, and the light is turned off and color development is carried out for 7-15min at room temperature.

(15) After completion of step (14), the 96-well plate was taken, washed 3 times with distilled water (for the purpose of stopping the reaction), and then the 96-well plate was put in an oven at 37 ℃ for drying.

(16) After step (15) is completed, the 96-well plates are counted using an immunoblotter S6 spot reader (CTL, cleveland, ohio); the results were analyzed using prism graphics software.

The amino acid sequence of the Rv1705c protein is shown as a sequence 1 in a sequence table.

The amino acid sequence of the Rv1485 protein is shown as a sequence 2 in a sequence table.

Example 1 high throughput purification of Mycobacterium tuberculosis 1781 protein and detection of its antigenicity

The inventors of the present invention used a high throughput method system to identify MTB protein antigens based on the ability of MTB protein antigens to induce IFN-gamma release from Peripheral Blood Mononuclear Cells (PBMC) isolated from patients with active tuberculosis, and then evaluated the protective effect of MTB protein antigens in BALB/c mice in a model of infection with tuberculosis. The experimental strategy for identifying MTB antigen protein and verifying the protective effect of MTB antigen protein in a model of BALB/c mice infected by tubercle bacillus is shown in FIG. 1.

1. High throughput purification of mycobacterium tuberculosis 1781 protein

1. Construction of recombinant plasmids

Mycobacterium tuberculosis clone library (described in Deng J, bi L, zhou L, guo SJ, fleming J, et al (2014) Mycobacterium tuberculosis proteome microarray for848global studies of protein function and immunogeneticity. Cell Rep 9:2317-2329; the library contains 3404H 37Rv and 437 CDC1551 sequencing ORF clones), each clone and vector pDEST17 were subjected to LR recombination reaction by LR clonase mixture to obtain the corresponding recombinant plasmid.

The vector pDEST17 and LR clonase mixture are both products of Invitrogen.

2. Expression of Mycobacterium tuberculosis proteins

(1) And (3) respectively introducing the recombinant plasmids constructed in the step (1) into escherichia coli Rosetta (DE 3) to obtain corresponding recombinant escherichia coli.

(2) After the step (1) is completed, the recombinant escherichia coli is respectively inoculated to 1mL of LB liquid medium containing 34 mug/mL of chloramphenicol and 100 mug/mL of ampicillin, and the corresponding culture broth 1 is obtained after shaking culture for 4 hours at 37 ℃ and 200 rpm.

(3) After the step (2) is completed, IPTG is respectively added into the culture bacteria liquid 1, so that the concentration of the IPTG in the system is 0.4mM, and then the culture bacteria liquid 2 is obtained after shaking culture for 6 hours at 37 ℃ and 200 rpm.

(4) After the step (3) is completed, the culture bacterial liquid 2 is respectively taken and centrifuged at 6000rpm for 15min, the supernatant is discarded, and the corresponding bacterial bodies are collected.

(5) After the completion of the step (4), the cells were resuspended in EDTA buffer having pH8.0 and 0.05M containing 50mM Tris-HCl, respectively, and then sonicated (sonication parameters: 12000rpm, total 10 min) to obtain a cell disruption solution. The cell disruption solution was centrifuged at 12000rpm for 10min to obtain a cell disruption supernatant and a cell disruption precipitate.

And (3) performing SDS-PAGE on the bacterial cell disruption supernatant and the bacterial cell disruption precipitate obtained in the step (5) respectively.

The experimental results show that each mycobacterium tuberculosis protein exists in inclusion bodies.

3. Purification of Mycobacterium tuberculosis proteins

The experiments were all performed at 4 ℃.

(1) The cells obtained in step 2 (5) were crushed and precipitated, and washed successively with 50mM Tris-HCl buffer, 2% (v/v) DOC-containing EDTA buffer of pH8.0, 0.05M and 1M urea aqueous solution.

(2) After completion of step (1), the suspension was resuspended in 50mM Tris-HCl buffer to give the corresponding resuspension.

