Method And Kit For Diagnosis Of Active Tuberculosis
20190025301 ยท 2019-01-24
Inventors
- Shengxiang GE (Xiamen, Fujian Province, CN)
- Yongliang LIU (Xiamen, Fujian Province, CN)
- Mengyuan CHEN (Xiamen, Fujian Province, CN)
- Jun ZHANG (Xiamen, Fujian Province, CN)
- Ningshao XIA (Xiamen, Fujian Province, CN)
Cpc classification
G01N2500/04
PHYSICS
G01N33/577
PHYSICS
International classification
Abstract
Disclosed are a method for diagnosing whether a subject is suffering from active tuberculosis, a method for determining the therapeutic effect of a therapy on active tuberculosis, a method for screening a candidate drug capable of treating active tuberculosis, and a kit comprising a specific stimulating agent and a reagent for detecting the level of IL-6, the method and kit belong to the fields of molecular biology, immunology and disease diagnosis.
Claims
1. A kit comprising one or more of RV0183, PlcD, or antigenic fragments thereof, and a reagent capable of detecting IL-6; preferably, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3; preferably, the kit comprises RV0183 and/or PlcD; preferably, the kit comprises one or more antigenic fragments of RV0183; more preferably, the antigenic fragment has an amino acid sequence selected from: SEQ ID NOs: 5-25; preferably, the kit comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19; optionally, the kit further comprises a combination of the following antigenic fragments: 1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11 and 22, 2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8 and 11-12, 3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-12, 22, and 24, or 4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12, 15, and 22-25; preferably, the kit comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, the reagent capable of detecting IL-6 is a substance that can specifically bind to IL-6, for example, an antibody, a targeting polypeptide or an aptamer; optionally, the reagent further comprises a detectable label; preferably, the reagent determines the level of IL-6 in the sample by immunologic assay; more preferably, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance; preferably, the reagent comprises an anti-IL-6 antibody or an antigen binding fragment thereof; more preferably, the reagent determines the level of IL-6 by ELISA; preferably, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-IL-6 antibody is an IgG antibody or an IgM antibody; preferably, the kit further comprises one or more devices or reagents selected from 1) to 5): 1) a blood collection device, e.g. a pyrogen-free vacuum blood collection tube; 2) an anticoagulant, e.g. heparin; 3) a culture solution or a culture medium; 4) a non-specific stimulating agent, e.g. phytohemagglutinin or Concanavalin A; 5) a diluent, e.g. phosphate buffer or physiological saline; preferably, the kit is useful for diagnosing active tuberculosis, determining the therapeutic effect of a therapy on active tuberculosis or screening a candidate drug capable of treating active tuberculosis.
2. Use of a specific stimulating agent in the manufacture of a kit for diagnosing active tuberculosis; wherein, the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; preferably, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3; preferably, the specific stimulating agent is selected from the group consisting of RV0183, PlcD, and a combination thereof; preferably, the specific stimulating agent is selected from one or more antigenic fragments of RV0183; more preferably, the antigenic fragment has an amino acid sequence selected from: SEQ ID NOs: 5-25; preferably, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19; optionally, the specific stimulating agent further comprises a combination of the following antigenic fragments: 1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11 and 22, 2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8 and 11-12, 3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-12, 22 and 24, or 4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12, 15 and 22-25; preferably, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, the kit comprises a reagent capable of detecting IL-6, such as an antibody, a targeting polypeptide or an aptamer that can specifically bind to IL-6; optionally, the reagent further comprises a detectable label; preferably, the reagent determines the level of IL-6 in the sample by immunologic assay; more preferably, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance; preferably, the reagent comprises an anti-IL-6 antibody or an antigen binding fragment thereof; more preferably, the reagent determines the level of IL-6 by ELISA; preferably, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-IL-6 antibody is an IgG antibody or an IgM antibody; preferably, the kit further comprises one or more devices or reagents selected from 1) to 5): 1) a blood collection device, e.g. a pyrogen-free vacuum blood collection tube; 2) an anticoagulant, e.g. heparin; 3) a culture solution or a culture medium; 4) a non-specific stimulating agent, e.g. phytohemagglutinin or Concanavalin A; 5) a diluent, e.g. phosphate buffer or physiological saline; preferably, the kit diagnoses whether the subject is suffering from active tuberculosis, by a method comprising the following steps: (1) stimulating at least one sample from the subject with a specific stimulating agent, and using the at least one sample as a test sample, and using a non-stimulated sample from the subject, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (2) determining the level of IL-6 in each of the samples in the step (1) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample; and (3) comparing the difference value with a reference value, or subjecting the difference value to statistical analysis so as to obtain a statistical analysis value, and comparing the statistical analysis value with a reference value, and determining whether the subject is suffering from active tuberculosis; wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat; preferably, in the step (3), statistical analysis of the difference value is carried out by using a statistical model selected from the group consisting of: Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM); more preferably, in the step (3), statistical analysis of the difference value is carried out by using Logistic regression model; preferably, in the step (1), stimulating one or more samples from the subject with at least two specific stimulating agents together or separately, and using the one or more samples as the test sample, wherein the specific stimulating agents are each independently selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; more preferably, in the step (1), stimulating at least two samples with RV0183 and PlcD separately, and using the at least two samples as the test sample; or, in the step (1), stimulating at least one sample with one or more antigenic fragments of RV0183 together, and using the at least one sample as the test sample; preferably, the step (1) further comprises: stimulating at least one sample with a non-specific stimulating agent, and using the at least one sample as a positive control sample; more preferably, the non-specific stimulating agent includes phytohemagglutinin or Concanavalin A; preferably, prior to the step (1), the method further comprises one or more of the following steps: (a) obtaining the sample from the subject by using a blood collection device; (b) treating the blood collection device or the sample from the subject with an anticoagulant; (c) treating the sample from the subject with a culture solution or a culture medium; and, (d) diluting the sample from the subject with a diluent.
3. Use of a specific stimulating agent in the manufacture of a kit for determining the therapeutic effect of a therapy on active tuberculosis; wherein, the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; preferably, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3; preferably, the specific stimulating agent is selected from the group consisting of RV0183, PlcD, and a combination thereof; preferably, the specific stimulating agent is selected from one or more antigenic fragments of RV0183; more preferably, the antigenic fragment has an amino acid sequence selected from: SEQ ID NOs: 5-25; preferably, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19; optionally, the specific stimulating agent further comprises a combination of the following antigenic fragments: 1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11 and 22, 2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8 and 11-12, 3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-12, 22 and 24, or 4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12, 15 and 22-25; preferably, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, the kit comprises a reagent capable of detecting IL-6, such as an antibody, a targeting polypeptide or an aptamer that can specifically bind to IL-6; optionally, the reagent further comprises a detectable label; preferably, the reagent determines the level of IL-6 in the sample by immunologic assay; more preferably, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance; preferably, the reagent comprises an anti-IL-6 antibody or an antigen binding fragment thereof; more preferably, the reagent determines the level of IL-6 by ELISA; preferably, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-IL-6 antibody is an IgG antibody or an IgM antibody; preferably, the kit further comprises one or more devices or reagents selected from 1) to 5): 1) a blood collection device, e.g. a pyrogen-free vacuum blood collection tube; 2) an anticoagulant, e.g. heparin; 3) a culture solution or a culture medium; 4) a non-specific stimulating agent, e.g. phytohemagglutinin or Concanavalin A; 5) a diluent, e.g. phosphate buffer or physiological saline; preferably, the kit determines the therapeutic effect of a therapy on active tuberculosis by a method comprising the following steps: (1) before administering the therapy to a subject, obtaining at least two samples from the subject, as a pre-therapy sample; (2) stimulating at least one pre-therapy sample from the subject with a specific stimulating agent, and using the at least one pre-therapy sample as a test sample, and using at least one non-stimulated pre-therapy sample from the subject, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (3) determining the level of IL-6 in each of the samples in the step (2) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a first difference value; (4) administering the therapy to the subject; (5) after administering the therapy to the subject, obtaining at least two samples from the subject, as a post-therapy sample; (6) stimulating at least one post-therapy sample from the subject with a specific stimulating agent, and using the at least one post-therapy sample as a test sample, and using at least one non-stimulated post-therapy sample from the subject, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (7) determining the level of IL-6 in each of the samples in the step (6) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a second difference value; and (8) comparing the second difference value with the first difference value, or separately subjecting the first difference value and the second difference value to statistical analysis so as to obtain a statistical analysis value of the first difference value and a statistical analysis value of the second difference value, and comparing the statistical analysis value of the second difference value with the statistical analysis value of the first difference value, and determining whether the therapy is effective in the treatment of active tuberculosis; wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat; preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using a statistical model selected from the group consisting of: Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM); more preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using Logistic regression model; preferably, in the steps (2) and (6), the pre-therapy sample and the post-therapy sample are subjected to the same treatment; preferably, in the steps (2) and (6), stimulating one or more samples from the subject with at least two specific stimulating agents together or separately, and using the one or more samples as the test sample, wherein the specific stimulating agents are each independently selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; more preferably, in the steps (2) and (6), stimulating at least two samples with RV0183 and PlcD separately, and using the at least two samples as the test sample; or, in the steps (2) and (6), stimulating at least one sample with one or more antigenic fragments of RV0183 together, and using the at least one sample as the test sample; preferably, the subject is a mammal, such as human; preferably, the therapy comprises administering an antitubercular agent to the subject, such as isoniazid, rifampicin, streptomycin, pyrazinamide, ethambutol or any combination thereof; preferably, the steps (2) and (6) further comprise: stimulating at least one sample with a non-specific stimulating agent, and using the at least one sample as a positive control sample; more preferably, the non-specific stimulating agent includes phytohemagglutinin or Concanavalin A; preferably, in the step (1), the pre-therapy sample from the subject is obtained by using a blood collection device; preferably, in the step (5), the post-therapy sample from the subject is obtained by using a blood collection device; preferably, prior to the step (1), the method further comprises one or more of the following steps: (a) treating the blood collection device or the sample from the subject with an anticoagulant; (b) treating the sample from the subject with a culture solution or a culture medium; and, (c) diluting the sample from the subject with a diluent; preferably, prior to the step (5), the method further comprises one or more of the following steps: (a) treating the blood collection device or the sample from the subject with an anticoagulant; (b) treating the sample from the subject with a culture solution or a culture medium; and, (c) diluting the sample from the subject with a diluent.
