CN114736276B

CN114736276B - CTL epitope peptide of mycobacterium tuberculosis LTBI-RD related protein antigen and application thereof

Info

Publication number: CN114736276B
Application number: CN202210569282.1A
Authority: CN
Inventors: 龚文平; 吴雪琼; 梁艳; 王杰; 刘银萍; 薛勇; 米洁; 李鹏川; 王小鸥; 王兰
Original assignee: 8th Medical Center of PLA General Hospital
Current assignee: 8th Medical Center of PLA General Hospital
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2023-08-01
Anticipated expiration: 2042-05-24
Also published as: CN114736276A

Abstract

The invention discloses CTL epitope peptide of a mycobacterium tuberculosis LTBI-RD related protein antigen and application thereof. In particular to an amino acid sequence of 10 CTL epitope peptides and application thereof in differential diagnosis of active tuberculosis and latent tuberculosis infection. The CTL epitope peptide of the invention can stimulate ATB, LTBI and UC group mouse models to generate immune response, so that IFN-gamma ⁺ The absolute count level of T lymphocytes and the secretion level of the Th1/Th2/Th9/Th17/Th22/Treg related cytokines have obvious differences among three groups, so that the differential diagnosis of ATB and LTBI can be better realized. Compared with the traditional detection technology TST and IGRAs, the CTL epitope peptide used as the ATB and LTBI diagnosis marker has the advantages of simple preparation method, low cost, high yield, higher sensitivity and specificity and the like.

Description

CTL epitope peptide of mycobacterium tuberculosis LTBI-RD related protein antigen and application thereof

Technical Field

The invention belongs to the field of immunology, and relates to CTL epitope peptide of a mycobacterium tuberculosis LTBI-RD related protein antigen and application thereof. In particular to the application of CTL epitope peptide derived from LTBI-RD related antigen in the differential diagnosis of active tuberculosis and latent tuberculosis infection caused by mycobacterium tuberculosis (Mycobacterium tuberculosis).

Background

Tuberculosis (TB) is a chronic infectious disease caused by mycobacterium Tuberculosis (Mycobacteria Tuberculosis, MTB), which can invade multiple organs and is most common in pulmonary involvement to form Tuberculosis, and has become one of the ten major infectious diseases in humans. Studies have shown that about 26% of the world's population is infected with tuberculosis, of which only 10% suffer from active tuberculosis (Active tuberculosis, ATB), meaning that nearly 90% are latent tuberculosis infections (Latent tuberculosis infection, LTBI). LTBI is a special state, i.e. an individual has been infected with mycobacterium tuberculosis but has not yet developed Active Tuberculosis (ATB), characterized by positive tuberculin skin test (Tuberculin Skin Test, TST), no clinical manifestations and no imaging changes of ATB. It is estimated that LTBI patients have a lifetime risk of 5-10% of ATB without timely diagnosis and intervention, which may be as high as 10% per year, well above HIV negative population if they are simultaneously infected with Human Immunodeficiency Virus (HIV). Epidemiological investigation has shown that about 85% -90% of new active tuberculosis develop from LTBI. Thus, early discovery and diagnosis of LTBI patients is the basis for controlling tuberculosis transmission.

TST is the only means for rapidly diagnosing the potential infection of the tubercle bacillus for a long time, but the method has low specificity and brings great difficulty to the diagnosis of tuberculosis. The antigen used in traditional TST assays is tuberculin (OT) or a purified protein derivative (Purified protein derivative, PPD) which has a partial common antigen with BCG and other mycobacteria and thus has a high false positive rate in the population vaccinated with BCG and cannot distinguish between LTBI and ATB patients. Recently, new TST detection methods have been developed, such as the Diaskin test, the C-Tb skin test, and the EC (recombinant tubercule bacillus fusion protein) test, which replace conventional PPD with the early secretion antigen target 6 (Early secretory antigenic target-6, ESAT-6) and culture filtrate protein10 (Culture filtrate protein, CFP-10) antigens of a virulent strain of Mycobacterium tuberculosis. With the development of immunological techniques, a method for diagnosing tuberculosis based on in vitro Interferon Gamma Release Assay (IGRA) has been established, which is to determine the infection of tubercle bacillus by detecting the amount of IFN-gamma generated by T lymphocytes in human peripheral blood after stimulation with a tuberculosis specific antigen, has stronger specificity and higher sensitivity than the TST method, and five commercial IGRA kits have been developed to overcome the drawbacks of the conventional TST, such as T-spot test (T-spot. TB), quantiFERON TB Gold In Tube (QFT-GIT), quantiFERON TB Gold Plus (QFT Plus), LIAISON QuantiFERON-TB Gold Plus (liaaison QFT-Plus), and LIOFeron TB/LTBI. Coincidentally, both these improved TST methods and the latest IGRAs technology employ ESAT-6 and CFP-10 as stimulating antigens. Both antigens are absent in BCG strains and most nontuberculous mycobacteria (Nontuberculous mycobacteria, NTM) and therefore do not readily cross-react and can eliminate the effects of BCG vaccination on tuberculosis diagnosis. Although the IGRA method can be used in detecting MTB infection, its sensitivity and specificity still lack stringent "gold standards" and are expensive, and the LTBI and ATB cannot be distinguished even when the IGRA detection is positive.

In view of the above, further development of a diagnosis method which has higher sensitivity and specificity and low cost, can distinguish LTBI and ATB, and early discovers and diagnoses LTBI patients, thereby having important significance and wide clinical application value for controlling tuberculosis epidemic situation.

Disclosure of Invention

The object of the present invention is to provide a group of CTL epitope peptides derived from LTBI-RD antigen and their use in the differential diagnosis of active tuberculosis and latent tuberculosis infection. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To achieve the above object, the present invention provides a polypeptide which can be a CTL epitope peptide of an LTBI-RD related protein antigen of mycobacterium tuberculosis, the polypeptide being any of the following:

a1 The amino acid sequence is the polypeptide at 268-276 of SEQ ID No.1 or the polypeptide with the same function obtained by substituting and/or deleting and/or adding the amino acid residues of the amino acid sequence at 268-276 of SEQ ID No. 1;

a2 The amino acid sequence is the polypeptide at 378-386 of SEQ ID No.1 or the polypeptide with the same function obtained by substituting and/or deleting and/or adding amino acid residues in the amino acid sequence at 378-386 of SEQ ID No. 1;

A3 The amino acid sequence is the polypeptide at the 23 rd to 31 th positions of SEQ ID No.2 or the polypeptide with the same function obtained by substituting and/or deleting and/or adding the amino acid residues of the amino acid sequence at the 23 rd to 31 th positions of SEQ ID No. 2;

a4 The amino acid sequence is the 92 th to 100 th polypeptides of SEQ ID No.2 or the polypeptides with the same functions obtained by substituting and/or deleting and/or adding the amino acid residues of the 92 th to 100 th amino acid sequences of SEQ ID No. 2;

a5 The amino acid sequence is polypeptide of 133 th to 141 th positions of SEQ ID No.3 or polypeptide with the same function obtained by substituting and/or deleting and/or adding amino acid residues of the amino acid sequence of 133 th to 141 th positions of SEQ ID No. 3;

a6 The amino acid sequence is polypeptide of 131 th to 139 th positions of SEQ ID No.3 or polypeptide with the same function obtained by substituting and/or deleting and/or adding amino acid residues of the amino acid sequence of 131 th to 139 th positions of SEQ ID No. 3;

a7 The amino acid sequence is the polypeptide at 202-210 of SEQ ID No.4 or the polypeptide with the same function obtained by substituting and/or deleting and/or adding amino acid residues in the amino acid sequence at 202-210 of SEQ ID No. 4;

a8 Polypeptide of 325 th to 333 th site of SEQ ID No.4 or polypeptide with the same function obtained by substituting and/or deleting and/or adding amino acid residues of the 325 th to 333 th site of the SEQ ID No. 4;

A9 The amino acid sequence is the polypeptide at the 47 th to 56 th positions of SEQ ID No.5 or the polypeptide with the same function obtained by substituting and/or deleting and/or adding the amino acid residues of the amino acid sequence at the 47 th to 56 th positions of SEQ ID No. 5;

a10 The amino acid sequence is the polypeptide at the 41 st to 49 th positions of SEQ ID No.5 or the polypeptide with the same function obtained by substituting and/or deleting and/or adding the amino acid residues of the amino acid sequence at the 41 st to 49 th positions of SEQ ID No. 5.