(3) After completion of step (2), the sample was washed with EDTA buffer at pH8.0, 1 mM.

(4) After completion of step (3), the resulting solution was dissolved in EDTA buffer of pH8.0 containing 15mM DTT and 8M urea at a concentration of 1mM to obtain a corresponding solution.

(5) After completion of step (4), the solution was dialyzed (water was added very slowly to solution 1 using a pump until solution 1 was diluted 10 times) against solution 1 (pH 10, 200mM Tris-HCl buffer containing 8M urea and 20mM L-Argine) to obtain the corresponding dialysate 1.

(6) After completion of step (5), dialysate 1 was dialyzed against solution 2 (pH 8.0 containing 20mM NaCl, 20mM Tris-HCl buffer) to give the corresponding dialysate 2.

The dialysate 2 was subjected to SDS-PAGE, respectively. Detection kit using GC endotoxin (Xiamen Limulus reagent Biotech Co., ltd.) the product of (2) to detect the purity of the corresponding Mycobacterium tuberculosis protein in each dialysate 2.

The results show that the purity of the corresponding mycobacterium tuberculosis proteins in the dialysate 2 is higher.

4. Renaturation

After step 3, dialysate 2 was taken separately and purified using standard protein denaturation and renaturation techniques to yield 1781 Mycobacterium tuberculosis proteins (including 1728H 37Rv and 53 CDC1551 proteins).

To demonstrate that the Mycobacterium tuberculosis protein purified using this strategy is equivalent to the protein purified after soluble expression, the inventors of the present invention evaluated the properties of Mycobacterium tuberculosis protein obtained in step 4 and commercial CFP10 purified recombinant CFP10 (Rv 3874) using IFN-gamma release assay. The results are shown in FIG. 2 (top panel is T-SPOT. TB kit, bottom panel is X.DOT-TB kit). The results showed that the results of the Mycobacterium tuberculosis protein obtained in step 4 and the recombinant CFP10 (Rv 3874) purified by commercial CFP10 were substantially identical, and this result suggests that the denatured/renatured protein can be used for antigen screening.

The above results indicate that the inventors of the present invention were able to purify 1781 proteins (1728H 37Rv and 53 CDC1551 proteins) to the appropriate standard for further analysis (> 80% purity and concentration of not less than 0.5 mg/mL). The molecular weight and purity of the proteins were assessed by SDS-PAGE (see Table 1 for partial proteins). Comparison of the functional class analysis of 1728 proteins (using the tuberculosis list) with the distribution of the entire proteome among the functional classes revealed that the 1781 proteins evaluated here were essentially randomly selected, and this subset was considered to be a good representation of the entire proteome. Over-expression of proteins and under-expression of cell wall and cellular process proteins in the intermediary metabolic and respiratory protein classes may reflect differences between proteins in terms of ease of purification.

TABLE 1

Rv No.	Gene name	Predicted molecular weight (kDa)	Actual molecular weight (kDa)
				Rv1705c	PPE22	38.444	41
Rv1485	hemZ	37.144	45

2. Antigenic detection of mycobacterium tuberculosis 1781 protein

1. Collection of clinical samples

Blood samples 2803 of patients with tuberculosis (determined specifically based on clinical symptoms, chest X-ray film, sputum smear microscope, bacteria culture results, IFN-. Gamma.ELISPOT test results and contact history) and 167 of healthy donors "clinically free from significant active tuberculosis symptoms and confirmed as tuberculosis negative by PPD test and IFN-. Gamma.ELISPOT test" were collected, respectively.

2. IFN-gamma ELISPOT detection

During Mycobacterium tuberculosis infection, T cells react to Mycobacterium tuberculosis antigens and play an important role in host-pathogen interactions. T cells are sensitized by mycobacterium tuberculosis antigens, and activated T cells, including cd4+ and cd8+ T cells, release IFN- γ when stimulated in vitro by different mycobacterium tuberculosis specific antigens, the levels of which reflect the intensity of the immune response induced by a given antigen. IFN-gamma release assays (ELISPOT or ELISA methods) are used in the clinical detection of latent Mycobacterium tuberculosis infection and as an aid in the diagnosis of active tuberculosis. Here, the 1781 Mycobacterium tuberculosis proteins obtained above were screened using the commercial IFN-. Gamma.ELISPOT method to assess the ability of each protein to stimulate IFN-. Gamma.release from PBMC derived from suspected tuberculosis patients.

