CN111551741A - Application of mycobacterium tuberculosis protein in preparing product for diagnosing latent tuberculosis infected person and/or active tuberculosis - Google Patents

Abstract

The present invention provides the use of Mycobacterium tuberculosis protein in preparing a product for diagnosing latent tuberculosis infection. The method provided by the invention can simultaneously detect the serum antibody response levels of three different Mycobacterium tuberculosis proteins, thereby better differential diagnosis of tuberculosis latent infection patients and active tuberculosis patients, with higher sensitivity and better specificity.

Description

Mycobacterium tuberculosis proteins in preparation for the diagnosis of latent and/or active tuberculosis infection Use in tuberculosis products

This application is a divisional application for an invention patent application with an application date of December 30, 2016 and an application number of 2016112595642.

technical field

The invention relates to the field of biomedicine, in particular to the use of Mycobacterium tuberculosis protein in preparing a product for diagnosing latent tuberculosis infection and/or active pulmonary tuberculosis.

Background technique

After the human body is infected with Mycobacterium tuberculosis, only about 10% of the people develop active tuberculosis, while 90% of the people do not develop the disease, but the tuberculosis in the body is not completely eliminated, but latent in the human body, called tuberculosis. Latent infection, these people do not have any clinical symptoms, most people do not develop disease in their lifetime, only 5% - 10% of people will lead to tuberculosis when their immunity is weakened. At present, about 1/3 of people in the world are infected with tuberculosis. It can be seen that there are a large number of latent infections. Therefore, it is necessary to monitor the latent infections and give timely drug treatment when the infection status changes, so as to achieve early detection and early detection. Treatment, which can not only prevent the deterioration of the disease, reduce the time of treatment and side effects caused by drug treatment, but also reduce the spread of TB. Therefore, the detection and identification of the two states after tuberculosis infection plays a very important role in the prevention and treatment of tuberculosis.

At present, there are many kinds of detection methods for tuberculosis infection, such as tuberculin skin test (TST), gamma interferon release test (IGRA), traditional sputum smear for acid-fast bacteria, rapid culture of mycobacteria, tuberculosis-specific nucleic acid fragments Amplification (quantitative and qualitative), etc. TST is a skin allergy. It has been used for a long time and is mainly used for outpatient screening. Although the test is simple and easy to perform, the BCG vaccination will produce the same results when detecting tuberculosis infection. It cannot identify natural infections and is not specific. In addition, latent tuberculosis infection cannot be differentiated from active tuberculosis. Interferon gamma release assay (IGRA) is mainly used to detect the cellular immune response. Although it can exclude the influence of BCG vaccination, it also cannot distinguish latent infection from active tuberculosis patients, and it cannot differentiate between treated patients and current infected patients. However, traditional sputum acid-fast bacteria smears and rapid culture of mycobacteria are mainly used to detect active tuberculosis patients, which are meaningless for the judgment of latent infection status. In addition, for active tuberculosis patients with atypical clinical symptoms, bacteria may not be found. study evidence. Therefore, it is of great value to find a method that can accurately identify latent infection and active tuberculosis.

Based on the different host protective immune responses induced by tuberculosis infection, finding serum markers to distinguish latent infection from active tuberculosis has a good application prospect. The serological diagnosis method of antigen-antibody reaction has attracted much attention because of its simplicity, rapidity, and easy access to specimens. Now some specific antigens of Mycobacterium tuberculosis have been used for the serological diagnosis of tuberculosis infection, such as Secreted protein antigens: antigen 85 complex antigens, namely Ag85A, Ag85B and Ag85C, etc.; lipoprotein antigens: 16kDa, 27kDa, 38kDa antigens, etc.; glycolipid antigens: lipoarabinomannan antigen, Mycobacterium tuberculosis glycolipid However, in actual use, these antigens have low positive detection rate and cross-immunity with other mycobacteria. Although they have certain value in high-risk population census, treatment effect monitoring, prognosis and prompting recurrence, However, it still cannot be used for the diagnosis of latent Mycobacterium tuberculosis infection.