Wherein: a1 In which the polypeptide name is CTL-Rv1737c-P1 and the amino acid sequence is RIAPRHVVL; a2 In which the polypeptide name is CTL-Rv1737c-P2 and the amino acid sequence is CTYTALHAR; a3 In the polypeptide name CTL-Rv2031c-P1, the amino acid sequence is AAFPSFAGL; a4 The polypeptide name is CTL-Rv2031c-P2, and the amino acid sequence is EFAYGSFVR; a5 The polypeptide name is CTL-Rv2626c-P1 and the amino acid sequence is KAICSPMAL; a6 The polypeptide name is CTL-Rv2626c-P2 and the amino acid sequence is FVKAICSPM; a7 In the polypeptide name CTL-Rv2659c-P1, the amino acid sequence is MAAWLAMRY; a8 In the polypeptide name CTL-Rv2659c-P2, the amino acid sequence is LAASTGATL; a9 In the polypeptide name CTL-Rv2660c-P1, the amino acid sequence is FTFSSRSPDF; a10 The polypeptide name is CTL-Rv2660c-P2, and the amino acid sequence is VVAPSQFTF.

The related antigen proteins of the mycobacterium tuberculosis LTBI-RD (the antigen belonging to the deletion region of the BCG and related to tuberculosis latent infection) are Rv1737c, rv2031c, rv2626c, rv2659c and Rv2660c. The amino acid sequence of the antigen protein Rv1737c is shown as SEQ ID No.1, the amino acid sequence of the antigen protein Rv2031c is shown as SEQ ID No.2, the amino acid sequence of the antigen protein Rv2626c is shown as SEQ ID No.3, the amino acid sequence of the antigen protein Rv2659c is shown as SEQ ID No.4, and the amino acid sequence of the antigen protein Rv2660c is shown as SEQ ID No. 5.

The invention also provides a polypeptide composition, which may be any of the following:

b1 The polypeptide composition consists of at least 2 or more of the following polypeptides: CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 or CTL-Rv2660c-P2;

b2 The polypeptide composition consists of polypeptides with amino acid sequences of 378-386 of SEQ ID No.1, 23-31 of SEQ ID No.2, 92-100 of SEQ ID No.2, 133-141 of SEQ ID No.3, 325-333 of SEQ ID No.4, 47-56 of SEQ ID No.5 and 41-49 of SEQ ID No. 5;

B3 The polypeptide composition consists of polypeptides with amino acid sequences of 378-386 of SEQ ID No.1 and 47-56 of SEQ ID No. 5.

B2 The polypeptide composition can be used to identify and distinguish between active tuberculosis patients and latent tuberculosis infected persons, healthy subjects and active tuberculosis patients or healthy subjects and latent tuberculosis infected persons.

B3 The polypeptide composition can be used to identify healthy subjects from active tuberculosis patients.

The invention also provides the polypeptide CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 or CTL-Rv2660c-P2, or any one of the following applications of the polypeptide composition:

d1 Use in the diagnosis and/or identification of diseases caused by mycobacterium tuberculosis;

d2 Use of a composition for diagnosing and/or identifying a disease caused by mycobacterium tuberculosis;

d3 Use in the identification of patients with active tuberculosis and those with latent tuberculosis;

d4 The use of said composition for the preparation of a product for the identification of patients suffering from active tuberculosis and patients suffering from latent tuberculosis;

d5 Use in the diagnosis of latent tuberculosis infection or in the manufacture of a product for the diagnosis of latent tuberculosis infection;

D6 Use in identifying a healthy subject from an active tuberculosis patient;

d7 Use of a composition for identifying a healthy subject from an active tuberculosis patient;

d8 Use in identifying a healthy subject from a latent tuberculosis infected person;

d9 Use of a composition for identifying a healthy subject from a latent tuberculosis infected person;

d10 For the preparation of tuberculosis vaccines.

The product may be a reagent, a kit (such as a diagnostic kit) or a medicament.

The active ingredients comprise the polypeptides CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and/or CTL-Rv2660c-P2, and the products with the functions as shown below also belong to the protection scope of the invention:

f1 Diagnosing and/or identifying a disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis);

f2 Identifying a distinction between active tuberculosis (Active tuberculosis, ATB) and latent tuberculosis infection (Latent tuberculosis infection, LTBI);

f3 Diagnosing latent tuberculosis infection (LTBI);

f4 Identifying and distinguishing healthy subjects, active tuberculosis patients and latent tuberculosis infected persons;

The active ingredient of the product consists of CTL epitope peptides derived from LTBI-RD antigen.

The epitope peptide may be synthesized artificially by conventional techniques.

The disease caused by mycobacterium tuberculosis described herein may be a human disease caused by mycobacterium tuberculosis or a mouse disease caused by mycobacterium tuberculosis, but is not limited thereto.

In the above application, the disease caused by mycobacterium Tuberculosis (Mycobacteria Tuberculosis, MTB) may be Tuberculosis (TB).

Further, the tuberculosis may be active tuberculosis (Active tuberculosis, ATB) or latent tuberculosis infection (Latent tuberculosis infection, LTBI).

The polypeptides CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and/or CTL-Rv2660c-P2 can be used as differential diagnosis biomarkers for active tuberculosis and latent tuberculosis infection.

In one embodiment of the invention, the biomarker is the polypeptide composition described in B2) herein, i.e. a polypeptide composition consisting of a polypeptide whose amino acid sequence is positions 378-386 of SEQ ID No.1, 23-31 of SEQ ID No.2, 92-100 of SEQ ID No.2, 133-141 of SEQ ID No.3, 325-333 of SEQ ID No.4, 47-56 of SEQ ID No.5 and 41-49 of SEQ ID No. 5;

In one embodiment of the invention, the biomarker is the polypeptide composition described in B3) herein, i.e. a polypeptide composition consisting of polypeptides whose amino acid sequence is positions 378-386 of SEQ ID No.1 and positions 47-56 of SEQ ID No. 5.

The invention also provides a vaccine, and the active ingredients of the vaccine can be polypeptides CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 or CTL-Rv2660c-P2, or the polypeptide composition.

Nucleic acid molecules encoding the polypeptides CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 or CTL-Rv2660c-P2 are also within the scope of the invention.

Biological materials containing the nucleic acid molecules are also within the scope of the invention, which may be recombinant vectors, expression cassettes, recombinant microorganisms or recombinant cells.

The invention also provides a method of identifying active tuberculosis from latent tuberculosis infection, the method comprising:

c1 Co-culturing a sample of the subject with a stimulus which is a polypeptide of CTL-Rv1737c-P1, CT L-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 or CTL-Rv2660c-P2, or the polypeptide composition;

C2 Detecting the level of IFN-gamma secreted in the sample, and distinguishing active tuberculosis from latent tuberculosis infection based on said IFN-gamma level.

