CN111948387A

CN111948387A - Application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of tuberculosis vaccine

Info

Publication number: CN111948387A
Application number: CN201910409579.XA
Authority: CN
Inventors: 毕利军; 张泓泰; 王雅果; 张先恩; 朱国峰
Original assignee: Tb Healthcare Co ltd; Institute of Biophysics of CAS
Current assignee: Tb Healthcare Co ltd; Institute of Biophysics of CAS
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2020-11-17
Anticipated expiration: 2039-05-16
Also published as: CN111948387B

Abstract

The invention discloses application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of a tuberculosis vaccine. The Rv1485 protein is a protein composed of an amino acid sequence shown in a sequence 2 in a sequence table. Experiments prove that the Rv1485 protein triggers effective Th1 type humoral and cellular immune responses, so that the control of tuberculosis is facilitated, and the Rv1485 protein can protect a host from being invaded 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 a tuberculosis vaccine.

Background

The implementation of the tuberculosis-terminating strategy of the world health organization by 2030 has required a new momentum in various aspects of tuberculosis prevention and control, including the development of new vaccines, diagnostics and drugs. The introduction of BCG vaccines nearly 100 years ago was ineffective, but was still the only licensed vaccine that increased resistance to anti-tuberculosis drugs, as well as the lethal synergy between tuberculosis and aids virus, causing this ancient disease to reappear, posing a global threat. Infectious pathogens are currently the leading cause of death in tuberculosis. Development of improved vaccines is likely to be the most effective strategy to achieve elimination of tuberculosis. Since the present era, a great deal of investment has been made in vaccine research, and 12 vaccine candidates have been generated in the current flow line at different clinical trial stages; however, the major bottleneck is the lack of antigen candidates that induce a protective immune response, which is a key determinant of vaccine efficacy. There is a need to develop a full system antigen screening study to identify broader, more effective immunodominant protective antigens.

Bcg is an attenuated live strain of mycobacterium bovis, widely used since 1921, and has been shown to prevent severe forms of tuberculosis in children, but its efficacy against adult tuberculosis is weak and inconsistent and may lead to bcg disease spread in the immunodeficient group. In the search for alternatives, the main immunization strategy being explored is the replacement of BCG with live recombinant BCG (rBCG) or genetically attenuated Mycobacterium Tuberculosis (MTB) vaccines (both live recombinant BCG (rBCG) and genetically attenuated Mycobacterium Tuberculosis (MTB) vaccines have higher safety and protective efficacy), the limited immunity conferred by primary vaccine and "immunotherapy" (vaccines that can shorten the treatment of active tuberculosis or potential tuberculosis infection (LTBI)) approaches using viral vectors or adjuvant-based subunit vaccines, with the aim of enhancing and amplifying the immune response originally induced by BCG (which can also be rBCG or attenuated MTB vaccines) vaccinated by administering subunit vaccines (consisting of proteins, peptides or DNA) before the infant is exposed to MTB. During childhood or adolescence, the protective effect of bcg begins to diminish. The protective efficacy of the antigen as a subunit is critical to the success of this immunization strategy. However, to date, only 11 antigens that induce a fully protective immune response (PepA, PPE18, Ag85A, Ag85B, TB10.4, PPE42, EsxV, EsxW, Rv1813, ESAT-6, and Rv2660c, respectively) have been widely used. Three protein/adjuvant vaccines are currently being tested in phase II, and three are currently being tested in phase IIa H56: IC31, combining Mycobacterium tuberculosis antigens Ag85B, ESAT6 and Rv2660c with IC31 adjuvant, M72/AS01E is currently undergoing stage IIb trials in Kenya, south Africa and Zanbia on AIDS virus negative adults infected with Mycobacterium tuberculosis, is a subunit vaccine containing tubercle bacillus antigens (32A and 39A) and adjuvant. Although the steady development of candidate vaccines through clinical trials is encouraging, the anti-tuberculosis protection provided by the promising candidate vaccines previously developed (e.g., MVA85A) does not significantly increase compared to vaccination with bcg alone (Refs), which is an important reminder for the development of tuberculosis vaccines and a challenge to develop effective tuberculosis vaccines.

Inadequate understanding of the nuclear immune response is an additional challenge facing the development of effective vaccines, particularly because there are no known immunologically relevant protective or therapeutic biomarkers available at present. It was reported that BCG induces T helper 1(Th1) -type responses, mainly involving IFN-. gamma.production (REFs) by CD4+ T cells. To be effective as a booster for the protection of tuberculosis, ideal candidate antigens should induce cellular immune responses at least as strong as BCG, including CD4+ and CD8+ T cells, as well as non-conventional T cells, such as gamma T cells and CD 1-restricted alpha beta T cells (Refs). IFN-gamma plays a key role in human and mouse tuberculosis control. TB diagnostic kits based on IFN- γ release in response to stimulation by MTB antigens 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 boosters for further development into protein subunit vaccines.