4. Use of a specific stimulating agent in the manufacture of a kit for screening a candidate drug capable of treating active tuberculosis; wherein, the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; preferably, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3; preferably, the specific stimulating agent is selected from the group consisting of RV0183, PlcD, and a combination thereof; preferably, the specific stimulating agent is selected from one or more antigenic fragments of RV0183; more preferably, the antigenic fragment has an amino acid sequence selected from: SEQ ID NOs: 5-25; preferably, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19; optionally, the specific stimulating agent further comprises a combination of the following antigenic fragments: 1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11 and 22, 2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8 and 11-12, 3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-12, 22 and 24, or 4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12, 15 and 22-25; preferably, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, the kit comprises a reagent capable of detecting IL-6, such as an antibody, a targeting polypeptide or an aptamer that can specifically bind to IL-6; optionally, the reagent further comprises a detectable label; preferably, the reagent determines the level of IL-6 in the sample by immunologic assay; more preferably, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance; preferably, the reagent comprises an anti-IL-6 antibody or an antigen binding fragment thereof; more preferably, the reagent determines the level of IL-6 by ELISA; preferably, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-IL-6 antibody is an IgG antibody or an IgM antibody; preferably, the kit further comprises one or more devices or reagents selected from 1) to 5): 1) a blood collection device, e.g. a pyrogen-free vacuum blood collection tube; 2) an anticoagulant, e.g. heparin; 3) a culture solution or a culture medium; 4) a non-specific stimulating agent, e.g. phytohemagglutinin or Concanavalin A; 5) a diluent, e.g. phosphate buffer or physiological saline; preferably, the kit screens a candidate drug capable of treating active tuberculosis by a method comprising the following steps: (1) before administering the candidate drug to an model animal, obtaining at least two samples from the animal, as a pre-therapy sample; (2) stimulating at least one pre-therapy sample from the animal with a specific stimulating agent, and using the at least one pre-therapy sample as a test sample, and using at least one non-stimulated pre-therapy sample from the animal, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (3) determining the level of IL-6 in each of the samples in the step (2) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a first difference value; (4) administering the candidate drug to the animal; (5) after administering the candidate drug to the animal, obtaining at least two samples from the animal, as a post-therapy sample; (6) stimulating at least one post-therapy sample from the animal with a specific stimulating agent, and using the at least one post-therapy sample as a test sample, and using at least one non-stimulated post-therapy sample from the animal, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (7) determining the level of IL-6 in each of the samples in the step (6) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a second difference value; and (8) comparing the second difference value with the first difference value, or separately subjecting the first difference value and the second difference value to statistical analysis so as to obtain a statistical analysis value of the first difference value and a statistical analysis value of the second difference value, and comparing the statistical analysis value of the second difference value with the statistical analysis value of the first difference value, and determining whether the therapy is effective in the treatment of active tuberculosis; wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat; preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using a statistical model selected from the group consisting of: Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM); more preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using Logistic regression model. preferably, in the steps (2) and (6), the pre-therapy sample and the post-therapy sample are subjected to the same treatment; preferably, in the steps (2) and (6), stimulating one or more samples from the animal with at least two specific stimulating agents together or separately, and using the one or more samples as the test sample, wherein the specific stimulating agents are each independently selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; more preferably, in the steps (2) and (6), stimulating at least two samples with RV0183 and PlcD separately, and using the at least two samples as the test sample; or, in the steps (2) and (6), stimulating at least one sample with one or more antigenic fragments of RV0183 together, and using the at least one sample as the test sample; preferably, the model animal is a non-human mammal, for example, a mouse, a guinea pig, a rabbit or a non-human primate (e.g. a cynomolgus monkey or a rhesus monkey); preferably, the subject is a mammal, such as human; preferably, the steps (2) and (6) further comprise: stimulating at least one sample with a non-specific stimulating agent, and using the at least one sample as a positive control sample; more preferably, the non-specific stimulating agent includes phytohemagglutinin or Concanavalin A; preferably, in the step (1), the pre-therapy sample from the animal is obtained by using a blood collection device; preferably, in the step (5), the post-therapy sample from the animal is obtained by using a blood collection device; preferably, prior to the step (1), the method further comprises one or more of the following steps: (a) treating the blood collection device or the sample from the animal with an anticoagulant; (b) treating the sample from the animal with a culture solution or a culture medium; and, (c) diluting the sample from the animal with a diluent; preferably, prior to the step (5), the method further comprises one or more of the following steps: (a) treating the blood collection device or the sample from the animal with an anticoagulant; (b) treating the sample from the animal with a culture solution or a culture medium; and, (c) diluting the sample from the animal with a diluent.
5. A method for diagnosing whether a subject is suffering from active tuberculosis, comprising the following steps: (1) providing at least two samples from the subject; (2) stimulating at least one sample from the subject with a specific stimulating agent, and using the at least one sample as a test sample, and using a non-stimulated sample, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (3) determining the level of IL-6 in each of the samples in the step (2), and calculating a difference value in the level of IL-6 between the test sample and the negative control sample; and (4) comparing the difference value with a reference value, or subjecting the difference value to statistical analysis so as to obtain a statistical analysis value, and comparing the statistical analysis value with a reference value, and determining whether the subject is suffering from active tuberculosis; wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat; preferably, in the step (4), the difference value is subjected to statistical analysis by using a statistical model selected from the group consisting of: Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM); more preferably, in the step (4), statistical analysis of the difference value is carried out by using Logistic regression model; preferably, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3; preferably, the antigenic fragments are antigenic fragments of RV0183; more preferably, the antigenic fragment has an amino acid sequence selected from: SEQ ID NOs: 5-25; preferably, the subject is a mammal, such as human; preferably, in the step (2), stimulating one or more samples from the subject with at least two specific stimulating agents together or separately, and using the one or more samples as the test sample, wherein the specific stimulating agents are each independently selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; preferably, in the step (2), stimulating at least two samples with RV0183 and PlcD separately, and using the at least two samples as the test sample; or, in the step (2), stimulating at least one sample with one or more of the antigenic fragments together, and using the at least one sample as the test sample; more preferably, in the step (2), stimulating at least one sample with a combination of the following antigenic fragments together, and using the at least one sample as the test sample: 1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19, 2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11, 13-14, 19 and 22, 3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8, 11-14 and 19, 4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-14, 19, 22 and 24, 5) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12-15, 19 and 22-25; or 6) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, in the step (3), the level of IL-6 in the samples is determined by immunologic assay; more preferably, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance; preferably, in the step (3), the level of IL-6 is determined by using an anti-IL-6 antibody or an antigen binding fragment thereof, for example, by ELISA; preferably, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-IL-6 antibody is an IgG antibody or an IgM antibody; preferably, the step (2) further comprises: stimulating at least one sample with a non-specific stimulating agent , and using the at least one sample as a positive control sample; more preferably, the non-specific stimulating agent includes phytohemagglutinin or Concanavalin A; preferably, prior to the step (1), the method further comprises one or more of the following steps: (a) obtaining the sample from the subject; (b) adding an anticoagulant such as heparin to the sample; (c) obtaining PBMC or a PBMC-containing blood component (e.g. peripheral blood buffy coat) from the sample; (d) adding a culture solution or a culture medium into the sample; and (e) diluting the sample.