The commercial IFN-. Gamma.ELISPOT assay involves antibody-based detection of IFN-. Gamma.release in response to the nuclear specific antigens ESAT6 and CF10, the results being reported as the number of spot forming cells, i.e., the number of IFN-. Gamma.producing lymphocytes detected. When the number of SFCs detected by ESAT6/CFP10 exceeded a certain standard (. Gtoreq.6 for the T-SPOT.TB kit and (. Gtoreq.11 for the X.DOT-TB kit), the test reading was considered positive for MTB infection. The test results may be compared to the final clinical diagnosis (or microbial culture results, known as the "gold standard" for tuberculosis diagnosis) to determine the sensitivity and specificity of the analysis.

The inventors of the present invention first conducted a rough primary screening in which PBMC samples from 8 suspected tuberculosis patients (including those who were ultimately diagnosed with active tuberculosis and those who were ultimately diagnosed with nontuberculous tuberculosis) were stimulated with ESAT6/CFP10 (control) and purified mycobacterium tuberculosis, respectively. Mycobacterium tuberculosis proteins were tested for IFN-gamma release levels using a commercial ELISPOT assay (T.SPOT-TB kit or X-DOT.TB kit, with separate clinical evaluations showing that these two commercial kits are essentially identical and are used interchangeably; see Table 3 and FIG. 3). While most of the MTB proteins tested did not induce detectable IFN- γ release levels, 369 MTB proteins stimulated detectable IFN- γ release levels in at least one of the 8 PBMC samples (see table 4 and fig. 4 a). The results were then compared to the final diagnosis obtained for each patient from whom PBMCs were obtained, and further analysis of 97 proteins that only caused IFN-gamma release from non-tubercular patients (see table 4) was excluded. To increase the likelihood of selecting the most promising antigen candidates for further evaluation, 188 MTB proteins (see table 4) that caused IFN- γ release in at least two active tuberculosis patients were selected for further analysis against another 20 PBMC samples from suspected patients. 49 of the 188 candidate antigens from screen 1 with tuberculosis showed strong antigen activity in screen 2 (IFN-. Gamma.release was stimulated in. Gtoreq.40% of the PBMC samples) and were continued for further analysis (see Table 5 and FIG. 4B). 49 antigen candidates caused more than 40% (i.e. more than 8) of IFN-gamma release from the PBMC sample.

The statistical information provided by the blood samples is shown in table 2.

TABLE 2

TABLE 3 Table 3

TABLE 4 Table 4

TABLE 5

The final round of IFN-gamma release screening was performed on the 49 candidate MTB antigens from screen 2, on 20 PBMC samples from active tuberculosis patients and 20 PBMC samples from healthy donors to select for sustained challenge of active tuberculosis (40%) and limited IFN-gamma immune response (30%) in healthy donors. 20 candidate antigens (see Table 6) were evaluated in a mouse challenge model, including 6 PPE protein family members (PPE 22, PPE23, PPE26, PPE28, PPE30, PPE 32), 5 intermediate metabolism and respiratory protein family members (HemZ, rv0082, adhA, nadA, rv 0187), 4 conserved hypothetical proteins (Rv 1352, rv1341, rv1482 and Rv 1147), regulatory protein class (Rv 3095, rv1151 c) 2, lipodystrophin class (Rv 1867, fadD 11.1) 2 and 1 member of the toxic, detoxification and adaptation protein class (Rv 2303 c).