Therefore, it is extremely important to find a new method and marker that can accurately diagnose latent tuberculosis infection and active tuberculosis patients.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to solve the problem that the products in the prior art for differential diagnosis of latent tuberculosis infection and/or active pulmonary tuberculosis have a low positive detection rate and cannot differentially diagnose latent Mycobacterium tuberculosis infection. Use of a Mycobacterium tuberculosis protein in preparing a product for diagnosing latent tuberculosis infection and/or active pulmonary tuberculosis.

In order to solve the above-mentioned technical problems, the technical scheme provided by one aspect of the present invention is:

Use of a product for detecting the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in a sample in the preparation of a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active pulmonary tuberculosis.

In the technical solution of the present invention, any product for detecting the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in a sample can be used for the preparation of identification, diagnosis and/or screening of latent tuberculosis infection and/or active pulmonary tuberculosis The product.

Preferably, the product for detecting the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in the sample includes Rv0284 protein, Rv2002 protein and/or Rv2421c protein.

More preferably, the Rv0284 protein is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.1 have been modified with the protein shown in SEQ ID NO.1. A protein with the same function; the Rv2002 protein is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are transformed with the protein shown in SEQ ID NO.2 A protein with the same function; the Rv2421c protein is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified and shown in SEQ ID NO.3 protein with the same function.

The term "engineering" as described above can use any prior art method for engineering proteins, including but not limited to, substitution and/or addition and/or deletion of amino acid residues to form derivatives of the original protein.

Preferably, the detection can be any suitable detection method in the prior art, but preferably, the detection method can be enzyme-linked immunosorbent assay, agglutination test, precipitation test, E rosette test, microphagocytosis test, immune Fluorescence experiments, colloidal gold immunochromatography experiments, spotted gold immunodiafiltration experiments and/or electrochemiluminescence experiments. More preferably, in one embodiment of the present invention, the detection method is an enzyme-linked immunosorbent assay.

Preferably, the sample can be any suitable sample obtained from the subject to be detected. But preferably, the sample includes serum, plasma, saliva, urine, pleural and ascites fluid and/or cerebrospinal fluid. More preferably, in one embodiment of the present invention, the sample is serum.

Another aspect of the present invention is to provide a marker composition for identifying, diagnosing and/or screening latent tuberculosis infection and/or active pulmonary tuberculosis, the marker composition is composed of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody composition;

Wherein, the Rv0284 is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.1 have the same function as the protein shown in SEQ ID NO.1 after being transformed. Protein; the Rv2002 is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 have the same function as the protein shown in SEQ ID NO.2 after being transformed. Protein; the Rv2421c is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 have the same function as the protein shown in SEQ ID NO.3 after being transformed. protein.

Another aspect of the present invention is to provide the use of the above marker composition in preparing a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active pulmonary tuberculosis.

Another aspect of the present invention provides a kit for identifying, diagnosing and/or screening latent tuberculosis infection and/or active pulmonary tuberculosis, the kit comprising Rv0284 protein, Rv2002 protein and/or Rv2421c protein.

Preferably, the Rv0284 protein is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.1 have been modified to have the same identity as the protein shown in SEQ ID NO.1 functional protein; the Rv2002 protein is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 have been modified with the protein shown in SEQ ID NO.2. A protein with the same function; the Rv2421c protein is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are transformed with the protein shown in SEQ ID NO.3. proteins with the same function.

The Rv0284 protein, Rv2002 protein and/or Rv2421c protein contained in the above kit can be in any form, including but not limited to direct protein samples, and proteins immobilized on a certain carrier, such as a detection chip.

In addition to the Rv0284 protein, the Rv2002 protein and/or the Rv2421c protein, the kit further includes at least kit instructions.

Criteria for the determination of the results described in the instructions: using the combined detection of the three proteins, if at least two of the anti-Rv0284 antibodies, anti-Rv2002 antibodies and/or anti-Rv2421c antibodies in the test results of the sample to be tested are positive, then It is determined that the sample to be tested is positive for active tuberculosis, otherwise it is latent tuberculosis infection.

Another aspect of the present invention is to provide the use of the above-mentioned kit in preparing a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active pulmonary tuberculosis.

The beneficial effects of the present invention are:

The invention can simultaneously detect the serum antibody response levels of three different Mycobacterium tuberculosis proteins, thereby better differentially diagnosing tuberculosis latent infection patients and active tuberculosis patients, with higher sensitivity and better specificity.