Disclosure of Invention

The object of the present invention is to prepare tuberculosis vaccines.

The invention firstly protects the application of the Rv1485 protein in the preparation of tuberculosis detection reagents, tuberculosis vaccines or antituberculosis drugs.

The Rv1485 protein may be a1) or a2) or a 3):

a1) protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

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

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

In order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with the label shown in the table 11.

TABLE 11 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a3), 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 of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by deleting one or several amino acid residues from the gene encoding the protein, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 11 above to the 5 'end and/or 3' end thereof.

The invention also protects a tuberculosis detection reagent which can comprise at least one of b1) -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 reagent for detecting tuberculosis, b1), b2) or b3) can be used as an effective component.

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- γ. The detection antibody can be another mouse monoclonal antibody resisting different epitopes 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 non-specific stimulating antigen of tuberculosis. The negative standard may be a cell culture fluid without antigen. The non-specific tubercular stimulating antigen may be phytohemagglutinin PHA.

Any of the kits described above may further comprise a readable carrier having recorded thereon: when the number of SFCs detected by ESAT6/CFP10 exceeds a certain standard (more than or equal to 6 for T-SPOT. TB kit and more than or equal to 11 for X.DOT-TB kit), the test reading is considered to be 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 b1) -b 3):

b1) the Rv1485 protein;

b2) a nucleic acid molecule encoding said Rv1485 protein;

In the tuberculosis vaccine, b1), b2) or b3) can be used as an effective component.

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

The invention also protects antituberculosis drugs, which may include antibodies that bind to the Rv1485 protein.

In the above anti-tuberculosis drugs, the antibody may be an antibody prepared by using the Rv1485 protein as an immunogen.

In the above anti-tuberculosis drugs, the antibody may be a1) or a 2).

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 immunogen to immunize animal (such as mouse, rat, rabbit, sheep, human) and the hybridoma technology or DNA recombination technology is adopted to prepare monoclonal antibody. The monoclonal antibody may be a humanized monoclonal antibody.

In the above anti-tuberculosis drugs, the antibody may be an active ingredient.

The anti-tuberculosis drug may specifically consist of any of the antibodies described above.

Experiments prove that the Rv1485 protein triggers effective Th1 type humoral and cellular immune responses, so that the control of tuberculosis is facilitated, and the Rv1485 protein can protect a host from being invaded by mycobacterium tuberculosis. The invention has important application value.

Drawings

FIG. 1 is an experimental strategy for identifying MTB antigen protein and verifying its protective effect in a BALB/c mouse tubercle bacillus infection model. (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) detecting the antigenicity of the protein by an IFN-gamma ELISPOT method: the proteins that induce IFN-. gamma.release in the first round of screening (PBMC samples from 8 suspected tuberculosis patients) were subjected to two additional screens (screening PBMC samples from 2: 20 suspected tuberculosis patients; screening PBMC samples from 3: 20 active tuberculosis patients and 20 healthy donors). (3) Immunizing BABL/c mice with 20 candidate antigens selected in the third screening, and then attacking with Mycobacterium tuberculosis H37 Rv; protection of candidate antigens was assessed by measuring pulmonary bacterial burden and histopathological analysis, by assessing cytokine secretion from splenocytes and IgG2 a: IgG1 ratio to evaluate host immune response, and the best Rv1705c and Rv1485 are obtained.

FIG. 2 is an IFN-. gamma.Release assay to evaluate the performance of the obtained Mycobacterium tuberculosis protein and commercial CFP10 purified recombinant CFP10(Rv 3874).

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

FIG. 4 shows that 49 candidate antigens associated with host immune response are selected from 1781 M.tuberculosis proteins by screening for IFN- γ release in PBMC samples from suspected tuberculosis patients. A is the antigenic activity of 1781 MTB proteins determined by the ELISPOT IFN- γ release assay, IFN- γ being released from 8 PBMC samples (from suspected tuberculosis patients), each column representing one MTB protein; clove style: MTB protein that stimulates IFN- γ release in a PBMC sample; light blue column: no MTB protein released by IFN- γ was detected; of the 188 putative antigen proteins identified in the first round of screening, 49 stimulated IFN- γ release in > 40% of PBMC samples. B is the selection of 20 PBMC samples (from patients suspected of having tuberculosis) for the second round of screening.