TABLE 6

3. Mycobacterium tuberculosis antigen immunization

BALB/c female mice of 6-8 weeks old purchased from Beijing Vitolihua Biotechnology Co., ltd, were not available at the Beijing thoracic Hospital animal center affiliated to the university of capital medical scienceAnd (3) feeding under special pathogen conditions. In total, 20 MTB antigens were evaluated in 3 experiments, 6 proteins in the first experiment, and 7 proteins in the two subsequent experiments, each including positive and negative controls. After one week of adaptation, mice were randomly divided into experimental and control groups (4 each), immunized 3 times subcutaneously (2 weeks apart), and immunized with 30 μg of Mycobacterium tuberculosis protein in 200 μl of incomplete Freund's adjuvant. Negative control mice were subcutaneously injected with saline and adjuvant 3 times (2 weeks apart), and BCG positive control mice were subcutaneously injected with single dose of BCG 1×10 ⁵ CFU. Mice were monitored daily by the animal care provider until the experiment was terminated.

4. Mycobacterium tuberculosis H37Rv infection

After 6 weeks from the last immunization in step 3, a bacterial suspension of Mycobacterium tuberculosis H37Rv (concentration 5X 10 ⁶ CFU/mL) was injected into the external vein of the tail of the mice (100. Mu.L of each mouse was inoculated at a dose of 5X 10 per mouse ⁵ CFU), the immunized mice were subjected to mycobacterium tuberculosis H37Rv vein infection. Mice were sacrificed after 6 weeks with cervical dislocation and lungs harvested. The lung organs were transferred to plastic Tekmar bags with 10mL of PBS buffer containing 0.1% (v/v) Tween-80. In FASTPRE-24 (MP biomedical), one leaf of each lung was homogenized in sterile 0.05% pbs-tween 80. Homogenized lung suspensions were plated in 10-fold serial dilutions on MedieBurk7H10 medium. Culturing at 37 deg.C for 3-4 weeks, humidifying air and 5% CO ₂ After that, colony Forming Units (CFU) scores. The other lung lobes were fixed in formaldehyde solution (4%) prior to hematoxylin-eosin (HE) and acid fast staining (AF) staining. Pathologists of histology slide exams and blindness scores are not associated with the study.

The inventors of the present invention evaluated the protective efficacy of the 20 candidate MTB antigens selected above in a BALB/c mouse challenge model. 7-9 week old BALB/c mice (three injections at 2 week intervals) were immunized with 30 μg of candidate antigen (in Freund's incomplete adjuvant) via lateral tail vein. After 6 weeks, mycobacterium tuberculosis H37Rv (5X 10 per mouse) ⁵ CFU) the mice were challenged intravenously and euthanized after six additional weeks. Will be used for waitingComparison of the bacterial load in the lungs of mice vaccinated with selected T cell antigens with those of control mice injected with saline or BCG (see fig. 5a and table 7) shows that the three protein antigens have significant protective efficacy; the pulmonary bacterial load of mice vaccinated with Rv1485 (4.86.+ -. 0.06log10 CFU) was significantly lower than that of the normal saline injected negative control mice (5.33.+ -. 0.04log10CFU, p<0.01, t-test) and can provide a slightly stronger protection than BCG control (5.00±0.04log10 cfu). Similarly, the lung bacterial load of Rv1705c and Rv1802 vaccinated mice was significantly lower than that of the negative control mice (Rv 1705c, 4.72.+ -. 0.07log10CFU versus saline, 5.33.+ -. 0.11log10CFU; rv1802, 4.90.+ -. 0.05log10CFU versus saline, 5.30.+ -. 0.07log10 CFUs). Rv1705c and Rv1485 provide substantially similar protection as BCG (see a and table 7 in fig. 5). Less tissue damage was observed during the lung microscopy of mice immunized with Rv1705c or Rv1485 relative to saline controls, and the lungs of mice immunized with these antigens were similar in appearance to those of mice immunized with BCG (see B in fig. 5). Consistent with this change in bacterial load, a significant difference in Hematoxylin and Eosin (HE) staining of the tissue was observed; similar to BCG controls, mice of Rv1705c or Rv1485 showed more severe interstitial pneumonia and inflammation throughout the lung than in saline controls. Infiltration of inflammatory cells into the alveolar wall was less pronounced and alveolar air gaps were not eliminated to the same extent as saline control (see C in fig. 5). The results of the acid quick staining showed that BCG, rv1705c and Rv1485 inoculated mice had no apparent mycobacterium tuberculosis bacterial cells in lung tissue compared to saline control.