6. A method for determining the therapeutic effect of a therapy on active tuberculosis, comprising the following steps: (1) before administering the therapy to a subject, obtaining at least two samples from the subject, as a pre-therapy sample; (2) stimulating at least one pre-therapy sample from the subject with a specific stimulating agent, and using the at least one pre-therapy sample as a test sample, and using at least one non-stimulated pre-therapy sample from the subject, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (3) determining the level of IL-6 in each of the samples in the step (2), and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a first difference value; (4) administering the therapy to the subject; (5) after administering the therapy to the subject, obtaining at least two samples from the subject, as a post-therapy sample; (6) stimulating at least one post-therapy sample from the subject with a specific stimulating agent, and using the at least one post-therapy sample as a test sample, and using at least one non-stimulated post-therapy sample from the subject, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (7) determining the level of IL-6 in each of the samples in the step (6) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a second difference value; and (8) comparing the second difference value with the first difference value, or separately subjecting the first difference value and the second difference value to statistical analysis so as to obtain a statistical analysis value of the first difference value and a statistical analysis value of the second difference value, and comparing the statistical analysis value of the second difference value with the statistical analysis value of the first difference value, and determining whether the therapy is effective in the treatment of active tuberculosis; wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat; preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using a statistical model selected from the group consisting of: Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM); more preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using Logistic regression model; preferably, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3; preferably, the antigenic fragments are antigenic fragments of RV0183; more preferably, the antigenic fragment has an amino acid sequence selected from: SEQ ID NOs: 5-25; preferably, the subject is a mammal, such as human; preferably, the therapy comprises administering an antitubercular agent to the subject, for example, isoniazid, rifampicin, streptomycin, pyrazinamide, ethambutol or any combination thereof; preferably, in the steps (2) and (6), the pre-therapy sample and the post-therapy sample are subjected to the same treatment; preferably, in the steps (2) and (6), stimulating one or more samples from the subject with at least two specific stimulating agents together or separately, and using the one or more samples as the test sample, wherein the specific stimulating agents are each independently selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; preferably, in the steps (2) and (6), stimulating at least two samples with RV0183 and PlcD separately, and using the at least two samples as the test sample; or, in the steps (2) and (6), stimulating at least one sample with one or more of the antigenic fragments together, and using the at least one sample as the test sample; more preferably, in the steps (2) and (6), stimulating at least one sample with a combination of the following antigenic fragments together, and using the at least one sample as the test sample: 1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19, 2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11, 13-14, 19 and 22, 3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8, 11-14 and 19, 4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-14, 19, 22 and 24, 5) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12-15, 19 and 22-25; or 6) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, in the step (3), the level of IL-6 in the samples is determined by immunologic assay; more preferably, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance; preferably, in the step (3), the level of IL-6 is determined by using an anti-IL-6 antibody or an antigen binding fragment thereof, for example, by ELISA; preferably, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-IL-6 antibody is an IgG antibody or an IgM antibody; preferably, the steps (2) and (6) further comprise: stimulating at least one sample with a non-specific stimulating agent, and using the at least one sample as a positive control sample; more preferably, the non-specific stimulating agent includes phytohemagglutinin or Concanavalin A; preferably, prior to the step (1), the method further comprises one or more of the following steps: (a) adding an anticoagulant such as heparin into the pre-therapy sample; (b) obtaining PBMC or a PBMC-containing blood component (e.g. peripheral blood buffy coat) from the pre-therapy sample; (c) adding a culture solution or a culture medium into the pre-therapy sample; and, (d) diluting the pre-therapy sample; preferably, prior to the step (5), the method further comprises one or more of the following steps: (a) adding an anticoagulant such as heparin to the post-therapy sample; (b) obtaining PBMC or a PBMC-containing blood component (e.g. peripheral blood buffy coat) from the post-therapy sample; (c) adding a culture solution or a culture medium to the post-therapy sample; and, (d) diluting the post-therapy sample.
7. A method for screening a candidate drug capable of treating active tuberculosis, comprising the following steps: (1) before administering the candidate drug to an model animal, obtaining at least two samples from the animal, as a pre-therapy sample; (2) stimulating at least one pre-therapy sample from the animal with a specific stimulating agent, and using the at least one pre-therapy sample as a test sample, and using at least one non-stimulated pre-therapy sample from the animal, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (3) determining the level of IL-6 in each of the samples in the step (2), and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a first difference value; (4) administering the candidate drug to the animal; (5) after administering the candidate drug to the animal, obtaining at least two samples from the animal, as a post-therapy sample; (6) stimulating at least one post-therapy sample from the animal with a specific stimulating agent, and using the at least one post-therapy sample as a test sample, and using at least one non-stimulated post-therapy sample from the animal, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; (7) determining the level of IL-6 in each of the samples in the step (6) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample, as a second difference value; and (8) comparing the second difference value with the first difference value, or separately subjecting the first difference value and the second difference value to statistical analysis so as to obtain a statistical analysis value of the first difference value and a statistical analysis value of the second difference value, and comparing the statistical analysis value of the second difference value with the statistical analysis value of the first difference value, and determining whether the therapy is effective in the treatment of active tuberculosis; wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat; preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using a statistical model selected from the group consisting of: Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM); more preferably, in the step (8), statistical analysis of the first difference value and the second difference value is carried out by using Logistic regression model; preferably, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3; preferably, the antigenic fragments are antigenic fragments of RV0183; more preferably, the antigenic fragment has an amino acid sequence selected from: SEQ ID NOs: 5-25; preferably, the model animal is a non-human mammal, for example, a mouse, a guinea pig, a rabbit or a non-human primate (e.g. a cynomolgus monkey or a rhesus monkey); preferably, the subject is a mammal, such as human; preferably, in the steps (2) and (6), the pre-therapy sample and the post-therapy sample are subjected to the same treatment; preferably, in the steps (2) and (6), stimulating one or more samples from the animal with at least two specific stimulating agents together or separately, and using the one or more samples as the test sample, wherein the specific stimulating agents are each independently selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof; preferably, in the steps (2) and (6), stimulating at least two samples with RV0183 and PlcD separately, and using the at least two samples as the test sample; or, in the steps (2) and (6), stimulating at least one sample with one or more of the antigenic fragments together, and using the at least one sample as the test sample; more preferably, in the steps (2) and (6), stimulating at least one sample with a combination of the following antigenic fragments together, and using the at least one sample as the test sample: 1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19, 2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11, 13-14, 19 and 22, 3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8, 11-14 and 19, 4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-14, 19, 22 and 24, 5) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12-15, 19 and 22-25; or 6) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, in the step (3), the level of IL-6 in the samples is determined by immunologic assay; more preferably, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance; preferably, in the step (3), the level of IL-6 is determined by using an anti-IL-6 antibody or an antigen binding fragment thereof, for example, by ELISA; preferably, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-IL-6 antibody is an IgG antibody or an IgM antibody; preferably, the steps (2) and (6) further comprise: stimulating at least one sample with a non-specific stimulating agent, and using the at least one sample as a positive control sample; more preferably, the non-specific stimulating agent includes phytohemagglutinin or Concanavalin A; preferably, prior to the step (1), the method further comprises one or more of the following steps: (a) adding an anticoagulant such as heparin into the pre-therapy sample; (b) obtaining PBMC or a PBMC-containing blood component (e.g. peripheral blood buffy coat) from the pre-therapy sample; (c) adding a culture solution or a culture medium into the pre-therapy sample; and, (d) diluting the pre-therapy sample; and; preferably, prior to the step (5), the method further comprises one or more of the following steps: (a) adding an anticoagulant such as heparin into the post-therapy sample; (b) obtaining PBMC or a PBMC-containing blood component (e.g. peripheral blood buffy coat) from the post-therapy sample; (c) adding a culture solution or a culture medium into the post-therapy sample; and, (d) diluting the post-therapy sample.
8. A polypeptide library, comprising: a first peptide having an amino acid sequence set forth in SEQ ID NO: 13; a second peptide having an amino acid sequence set forth in SEQ ID NO: 14; and a third peptide having an amino acid sequence set forth in SEQ ID NO: 19; optionally, the polypeptide library further comprises a combination of the following polypeptides: 1) polypeptides having the amino acid sequences set forth in SEQ ID NOs: 5, 11 and 22, 2) polypeptides having the amino acid sequences set forth in SEQ ID NOs: 7-8 and 11-12, 3) polypeptides having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-12, 22 and 24, or 4) polypeptides having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12, 15 and 22-25; preferably, the polypeptide library comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25; preferably, the polypeptide library can induce the generation of IL-6 in a sample; wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat; preferably, the polypeptide library is useful for diagnosing active tuberculosis, determining the therapeutic effect of a therapy on active tuberculosis or screening a candidate drug capable of treating active tuberculosis.
Description
DESCRIPTION OF THE DRAWINGS
[0299]
[0300]
[0301]
[0302]
[0303]
[0304]
[0305]
[0306]
[0307]
SEQUENCE INFORMATION
[0308] The information of the sequences involved in the invention are provided in the following Table 1.