FIG. 5 is a graph demonstrating the protective efficacy of 20 candidate M.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: brine. BALB/c mice were inoculated subcutaneously with saline, BCG or one of the 20 candidate MTB T cell antigens and then 6 weeks later with 5X 10⁵CFU mycobacterium tuberculosis H37Rv intravenous infection. Mice were euthanized after another 8 weeks. Bacterial load in the lungs was assessed by scoring CFU. One-way analysis of variance (ANOVA) was used for group comparisons. Data shown are log10 for ± SEM. B is a representative image of lungs from BALB/c mice vaccinated with Rv1705c or Rv1485 6 weeks after infection with M.tuberculosis H37 Rv. Lungs from BCG and PBS vaccinated mice served as controls. C. Pathological changes in the lungs of vaccinated mice. The following figures: representative images (scale: 200 μm) of hematoxylin-eosin stained sections of lung tissue from mice vaccinated with candidate MTB T cell antigen and control mice. Upper left inset panel: acid-fast (AF) staining of lung tissue sections showed bacterial load; arrows indicate AF-positive bacteria (scale bar, 50 μm).

Figure 6 is the immune response of Rv1705c or Rv1485 immunized mice. A is Rv1705c or Rv1485 specific IgG, IgG1 and IgG2a antibody levels in immunized mice. Sera from mice immunized with Rv1705c or Rv1485 were collected three weeks after final immunization and detected by ELISA for specific IgG, IgG1 and IgG2a antibodies. Results shown are mean log10 endpoint titers (± SEM), n-3. IgG2 a: 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. Mouse splenocytes were harvested from immunized mice four weeks after the last immunization. Cells were seeded into 24-well plates and incubated with 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 for 72h (assay for all other cytokines) at 37 ℃. 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. Results presented are from two independent experiments.

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

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

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

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

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

The Mycobacterium tuberculosis H37Rv strain was provided by the Beijing thoracic Hospital, affiliated with the university of capital medicine. The Mycobacterium tuberculosis H37Rv strain is hereinafter referred to as Mycobacterium tuberculosis.

Escherichia coli Rosetta (DE3) is a product of Beijing Quanjin Biotechnology Ltd. LB liquid medium is a product of Oxoid, England. The kit of the X.DOT-TB is a product of Guangdong body Bi kang Biotech limited company.

Tb kit is a product of Oxford Immunotec, uk. The IFN-gamma monoclonal capture antibody and the IFN-gamma detection antibody are both components of a T-SPOT.

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

Medias of MedieBurk7H 10: middlebrook7H10 medium containing 10% (v/v) OADC. The medium Middlebrook7H10 is a product of BD company.

In the examples described below, statistical analysis was performed using a patterned pad prism (version 6) and Excel. Data are presented as ± SEM. Statistical significance of the comparison data between groups was determined using one-way analysis of variance (ANOVA) × P < 0.05; p < 0.01; p < 0.001. The significance of the differences between samples was analyzed using paired t-test, with statistical treatment at P < 0.05.

The IFN- γ release assay in the examples below was performed using the X.DOT-TB kit or the T-SPOT.TB kit.

Tb kit the procedure for detection using the T-spot.tb kit was as follows:

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

(2) A96-well plate was taken, 100. mu.L of IFN-. gamma.monoclonal capture antibody was added to each well, and the plate was coated overnight at 4 ℃.

(3) After the step (2) was completed, the 96-well plate was taken out, the liquid phase was discarded, and PBS buffer solution (pH7.4, 0.01M) was added to wash twice (1 min each time), and patted dry.

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

(5) And (4) after the step (4) is finished, taking the 96-well plate, discarding the liquid phase, and adding RPMI1640 culture solution for rinsing once.

(6) Taking the 96-well plate which completes the step (5), and adding 50 μ L of an aqueous solution of M.tuberculosis protein (with a concentration of 200pmol) to each detection well; add 50. mu.L of an aqueous solution of the fusion protein CFP10-ESAT6 (200 pmol concentration) to each control well; add 50. mu.L of serum-free medium to each negative control well; add 50. mu.L of phytohemagglutinin solution (phytohemagglutinin dissolved in AIM V) to each positive control well^TMMedium serum-free Medium at a concentration of 200 pmol).

(7) After completion of step (6), 100. mu.L of PBMC collected in step (1) (about 2.5X 10) was added to each well⁶Individual lymphocytes).

(8) After completion of step (7), the 96-well plate was placed in an incubator at 37 ℃ with 5% CO₂Culturing for 18-20 h.