The level of IFN-gamma secreted in the test sample may be the number of cells that test IFN-gamma produced by stimulation by the polypeptide.

Further, the detection of secreted IFN-gamma levels in a sample can be performed by detecting the number of IFN-gamma cells produced by stimulation by the polypeptide using an ELISPOT kit for detecting IFN-gamma.

Specifically, step C1) is co-culturing the subject sample with a stimulus having the amino acid sequence of the polypeptides at positions 378-386 of SEQ ID No.1, 23-31 of SEQ ID No.2, 92-100 of SEQ ID No.2, 133-141 of SEQ ID No.3, 325-333 of SEQ ID No.4, 47-56 of SEQ ID No.5 and 41-49 of SEQ ID No. 5.

The present invention also provides a method of identifying a healthy subject from an active tuberculosis patient, the method comprising:

m1) co-culturing a subject sample with a stimulus which is a polypeptide CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 or CTL-Rv2660c-P2, or the polypeptide composition;

M2) detecting the level of secreted IFN- γ in the sample, and discriminating between active tuberculosis and latent tuberculosis infection based on said IFN- γ level.

Specifically, step M1) is co-culturing the subject sample with a stimulus having the amino acid sequence of the polypeptides at positions 378-386 of SEQ ID No.1 and positions 47-56 of SEQ ID No. 5.

The invention also provides a kit for distinguishing active tuberculosis from latent tuberculosis infection, the kit comprises polypeptides CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 or CTL-Rv2660c-P2, or the polypeptide composition.

Further, the kit also comprises an IFN-gamma detection reagent.

The subject sample described herein may be a blood sample or a tissue sample.

The purpose of the above-described applications and methods may be for disease diagnosis purposes, disease prognosis purposes and/or disease treatment purposes, as well as for non-disease diagnosis purposes, non-disease prognosis purposes and non-disease treatment purposes; their direct purpose may be information of intermediate results of obtaining disease diagnosis results, disease prognosis results and/or disease treatment results, and their direct purpose may be non-disease diagnosis purpose, non-disease prognosis purpose and/or non-disease treatment purpose.

The invention uses five antigen proteins (Rv 1737c, rv2031c, rv2626c, rv2659c and Rv2660 c) as target antigens, and predicts potential epitopes recognized by cytotoxic T Cells (CTL) by using a bioinformatics technology. Predicted dominant CTL peptides were synthesized in vitro and evaluated for their potential ability to distinguish LTBI from ATB in animal models by enzyme-linked immunospot (ELISPOT) and high throughput liquid protein microarray detection techniques. In addition, sensitivity and specificity of these peptides and combinations thereof were determined using a subject-operator profile (ROC). The invention provides a new differential diagnosis candidate target for differential diagnosis of LTBI and ATB, and emphasizes the potential value of the LTBI-RD antigen derived peptide as a new method for diagnosing the LTBI and the ATB.

The present invention for the first time found that 10 CTL epitope peptides (CTL-Rv 1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and CTL-Rv2660 c-P2) of Mycobacterium tuberculosis are capable of stimulating the immune response of ATB, LTBI and Uninfected Control (UC) BALB/c mouse models, such that IFN-gamma ⁺ The absolute count level of T lymphocyte and the secretion level of Th1/Th2/Th9/Th17/Th22/Treg relative cytokine have obvious difference between three groups, and can be better identified on a mouse model Diagnosis of ATB and LTBI. The 10 CTL epitope peptides of the mycobacterium tuberculosis can be artificially prepared by a polypeptide synthesis technology, and the CTL epitope is used as an ATB and LTBI differential diagnosis biomarker. The invention has great value for diagnosis or differential diagnosis of active tuberculosis and tuberculosis latent infection.

Drawings

FIG. 1 is a flow chart of ATB and LTBI animal model construction.

FIG. 2 is an evaluation of ATB and LTBI mouse models. Mice in the ATB and LTBI groups were infected with Mycobacterium tuberculosis 4 weeks later, and the LTBI group was given isoniazid and pyrazinamide treatment for 12 weeks. Survival of each group of mice was observed and recorded (a in fig. 2). After week 29, all three groups of mice were sacrificed and analyzed for lung weight (B in fig. 2) and CFU (C in fig. 2). Right lung lobes of each group of mice were then HE stained and observed under a microscope at 40 x magnification (D in fig. 2). Each group was displayed with 2 representative images (D in fig. 2), and the lesion area of each mouse was observed and counted by software (E in fig. 2). Data are expressed in mean±sem and compared using one-way variance analysis (ANOVA) or Kruskal-Wallis test based on the normality and variance of the data. P <0.05 is a significant difference.

FIG. 3 shows that CTL epitope dominant peptide induces mouse IFN-gamma ⁺ Detection of T lymphocyte numbers. CTL epitope dominant peptides were used to stimulate spleen cells of ATB, LTBI or UC group mice in vitro. Detection every 3×10 using mouse ELISPOT kit ⁵ IFN-gamma in cells ⁺ T lymphocyte number (expressed as SFC). Based on the normality and alignment of the variances, statistical analysis of the results was performed using the Kruskal-Wallis test or one-way analysis of variance (ANOVA). Data are expressed as mean+sem (n=3), P<A difference of 0.05 is statistically significant.

FIG. 4 shows induction of IFN-gamma by ATB, LTBI and UC mouse polypeptides ⁺ ROC curve of T lymphocytes. Detection of CTL epitope peptide-induced IFN-gamma by ROC Curve Using Wilson/Brown detection ⁺ Diagnosis of ATB, LTBI and UC by T lymphocytesSensitivity and specificity of the break. AUC values and P values are shown in each figure. P (P)<0.05 is a significant difference.

FIG. 5 is a schematic of the dilution of the standard in example 5.

FIG. 6 shows a cytokine study induced by CTL dominant peptide. Spleen cells from ATB, LTBI and UC mice were stimulated with 10 CTL-dominant peptides for 48 hours. The supernatant was assayed for IFN-gamma, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF-alpha, GM-CSF, IL-18, IL-10, L-17A, IL-22, IL-23, IL-27, IL-9 cytokine levels using the mouse Th1/Th2/Th9/Th17/Th22/Treg cytokine kit. Differences in cytokines were compared for each group using a Tukey test modified two-way analysis of variance (ANOVA). The P-values of the cytokine differences (ATB vs LTBI, ATB vs UC, LTBI vs UC) of each group are shown in the heat map (FIG. 6A). The significant difference is represented by a light gray box with a P value <0.05, and the dark gray box with a P value of 0.05 or more. In addition, the P-values of the polypeptide-induced cytokines between ATB and LTBI, ATB and UC, and LTBI and UC were all less than 0.05, represented by white diagonal grids, and detailed information was represented by violin (B in fig. 6).

FIG. 7 is a ROC curve of CTL dominant peptide induced cytokines in ATB, LTBI and UC mouse identification. Sensitivity and specificity of CTL epitope peptide-induced IFN-gamma and IL-6 cytokines to ATB, LTBI and UC diagnostics were detected by ROC curve using Wilson/Brown assay. AUC values and P values are shown in each figure. P <0.05 is significant.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative tests in the following examples were all performed in triplicate, and the results were averaged.

Wild type BALB/c mice (6-7 week old, female) in the examples below were produced by Peking Vitrenia laboratory animal technologies Co., ltd (www.vitalriver.com).

All epitope peptides in the following examples were synthesized by Hangzhou Dangang biotechnology Co.

The Roche medium (product name: roche tube (acid)) in the examples described below is a product of Pinctada martensii Biotechnology Co., ltd (Baso Biotechnology Co., LTD, zhuhai, guangdong province, china) under the product designation BA7005E.