TABLE-US-00001 TABLE 1 Sequence information SEQ ID NO: Description 1 the amino acid sequence of recombinant protein RV0183 2 the nucleotide sequence of recombinant protein RV0183 3 the amino acid sequence of recombinant protein Plcd 4 the nucleotide sequence of recombinant protein Plcd 5~25 the peptide sequences (P1-P21) of a polypeptide library of RV0183 26~29 primers
[0309] The amino acid sequence of recombinant protein RV0183 (SEQ ID NO: 1)
TABLE-US-00002 MTTTRTERNFAGIGDVRIVYDVWTPDTAPQAVVVLAHGLGEHARRYDHVA QRLGAAGLVTYALDHRGHGRSGGKRVLVRDISEYTADFDTLVGIATREYP GCKRIVLGHSMGGGIVFAYGVERPDNYDLMVLSAPAVAAQDLVSPVVAVA AKLLGVVVPGLPVQELDFTAISRDPEVVQAYNTDPLVHHGRVPAGIGRAL LQVGETMPRRAPALTAPLLVLHGTDDRLIPIEGSRRLVECVGSADVQLKE YPGLYHEVFNEPERNQVLDDVVAWLTERL
[0310] The nucleotide sequence of recombinant protein RV0183 (SEQ ID NO: 2)
TABLE-US-00003 GGATCCATGACTACCACCCGGACTGAACGGAATTTCGCGGGCATCGGCGA TGTGCGCATCGTCTACGACGTCTGGACGCCGGACACCGCGCCGCAAGCGG TGGTCGTGCTGGCCCATGGTCTGGGCGAGCATGCCCGCCGCTACGACCAT GTCGCGCAGCGGCTCGGCGCGGCCGGCCTGGTCACCTATGCGCTTGACCA CCGCGGGCATGGCCGCTCGGGTGGCAAACGGGTGCTAGTGAGAGACATCT CCGAGTACACCGCTGACTTCGACACCCTCGTTGGGATCGCCACCCGGGAA TATCCCGGGTGCAAGCGCATCGTGCTCGGGCACAGCATGGGCGGCGGCAT TGTGTTCGCTTACGGTGTCGAACGTCCAGACAACTACGACCTGATGGTGC TTTCGGCGCCGGCGGTGGCGGCACAGGACCTGGTGAGCCCGGTAGTGGCG GTTGCCGCCAAGCTTCTGGGCGTCGTGGTGCCCGGCCTGCCGGTGCAGGA ACTGGATTTTACTGCCATCTCTCGCGACCCTGAGGTGGTCCAGGCTTACA ACACCGACCCACTCGTGCACCACGGACGGGTTCCGGCCGGGATTGGCCGC GCGCTGCTGCAGGTGGGCGAGACCATGCCGCGGCGAGCACCGGCATTGAC CGCGCCGCTGCTAGTGCTGCACGGCACCGATGACCGGCTGATCCCCATCG AGGGCAGCCGTCGCCTGGTCGAATGTGTGGGATCGGCCGACGTGCAGCTG AAGGAGTATCCCGGGCTGTACCACGAGGTGTTCAACGAGCCGGAGCGCAA CCAGGTGCTCGACGATGTGGTCGCCTGGCTCACCGAGCGGTTGTAAGAAT TC
[0311] The amino acid sequence of recombinant protein Plcd (SEQ ID NO: 3)
TABLE-US-00004 DAGVSWKVYRNKTLGPISSVLTYGSLVTSFKQSADPRSDLVRFGVAPSYP ASFAADVLANRLPRVSWVIPNVLESEHPAVPAAAGAFAIVNILRILLANP AVWEKTALIVSYDENGGFFDHVVPATAPAGTPGEYVTVPDIDQVPGSGGI RGPIGLGFRVPCFVISPYSRGPQMVHDTFDHTSQLRLLETRFGVPVPNLT AWRRSVTGDMTSTFNFAVPPNSSWPNLDYPGLHALSTVPQCVPNAALGTI NRGIPYRVPDPQIMPTQETTPTRGIPSGPC
[0312] The nucleotide sequence of recombinant protein Plcd (SEQ ID NO: 4)
TABLE-US-00005 GATGCCGGCGTCAGCTGGAAGGTGTATCGCAACAAGACACTCGGGCCCAT CTCCTCGGTTCTTACTTACGGCTCGCTTGTGACGTCTTTCAAACAGTCAG CCGATCCCAGGTCAGATCTTGTCCGCTTTGGCGTGGCACCAAGCTATCCC GCGAGCTTCGCGGCCGACGTCTTAGCCAATAGACTGCCGCGGGTCTCCTG GGTGATTCCCAATGTTCTCGAATCCGAACATCCTGCGGTTCCAGCCGCGG CCGGGGCTTTCGCAATCGTCAACATCTTAAGAATATTGCTTGCCAATCCT GCGGTGTGGGAAAAGACGGCGCTGATCGTCAGCTACGACGAAAACGGCGG CTTTTTCGACCACGTTGTTCCTGCTACCGCGCCGGCCGGGACTCCCGGCG AATATGTCACGGTGCCTGACATCGATCAGGTGCCGGGCTCCGGCGGAATA CGCGGGCCGATCGGTTTGGGCTTTCGCGTTCCCTGCTTCGTCATTTCGCC GTACAGCCGTGGCCCGCAGATGGTTCACGACACGTTTGACCACACCTCAC AGCTGAGATTGCTCGAAACTCGGTTCGGGGTGCCAGTTCCCAACCTCACG GCTTGGCGGCGCAGTGTGACCGGCGACATGACGTCAACGTTCAACTTCGC TGTCCCGCCCAACTCATCATGGCCCAACCTGGATTATCCCGGGCTGCACG CGCTATCAACGGTGCCGCAGTGCGTGCCCAACGCGGCGCTGGGCACGATA AACCGTGGAATCCCGTATCGGGTTCCTGATCCACAGATCATGCCCACGCA GGAAACCACGCCTACCCGTGGTATTCCGAGCGGTCCGTGCTAA
[0313] The peptide sequences (PI-P21) (SEQ ID NO: 5-25) of a polypeptide library of RV0183
TABLE-US-00006 RV0183-p1: (SEQ ID NO: 5) MTTTRTERNFAGIGDVRIVY RV0183-p2: (SEQ ID NO: 6) GDVRIVYDVWTPDTAPQAVV RV0183-p3: (SEQ ID NO: 7) TAPQAVVVLAHGLGEHARRY RV0183-p4: (SEQ ID NO: 8) GEHARRYDHVAQRLGAAGLV RV0183-p5: (SEQ ID NO: 9) LGAAGLVTYALDHRGHGRSG RV0183-p6: (SEQ ID NO: 10) RGHGRSGGKRVLVRDISEYT RV0183-p7: (SEQ ID NO: 11) RDISEYTADFDTLVGIATRE RV0183-p8: (SEQ ID NO: 12) VGIATREYPGCKRIVLGHSM RV0183-p9: (SEQ ID NO: 13) IVLGHSMGGGIVFAYGVERP RV0183-p10: (SEQ ID NO: 14) AYGVERPDNYDLMVLSAPAV RV0183-p11: (SEQ ID NO: 15) VLSAPAVAAQDLVSPVVAVA RV0183-p12: (SEQ ID NO: 16) SPVVAVAAKLLGVVVPGLPV RV0183-p13: (SEQ ID NO: 17) VVPGLPVQELDFTAISRDPE RV0183-p14: (SEQ ID NO: 18) AISRDPEVVQAYNTDPLVHH RV0183-p15: (SEQ ID NO: 19) TDPLVHHGRVPAGIGRALLQ RV0183-p16: (SEQ ID NO: 20) IGRALLQVGETMPRRAPALT RV0183-p17: (SEQ ID NO: 21) RRAPALTAPLLVLHGTDDRL RV0183-p18: (SEQ ID NO: 22) HGTDDRLIPIEGSRRLVECV RV0183-p19: (SEQ ID NO: 23) RRLVECVGSADVQLKEYPGL RV0183-p20: (SEQ ID NO: 24) LKEYPGLYHEVFNEPERNQV RV0183-p21: (SEQ ID NO: 25) EPERNQVLDDVVAWLTERL Primers (5-3) (SEQ ID NOs: 26~29) plcD-1-F: (SEQ ID NO: 26) TTCAACCATCGCCGCCTCTACCA plcD-1-R: (SEQ ID NO: 27) CCATCGCCGCCTCTACCAGT plcD-2-F: (SEQ ID NO: 28) GGATCCATGGATGCCGGCGTCAG plcD-2-R: (SEQ ID NO: 29) AAGCTTTTAGCACGGACCGCTCG
Specific Modes for Carrying Out the Invention
[0314] The present invention is illustrated by reference to the following examples (which are not intended to limit the protection scope of the present invention).
[0315] Unless indicated otherwise, the experiments and methods (for example, molecular biological experimental methods and immunological assays) as described in Examples are substantively carried out by conventional methods as well known in the art and as described in various reference documents. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), which are incorporated herein by reference. Enzymatic reactions and purification technologies are carried out in accordance with the instructions of manufacturers, for example, as generally used in the art or described herein. Reagents or instruments, the manufacturers of which are not indicated, are the conventional products that can be purchased on the market. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the invention. All the disclosed solutions and other reference documents as mentioned here are incorporated herein by reference.
EXAMPLE 1
Cloning, Expression and Endotoxin-Removal Purification of Recombinant Protein RV0183
[0316] 1. Construction of an Expression Vector Comprising a Sequence Encoding Recombinant Protein RV0183
[0317] 1.1.1 Obtainment of a Gene Fragment of Interest
[0318] A gene fragment of interest was artificially synthesized. In order to introduce an expression vector conveniently, when the gene fragment of interest was synthesized, an enzyme cleavage site for BamHI was added at 5 end, and an enzyme cleavage site for EcoRI was added at 3 end. The vector used was pMD18T. Therefore, a RV0183 plasmid containing enzyme cleavage sites at two ends was obtained.