(9) And (3) after the step (8) is finished, taking the 96-well plate, removing the supernatant, adding 200 mu L of precooled ice water, placing for 5min at the temperature of minus 20 ℃ (for cracking cells), and then placing for 5min at the temperature of 4 ℃ (for cracking cells).

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

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

(12) After the step (11) is completed, the 96-well plate is taken out, the supernatant is discarded, and the plate is washed 5 times with washing liquid (200. mu.L of washing liquid is added each time, and the washing time is 1min each time), and the plate is dried.

(13) After completion of step (12), the 96-well plate was taken, 100. mu.L of a dilution of alkaline phosphatase-labeled streptavidin (consisting of 999 parts by volume of a PBS buffer solution of pH7.4 and 0.01M and 1 part by volume of alkaline phosphatase-labeled streptavidin mixed together) was added to each well, and incubated at 37 ℃ for 30 min.

(13) After the step (12) is completed, the 96-well plate is taken out, the supernatant is discarded, and the plate is washed 5 times with washing liquid (200. mu.L of washing liquid is added each time, and the washing time is 1min each time), and the plate is dried.

(14) And (4) after the step (13) is completed, taking the 96-well plate, adding 100 mu L of BCIP/NBT substrate solution into each well, and carrying out light-blocking color development 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 placed in an oven at 37 ℃ to dry.

(16) After step (15) was completed, the 96-well plate was taken and counted using an immunospot 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 antigenicity thereof

The inventors of the present invention systematically identified the MTB protein antigen using a high throughput method, based on its ability to induce IFN- γ release from Peripheral Blood Mononuclear Cells (PBMCs) isolated from patients with active tuberculosis, and then evaluated the protective effect of the MTB protein antigen in a BALB/c mouse model of infection with tubercle bacillus. The experimental strategy for identifying MTB antigenic protein and verifying its protective effect in a model of BALB/c mice infected with tubercle bacillus is shown in FIG. 1.

High-throughput purification of mycobacterium tuberculosis 1781 protein

1. Construction of recombinant plasmid

A Mycobacterium tuberculosis clone library (described in Deng J, Bi L, Zhou L, Guo SJ, Fleming J, et al (2014) Mycobacterium tuberculosis genome microarray for848 viral studios of protein function and immunogenicity. cell Rep 9: 2317-2329; the library contains 3404H 37Rv and 437 CDC1551 sequenced ORF clones), and each clone and the vector pDEST17 are subjected to LR recombination reaction by LR clonase mixture to obtain a corresponding recombinant plasmid.

Both the vector pDEST17 and the LR clonase mixture were products of Invitrogen.

2. Expression of Mycobacterium tuberculosis proteins

(1) The recombinant plasmids constructed in the step 1 are respectively introduced into Escherichia coli Rosetta (DE3) 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 culture medium containing 34 mug/mL of chloramphenicol and 100 mug/mL of ampicillin, and the mixture is subjected to shaking culture at 37 ℃ and 200rpm for 4h to obtain corresponding culture solution 1.

(3) After the step (2) is finished, adding IPTG into the culture bacterial liquid 1 respectively to enable the concentration of the IPTG in a system to be 0.4mM, and then carrying out shaking culture at 37 ℃ and 200rpm for 6h to obtain corresponding culture bacterial liquid 2.

(4) And (4) after the step (3) is finished, respectively taking the culture bacterial liquid 2, centrifuging at 6000rpm for 15min, discarding the supernatant, and collecting corresponding thalli.

(5) After the step (4) is finished, respectively taking the thalli, using EDTA buffer solution with 50mM Tris-HCl, pH8.0 and 0.05M to resuspend, and then carrying out ultrasonic disruption (the ultrasonic parameter is 12000 r/min, and the total time is 10min) to obtain a thalli 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 cell disruption supernatant and the cell disruption precipitate obtained in the step (5) respectively.

The experimental result shows that each mycobacterium tuberculosis protein exists in the inclusion body.

3. Purification of mycobacterium tuberculosis proteins

The experiments were all carried out at 4 ℃.

(1) The disrupted bacterial cell precipitate obtained in step 2 (5) was washed with 50mM Tris-HCl buffer, 2% (v/v) DOC-containing EDTA buffer (pH 8.0, 0.05M) and 1M urea aqueous solution in this order.

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

(3) After completion of step (2), the reaction mixture was washed with 1mM EDTA buffer solution having a pH of 8.0.

(4) After completion of step (3), the resulting solution was dissolved in EDTA buffer (pH 8.0, 1 mM) containing 15mM DTT and 8M urea to obtain a corresponding solution.