Gibco in the examples below ^TM The Advanced RPMI 1640 medium is manufactured by Siemens Fei China Co., ltd, the product number is 12633012, and the Advanced RPMI 1640 medium is called for short.

The Mouse IFN-. Gamma.ELISPot in the examples below ^PLUS kit (ALP) is available from MABTECH company, sweden under the designation 3321-4APT-2.

Th1/Th2/Th9/Th17/Th22/Treg Cytoikine 17-Plex Mouse ProcartaPlex in the examples below ^TM Panel is product of Siemens Fei China, inc., product number EPX170-26087-901.

The Chinese population dominant HLA molecular sieves in the examples below were selected from the Allele Frequency Net Database (AFND) database (http:// www.allelefrequencies.net/default. Asp).

Prediction of CTL epitopes in the following examples utilized an online epitope prediction website: immune Epitope Data Base (IEDB) database (http:// tools. IEDB. Org/mhcii/or http:// tools. Immuneepitube. Org/mhci /) for prediction.

Mycobacterium tuberculosis (Mycobacterium tuberculosis) used in the examples below are all Mycobacterium tuberculosis standard strains H37Rv (Mycobacterium tuberculosis, H37Rv strain) reported in the literature "Yan L, xiaoyan Z, li X, et al, immunology and Therapeutic Effects of pVAX-Rv 1419 DNA from Mycobacterium tuberculosis [ J ]. Current Gene Therapy,2016,16 (4): 249-255 ], and in compliance with biosafety operating specifications, the public is available from the applicant and can only be used for repeating the experiments of the present invention.

Example 1, chinese population-specific HLA I allele screening

1. A Allele Frequency Net Database database is entered and a HLA Allele frequency Classical submenu is selected. Parameter selection Country, namely China, sample size is more than or equal to 500, and other parameters are selected as default values.

2. Clicking the Search button starts the Search. Alle with an Alle Frequency of 0.10 or more (critical value of 0.10 or more, see reference Paul S, lindestam Arlehamn C S, scriba T J, et al Development and validation of a broad scheme for prediction of HLA class II restricted T cell epitopes [ J ]. Journal of Immunological Methods,2014, 422:733-738.) was selected as the dominant HLA I restriction Allele of the Chinese population.

3. By searching Allele Frequency Net Database database, we select the Allele with the Allele Frequency more than or equal to 0.10 as the HLA I restriction Allele of Chinese crowd, and finally screen out 5 HLA-A alleles of Chinese crowd, which are HLA-A 11:01, HLA-A 02:01, HLA-A 24:02, HLA-A 33:03 and HLA-A 30:01 respectively; a total of 3 HLA-B alleles, HLA-B15:01, HLA-B15:02 and HLA-B15:11, respectively; a total of 4 HLA-C alleles are HLA-C01:02, HLA-C07:02, HLA-C03:04 and HLA-C06:02, respectively.

EXAMPLE 2 CTL epitope prediction and screening of Chinese crowd-specific LTBI-RD-related antigen

In previous studies by the applicant (Gong W and Wu X, (2021), differential Diagnosis of Latent Tuberculosis Infection and Active Tuberculosis: A Key to a Successful Tuberculosis Control Strategy. Front. Microbiol.12:745592.Doi: 10.3389/fmib. 2021.745592), 21 candidate antigens associated with LTBI-RD (antigens belonging to both the tuberculosis latent infection-associated and BCG deletion region) have been identified. In the present invention, five antigens (Rv 1737c, rv2031c, rv2626c, rv2659c and Rv2660 c) were selected as target antigens from the 21 candidate antigens, and potential epitopes recognized by cytotoxic T Cells (CTLs) were predicted using bioinformatics techniques. The amino acid sequence of the antigen protein Rv1737c is shown as SEQ ID No.1, the amino acid sequence of the antigen protein Rv2031c is shown as SEQ ID No.2, the amino acid sequence of the antigen protein Rv2626c is shown as SEQ ID No.3, the amino acid sequence of the antigen protein Rv2659c is shown as SEQ ID No.4, and the amino acid sequence of the antigen protein Rv2660c is shown as SEQ ID No. 5.

1. Amino acid sequences of target proteins such as Rv1737c, rv2031c, rv2626c, rv2659c, rv2660c and the like are obtained from NCBI database, and epitope conditions are predicted.

TABLE 1 list of selected RD-LTBI related antigenic characteristics

2. Into the CTL epitope prediction page (http:// tools. Immuneepitope. Org/mhci /). The amino acid sequence of the target protein is filled in the column of ' Enter protein sequence(s) in FASTA format ' of the prediction page, the screened MHC I restriction alleles which exist in high frequency in Chinese population are filled in the column of ' Select MHC allele(s) through Upload allele file, and other parameters are selected as default values.

3. And adopting an IEDB database to predict dominant CTL epitopes of Chinese population. There are 9 prediction methods for the IEDB database, artificial Neural Network (ANN), stabilized Matrix Method (SMM), SMM with a Peptide: MHC Binding Energy Covariance matrix (SMMPMBEC), scoring Matrices derived from Combinatorial Peptide Libraries (comblib_Sidney 2008), consensus, netMHCpan, netMHCcons, pickPocket, and NetMHCstabpan, respectively. The system defaults to choose the IEDB recommended2.19 method to predict, which uses Consensus, ANN, SMM and combLibif to predict preferentially, and uses NetMHCIIpan when the condition does not allow. The priority of these methods is from high to low, respectively Consensu > ANN > SMM > NetMHCpan > CombLib.

4. Epitopes predicted by the IEDB database are ranked from small to large with an integrated score of percentile_rank (smaller scores indicate higher affinities). An epitope with a Percentile_rank score less than or equal to 10 is selected from each target protein epitope as a candidate epitope, 70 candidate epitopes are obtained, wherein Rv1737c, rv2031c, rv2626c, rv2659c and Rv2626c respectively contain 20, 13, 14, 17 and 6 candidate epitopes, and specific epitope information is shown in table 2.

TABLE 2 IEDB database predicts dominant CTL epitope results for LTBI-RD related antigen sources

5. The predicted dominant C TL epitope peptide is synthesized in vitro by adopting a solid phase synthesis method by entrusting Hangzhou Dangang biotechnology limited company, and then the synthesized dominant CTL epitope peptide is purified by adopting a high-pressure liquid chromatography method, so that the purified dominant CTL epitope peptide is finally prepared. The first two epitope peptides with highest ranking per antigen synthesis, if not synthesized, proceed back to the next. Finally, 10 LTBI-RD related CTL epitope peptides, namely CTL-Rv17 c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and CTL-Rv2660 c-P2, are synthesized. Specifically 10 LTBI-RD related CTL epitope peptides have the following amino acid sequences:

The amino acid sequence of CTL epitope peptide CTL-Rv1737c-P1 is shown in 268-276 (RIAPRHVVL) of SEQ ID No. 1.

The amino acid sequence of CTL epitope peptide CTL-Rv1737c-P2 is shown in positions 378-386 (CTYTALHAR) of SEQ ID No. 1.

The amino acid sequence of CTL epitope peptide CTL-Rv2031c-P1 is shown in 23 rd to 31 rd (AAFPSFAGL) of SEQ ID No. 2.

The amino acid sequence of CTL epitope peptide CTL-Rv2031c-P2 is shown in 92 th to 100 th (EFAYGSFVR) of SEQ ID No. 2.

The amino acid sequence of CTL epitope peptide CTL-Rv2626c-P1 is shown in the 133 th to 141 th (KAICSPMAL) positions of SEQ ID No. 3.