[0319] Enzyme cleavage: the artificially constructed RV0183 plasmid was subjected to double enzyme digestion by BamHI/EcoRI. The enzyme cleavage product was recovered by agarose gel electrophoresis.
[0320] 1.1.2 Fusion of a Gene Fragment of Interest and Construction of an Expression Vector
[0321] The pTO-T7 vector (the information of which can be found in Luo Wenxin, Zhang Jun, Yang Haijie, et al., Construction and application of an Escherichia coli high effective expression vector with an enhancer, Chinese Journal of Biotechnology, 2000, 16(5): 578-581), which had been subjected to double enzyme digestion by BamHI/EcoRI, was ligated to the enzyme cleavage product encoding RV0183, thereby obtaining an expression vector pTO-T7-RV0183 comprising the gene fragment of RV0183.
[0322] 2. Expression and Endotoxin-Removal Purification of Recombinant Protein RV0183
[0323] E. coli ER2566 (preserved in laboratory) transformed with the constructed expression vector pTO-T7-RV0183, were spread on a solid LB medium (the components of the LB medium: 10 g/L peptone, 5 g/L yeast powder, and 10 g/L NaCl, the same hereinafter) containing kanamycin (Kan, at a final concentration of 100 g/ml). Stationary culture was carried out at 37 C. until single colonies were observed clearly. Single colonies were picked and transferred to a liquid LB medium (containing 100 g/ml kanamycin). The cultures were incubated in a shaking incubator at 180 rpm at 37 C. for 8 h. Then the bacteria solution was transferred to 500 mL of liquid LB medium (containing 100 g/ml kanamycin) in a culture flask, and incubated in a shaking incubator at 180 rpm at 37 C. When the OD.sub.600 of the liquid in the culture flask reached about 0.6-0.8, IPTG was added at a final concentration of 0.2 mM/L, and the incubation was further performed at 180 rpm for 4 h at 37 C. After centrifugation of the cultures at 5000 rpm for 10 min, the bacteria were collected.
[0324] Since recombinant protein RV0183 was used to stimulate whole blood, endotoxin needed to be removed from the protein product. Methods for purifying a recombinant protein and removing endotoxin from bacterial proteins are well known by a person skilled in the art. In the Example, the following exemplary method was used.
[0325] The collected bacteria were suspended in a buffer (50 mM TB8.0), placed in an ice bath, disrupted by ultrasonic waves, and then centrifuged at 12000 rpm for 10 min, and inclusion bodies were collected.
[0326] Ni-NTA column purification: the supernatant of the expressed protein of interest was purified by self-assembled Ni-NTA column (manufacturer of media: Qiagen). In brief, the sample was loaded to a Ni-NTA column, and eluted with 0.2% sodium deoxycholate, 50 mM TB8.0 to remove a part of endotoxin, followed by elution with 200 ml 50 mM TB8.0 to remove sodium deoxycholate; finally, the protein of interest was eluted with 100 ml eluent (150 mM imidazole, 50 mM TB8.0).
[0327] DEAE column purification: the eluted protein was further purified by DEAE column. The loading buffer was 50 mM TB8.0, and the eluent was 400 mM NaCl, 50 mM TB8.0. The eluted protein was dialyzed to 50 mM TB8.0 and stored.
[0328] During purification of the protein, the buffers used were prepared with water for injection, and the experimental containers used were dried and baked at 200 C. for more than 2 h.
[0329] The recombinant protein RV0183 obtained by two-step purification was detected by SDS-PAGE, and the result was shown in
[0330] A limulus reagent was used to detect endotoxin in the purified recombinant protein RV0183. The result showed that after the two-step purification, the recombinant protein RV0183 had an endotoxin content of below 100 EU/mg.
EXAMPLE 2
Cloning, Expression and Endotoxin-Removal Purification of Recombinant Protein Plcd
[0331] 2.1 Construction of an Expression Vector Comprising a Sequence Encoding Recombinant Protein PlcD
[0332] The gene fragment of interest was obtained by PCR amplification.
[0333] Since the genomic fragment of M. tuberculosis was long and the GC-base content was higher, it was difficult to amplify a fragment of interest directly. Therefore, a fragment of interest was obtained by a Nested PCR method through two rounds of PCR amplification using the genomic DNA of M. tuberculosis H37Rv as template.
[0334] 2.1.1 Design of PCR Primers
[0335] According to the gene sequence corresponding to the PlcD protein of M. tuberculosis H37Rv as recorded in NCBI, primers were evaluated and screened by software, and the corresponding enzyme cleavage sites were added. The designed primers were shown as followed:
TABLE-US-00007 TABLE 2 Primer sequence (SEQ ID NOs: 26-29) SEQ Enzyme ID Primer cleavage NO: name Primer sequence site 26 plcD-1-F 5-TTCAACCATCGCCGCCTCTACCA-3 no 27 plcD-1-R 5-CCATCGCCGCCTCTACCAGT-3 no 28 plcD-2-F 5-GGATCCATGGATGCCGGCGTCAG-3 BamH I 29 plcD-2-R 5-AAGCTTTTAGCACGGACCGCTCG-3 Hind III
[0336] 2.1.2 First Round of PCR Amplification Reaction
[0337] In accordance with the following table, the components were homogeneously mixed in a 0.5 ml Ependorf tube, and a 20 l PCR reaction system was established.
TABLE-US-00008 First-Round PCR reaction system for plcD protein-coding gene template plcD-1-F plcD-1-R 10xBuffer dNTP DMSO glycerol rTaq DNA ddH.sub.2O 0.4 L 0.4 L 2 L 0.8 L 1.5 l 1.5 l 0.8 L 0.4 L 12.2 L
[0338] Amplification program: pre-denaturation at 95 C. for 10 min; after hot start, 30 cycles of denaturation at 95 C. for 1 min, annealing at 55 C. for 1 min, and elongation at 72 C. for 3 min; elongation at 72 C. for 10 min.
[0339] 2.1.3 Second Round of PCR Amplification Reaction
[0340] The First-Round PCR product was used as template, and in accordance with the following table, the components were homogeneously mixed in a 0.5 ml Ependorf tube to form a 50 l PCR reaction system.
TABLE-US-00009 Second-Round PCR reaction system for plcD protein-coding gene template plcD-2-F plcD-2-R 10xBuffer dNTP DMSO glycerol rTaq DNA ddH.sub.2O 1 l 1 l 5 l 2 L 2.5 l 2.5 l 2 L 1 l 33 L
[0341] Amplification program: pre-denaturation at 95 C. for 10 min; 30 cycles of denaturation at 95 C. for 1 min, annealing at 55 C. for 1 min, and elongation at 72 C. for 3 min; elongation at 72 C. for 10 min. The amplification product was identified and recovered by 1% agarose gel electrophoresis.
[0342] 2.1.4 Construction of a Cloning Vector
[0343] The following components were added and mixed in a 0.5 ml Ependorf tube, and reacted at 16 C. for 12 h, thereby obtaining a pMD18-T cloning vector ligated to the gene fragment of interest.
TABLE-US-00010 Gene fragment of pMD18-T Vector interest Solution I Total reaction volume 0.5 l 5 l 4.5 l 10 l
[0344] 2.1.5 Transformation with the Cloning Vector-Ligated Product
[0345] 40 l competent DH5 cell and 10 L of the cloning vector-ligated product were mixed, and incubated on ice for 15 min to allow adsorption. After heat shock at 42 C. for 90 s, the reaction was immediately stopped by virtue of an ice bath. 200 L antibiotic-free LB medium was added, and the cultures were incubated in a shaking incubator at 37 C. for 45 min. 200 L culture solution was spread onto a solid medium containing ampicillin, and the cultures were incubated at 37 C. for 12 h.
[0346] 2.1.6 Recovery of the Cloning Vector Plasmid
[0347] Single clone colonies were picked and seeded in 5 mL liquid LB medium containing ampicillin, and incubated at 37 C. in a shaking incubator overnight. 1.5 mL bacterial solution was placed in a 1.5 mL Eppendorf tube, and centrifuged at 12000 g for 1 min, and the supernatant was discarded. 250 L Solution I was added, and the precipitate was completely suspended under shaking. 250 L Solution II was added, and the tube was gently turned upside down for several times so that bacteria were lysed. 350 L Solution III was added, and the resultant mixture was mixed homogeneously, and placed in an ice bath for 15 min. After centrifugation at 12000 rpm for 10 min, the supernatant was transferred to an equilibrated adsorption column for plasma-recovery, and was left to stand at room temperature for 5 min. The subsequent recovery operations were the same as the steps in gel extraction.
[0348] The recovered product was identified by 1% agarose gel electrophoresis, and it was confirmed that the recovery was successful. The obtained pMD18-T containing the gene fragment of interest was cleaved by enzyme, to recover the cloned fragment of interest, and a part of cloning vectors was sent to be sequenced.