By further assessing 20 of the 49 proteins that induced a strong specific cellular antigen response in the BALB/c mouse challenge model, it was shown that the Rv1705c protein and Rv1485 protein had approximately equivalent protective effects to bcg and significantly reduced bacterial burden in the lungs of infected mice.

TABLE 7

5. Mouse spleen cell culture and cytokine detection

(1) Spleen cells were isolated from mice 3 weeks after the last immunization of step 3. The spleen from harvest was homogenized and filtered through a nylon cell filter (BD Pharmingen company product, pore size 100 μm). The obtained spleen cells were washed twice with RPMI1640 medium (Invitrogen corporation product), centrifuged at 1000rpm for 5min, and the cell pellet was resuspended in the medium (RPMI 1640 medium containing 10% (v/v) FBS) and then inoculated into 96-well plates (about 2.5X10) ⁵ Cells/wells).

(2) To the 96-well plate, an aqueous solution of Mycobacterium tuberculosis protein (concentration: 10. Mu.g/mL), serum-free medium or PPD (concentration: 10. Mu.g/mL) was added, mixed, and then incubated at 37℃for 24 hours (for detecting IL-2) or 72 hours (for detecting IFN-. Alpha., TNF-. Alpha.IL-4, IL-6 or IL-10).

(3) After completion of step (2), the supernatant was collected by centrifugation at 1000rpm for 5min, and the IFN-. Alpha.s, TNFα, IL-2, IL-4, IL-6 and IL-10 concentrations were detected using ELISA kit (bioleged, san Diego).

6. Antigen-specific antibody titre

After 1 week after the last immunization of step 3, specific serum IgG2a and IgG1 isotype antibody responses were detected by ELISA. The method comprises the following specific steps:

(1) A96-well plate was prepared, and 100. Mu.L of a buffer solution containing Mycobacterium tuberculosis protein at a concentration of 1. Mu.g/mL and having a pH of 9.6 and 0.05M was added to each well, and the mixture was coated overnight at 4 ℃.

(2) After completion of step (1), the 96-well plate was first buffered with PBS-T buffer (containing 137mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ 、2mM KH ₂ PO ₄ And 0.05% (v/v) Tween-20) for 3 times; then placing in a blocking buffer (PBS-T buffer containing 3% (v/v) BSA) and standing at 37 ℃ for 2h; finally, the mixture is washed 3 times by PBS-T buffer solution, and then the diluted solution of mouse serum is added for standing reaction for 1h at 37 ℃.

Mouse serum dilution: the mouse serum was diluted to 1000-fold with incubation buffer (PBS-T buffer containing 1% (v/v) BSA).

(3) After the step (2) is completed, the 96-well plate is taken and washed with PBS-T buffer solution for 3 times; horseradish peroxide is then added to each wellEnzyme (HRP) -conjugated goat anti-mouse IgG1 and IgG2a antibodies (Bethyl Laboratories, montary, texas) (working concentration: 10000-fold diluted with incubation buffer), standing for 1h at 37 ℃; then washing 3 times with PBS-T buffer, adding TMB (3, 3', 5' -tetramethylbenzidine) aqueous solution, and incubating the 96-well plate at room temperature for 15min; finally add 2N H ₂ SO ₄ The reaction was terminated, the light emission at 450nm was read using a microplate reader (Tecan, switzerland) and IgG2a was calculated: igG1 endpoint cost performance.