The amino acid sequence of CTL epitope peptide CTL-Rv2626c-P2 is shown in 131-139 (FVKAICSPM) of SEQ ID No. 3.

The amino acid sequence of CTL epitope peptide CTL-Rv2659c-P1 is shown in positions 202-210 (MAAWLAMRY) of SEQ ID No. 4.

The amino acid sequence of CTL epitope peptide CTL-Rv2659c-P2 is shown in 325 th-333 rd (LAASTGATL) of SEQ ID No. 4.

The amino acid sequence of CTL epitope peptide CTL-Rv2660c-P1 is shown in positions 47-56 (FTFSSRSPDF) of SEQ ID No. 5.

The amino acid sequence of CTL epitope peptide CTL-Rv2660c-P2 is shown in the 41 st to 49 th (VVAPSQFTF) of SEQ ID No. 5.

Example 3, ATB and LTBI mouse model construction

1. Grouping mice: 30 female BALB/c mice of 6-7 weeks of age were stratified by body weight, randomly divided into 3 groups (ATB group, LTBI group and control UC group), 10 in each group, and the body weights of the mice in each group were as close as possible.

2. Intervention measures: as shown in FIG. 1, the ATB group and LTBI group were intravenously injected with 0.4mL of H37Rv Mycobacterium tuberculosis suspension per mouse tail to give a dose of 3.6X10 per mouse ⁵ CFU. From week 4 to week 17, each mouse of the LTBI group consumed drinking water containing 0.12g/L isoniazid and 8g/L pyrazinamide, and from week 17 to week 29, the LTBI group mice did not undergo any intervention, and were given normal drinking water (drinking water without 0.12g/L isoniazid and 8g/L pyrazinamide). The Uninfected Control (UCs) group of mice was a negative control group, fed normally, given normal water without any intervention. The ATB group was fed normally after injection of H37Rv Mycobacterium tuberculosis suspension, and given normal drinking water.

3. Model evaluation: groups of mice were sacrificed after week 29 and spleen and lung were taken for observation of infection models. Briefly, the lung is weighed and then homogenized in physiological saline. Subsequently, 0.1mL of diluted solution for each lung specimen was inoculated into roche medium, double inoculated, and incubated at 37 ℃. After 28 days, CFU counts were performed for each plate. In addition, pathology was analyzed using the right lung. Lesion area rates were calculated using Image-Pro Plus software (Version 6.0,Media Cybernetics,Inc: bethesda, MD, USA). The flow chart of infection, treatment, activation and evaluation of animal model is shown in figure 1.

4. Experimental results: the results show that the ATB group 8 mice die after infection with mycobacterium tuberculosis, while LTBI and UC group mice survive. ATB group survival was significantly lower than LTBI and UC groups (p=0.0003, a in fig. 2). The lung weight (p=0.0032, B in fig. 2) and CFU load (p=0.0007, C in fig. 2) were significantly higher in ATB mice than in UC mice. Although the lung weights and CFU loads of LTBI group mice were not statistically significantly different from those of ATB and UC groups, the average of the lung weights and CFU loads of LTBI group mice was much higher than that of UC group but much lower than that of ATB group (B and C in fig. 2). The lung lesions of each group of mice were observed at 40-fold visual field (D in fig. 2), and statistics showed that the ATB group lung injury area was significantly greater than that of LTBI group (P < 0.0001) and UC group (P < 0.0001), and that LTBI group lung injury area was significantly greater than UC group (P <0.0001, E in fig. 2). These data indicate that ATB and LTBI mouse models have been successfully constructed, resulting in ATB model mice, LTBI model mice, and control mice.

Example 4 ELISPOT experiments on ATB and LTBI mouse models

30 female BALB/c mice, 16-18g in weight, 6-7 weeks old, were randomly divided into 3 groups (ATB group, LTBI group and control UC group) according to the method of example 3, 10 in each group, and ATB model mice, LTBI model mice and control mice were constructed according to the method of example 3.

The present example uses Mouse IFN-. Gamma.ELISPot ^PLUS kit (ALP) IFN-. Gamma.ELISPOT assay was performed to test the ability of 10 LTBI-RD-related CTL epitope peptides of example 2 to induce IFN-. Gamma.secretion by T cells.

The method comprises the following specific steps:

1. preparation of spleen cell suspension: mice were sacrificed, and the mice were sterilized in 75% alcohol for 10min and then removed, and the spleens were dissected. 10mL of Advanced RPMI 1640 culture was previously aspirated into a sterile petri dish, a sterile 200 mesh copper mesh was placed in the petri dish, and then the spleen was placed in the 200 mesh copper mesh, and the spleen cells were dispersed by gentle extrusion with a syringe push rod head (sterile). ELISPOT plate (Mouse IFN-. Gamma.ELISPot) was prepared in advance ^PLUS kit (ALP): PBS was washed four times (200 μl/well) and then incubated with Advanced RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS) for at least 30min at 200 μl/well to give a spleen cell suspension.

2. Erythrocyte lysis: the spleen cell suspension obtained above was centrifuged at 500g for 5min at 4℃and the supernatant was discarded. Adding erythrocyte lysate according to the amount of 20-30 mL/spleen, gently blowing and mixing, and cracking for 4-5min at room temperature. During which the sample was gently shaken every one minute. Centrifuge at 500g for 5min at 4℃and discard the red supernatant. If incomplete red blood cell lysis is found, the above steps may be repeated once. Usually very small amounts of red blood cells do not affect some subsequent detection. Washing for 1-2 times: adding appropriate amount of Advanced RPMI 1640 medium, suspending, precipitating, centrifuging at 4deg.C for 2-3 min at 500g, and discarding supernatant. Repeating for 1 time, and washing for 1-2 times to obtain lymphocyte precipitate. The amount of washing liquid used should generally be at least 5 times the volume of the cell pellet.

3. Counting and plating: the lymphocyte pellet was resuspended in Advanced RPMI 1640 medium, counted and the cell concentration was adjusted to 3X 10 ⁶ And (3) one/mL for later use. The wells were plated at 100. Mu.L/well to give a final concentration of 3X 10 cells per well ⁵ And/or holes. Solutions of the corresponding CTL epitope peptides were added at 10 μl/well, respectively: CTL-Rv1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2626c-P2, CTL-Rv2659c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and CTL-Rv2660c-P2 (in solution, the concentration of CTL epitope peptide was 100. Mu.g/mL, 3 wells per solution of CTL epitope peptide, PHA (40. Mu.g/mL) was used as positive control well, advanced RPMI 1640 medium was used as negative control well), 96-well ELISPOT plates were placed in a solution containing 5% CO ₂ During incubation of the cell culture incubator at 37℃for 12-48h, the ELISPOT plate was not removed.

4. ELISPot assay mouse IFN-gamma response spots: cells in the ELISPOT plate were gently removed and washed 5 times with PBS in an amount of 200 μl/well. R4-6A 2-labeled monoclonal antibody (1 mg/mL, mouse IFN-. Gamma.ELISPot.) was conjugated with PBS containing 0.5% FBS ^PLUS The kit (ALP) was diluted to a final concentration of 1. Mu.g/mL, and ELISPOT plates were added in an amount of 100. Mu.L/well and incubated for 2h at room temperature. PBS was washed 5 times, 200. Mu.L/well. streptavidin-ALP (Mouse IFN-. Gamma.ELISPot) was performed using PBS containing 0.5% FBS ^PLUS kit (ALP) was diluted 1:1000, and ELISPOT plates were added in an amount of 100. Mu.L/well and incubated for 1h at room temperature. PBS was washed 5 times, 200. Mu.L/well. The color-developing solution was filtered by using a 0.45 μm filter, and then ELISPOT plates were added in an amount of 100. Mu.L/well, and the change in spots in the wells was observed. After the spots reach the requirements, a large amount of tap water is used for flushing, the water is taken for drying, and the spots are naturally dried in a dark place. And (5) reading a plate, scanning and counting results.