Description of drawings

Figure 1 is a statistical graph of the expression levels of anti-Rv0284 antibody, anti-Rv2002 antibody and anti-Rv2421c antibody in the serum of latent tuberculosis infection patients (n=93) and active tuberculosis patients (n=92) detected by ELISA;

Fig. 2 is the ROC curve diagram of the antibody corresponding to the combined detection of three antigens;

sequence description

SEQ ID NO.1 is the amino acid sequence of the Rv0284 protein in the examples of the present invention;

SEQ ID NO.2 is the amino acid sequence of the Rv2002 protein in the examples of the present invention;

SEQ ID NO. 3 is the amino acid sequence of the Rv2421c protein in the examples of the present invention.

Detailed ways

The invention discloses the use of a product for detecting the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in a sample in preparing a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active pulmonary tuberculosis. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be specially pointed out that all similar substitutions and modifications are obvious to those skilled in the art, they are all considered to be included in the present invention, and relevant persons can obviously do so without departing from the content, spirit and scope of the present invention. The content described herein can be modified or appropriately changed and combined to realize and apply the technology of the present invention.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

Example 1: Collection and processing of samples

The sample detected by the present invention is serum, and the collection method is to collect blood from subjects on an empty stomach, use ordinary biochemical test tubes without anticoagulant, stand at room temperature for natural coagulation, and centrifuge at 3000 rpm for 10 minutes at room temperature to collect serum fractions. Packed in a cryopreservation tube and stored in a -80°C low-temperature freezer until used in experiments.

The samples used in the present invention are divided into two groups: a latent infection group and an active tuberculosis patient group; wherein, the latent infection group refers to those who have no clinical symptoms, positive TST test and T-SPOT test, and negative chest X-ray; The active tuberculosis patient group refers to the confirmed tuberculosis patients who were positive by smear or culture and who had not started or just started systemic chemotherapy. Diabetes, HIV infection and other autoimmune diseases were excluded from both groups.

The latent infection group specimens used in the present invention are from the epidemiological investigation project of the Epidemiology Research Office of the Beijing Institute of Tuberculosis and Thoracic Tumors, and the active tuberculosis patient group specimens are from inpatients in various wards of the Tuberculosis Department of the Beijing Chest Hospital Affiliated to Capital Medical University. This project has been approved by the Research Ethics Committee of Beijing Chest Hospital Affiliated to Capital Medical University, and all subjects gave informed consent.

Example 2: Detection of samples by ELISA experiment

1. Various buffers, diluents, reaction solutions, and stop solutions required to set up an ELSIA experiment:

(1) Coating buffer (pH9.6, 0.05mol/L carbonate buffer):

Na ₂ CO ₃ 1.59g, NaHCO ₃ 2.93g, add double distilled water to 1000ml, adjust pH to 9.6;

(2) PBS solution at pH 7.4:

NaCl 8.0g, KCl 0.2g, KH ₂ PO ₄ 0.2g, Na ₂ HPO ₄ ·12H ₂ O 2.9g, add double distilled water to 1000ml, adjust pH to 7.4;

(3) PBST washing solution at pH 7.4:

Add 0.5ml of Tween-20 to 1000ml of PBS solution and adjust the pH to 7.4;

(4) Blocking solution (PBS solution containing skim milk powder at pH 7.4):

5g of skim milk powder was added to 100ml of PBS solution;

(5) Substrate buffer: 25.7 ml of 0.2M NaH ₂ PO ₄ (28.4 g/L), 24.3 ml of 0.1 M citric acid (19.2 g/L), and 50 ml of distilled water.

(6) TMB (tetramethylbenzidine) using solution: TMB (10mg/5ml absolute ethanol) 0.5ml substrate buffer (pH5.5) 10ml 0.75% H ₂ O ₂ 32 μl;

(7) Stop solution (2M H ₂ SO ₄ ): 21.32 ml H ₂ SO ₄ , 178.68 ml H ₂ O.