Potential subunit vaccine candidates must be able to induce humoral responses to provide protection against tuberculosis. To assess the humoral response elicited by Rv1705c and Rv1485, BALB/c mice were immunized (three injections at 2 week intervals) with Rv1705c or Rv1485 (in incomplete freund's adjuvant) via lateral tail veins. Titers of Rv1705c or Rv1485 specific IgG, igG1 and IgG2a were measured in serum obtained three weeks after the last immunization with ELISA. High titers of specific IgG, igG1 and IgG2a for Rv1705c or Rv1485 were observed, and no IgG/IgG1/IgG2a was detected in the vaginal control group (see fig. 6 a and table 8). Th1 immune responses have been reported to have an important protective effect on Mycobacterium tuberculosis infection (Refs), while Th2 immune responses are thought to impair host protective immunity. The ratio of IgG2a/IgG1 (IgG 2a is a Th 1-type antibody and IgG1 is a Th 2-type antibody) was plotted to indicate whether Th1 or Th2 profile was induced by Rv1705c or Rv1485. The IgG2a/IgG1 ratio in Rv1705c group was 3.33 and BCG was 1.33, indicating that a moderate Th1 type immune response was induced in response to Rv1705c antigen. The IgG2a/IgG1 ratio in the Rv1485 group was 1.67 and BCG was 1.33, indicating that a moderate Th1 type immune response was induced in response to the Rv1485 antigen.

TABLE 8

Since mycobacterium tuberculosis is an intracellular pathogen, the importance of the cellular response is at least as important as the humoral response. To evaluate cellular responses, secretion of typical Th1 cytokines IL-2, IL-6, TNF- α and IFN- α were measured, which not only activate macrophages, but also promote polarization of effector Th1 cells and Th2 cytokines, IL-4 and IL-10, which are responsible for inhibiting macrophage function and promoting antibody responses. Mice immunized with Rv1705c or Rv1485 (3 times, 2 weeks apart) were euthanized 4 weeks after the third round of immunization, and spleen cells were harvested. Cytokine expression levels in culture supernatants of isolated spleen cells stimulated with antigen and PPD (purified protein derivative), respectively, were measured by ELISA. The results showed that Th1 cytokines IL-2, IL-6, TNF-. Alpha.and IFN-. Gamma.were all present at high concentrations after stimulation with either Rv1705c or Rv1485, whereas Th2 cytokines IL-4 and IL-10 were not detected (see FIG. 6, B and Table 9). The levels of cytokine detected were significantly higher for Rv1705c or Rv1485 immunized mice than for BCG immunized mice, probably because these mice received only one BCG immunization rather than three immunizations of Rv1705 or Rv1485 immunized mice. In addition, the time between BCG immunization and euthanasia was 8 weeks for BCG immunized mice and 4 weeks for mice immunized with Rv1705c or Rv1485, and no spleen cells harvested were stimulated with any immunogen prior to measuring cytokine release. Th1 cytokines such as IL-2, TNF- α and the well known anti-tuberculosis cytokine IFN- γ promote Th1 immune responses, play an important role in combating pathogens, controlling tuberculosis and effectively protecting the host. The results indicate that Rv1705c or Rv1485 elicit potent Th 1-type humoral and cellular immune responses, favoring tuberculosis control, possibly explaining why Rv1705c protein or Rv1485 protein may protect the host from mycobacterium tuberculosis.

TABLE 9

<110> biological physical institute of sciences of China academy of sciences of biological sciences of Guangdong Libikang

<120> application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of tuberculosis vaccine