5. Experimental results: as a result, it was found that 7 CTL epitope peptides (CTL-Rv 1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and CTL-Rv2660 c-P2) stimulated IFN-. Gamma.produced by mouse spleen cells ⁺ The counts of T lymphocytes were significantly higher in ATB mice than in LTBI and UC mice (FIG. 3), indicating that these 7 CTL epitope peptides (CTL-Rv 1737c-P2 (positions 378-386 of SEQ ID No. 1), CTL-Rv2031c-P1 (positions 23-31 of SEQ ID No. 2), CTL-Rv2031c-P2 (positions 92-100 of SEQ ID No. 2), CTL-Rv2626c-P1 (positions 133-141 of SEQ ID No. 3), CTL-Rv2659c-P2 (positions 325-333 of SEQ ID No. 4), CTL-Rv2660c-P1 (positions 47-56 of SEQ ID No. 5) and CTL-Rv2660c-P2 (positions 41-49 of SEQ ID No. 5)) could excite a stronger specific T cell immune response in vivo. Thus, these immunodominant peptides were selected for ROC curve analysis (fig. 4). 7. IFN-gamma induced by CTL epitope peptide ⁺ Number of T lymphocytes ATB mice can be isolated from UC group mice (fig. 4, auc=1, p<0.0001 Or LTBI mice (auc=1, p)<0.0001 In addition, LTBI mice can be distinguished from UC mice (auc=0.8912, p)<0.0001). Further analysis found that the sensitivities of the above 7 epitope peptides in combination with differential diagnosis of ATB vs UC, ATB vs LTBI and UC vs LTBI were 100%, 100% and 76.19%, respectively, and the specificities were 95.24%, 95.24% and 85.71%, respectively (table 3).

TABLE 3 IFN-gamma epitope-induced by CTL ⁺ T lymphocyte combined diagnosis of sensitivity and specificity of ATB and LTBI

Wherein: AUC represents area under the curve (area under the curve); p value represents a P value; 95% ci represents 95% confidence interval (95%confidence interval); the Cutoff value represents a Cutoff value (threshold); sensitivity represents Sensitivity; specificity means Specificity.

Subject working curves ROC for candidate diagnostic markers were plotted using GraphPad Pirsm 9.3.1 version of software and statistically analyzed using Wilson/Brown test. The stimulation data for each polypeptide was entered into the software and the analysis consisted of three parts: the first part is the ROC graph; the second part is the overall statistics, including AUC values and their 95% ci and P values; the third part is to demonstrate sensitivity and specificity, choosing the best CUTOFF value based on likelihood ratio values.

Example 5, ATB and LTBI mouse model cytokine detection

1. Preparation of spleen cell suspension: the procedure is as in example 4, step 1.

2. Erythrocyte lysis: the procedure is as in example 4, step 2.

3. Counting and plating: the procedure is as in example 4, step 3.

4. Luminex 200 detection of 17 cytokines in mice

Adopts a kit (Th 1/Th2/Th9/Th17/Th22/Treg Cytokine 17-Plex Mouse ProcartaPlex) ^TM Panel) 17 cytokines (IFN-gamma, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF- α, GM-CSF, IL-18, IL-10, IL-17A, IL-22, IL-23, IL-27, IL-9) were detected. The method comprises the following specific steps:

(1) Reagent dilution

Wash buffer (10X-1X): 10 times of wash buffers are used for buffering: ddH ₂ O=9:1 was diluted to 1X Wash Buffer.

Beads (50X-1X): the Beads (microspheres) were vortexed for 30s, 100. Mu.L each of 50 XBeads per tube was removed, and 1 XWash Buffer was added to a final volume of 5mL and mixed well.

Detection Antibody (50X-1X): each of the tubes 50 and X Detection Antibody (detection antibody) was taken out in an amount of 60. Mu.L, detection antibodydiluent (detection antibody diluent) was added to a final volume of 3mL, and the mixture was homogenized to obtain a 1X detection antibody mixture.

(2) Dissolving standard

Taking out the standard substance, and centrifuging for 10s at 2000 Xg; 50. Mu.L of Universal Assay Buffer was added to each of the standard tubes; gently mixing for 30s; placing on ice for 5-10min; the standard was mixed into a tube, and Universal Assay Buffer was added to obtain 250. Mu.L of the mixed standard. See table 4 for standard configuration details.

Table 4 list of standard configuration

(3) Dilution of Standard substance (4 times)

Taking out the PCR 8 tube provided in the kit for diluting the standard substance; as shown in fig. 5, 200 μl of the mixed standard was added to the first tube (tube 1) as a standard 1; 150. Mu.L of Universal Assay Buffer was added to each of the tubes 2-8; adding 50 mu L of mixed standard substance into a tube 2 from the tube 1, blowing up and down for 10 times, and uniformly mixing to avoid bubbles as much as possible; the new gun head was replaced, 50. Mu.L of the diluted standard substance was sucked from the tube 2 and transferred to the tube 3, and the mixture was blown up and down 10 times and mixed uniformly. Transferring sequentially to finish gradient dilution of the mixed standard substance; placing on ice for standby.

(4) Preparation of microspheres

Vortex microsphere 30s; 50 μl of the premix microspheres were added to each well in a 96-well plate. The 96-well plate was placed in a magnetic separation plate to ensure that the well plate was firmly clamped. And standing the plate for 2min, and allowing the microspheres to sink. The magnetic plate is then quickly inverted and the liquid in the well plate is poured out. In the process, the 96-well plate cannot be taken out from the magnetic separation plate; 150 mu L of 1 XWash Buffer is added into each hole, the mixture is stood for 30s, then the magnetic plate is inverted, and the liquid in the hole plate is poured out; in the inverted state, the remaining liquid on the surface of the orifice plate is adsorbed with a paper towel.

(5) Microsphere incubation with sample

Add 50. Mu.L of sample or standard, respectively, to the designated wells; 50 μ LUniversal Assay Buffer was added to the blank; the well plate was sealed and incubated with shaking at 500rpm for 30min at room temperature and allowed to stand overnight at 4 ℃. The next day was removed and incubated with shaking at 500rpm for 30min at room temperature.

(6) Washing plate

Placing the 96-well plate in a magnetic separation plate, and standing for 2min; the sealing film is removed lightly, so that liquid splashing is avoided; inverting the liquid in the orifice plate; to each well, 150. Mu.L of 1 XWash Buffer was added, and the well plate was left to stand for 30 seconds, and the liquid in the well plate was removed upside down. Repeating the steps, and washing for 3 times; at the end of the last wash, the residual liquid was adsorbed with paper towels.

(7) Adding detection antibody

Adding 25 mu L of 1X detection antibody mixture to each well; sealing the aperture plate using a new sealing membrane; the 96-well plate was removed from the magnetic separation plate and placed in a well plate shaker at 500rpm for 30min at room temperature.

(8) Washing plate

Placing the 96-well plate in a magnetic separation plate, and standing for 2min; the sealing film is removed lightly, so that liquid splashing is avoided; inverting the liquid in the orifice plate; 150 mu L of 1 XWash Buffer is added into each hole, the mixture is stood for 30s, the liquid in the pore plate is removed in an inverted way, and the mixture is washed for 3 times; at the end of the last wash, the residual liquid was adsorbed with paper towels.

(9) Adding SA-PE

Add 50. Mu.L SA-PE to each well; sealing the aperture plate using a new sealing membrane; the 96-well plate was removed from the magnetic separation plate and placed in a well plate shaker at 500rpm for 30min at room temperature.