(5) After completion of step (4), the solution was dialyzed (water was very slowly added 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), the dialysate 1 was dialyzed against solution 2 (20 mM Tris-HCl buffer, pH8.0, containing 20mM NaCl) to give the corresponding dialysate 2.

Dialysate 2 was subjected to SDS-PAGE, respectively. The purity of the corresponding Mycobacterium tuberculosis protein in each dialysate 2 was examined by GC endotoxin detection kit (product of Limulus xiamenensis reagent Biotech Co., Ltd.).

The results show that the purity of the corresponding M.tuberculosis proteins in the dialysate 2 is high.

4. Renaturation

After step 3 was completed, dialysate 2 was taken and purified using standard protein denaturation and renaturation techniques to obtain 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 performance of the Mycobacterium tuberculosis protein obtained in step 4 and the commercial CFP10 purified recombinant CFP10(Rv3874) with IFN-. gamma.release assay. The results are shown in FIG. 2 (upper panel is T-SPOT. TB kit, lower panel is X.DOT-TB kit). The results showed that the mycobacterium tuberculosis protein obtained in step 4 was substantially identical to the results of the commercial CFP10 purified recombinant CFP10(Rv3874), indicating that the denatured/renatured protein could be used for antigen screening.

The above results show that the inventors of the present invention were able to purify 1781 proteins (1728H 37Rv and 53 CDC1551 proteins) to appropriate standards 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 some 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 1781 proteins evaluated here were essentially randomly selected, a subset that was considered a good representation of the entire proteome. The over-expression of proteins in the intermediary metabolic and respiratory protein classes and the low expression of cell wall and cellular process proteins may reflect the differences between proteins in ease of purification.

TABLE 1

Rv No.	Name of Gene	Predicted molecular weight (KDa)	Actual molecular weight (KDa)
				Rv1705c	PPE22	38.444	41
Rv1485	hemZ	37.144	45

Second, antigenicity detection of Mycobacterium tuberculosis 1781 protein

1. Collection of clinical specimens

2803 blood samples of tuberculosis patients (specifically determined according to clinical symptoms, chest X-ray films, sputum smear microscopes, bacterial culture results, IFN-gamma ELISPOT detection results and contact history) and 167 blood samples of healthy donors who have no obvious active tuberculosis symptoms and are proved to be negative in tuberculosis through PPD tests and IFN-gamma ELISPOT tests in China are respectively collected in recent years of clinical diagnosis in certain hospitals.

2. IFN-gamma ELISPOT assay

During a mycobacterium tuberculosis infection, T cells respond to mycobacterium tuberculosis antigens and play an important role in host-pathogen interactions. T cells are primed by mycobacterium tuberculosis antigens and activated T cells, including CD4+ and CD8+ T cells, when stimulated in vitro by different mycobacterium tuberculosis-specific antigens, release IFN- γ at levels reflecting the magnitude of the immune response induced by a given antigen. The IFN-gamma release test (ELISPOT or ELISA method) is used for clinically detecting latent infection of mycobacterium tuberculosis and is used as an auxiliary tool for diagnosing active tuberculosis. Here, 1781 M.tuberculosis proteins obtained as described above were screened using a commercial IFN-. gamma.ELISPOT method to assess the ability of each protein to stimulate IFN-. gamma.release from PBMCs derived from patients suspected of having tuberculosis.

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

The inventors first performed a rough preliminary screening in which PBMC samples from 8 suspected tuberculosis patients (including patients ultimately diagnosed as active tuberculosis and patients ultimately diagnosed as non-tuberculosis) were stimulated with ESAT6/CFP10 (control) and purified mycobacterium tuberculosis, respectively. Mycobacterium tuberculosis protein, using a commercial ELISPOT assay to detect IFN- γ release levels (T.SPOT-TB kit or X-DOT.TB kit, clinical evaluation alone indicates that the two commercial kits are essentially equivalent and can be used interchangeably; see Table 3 and FIG. 3). Although most of the MTB proteins tested did not induce detectable levels of IFN- γ release, 369 MTB proteins elicited detectable levels of IFN- γ release in at least one of the 8 PBMC samples (see table 4 and a in figure 4). The results were then compared with the final diagnosis obtained for each patient from whom PBMCs were obtained, and further analysis of 97 proteins causing only IFN- γ release in non-tuberculous patients was excluded (see table 4). To increase the likelihood of selecting the most promising antigen candidates for further evaluation, 188 MTB proteins causing IFN- γ release in at least two active tuberculosis patients (see table 4) were selected for further analysis against another 20 PBMC samples from suspected patients. The 188 candidate antigens from Screen 1 with tuberculosis, 49 of which showed strong antigenic activity in Screen 2 (IFN-. gamma.release was stimulated in ≧ 40% of PBMC samples) and were continued for further analysis (see Table 5 and B in FIG. 4). 49 antigen candidates caused greater than 40% (i.e., greater than 8) IFN- γ release from PBMC samples.