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 385

<212> PRT

<213> Artificial sequence

<400> 1

Met Asp Phe Gly Ala Leu Pro Pro Glu Val Asn Ser Gly Arg Met Tyr

1 5 10 15

Cys Gly Pro Gly Ser Ala Pro Met Val Ala Ala Ala Ser Ala Trp Asn

20 25 30

Gly Leu Ala Ala Glu Leu Ser Val Ala Ala Val Gly Tyr Glu Arg Val

35 40 45

Ile Thr Thr Leu Gln Thr Glu Glu Trp Leu Gly Pro Ala Ser Thr Leu

50 55 60

Met Val Glu Ala Val Ala Pro Tyr Val Ala Trp Met Arg Ala Thr Ala

65 70 75 80

Ile Gln Ala Glu Gln Ala Ala Ser Gln Ala Arg Ala Ala Ala Ala Ala

85 90 95

Tyr Glu Thr Ala Phe Ala Ala Ile Val Pro Pro Pro Leu Ile Ala Ala

100 105 110

Asn Arg Ala Arg Leu Thr Ser Leu Val Thr His Asn Val Phe Gly Gln

115 120 125

Asn Thr Ala Ser Ile Ala Ala Thr Glu Ala Gln Tyr Ala Glu Met Trp

130 135 140

Ala Gln Asp Ala Met Ala Met Tyr Gly Tyr Ala Gly Ser Ser Ala Thr

145 150 155 160

Ala Thr Lys Val Thr Pro Phe Ala Pro Pro Pro Asn Thr Thr Ser Pro

165 170 175

Ser Ala Ala Ala Thr Gln Leu Ser Ala Val Ala Lys Ala Ala Gly Thr

180 185 190

Ser Ala Gly Ala Ala Gln Ser Ala Ile Ala Glu Leu Ile Ala His Leu

195 200 205

Pro Asn Thr Leu Leu Gly Leu Thr Ser Pro Leu Ser Ser Ala Leu Thr

210 215 220

Ala Ala Ala Thr Pro Gly Trp Leu Glu Trp Phe Ile Asn Trp Tyr Leu

225 230 235 240

Pro Ile Ser Gln Leu Phe Tyr Asn Thr Val Gly Leu Pro Tyr Phe Ala

245 250 255

Ile Gly Ile Gly Asn Ser Leu Ile Thr Ser Trp Arg Ala Leu Gly Trp

260 265 270

Ile Gly Pro Glu Ala Ala Glu Ala Ala Ala Ala Ala Pro Ala Ala Val

275 280 285

Gly Ala Ala Val Gly Gly Thr Gly Pro Val Ser Ala Gly Leu Gly Asn

290 295 300

Ala Ala Thr Ile Gly Lys Leu Ser Leu Pro Pro Asn Trp Ala Gly Ala

305 310 315 320

Ser Pro Ser Leu Ala Pro Thr Val Gly Ser Ala Ser Ala Pro Leu Val

325 330 335

Ser Asp Ile Val Glu Gln Pro Glu Ala Gly Ala Ala Gly Asn Leu Leu

340 345 350

Gly Gly Met Pro Leu Ala Gly Ser Gly Thr Gly Thr Gly Gly Ala Gly

355 360 365

Pro Arg Tyr Gly Phe Arg Val Thr Val Met Ser Arg Pro Pro Phe Ala

370 375 380

Gly

385

<210> 2

<211> 344

<212> PRT

<213> Artificial sequence

<400> 2

Met Gln Phe Asp Ala Val Leu Leu Leu Ser Phe Gly Gly Pro Glu Gly

1 5 10 15

Pro Glu Gln Val Arg Pro Phe Leu Glu Asn Val Thr Arg Gly Arg Gly

20 25 30

Val Pro Ala Glu Arg Leu Asp Ala Val Ala Glu His Tyr Leu His Phe

35 40 45

Gly Gly Val Ser Pro Ile Asn Gly Ile Asn Arg Thr Leu Ile Ala Glu

50 55 60

Leu Glu Ala Gln Gln Glu Leu Pro Val Tyr Phe Gly Asn Arg Asn Trp

65 70 75 80

Glu Pro Tyr Val Glu Asp Ala Val Thr Ala Met Arg Asp Asn Gly Val

85 90 95

Arg Arg Ala Ala Val Phe Ala Thr Ser Ala Trp Ser Gly Tyr Ser Ser

100 105 110

Cys Thr Gln Tyr Val Glu Asp Ile Ala Arg Ala Arg Arg Ala Ala Gly

115 120 125

Arg Asp Ala Pro Glu Leu Val Lys Leu Arg Pro Tyr Phe Asp His Pro

130 135 140

Leu Phe Val Glu Met Phe Ala Asp Ala Ile Thr Ala Ala Ala Ala Thr

145 150 155 160

Val Arg Gly Asp Ala Arg Leu Val Phe Thr Ala His Ser Ile Pro Thr

165 170 175

Ala Ala Asp Arg Arg Cys Gly Pro Asn Leu Tyr Ser Arg Gln Val Ala

180 185 190

Tyr Ala Thr Arg Leu Val Ala Ala Ala Ala Gly Tyr Cys Asp Phe Asp

195 200 205

Leu Ala Trp Gln Ser Arg Ser Gly Pro Pro Gln Val Pro Trp Leu Glu