(10) Washing plate

(11) On-machine detection

120 μl of Reading Buffer was added to each well; sealing the aperture plate using a new sealing membrane; taking out the 96-well plate from the magnetic separation plate, and placing the 96-well plate into a well plate oscillator to oscillate for 5min at the room temperature of 500 rpm; the sealing film was gently removed and placed into a Luminex 200 instrument for reading. And fitting a standard curve by adopting a five-parameter nonlinear regression mode, and calculating a concentration value.

(12) Experimental results

To further elucidate the potential value of 10 dominant CTL epitope peptides in differential diagnosis of LTBI, ATB and UC mice, spleen cells of LTBI, ATB and UC mice were stimulated in vitro with the above epitope peptides (CTL-Rv 1737c-P1, CTL-Rv1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and CTL-Rv2660 c-P2), respectively, and expression levels of 17 cytokines in spleen cell culture supernatants were examined. To simplify and visualize the data, P-value heatmaps were plotted from the differences in P-values of the individual epitope peptide-induced cytokines in the three groups (fig. 6). As a result, it was found that 4 dominant peptides (CTL-Rv 1737c-P2, CTL-Rv2031c-P1, CTL-Rv2626c-P1 and CTL-Rv2660 c-P1) stimulated ATB, and that cytokine levels produced by spleen cells of mice in LTBI and UC groups were significantly different among the three groups (shown in white grids in FIG. 6A).

Specifically (B in fig. 6): (1) for CTL-Rv1737c-P2 peptide, its induced IFN- γ levels were significantly higher in ATB mice than in LTBI (p=0.0155) or UC (P < 0.0001) group mice, and their induced IFN- γ levels were significantly higher in LTBI group mice than in UC group (p=0.0016); (2) for CTL-RV2031c-P1 peptide, its induced IL-6 levels were significantly lower in ATB mice than in LTBI (p=0.0012) or UC (p=0.0002) group mice, and its induced IL-6 levels were significantly lower in LTBI group mice than in UC group (p=0.0027); (3) for CTL-RV2626c-P1 peptide, its induced TNF- α levels were significantly higher in ATB mice than in LTBI (p= 0.0226) and lower in mice of the UC group (p=0.0062), TNF- α levels in LTBI mice were significantly lower than in the UC group (p=0.0040); (4) for the CTL-RV2660c-P1 peptide, the level of IFN-gamma induced was significantly higher in the ATB group mice than in the LTBI (P=0.0067) or UC group (P=0.0097) mice, and the level of IFN-gamma was significantly lower in the mouse LTBI than in the UC group (P= 0.0334).

Based on the above data, IFN-gamma cytokines were selected for ROC curve analysis (FIG. 7 and Table 5). The combined detection of the levels of the CTL epitope peptides CTL-Rv1737c-P2 and CTL-Rv2660c-P1 induced IFN- γ can distinguish ATB mice from UC group mice (fig. 7, auc=1.000, p=0.0039), but cannot distinguish LTBI mice from ATB (auc=0.7500, p= 0.1495) and UC group mice (auc=0.5000, P > 0.9999). Further analysis found that the sensitivity of the above 2 epitope peptides in combination with differential diagnosis of ATB vs UC was 100% and the specificity was 83.33% (Table 5).

TABLE 5 sensitivity and specificity of diagnosis of ATB and LTBI in combination with CTL epitope-induced cytokines

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To sum up:

(1) The invention discovers for the first time that 7 CTL epitope peptides (CTL-Rv 1737c-P2, CTL-Rv2031c-P1, CTL-Rv2031c-P2, CTL-Rv2626c-P1, CTL-Rv2659c-P2, CTL-Rv2660c-P1 and CTL-Rv2660 c-P2) stimulate IFN-gamma generated by mouse spleen cells ⁺ The T lymphocyte count was significantly higher in ATB mice than LTBI and UC mice. IFN-gamma induced by 7 CTL epitope peptides ⁺ The number of T lymphocytes can distinguish ATB mice from UC group mice or LTBI mice, and can also distinguish LTBI mice from UC mice. Further analysis found that the sensitivities of the above 7 epitope peptides in combination with differential diagnosis of ATB vs UC, ATB vs LTBI and UC vs LTBI were 100%, 100% and 76.19% respectively, and the specificities were 95.24%, 95.24% and 85.71% respectively.

(2) The level of IFN-gamma induced by CTL-Rv1737c-P2 peptide is significantly higher in ATB mice than in LTBI or UC mice, and the level of IFN-gamma induced by it is significantly higher in LTBI mice than in UC mice; the level of IL-6 induced by CTL-RV2031c-P1 peptide is significantly lower in ATB mice than in LTBI or UC mice, and the level of IL-6 induced by it is significantly lower in LTBI mice than in UC mice; CTL-RV2626c-P1 peptide induced TNF- α levels in ATB mice were significantly higher than those in LTBI and lower than those in UC groups, and in LTBI mice; CTL-RV2660c-P1 peptide induced IFN-gamma levels were significantly higher in ATB mice than in LTBI or UC mice, and IFN-gamma levels were significantly lower in mouse LTBI than in UC mice. Based on the above data, we selected IFN-gamma cytokines for ROC curve analysis. The combined detection of the levels of IFN-gamma induced by CTL epitope peptides CTL-Rv1737c-P2 and CTL-Rv2660c-P1 can distinguish ATB mice from UC mice, but cannot distinguish LTBI mice from ATB and UC mice. Further analysis shows that the sensitivity of the combined differential diagnosis ATB vs UC of the 2 epitope peptides is 100 percent, and the specificity is 83.33 percent.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

SEQUENCE LISTING

<110> eighth medical center of general Hospital for liberation of Chinese people

<120> CTL epitope peptide of mycobacterium tuberculosis LTBI-RD related protein antigen and application thereof

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 395

<212> PRT

<213> Mycobacterium tuberculosis

<400> 1

Met Arg Gly Gln Ala Ala Asn Leu Val Leu Ala Thr Trp Ile Ser Val

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

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Ala Arg Asp Met Ser Leu Ser Ser Ala Glu Ala Ser Leu Leu Val Ala

35 40 45

Thr Pro Ile Leu Val Gly Ala Leu Gly Arg Ile Val Thr Gly Pro Leu

50 55 60

Thr Asp Arg Phe Gly Gly Arg Ala Met Leu Ile Ala Val Thr Leu Ala

65 70 75 80

Ser Ile Leu Pro Val Leu Ala Val Gly Val Ala Ala Thr Met Gly Ser

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Tyr Ala Leu Leu Val Phe Phe Gly Leu Phe Leu Gly Val Ala Gly Thr

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Ile Phe Ala Val Gly Ile Pro Phe Ala Asn Asn Trp Tyr Gln Pro Ala

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Arg Arg Gly Phe Ser Thr Gly Val Phe Gly Met Gly Met Val Gly Thr

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Ala Leu Ser Ala Phe Phe Thr Pro Arg Phe Val Arg Trp Phe Gly Leu

145 150 155 160

Phe Thr Thr His Ala Ile Val Ala Ala Ala Leu Ala Ser Thr Ala Val

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Val Ala Met Val Val Leu Arg Asp Ala Pro Tyr Phe Arg Pro Asn Ala

180 185 190

Asp Pro Val Leu Pro Arg Leu Lys Ala Ala Ala Arg Leu Pro Val Thr

195 200 205

Trp Glu Met Ser Phe Leu Tyr Ala Ile Val Phe Gly Gly Phe Val Ala

210 215 220

Phe Ser Asn Tyr Leu Pro Thr Tyr Ile Thr Thr Ile Tyr Gly Phe Ser

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

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

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His Val Val Leu Ala Ser Leu Ala Gly Thr Ala Leu Leu Ala Phe Ala