The blood samples provided statistical information as shown in table 2.

TABLE 2

TABLE 3

TABLE 4

TABLE 5

For 49 candidate MTB antigens from

screen

2, 20 PBMC samples from active tuberculosis patients and 20 PBMC samples from healthy donors were subjected to a final round of IFN- γ release screening to select for persistent challenge of healthy donor active tuberculosis (40%) and limited IFN- γ immune response (30%). 20 candidate antigens (see table 6) were evaluated in a mouse challenge model, including 6 PPE protein family members (PPE22, PPE23, PPE26, PPE28, PPE30, PPE32), 5 intermediate metabolic and respiratory protein family members (HemZ, Rv0082, AdhA, NadA, Rv0187), 4 conserved hypothetical proteins (Rv1352, Rv1341, Rv1482 and Rv1147), regulatory protein classes (Rv3095, Rv1151c)2, lipocalin classes (Rv1867, faddd 11.1)2 and 1 member of the toxic, detoxified and adaptated protein class (Rv2303 c).

TABLE 6

3. Mycobacterium tuberculosis antigen immunization

BALB/c female mice, 6-8 weeks old, purchased from Beijing Wintolite Biotechnology Limited, were housed without special pathogens at the animal center of the Beijing thoracic Hospital, affiliated with the university of capital medical sciences. In total 20 MTB antigens were evaluated in 3 experiments, 6 proteins in the first experiment and 7 proteins in the next two experiments, respectively, with positive and negative controls included in each experiment. After one week of acclimation, mice were randomly divided into experimental and control groups (4 per group), immunized 3 times subcutaneously (2 weeks apart) with 30 μ g of M.tuberculosis protein in 200 μ L of incomplete Freund's adjuvant. The mice of the negative control group are injected with physiological saline and adjuvant 3 times (2 weeks apart), and the mice of the positive control group of BCG are injected with single dose of BCG 1 multiplied by 10⁵And (4) CFU. Mice were monitored daily by animal care personnel until termination of the experiment.

4. Infection with mycobacterium tuberculosis H37Rv

6 weeks after the last immunization in step 3, a suspension of M.tuberculosis H37Rv (concentration 5X 10)⁶CFU/mL) was injected into the mouse tail vein (100. mu.L per mouse at the dose per mouseMouse 5X 10⁵CFU), the immunized mice were subjected to intravenous infection with mycobacterium tuberculosis H37 Rv. After 6 weeks, the mice were sacrificed by cervical dislocation and lungs were harvested. The lung organs were transferred to plastic Tekmar bags containing 10mL of PBS buffer containing 0.1% (v/v) Tween-80. In FASTPRE-24(MP biomedicine), one leaf of each lung was homogenized in sterile 0.05% PBS-Tween 80. Homogenized lung suspension was plated in 10-fold serial dilutions on MedieBurk7H10 medium. Culturing at 37 deg.C for 3-4 weeks, humidifying with air and 5% CO₂Thereafter, Colony Forming Units (CFU) were scored. The other lung lobe was fixed in formaldehyde solution (4%) before hematoxylin-eosin (HE) and acid fast stain (AF) staining. Pathologists of histopathology slide examination and blind scoring are not associated with the study.