[0349] 2.1.7 Construction of the Cloning Vector Expressing PlcD
[0350] The gene fragment of interest obtained by enzyme cleavage of pMD-18T was ligated to the enzymatically cleaved PTO-T7 vector, and the ligation system was as followed: PTO-T7 vector (1 l), the gene fragment of interest (6 l), 10T4 DNA Ligase Buffer (1 l), and T4 DNA ligase (1 l), at a total reaction volume of 10 l. The time for ligation was 2 h.
[0351] 2.2 Transformation with the Cloning Vector for Expression and Protein Expression and Purification
[0352] 2.2.1 Transformation with the Expression Vector-Ligated Product
[0353] The steps were the same as the above-mentioned ligation steps, ER2566 competent cells were used for transformation, and the transformed ER2566 was spread onto a kanamycin-containing LB resistant medium.
[0354] 2.2.2 Expression and Purification of Plcd Protein
[0355] Extraction of the Protein of Interest
[0356] The bacteria, which were identified to express the correct protein of interest in a small amount, were activated. After incubation at 37 C. for 4 h, 1 mmol/L IPTG was added, and the culture was maintained at 37 C. for 4 h. The bacteria were collected by centrifugation at 9000 g for 6 min. The bacteria were rinsed and then re-centrifuged to collect the precipitate. The precipitate was re-suspended at a ratio of 200 mL bacterial solution to 5 mL TB 8.0 buffer. The re-suspended bacterial solution was disrupted by ultrasonic treatment, and the time for treatment was calculated at 3 min per 500 mL bacterial solution. The sample after ultrasonic treatment was centrifuged at 12000 g for 10 min to collect the disrupted precipitate. The disrupted product precipitate was rinsed with TB 8.0 solution repeatedly for three times, and was subjected to shaking in a 37 C. incubator for 15 min after each suspension. After re-suspending in 15 mL TB 8.0 solution containing 6 M urea, the precipitate was blew up sufficiently. When most of the precipitate was blew up, centrifugation at 12000 g for 10 min was performed to collect the supernatant, which was used in the following purification step.
[0357] Nickel Ion Column Affinity Chromatography
[0358] Ni-NTA column purification: the supernatant for expression of the protein of interest was purified by a self-assembled Ni-NTA column (manufacturer of media: Qiagen). The sample was loaded onto a Ni-NTA column, eluted with 0.2% sodium deoxycholate, 50 mM TB8.0+6 M urea to remove a part of endotoxin, and then washed with 200 ml 50 mM TB8.0+6 M urea to remove sodium deoxycholate; and finally, the protein of interest was eluted with 100 ml of the eluent (150 mM imidazole, 50 mM TB8.0, 6 M urea).
[0359] Renaturation of Protein
[0360] plcD protein was expressed in the form of inclusion bodies. Therefore, in the experiment, 6 M urea was used to denaturalize and dissolve the inclusion bodies, and after affinity-chromatographic purification, the denaturant was gradually removed by using a gradient dialysis method. During the dialysis, dithiothreitol (DTT) was added to prevent the formation of wrong disulfide bonds, help the correct folding of protein, and retain the soluble state. The dialysis steps were shown in the following table. The result was shown in
Steps for Dialysis and Renaturation of PlcD Protein
[0361]
TABLE-US-00011 Dialysis Solution Components Dialysis time Dialysis Solution I 4M urea in TB 8.0, 2 mmol/L DTT 4 h Dialysis Solution II 2M urea in TB8.0, 2 mmol/L DTT 12 h Dialysis Solution III 1M urea in TB8.0, 2 mmol/L DTT 4 h Dialysis Solution IV TB 8.0, 1 mmol/L DTT 4 h Dialysis Solution V TB 8.0 4 h
[0362] Removal and Detection of Endotoxin
[0363] According to the method of Shigui Liu et al. (Liu S G, Tobias R. Removal of endotoxin from recombinant protein preparations [J]. Clinical Biochem, 1997, 30: 455-463), endotoxin was removed from the recombinant protein by extraction using a non-ionic detergent in the experiment
[0364] Triton X-114 was added into the protein solution at a final concentrating of 1% (w/v), and the two phases were mixed homogeneously at 4 C. After shaking for 15 min, the protein solution containing Triton X-114 was placed in a 37 C. water bath for 10 min, a turbid system was observed, and oil droplets appeared in the solution. After centrifugation at 25 C. 12000 g for 10 min, water phase was removed carefully. The above steps were repeated once to collect water phase. The endotoxin content in the treated protein solution was determined by limulus reagent gel method.
EXAMPLE 3
Screening of Markers for RV0183-Specific T Cell Response
[0365] 3.1 Collection and Treatment of Stimulated Samples
[0366] 3.1.1 Collection of Samples for In Vitro Stimulation
[0367] Heparin-anticoagulation tubes were used to collect 5 ml peripheral blood from 4 hospitalized aTB patients who were tested positive in IGRAs, and collect 5 ml peripheral blood from 4 latently infected individuals who were tested positive in IGRAs and had no clinical symptoms.
[0368] 3.1.2 Stimulation and Collection of Samples
[0369] The samples were dispensed into 9 endotoxin-free 2 ml EP tubes, at 500 l peripheral blood per EP tube, and to each of the EP tubes, a culture solution (15 l) was added (the culture solution was prepared as followed: to 30 l PBS, 133.33 mg/ml D-glucose and 166.67 mM KCl were added; wherein PBS was prepared as followed: to Na.sub.2HPO4.12 H.sub.2O 2.9 g, KH.sub.2PO4 0.24 g, NaCl 8 g, and KCl 0.2 g, ultrapure water was added to a final volume of 1 L.). The EP tubes were designated as N, Ta, and P, wherein the N tube did not contain any stimulating agent, the Ta tube contained the stimulating antigen RV0183 (1 g), and the P tube contained PHA (20 g).
[0370] After adding peripheral blood to the EP tube, the resultant mixture was mixed homogenously by turning the tube upside down, and was incubated in a 37 C. incubator for 20-24 h; plasma samples were collected at 5000 rpm, and the cytokine contents in plasma were determined.
[0371] 3.2 Determination of Cytokine Contents in Plasma
[0372] The cytokines in the collected plasma samples were quantitated by using the commercialized Milliplex Kit (Merck Millipore, St.Charles, Mo., USA) (Batch No: HCYTMAG-60K-PX38, HCP2MAG-62K-PX23, HCP3MAG-63K-PX11), wherein the detection indexes comprised 69 cytokines including EGF (see Table 2), and the test platform was Luminex 200 (the washing solution, standard sample, reference sample, and magnetic beads in the following steps were obtained from the Kit).
[0373] The test steps comprised: 1) adding a washing solution (200 l) to each well in a 96-well test plate, shaking at room temperature for 10 min, and removing the washing solution; 2) to the corresponding wells, separately adding a standard sample (25 l) and a reference sample (25 l), wherein two wells were set for each sample; 3) adding an assay buffer (25 l) to each sample well; 4) to each of the standard sample wells, the reference sample wells and the blank control sample wells, adding a plasma matrix (25 l) (the matrix refers to a sample diluent in the Kit); 5) adding a plasma sample (25 l) to each well; 6) adding magnetic beads (25 l) to each well, and incubating at 2-8 C. overnight; 7) removing the liquid in the well, washing the well twice at 200 l washing solution per well; 8) adding a detection antibody (25 l) to each well, and incubating at room temperature for 1 h; 9) adding Steptavidin-Phycoerythrin (25 l) to each well, and reacting at room temperature for 30 min; 10) removing the liquid in the well, and washing the plate twice; 11) adding a sheath fluid (150 l) to each well, and mixing homogeneously under shaking; 11) reading the plate in Luminex 200 platform; and 12) for each sample, subtracting the actually determined value of the N tube from the actually determined value of the Ta tube to obtain a difference value as a final result (i.e. the actually increased value of cytokine, represented by TaN). (The method was a double antibody sandwich method)
[0374] The result was shown in Table 3. The cytokines were analyzed for their different responses in aTB patients and a latently infected population, and it was found by the screening that IL-6 could effectively determine whether individuals with TB infection were in a state of active tuberculosis (aTB) or in a state of Latent Tuberculosis Infection (LTBI) (the hospitalized TB patients with clinical symptoms were aTB patients, and the subjects, which were tested positive in IGRA and had no clinical symptoms, were the latently infected individuals).