210 215 220

Pro Asp Val Thr Asp Gln Leu Thr Gly Leu Ala Gly Ala Gly Ile Asn

225 230 235 240

Ala Val Ile Val Cys Pro Ile Gly Phe Val Ala Asp His Ile Glu Val

245 250 255

Val Trp Asp Leu Asp His Glu Leu Arg Leu Gln Ala Glu Ala Ala Gly

260 265 270

Ile Ala Tyr Ala Arg Ala Ser Thr Pro Asn Ala Asp Pro Arg Phe Ala

275 280 285

Arg Leu Ala Arg Gly Leu Ile Asp Glu Leu Arg Tyr Gly Arg Ile Pro

290 295 300

Ala Arg Val Ser Gly Pro Asp Pro Val Pro Gly Cys Leu Ser Ser Ile

305 310 315 320

Asn Gly Gln Pro Cys Arg Pro Pro His Cys Val Ala Ser Val Ser Pro

325 330 335

Ala Arg Pro Ser Ala Gly Ser Pro

340

Claims

Application of Rv1485 protein in preparing tuberculosis detection reagent, tuberculosis vaccine or antitubercular drug;

the Rv1485 protein is a protein composed of an amino acid sequence shown as a sequence 2 in a sequence table;

the application is realized only by the Rv1485 protein;

the application was not achieved by any mycobacterium tuberculosis antigen other than Rv1485 protein.
Application of Rv1485 protein in preparing T-SPOT.TB kit or X.DOT-TB kit;

the Rv1485 protein is a protein composed of an amino acid sequence shown as a sequence 2 in a sequence table;

the application was not achieved by any mycobacterium tuberculosis antigen other than Rv1485 protein.
3. The use according to claim 2, characterized in that: the kit also contains a capture antibody and/or a detection antibody;

the capture antibody is a mouse monoclonal antibody against human or animal IFN-gamma;

the detection antibody is another mouse monoclonal antibody of different epitopes of anti-human or animal IFN-gamma.
4. A use according to claim 2 or 3, characterized in that: the kit also contains a positive standard substance and/or a negative standard substance;

the positive standard is tuberculosis non-specific stimulating antigen;

the negative standard is an antigen-free cell culture solution.
5. A tuberculosis vaccine comprising an Rv1485 protein of claim 1 and excluding any mycobacterium tuberculosis antigen other than the Rv1485 protein.
6. A tuberculosis vaccine is obtained by modifying recombinant BCG; the method is characterized in that: the recombinant BCG contains the Rv1485 protein of claim 1 and does not contain any mycobacterium tuberculosis antigens other than the Rv1485 protein.
7. An antitubercular drug, an antibody that binds to the Rv1485 protein of claim 1; the method is characterized in that: the antibody binds to an Rv1485 protein of claim 1 and does not bind to any mycobacterium tuberculosis antigen other than the Rv1485 protein.
8. An antitubercular drug according to claim 7, wherein: the antibody is prepared by using the Rv1485 protein of claim 1 as an immunogen.
9. An antitubercular drug according to claim 8, wherein: the antibody is A1) or A2):

a1 Polyclonal antibodies prepared by immunizing animals with the Rv1485 protein as an immunogen;

a2 Using the Rv1485 protein as immunogen to immunize animals, and adopting a hybridoma technology or a DNA recombination technology to prepare the monoclonal antibody.