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Ala Ala Leu Gln Pro Pro Pro Glu Val Trp Ser Ala Ala Thr Phe Ile

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Thr Leu Ala Val Cys Leu Gly Val Gly Thr Gly Gly Val Phe Ala Trp

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Val Ala Arg Arg Ala Pro Ala Ala Ser Val Gly Ser Val Thr Gly Ile

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Val Ala Ala Ala Gly Gly Leu Gly Gly Tyr Phe Pro Pro Leu Val Met

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Gly Ala Thr Tyr Asp Pro Val Asp Asn Asp Tyr Thr Val Gly Leu Leu

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Leu Leu Val Ala Thr Ala Leu Val Ala Cys Thr Tyr Thr Ala Leu His

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Ala Arg Glu Pro Val Ser Glu Glu Ala Ser Arg

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<213> Mycobacterium tuberculosis

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Met Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro

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Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu Arg

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Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu

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Gly Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys

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Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala Glu

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Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly

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Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp Asp

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Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val

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Ser Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn

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<213> Mycobacterium tuberculosis

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Met Thr Thr Ala Arg Asp Ile Met Asn Ala Gly Val Thr Cys Val Gly

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Glu His Glu Thr Leu Thr Ala Ala Ala Gln Tyr Met Arg Glu His Asp

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Ile Gly Ala Leu Pro Ile Cys Gly Asp Asp Asp Arg Leu His Gly Met

35 40 45

Leu Thr Asp Arg Asp Ile Val Ile Lys Gly Leu Ala Ala Gly Leu Asp

50 55 60

Pro Asn Thr Ala Thr Ala Gly Glu Leu Ala Arg Asp Ser Ile Tyr Tyr

65 70 75 80

Val Asp Ala Asn Ala Ser Ile Gln Glu Met Leu Asn Val Met Glu Glu

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His Gln Val Arg Arg Val Pro Val Ile Ser Glu His Arg Leu Val Gly

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Ile Val Thr Glu Ala Asp Ile Ala Arg His Leu Pro Glu His Ala Ile

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Val Gln Phe Val Lys Ala Ile Cys Ser Pro Met Ala Leu Ala Ser

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<213> Mycobacterium tuberculosis

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Val Thr Gln Thr Gly Lys Arg Gln Arg Arg Lys Phe Gly Arg Ile Arg

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Gln Phe Asn Ser Gly Arg Trp Gln Ala Ser Tyr Thr Gly Pro Asp Gly

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Arg Val Tyr Ile Ala Pro Lys Thr Phe Asn Ala Lys Ile Asp Ala Glu

35 40 45

Ala Trp Leu Thr Asp Arg Arg Arg Glu Ile Asp Arg Gln Leu Trp Ser

50 55 60

Pro Ala Ser Gly Gln Glu Asp Arg Pro Gly Ala Pro Phe Gly Glu Tyr

65 70 75 80

Ala Glu Gly Trp Leu Lys Gln Arg Gly Ile Lys Asp Arg Thr Arg Ala

85 90 95

His Tyr Arg Lys Leu Leu Asp Asn His Ile Leu Ala Thr Phe Ala Asp

100 105 110

Thr Asp Leu Arg Asp Ile Thr Pro Ala Ala Val Arg Arg Trp Tyr Ala

115 120 125

Thr Thr Ala Val Gly Thr Pro Thr Met Arg Ala His Ser Tyr Ser Leu

130 135 140

Leu Arg Ala Ile Met Gln Thr Ala Leu Ala Asp Asp Leu Ile Asp Ser

145 150 155 160

Asn Pro Cys Arg Ile Ser Gly Ala Ser Thr Ala Arg Arg Val His Lys

165 170 175

Ile Arg Pro Ala Thr Leu Asp Glu Leu Glu Thr Ile Thr Lys Ala Met

180 185 190

Pro Asp Pro Tyr Gln Ala Phe Val Leu Met Ala Ala Trp Leu Ala Met

195 200 205

Arg Tyr Gly Glu Leu Thr Glu Leu Arg Arg Lys Asp Ile Asp Leu His

210 215 220

Gly Glu Val Ala Arg Val Arg Arg Ala Val Val Arg Val Gly Glu Gly

225 230 235 240

Phe Lys Val Thr Thr Pro Lys Ser Asp Ala Gly Val Arg Asp Ile Ser

245 250 255

Ile Pro Pro His Leu Ile Pro Ala Ile Glu Asp His Leu His Lys His

260 265 270

Val Asn Pro Gly Arg Glu Ser Leu Leu Phe Pro Ser Val Asn Asp Pro

275 280 285

Asn Arg His Leu Ala Pro Ser Ala Leu Tyr Arg Met Phe Tyr Lys Ala

290 295 300

Arg Lys Ala Ala Gly Arg Pro Asp Leu Arg Val His Asp Leu Arg His

305 310 315 320

Ser Gly Ala Val Leu Ala Ala Ser Thr Gly Ala Thr Leu Ala Glu Leu

325 330 335

Met Gln Arg Leu Gly His Ser Thr Ala Gly Ala Ala Leu Arg Tyr Gln

340 345 350

His Ala Ala Lys Gly Arg Asp Arg Glu Ile Ala Ala Leu Leu Ser Lys

355 360 365

Leu Ala Glu Asn Gln Glu Met

370 375

<210> 5

<211> 75

<212> PRT

<213> Mycobacterium tuberculosis

<400> 5

Val Ile Ala Gly Val Asp Gln Ala Leu Ala Ala Thr Gly Gln Ala Ser

1 5 10 15

Gln Arg Ala Ala Gly Ala Ser Gly Gly Val Thr Val Gly Val Gly Val

20 25 30

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

35 40 45

Phe Ser Ser Arg Ser Pro Asp Phe Val Asp Glu Thr Ala Gly Gln Ser

50 55 60

Trp Cys Ala Ile Leu Gly Leu Asn Gln Phe His

65 70 75

Claims

1. The polypeptide composition is characterized by comprising 7 polypeptides, wherein the amino acid sequences of the 7 polypeptides are respectively shown as 378-386 of SEQ ID No.1, 23-31 of SEQ ID No.2, 92-100 of SEQ ID No.2, 133-141 of SEQ ID No.3, 325-333 of SEQ ID No.4, 47-56 of SEQ ID No.5 and 41-49 of SEQ ID No. 5.

2. Use of the polypeptide composition of claim 1 for the preparation of a product for diagnosing a disease caused by mycobacterium tuberculosis.

3. Use of the polypeptide composition of claim 1 for any of the following:

d1 The use of said composition for the preparation of a product for the identification of patients suffering from active tuberculosis and patients suffering from latent tuberculosis;

d2 Use of a composition for identifying a healthy subject from an active tuberculosis patient;

d3 Use of a composition for identifying a healthy subject from a latent tuberculosis infected person;

d4 For the preparation of tuberculosis vaccines.

4. The use according to claim 2, wherein the disease caused by mycobacterium tuberculosis is tuberculosis.

5. A vaccine, characterized in that the active ingredient of the vaccine is the polypeptide composition of claim 1.

6. A nucleic acid molecule composition comprising nucleic acid molecules encoding 7 polypeptides in the polypeptide composition of claim 1.

7. A biological material comprising the nucleic acid molecule composition of claim 6, said biological material being a recombinant vector or a recombinant cell.

8. A kit for identifying active tuberculosis from latent tuberculosis infection, said kit comprising the polypeptide composition of claim 1.