TABLE-US-00012 TABLE 3 Sample Sample Sample Sample Sample Sample Sample Sample 1(aTB) 2(aTB) 3(aTB) 4(aTB) 5(LTBI) 6(LTBI) 7(LTBI) 8(LTBI) Stimulating antigen RV0183 EGF pg/ml 15 47 15.29 16.45 19 32 10 5 FGF-2 pg/ml 3.42 21.6 25.42 7.51 1.93 15.95 1 2.7 EOTAXIN pg/ml 13 7 3 7 18 10 10 36 TGF-a pg/ml 0.97 5.42 5.71 2.9 0.1 0.97 7.5 0.09 G-CSF pg/ml 146.96 282.14 125 110.96 118.4 134.96 65 22.21 Flt-3L pg/ml 0 0 0 0 0 0 0 0 GM-CSF pg/ml 18.82 71.82 30.3 16.75 17.17 26.81 16.41 26.15 Fractalkine pg/ml 103 174 152.3 183.68 160.64 8 50 81.64 IFNa2 pg/ml 20.11 43.99 0 14.08 3.45 17.61 13.32 17.21 IFN-g pg/ml 14.74 16.89 2.03 5.81 0.42 1.5 5.54 57.59 GRO pg/ml 3362 2571 2792 461 344 3095 4242 179 IL-10 pg/ml 55.65 70.29 0 9.39 4.07 0 5.4 0 MCP-3 pg/ml 494.02 9772.1 763 294.42 93.3 440.39 2137 192.95 IL-17P40 pg/ml 0 0 0 0 0 0 0 0 MDC pg/ml 100 57 9.24 34.53 255 115 12 40 IL-12P70 pg/ml 0 1.07 0.42 0 0 0.21 0.11 0 IL-13 pg/ml 0 0 0 0 0 0 0 0 IL-15 pg/ml 0 0 0 0 0 0 0 0 sCD40L pg/ml 885 1481 1567 636 636 1707 2639 2161 IL-17A pg/ml 0 1.35 0 0 0 0 0.4 0 IL-1RA pg/ml 0 45.6 0 0 0.39 0 15.05 0 IL-1a pg/ml 29.62 83.48 66.36 31.88 0 24.77 38.26 3.61 IL-9 pg/ml 0 0 0 0 0 0 0 0 IL-1b pg/ml 13.78 132.92 47.86 78.77 59.26 53.93 5.65 29.01 IL-2 pg/ml 0 0 0 0 0 0 61.6 0 IL-3 pg/ml 0 0 0 0 0 0 0 0 IL-4 pg/ml 9.92 0 0 0 0 0 0 0 IL-5 pg/ml 0 0 0 0 0 0 2.76 0 IL-6 pg/ml 790 6254.3 761.01 1098.7 403.11 241.52 329 248.01 IL-7 pg/ml 7.81 19.08 16.88 15.1 7.53 11.7 2.8 2.08 IL-8 pg/ml 5505.88 5694.9 5889.9 6290.9 6002.9 6015.9 0 460 IP-10 pg/ml 7761.01 0 0 0 589 4975 0 9317 MCP-1 pg/ml 6668.62 0 0 6099.6 0 6729.6 0 6968.6 MIP-1a pg/ml 412 3062.9 917.75 152.9 248.53 285 913 138.26 MIP-1b pg/ml 515 2091 422 480 386 394 240 418 TNF-a pg/ml 12.67 159.5 43 56.05 63.51 39.22 49 39.33 TNF-b pg/ml 0 0 0 0 0 0 0 0 VEGF pg/ml 153 422 168 107.13 19.56 104 147 96.24 CXLC6 pg/ml 58 267 48 38.46 27 240 1 24.21 CXCL9 pg/ml 543 1447 904 10528 227 779 1340 1041 CXCL11 pg/ml 176 87 144 445.8 37 150 203 99 CXCL19 pg/ml 21.31 9.05 28 39 1.17 15.7 9.31 4.99 CXCL20 pg/ml 47.89 107.51 227.32 243.33 79.97 360.95 1.52 49.84 IL-11 pg/ml 0 5.23 0.07 0.84 1.74 1.7 2.67 0 IL-29 pg/ml 33 217 113.5 124 0 0 0 0 M-CSF pg/ml 0 0 0 0 0 0 0 0 XCL1 pg/ml 24.55 9.55 19.74 33.12 4.98 46.29 19.3 0 6Ckine pg/ml 206 397 424 33 64 94 518 30.9 BCA-1 pg/ml 0.83 39.21 6.69 3.15 6.32 4.77 5.54 8.84 CTACK pg/ml 167 21 30 29 20 4 18 20 ENA-78 pg/ml 1853 15438 2397 4530 798 13933 26926 256 Eotaxin-2 pg/ml 39 1498 170 1087 576 194 272 412 Eotaxin-3 pg/ml 17.91 63 142 54 88 8 8 27 I-309 pg/ml 1.66 8.86 3.39 0.21 3.32 4.9 7.54 1.35 IL-16 pg/ml 25.14 35.82 65.44 63.86 29.7 289 95.3 10.09 IL-20 pg/ml 73 49 917.62 113 63 83 446 370 IL-21 pg/ml 0 0 0 0 0 0 0 0 IL-23 pg/ml 1.56 23.47 174 0 0 4.67 60.74 0 IL-28a pg/ml 0 18.63 9.58 2.55 0 2.55 7.7 22.32 IL-33 pg/ml 0 0 60.02 0 2.43 0 0 0 LIF pg/ml 0 0 0 13.58 0 3.76 16.1 0 MCP-2 pg/ml 103.43 813 5.5 70.97 36.72 12.29 19.4 133.34 MCP-4 pg/ml 10.17 43 19 9.38 7.69 10.81 65 9.96 MIP-1d pg/ml 191 4245 119 375 89 1152 9 564 SCF pg/ml 0 8.66 0.82 0 5.14 0 5.77 0 SDF-1a pg/ml 298 1086 1564 511 519 0 507 410 TARC pg/ml 0.4 1.33 0 4.88 2.55 4.64 10.1 1.11 TPO pg/ml 47 102 169 47 61 71 30 7 TRAIL pg/ml 0 31.1 0 0 0 0.43 3.55 0
EXAMPLE 4
Screening of aTB Patients from a TB Infection Population by Using RV0183
[0375] 4.1 Cohort Subject Information
[0376] 4.1.1 aTB patients: 79 patients clinically diagnosed with active tuberculosis; 2) a latent tuberculosis infection (LTBI) population and a healthy population: 37 health care workers participated into the cohort, wherein 14 heath care workers, who were tested positive in IGRAs, but had no clinical symptoms of tuberculosis and no history of tuberculosis, were diagnosed as latently infected individuals; and the other 23 heath care workers, who were tested negative in IGRAs, were diagnosed as healthy individuals not infected with M. tuberculosis.
[0377] 4.2 Sample Collection and a System for Sample Stimulation and Culture
[0378] 4.2.1 Whole blood samples were dispensed into three 2 ml EP tubes, at 500 l whole blood per EP tube; the 3 EP tubes were designated as N, Ta and P; the N tube comprised a culture solution (15 l) (which was prepared as described in Example 4) containing no antigen; the Ta tube comprised a culture solution (15 l) containing the recombinant antigen RV0183 (1 g); and the P tube comprised a culture solution (15 l) containing PHA (20 g).
[0379] 4.2.2 After dispensing, the whole blood was mixed homogeneously by turning the tube upside down gently, and was incubated in a 37 C. incubator for 202 h.
[0380] 4.2.3 After incubation, plasma was collected by centrifugation at 5000 rpm for 10 min, and the IL-6 level was determined.
[0381] 4.3 Determination of IL-6 Level in Plasma
[0382] IL-6 ELISA Kit (Xiamen Innovax Biotech CO., LTD.) was used to determine the IL-6 level in plasma.
[0383] 1) Treatment of plasma samples: the sample was 2-fold diluted with 20% newborn bovine serum (NBS); 2) Sample addition and incubation: to each well of a 96-well IL-6 test plate, the treated plasma (100 l) was added, the reaction was carried out at 37 C. for 40 min, the plate was washed for five times, and then remove the wash; 3) Enzyme addition and incubation: an enzyme-labeled secondary antibody (100 1) was added to each well, the reaction was carried out in a 37 C. incubator for 40 min, the plate was washed for five times, and then remove the wash; 4) Color development: a color developing solution was added, the reaction was carried for 10 min, and a stop solution (50 l) was added to stop the reaction; 5) Determination: the absorption value at 450 nm was determined by an ELISA instrument; and 6) for each sample, subtracting the actually determined value of the N tube from the actually determined value of the Ta tube to obtain a difference value as a final result (i.e. the actually increased value of cytokine, represented by TaN).
[0384] The result was shown in
EXAMPLE 5
Screening of aTB Patients from a General Population by Using RV0183
[0385] 5.1 Cohort Subject Information
[0386] 5.1.1 aTB patients: 207 patients who were clinically diagnosed with active tuberculosis;
[0387] 5.1.2 Subjects undergoing a health check-up and health care workers: 65 subjects who underwent a health check-up in general department, wherein the subjects, who were tested positive in IGRAs and had no clinical symptoms, were diagnosed as the individuals latently infected with M. tuberculosis; and the subjects, who were tested negative in IGRAs, were diagnosed as the individuals not infected with M. tuberculosis. Among 82 health care workers, the workers, who were tested positive in IGRAs and had no clinical symptoms, were diagnosed as the individuals latently infected with M. tuberculosis; and the workers, who were tested negative in IGRAs, were diagnosed as the individuals not infected with M. tuberculosis.