The inventors of the present invention evaluated the protective efficacy of the above selected 20 candidate MTB antigens in a BALB/c mouse challenge model. 7-9 week old BALB/c mice (three injections at 2 week intervals) were immunized via the lateral tail vein with 30 μ g of candidate antigen (in Freund's incomplete adjuvant). 6 weeks later, the mice were treated with M.tuberculosis H37Rv (5X 10 per mouse)⁵CFU) mice were challenged intravenously and euthanized after another six weeks. Comparison of bacterial load in the lungs of mice vaccinated with candidate T cell antigens with that of control mice injected with saline or BCG (see A in FIG. 5 and Table 7) indicated that the three protein antigens had significant protective efficacy; the pulmonary bacterial load of mice vaccinated with Rv1485 (4.86. + -. 0.06log10CFU) is significantly lower than that of negative control mice injected with physiological saline (5.33. + -. 0.04log10 CFU; p)<0.01, t-test) and can provide slightly greater protection than BCG control (5.00 ± 0.04log10 CFU). Similarly, the lung bacterial load of Rv1705c and Rv1802 vaccinated mice was significantly lower than negative control mice (Rv1705c, 4.72. + -. 0.07log10CFU versus physiological saline, 5.33. + -. 0.11log10 CFU; Rv1802, 4.90. + -. 0.05log10CFU versus physiological saline, 5.30. + -. 0.07log10 CFU). Rv1705c and Rv1485 provide protection substantially similar to BCG (see a in fig. 5 and table 7). Minor tissue damage relative to saline controls was observed during lung microscopy of mice immunized with Rv1705c or Rv1485, and the lungs of mice immunized with these antigens were similar in appearance to the lungs of mice immunized with BCG (see fig. 5, B). And thinThis change in bacterial load was consistent, and a clear difference in Hematoxylin and Eosin (HE) staining of the tissues was observed; similar to the BCG control, mice with Rv1705c or Rv1485 showed more severe interstitial pneumonia and inflammation throughout the lungs than in the saline control. Infiltration of inflammatory cells into the alveolar wall was less pronounced and alveolar air space was not eliminated to the same extent as the saline control (see C in fig. 5). The acidic rapid staining results showed no significant mycobacterium tuberculosis bacterial cells in lung tissue of BCG, Rv1705c and Rv1485 vaccinated mice compared to saline controls.

By further evaluating 20 of the 49 proteins that induce strong specific cellular antigen responses in the BALB/c mouse challenge model, it was shown that Rv1705c protein and Rv1485 protein have approximately comparable protective effects to bcg and can significantly reduce the bacterial burden of infecting the mouse lungs.

TABLE 7

5. Mouse spleen cell culture and cytokine detection

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

(2) An aqueous solution of M.tuberculosis protein (10. mu.g/mL), serum-free medium or PPD (10. mu.g/mL) was added to the 96-well plate, mixed and then incubated at 37 ℃ for 24h (for detection of IL-2) or 72h (for detection of IFN-. alpha., TNF. alpha. IL-4, IL-6 or IL-10).

(3) After completion of step (2), centrifugation was carried out at 1000rpm for 5min, and the supernatant was collected and assayed for IFN-. alpha., TNF. alpha., IL-2, IL-4, IL-6 and IL-10 concentrations using an ELISA kit (Biolegend, San Diego).

6. Antigen specific antibody titer

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

(1) a96-well plate was prepared, and 100. mu.L of 0.05M carbonate buffer pH9.6 containing 1. mu.g/mL of Mycobacterium tuberculosis protein was added to each well and coated overnight at 4 ℃.

(2) After completion of step (1), the 96-well plate was first washed with PBS-T buffer (containing 137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄、2mM KH₂PO₄And 0.05% (v/v) Tween-20 in water) 3 times; then placing the mixture in a blocking buffer (PBS-T buffer containing 3% (v/v) BSA), and standing the mixture for 2 hours at 37 ℃; and finally, washing the mixture for 3 times by using PBS-T buffer solution, adding mouse serum diluent, and standing the mixture for reaction for 1h at 37 ℃.

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

(3) After the step (2) is finished, taking the 96-pore plate, and washing the 96-pore plate for 3 times by using PBS-T buffer solution; horse Radish Peroxidase (HRP) -conjugated goat anti-mouse IgG1 and IgG2a antibodies (Bethy Laboratories, Montgomery, Texas) (working concentration: 10000 times diluted with incubation buffer) were then added to each well and allowed to stand at 37 ℃ for 1 h; then washing with PBS-T buffer solution for 3 times, adding TMB (3,3', 5,5' -tetramethylbenzidine) water solution, and incubating 96-well plate for 15min at room temperature; finally 2N H was added₂SO₄The reaction was stopped, the light emission at 450nm was read using a microplate reader (Tecan, Switzerland) and the IgG2 a: IgG1 endpoint titer ratio.