[0388] 5.2 Sample Collection and the System for Sample Stimulation and Culture
[0389] 5.2.1 3 ml peripheral blood was collected from each subject, and the whole blood was dispensed to three 2 ml EP tubes, at 1 ml whole blood per tube; the three EP tubes were designated as N, Ta and P. To each EP tube, a culture solution (30 l) (which was prepared as described in Example 4) was added; wherein to the N tube, a culture solution containing no antigen was added; to the Ta tube, a culture solution (30 l) containing the recombinant antigen RV0183 (2 g) was added; and, to the P tube, a culture solution (30 l) containing PHA (40 g) was added;
[0390] 5.2.2 After dispensing, the whole blood was mixed homogenously by turning the tube upside down, and incubated statically in a 37 C. incubator for 202 h;
[0391] 5.2.3 After incubation, the EP tubes were placed in a centrifugal machine, plasma samples were collected by centrifugation at 5000 rpm for 10 min, and the IL-6 secretion level was determined;
[0392] 5.3 Determination of IL-6 Secretion Level in Plasma
[0393] IL-6 ELISA Kit (Xiamen Innovax Biotech CO., LTD.) was used to determine the IL-6 level in plasma.
[0394] The method for determining IL-6 was the same as the one described in Example 4.
[0395] The result was shown in
Example 6
Screening of aTB Patients by Using Antigenic Fragments of RV0183
[0396] 6.1 Cohort Subject Information
[0397] The same as Example 5.
[0398] 6.2 Sample Collection and the System for Sample Stimulation and Culture
[0399] 6.2.1 aTB subjects in clinic were collected, 15 ml whole blood was collected from each subject, and the whole blood was dispensed to 22 EP tubes, at 500 l whole blood per tube. Among them, one EP tube was used as a negative control tube (N), and the other 21 EP tubes were designated as P 1, P2, P3 . . . P21. To the P1 tube, the peptide fragment RV0183-p1 (1 g) was added; to the P2 tube, the peptide fragment RV0183-p2 (1 g) was added, and so on, until the peptide fragment RV0183-p21 (1 g) was added to the P21 tube.
[0400] 6.2.2 After dispensing, the whole blood was mixed homogenously by turning the tube upside down, and incubated statically in a 37 C. incubator for 202 h;
[0401] 6.2.3 After incubation, the EP tubes were placed in a centrifugal machine, plasma samples were collected by centrifugation at 5000 rpm for 10 min, and the IL-6 secretion level was determined;
[0402] 6.3 Determination of IL-6 Secretion Level in Plasma
[0403] IL-6 ELISA Kit (Xiamen Innovax Biotech CO., LTD.) was used to determine the IL-6 level in plasma.
[0404] The method for determining IL-6 was the same as the one described in Example 4.
[0405] The result was shown in
EXAMPLE 7
Screening of aTB Patients by Using RV0183 and PlcD in Combination
[0406] 7.1 Cohort Subject Information
[0407] aTB(IGRA+) represented patients who were tested positive in TB-IGRA and were diagnosed with active tuberculosis, and aTB(IGRA) represented patients who were tested negative in TB-IGRA and were diagnosed with active tuberculosis. HC(IGRA+) represented healthy subjects who were tested positive in TB-IGRA, and HC(IGRA) represented healthy subjects who were tested negative in TB-IGRA. There were 55 subjects for aTB(IGRA+), 9 subjects for aTB(IGRA), 13 subjects for HC(IGRA+), and 52 subjects for HC(IGRA).
[0408] 7.2 Sample Collection and the System for Sample Stimulation and Culture
[0409] 7.2.1 4 ml peripheral blood was collected from each subject, and whole blood was dispensed to four 2 ml EP tubes, at 1 ml whole blood per EP tube; the 4 EP tubes were designated as N, Ta1, Ta2, and P, a culture solution (30 l) (which was prepared as described in Example 4) was added to each EP tube; wherein to the N tube, a culture solution containing no antigen was added; to the Ta1 tube, a culture solution (30 l) containing the recombinant antigen RV0183 (2 g) was added; to the Ta2 tube, a culture solution (30 l) containing recombinant antigen Plcd (2 g) was added; and to the P tube, a culture solution (30 l) containing PHA (40 g) was added;
[0410] 7.2.2 After dispensing, the whole blood was mixed homogenously by turning the tube upside down, and incubated statically in a 37 C. incubator for 202 h;
[0411] 7.2.3 After incubation, the EP tubes were placed in a centrifugal machine, plasma samples were collected by centrifugation at 5000 rpm for 10 min, and the IL-6 secretion level was determined;
[0412] 7.3 Determination of IL-6 Secretion Level in Plasma
[0413] IL-6 ELISA Kit (Xiamen Innovax Biotech CO., LTD.) was used to determine the IL-6 level in plasma.
[0414] The method for determining IL-6 was the same as the one described in Example 4.
[0415] The results were shown in
[0416] As seen from the results above, the actually changed IL-6 levels, resulted from the stimulation with RV0183 or Plcd, could be used alone or in combination for the detection of aTB patients, and had high sensitivity and specificity.
EXAMPLE 8
Evaluation of the Ability of Other Antigens of M. tuberculosis to Screen or Diagnose of Active Tuberculosis
[0417] 8.1 Cohort Subject Information
[0418] Whole blood samples were collected from 5 patients who were clinically diagnosed with active tuberculosis, and 5 latently infected individuals who had no clinical symptoms.
[0419] 8.2 Sample Collection and the System for Sample Stimulation Culture
[0420] 8.2.1 10 ml whole blood sample was collected, and dispensed to 9 EP tubes, at 1 ml whole blood per tube. Among them, 1 EP tube was used as negative control tube (N), to the other 8 EP tubes, one of the 7 antigens as listed in Table 3 was added, at 2 g per tube;
[0421] 8.2.2 After dispensing, the whole blood was mixed homogenously by turning the tube upside down, and incubated statically in a 37 C. incubator for 202 h;
[0422] 8.2.3 After incubation, the EP tubes were placed in a centrifugal machine, plasma samples were collected by centrifugation at 5000 rpm for 10 min, and the IL-6 secretion level was determined;
[0423] 8.3 Determination of IL-6 Secretion Level in Plasma
[0424] IL-6 ELISA Kit (Xiamen Innovax Biotech CO., LTD.) was used to determine the IL-6 level in plasma.
[0425] The method for determining IL-6 was the same as the one described in Example 4.
[0426] The result was shown in Table 4. After the stimulation with the antigens such as ESAT-6, CFP-10, RV1009, RV1884c, RV2389c, RV2450c, RV3097c, and RV3542, there was no significant change in the IL-6 level between the aTB samples and the LTBI samples, that is, these antigens could not be used to distinguish active tuberculosis from latent tuberculosis infection.
TABLE-US-00013 TABLE 4 aTB aTB aTB aTB aTB LTBI LTBI LTBI LTBI LTBI ESAT-6 0.085 0.162 0.132 0.203 0.162 0.051 0.069 0.097 0.071 0.125 CFP-10 0.038 0.074 0.139 0.12 0.074 0.044 0.075 0.1 0.079 0.061 RV1009 0.054 0.086 0.157 0.094 0.086 0.057 0.072 0.074 0.066 0.065 RV1884C 0.172 0.062 0.135 0.121 0.062 0.037 0.074 0.106 0.096 0.054 RV2389C 0.205 0.053 0.282 0.132 0.053 0.081 0.065 0.1 0.198 0.055 RV2450C 0.07 0.075 0.818 0.379 0.075 0.148 0.082 0.282 0.094 0.059 RV3542 0.067 0.171 0.251 0.279 0.171 0.054 0.023 0.091 0.136 0.129 RV3097C 0.073 0.067 0.032 0.207 0.067 0.064 0.136 0.086 0.284 0.057
EXAMPLE 9
Screening of aTB Patients from Patients with Different Pulmonary Diseases by Using a Polypeptide Library of RV0183
[0427] 9.1 Cohort Subject Information
[0428] 9.1.1 Active TB cases: 101 patients, who were clinically diagnosed with active tuberculosis;
[0429] 9.1.2 Cases with old tuberculosis: 19 patients with old tuberculosis, who had no clinical symptoms;
[0430] 9.1.3 Cases with a non-TB pulmonary disease in clinic: the cases, who were tested positive in IGRA, were the individuals latently infected with M. tuberculosis; the cases, who were tested negative in IGRA, were the individuals not infected with M. tuberculosis.
[0431] 9.2 Sample Collection and the System for Sample Stimulation and Culture
[0432] 9.2.1 Sample collection: Sample collection and treatment were the same as those described in Example 5.
[0433] 9.2.2 Sample stimulation and culture: a polypeptide library comprising 21 antigenic fragments of RV0183 (SEQ ID NOs: 5-25) was used as a stimulating agent (Ta), to stimulate whole blood. Each polypeptide of the polypeptide library was formulated as a solution at a concentration of 1 g/mL, and after mixing these solutions in an equal amount, 30 L was used in the experiment. The other operations were the same as those described in Example 5.
[0434] 9.3 Determination of IL-6 Level
[0435] The same as Example 5.
[0436] The result was shown in
[0437] Although the specific embodiments of the invention have been described in details, those skilled in the art would understand that, according to all the disclosed teachings, various modifications and changes can be made, and that such modifications and changes are within the scope of the present invention. The scope of the present invention is given by the appended claims and any equivalents thereof.