Potential subunit vaccine candidates must be able to induce a humoral response to provide protection against tuberculosis. To assess the humoral response elicited by Rv1705c and Rv1485, BALB/c mice were immunized with Rv1705c or Rv1485 (in incomplete freund's adjuvant) via the lateral tail vein (three injections at 2 week intervals). Titers of Rv1705c or Rv1485 specific IgG, IgG1 and IgG2a were measured in sera obtained three weeks after the last immunization with ELISA. High titers of IgG, IgG1 and IgG2a specific to Rv1705c or Rv1485 were observed, and no IgG/IgG1/IgG2a was detected in the vaginal control group (see a in fig. 6 and table 8). The Th1 immune response has been reported to have an important protective effect against mycobacterium tuberculosis infection (Refs), whereas the Th2 immune response is thought to impair host protective immunity. The ratio of IgG2a/IgG1(IgG2a is a Th1 type antibody and IgG1 is a Th2 type antibody) was plotted to indicate whether the Th1 or Th2 profile was induced by Rv1705c or Rv 1485. The IgG2a/IgG1 ratio in the Rv1705c group was 3.33, whereas BCG was 1.33, indicating induction of a moderate Th 1-type immune response in response to Rv1705c antigen. The ratio IgG2a/IgG1 in the Rv1485 group was 1.67 and BCG was 1.33, indicating induction of a moderate Th 1-type immune response 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 the cellular response, secretion of typical Th1 cytokines IL-2, IL-6, TNF- α and IFN- α was measured, which not only activated macrophages but also promoted polarization of the effector Th1 and Th2 cytokines, IL-4 and IL-10, which were 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 splenocytes harvested. Cytokine expression levels in culture supernatants of isolated splenocytes stimulated with antigen and PPD (purified protein derivative), respectively, were measured by ELISA. The results show that the Th1 cytokines IL-2, IL-6, TNF- α and IFN- γ were present in high concentrations after stimulation with Rv1705c or Rv1485, whereas the Th2 cytokines IL-4 and IL-10 were not detected (see B in FIG. 6 and Table 9). The cytokine levels detected were significantly higher for the Rv1705c or Rv1485 immunized mice than for the BCG immunized mice, probably because these mice received only one BCG immunization instead of three immunizations given to Rv1705 or Rv1485 immunized mice. Furthermore, the time between BCG immunization of mice and euthanasia was 8 weeks for BCG immunized mice and 4 weeks for mice immunized with Rv1705c or Rv1485, and harvested splenocytes were not stimulated with any immunogen prior to measuring cytokine release. Th1 cytokines, such as IL-2, TNF-alpha and well-known anti-tubercular cytokine IFN-gamma, promote Th1 immune response, in resisting pathogens, control tuberculosis and effectively protect the host. The results indicate that Rv1705c or Rv1485 elicited potent Th 1-type humoral and cellular immune responses, in favor of tuberculosis control, which may explain why Rv1705c protein or Rv1485 protein may protect the host from mycobacterium tuberculosis.

TABLE 9

<110> institute of biophysical research of the Chinese academy of sciences of Guangdong BODIKANG Biotechnology GmbH

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

<160> 2

<170> PatentIn version 3.5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Claims

The application of the Rv1485 protein in the preparation of tuberculosis detection reagents, tuberculosis vaccines or antituberculosis drugs;

the Rv1485 protein is a1) or a2) or a 3):

a1) protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table and has the same functions as the protein of a 1).
2. A tuberculosis detection reagent comprising at least one of b1) -b 3):

b1) the Rv1485 protein of claim 1;

b2) a nucleic acid molecule encoding said Rv1485 protein of claim 1;

b3) a recombinant protein expressed by a recombinant bacterium comprising a nucleic acid molecule encoding an Rv1485 protein of claim 1.
3. A T-spot.tb kit or x.dot-TB kit containing the tuberculosis detecting reagent of claim 2.
4. The kit of claim 3, wherein: the kit further comprises a capture antibody and/or a detection antibody;

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

the detection antibody is another mouse monoclonal antibody resisting different epitopes of human or animal IFN-gamma.
5. The kit of claim 3 or 4, wherein: the kit also contains a positive standard substance and/or a negative standard substance;

the positive standard substance is a tuberculosis non-specific stimulation antigen;

the negative standard is a cell culture solution without antigen.
6. A tuberculosis vaccine comprising at least one of b1) -b 3):

b1) the Rv1485 protein of claim 1;

b2) a nucleic acid molecule encoding said Rv1485 protein of claim 1;

b3) a recombinant protein expressed by a recombinant bacterium comprising a nucleic acid molecule encoding an Rv1485 protein of claim 1.
7. A tuberculosis vaccine, which is obtained by modifying recombinant BCG containing the Rv1485 protein in claim 1.
8. An anti-tuberculosis drug comprising an antibody that binds to Rv1485 protein as claimed in claim 1.
9. The anti-tuberculosis drug according to claim 8, wherein: the antibody is prepared by taking the Rv1485 protein as an immunogen in claim 1.
10. The anti-tuberculosis drug according to claim 9, wherein: the antibody is a1) or a 2):

A1) polyclonal antibodies prepared by immunizing animals with said Rv1485 protein as an immunogen;

A2) the Rv1485 protein is used as immunogen to immunize animal, and hybridoma technology or DNA recombination technology is adopted to prepare monoclonal antibody.