(8) Secondary antibody: goat anti-human IgG labeled with horseradish peroxidase (HRP);

(9) 96-well microtiter plate; Nunc Incorporated (Theromo, Gebenhagen, Denmark)

2. Detection steps:

(1) Coating: Rv0284, Rv2002 and Rv2421c antigens were dissolved in coating buffer (5ug/ml) respectively; 100ul/well was added to 96-well microtiter plate, 4°C overnight;

(2) Wash the plate: PBST washing solution 300ul/well, 3min, wash 3 times;

(3) Blocking: blocking solution, 300ul/well, incubated at room temperature for 1h;

(4) plate washing: washing solution 300ul/well, 3min, wash 3 times;

(5) Add serum: 1:400 diluted serum to be tested, 100ul/well, incubated at room temperature for 1h;

(6) plate washing: washing solution 300ul/well, 3min, wash 5 times;

(7) Add enzyme-labeled secondary antibody: freshly diluted enzyme-labeled antibody (1:30000) 100ul/well, incubate at room temperature for 1h;

(8) plate washing: washing solution 300ul/well, 3min, wash 5 times;

(9) Color development: freshly prepared TMB color developing solution, 100ul/well, incubate at room temperature for 5-10min in the dark;

(10) Termination: stop solution 2M H ₂ SO ₄ , 50ul/well;

(11) Determination: The microplate reader was adjusted to 450 nm, and the OD value of each well was measured after the first blank PBS control well was adjusted to zero.

3. Results Analysis and Judgment Criteria:

Use SPSS22.0 software to draw the ROC curve, according to the coordinate values, get the specificity and sensitivity corresponding to different critical points, and then calculate the Youden index (Youden index = specificity + sensitivity-1), get three antigens The critical point OD values of Rv0248 are 0.3475, Rv2002 is 0.3048, and Rv2421c is 0.4173;

The results are shown in Figure 1. The results show that all three proteins have the function of distinguishing latent tuberculosis infection from active tuberculosis.

judgement standard:

Using the combined detection of the three proteins, if the test results of the test sample are positive for at least two antibodies among the anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody, the test sample is determined to be positive for active tuberculosis , otherwise latent tuberculosis infection.

Example 3: Specificity and Sensitivity of the Method for Differential Diagnosis of Active Tuberculosis Patients and Latent Infections Using ELISA

92 sera from patients clinically diagnosed with active pulmonary tuberculosis and 93 sera from patients with latent infection were used for detection; the ELISA method in Examples 1 and 2 was used for detection, and whether the tested person was positive for active pulmonary tuberculosis was determined by the judgment standard of Example 2 , the results are shown in Figure 2, the specificity of the analysis was 83.87%, and the sensitivity was 79.35%. In Figure 2, the abscissa 1-specificity represents the false positive rate, and the ordinate sensitivity represents the true positive rate.

The above results prove that the present invention detects the serum antibody response levels of three different Mycobacterium tuberculosis proteins, and can better differentiate and diagnose patients with latent tuberculosis infection and active tuberculosis patients, with higher sensitivity and better specificity.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

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Claims (10)

1. Use of a product for detecting the levels of anti-Rv0284 antibody, anti-Rv2002 antibody and anti-Rv2421c antibody in a sample in the preparation of a product for identification, diagnosis and/or screening of latent tuberculosis infection. 2. The use according to claim 1, wherein the product for detecting the levels of anti-Rv0284 antibody, anti-Rv2002 antibody and anti-Rv2421c antibody in a sample comprises Rv0284 protein, Rv2002 protein and Rv2421c protein. 3. The use according to claim 2, wherein the Rv0284 protein is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified Then the protein with the same function as the protein shown in SEQ ID NO.1; the Rv2002 protein is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO. A protein with the same function as the protein shown in SEQ ID NO.2 after the transformation; the Rv2421c protein is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 The modified protein has the same function as the protein shown in SEQ ID NO.3. 4. The purposes according to any one of claims 1-3, wherein the detection method comprises enzyme-linked immunosorbent assay, agglutination experiment, precipitation experiment, E rosette experiment, small phagocytosis experiment, immunofluorescence experiment , colloidal gold immunochromatography experiments, spotted gold immunodiafiltration experiments and/or electrochemiluminescence experiments. 5. The use according to any one of claims 1-3, wherein the sample comprises serum, plasma, saliva, urine, pleural ascites and/or cerebrospinal fluid. 6. A marker composition for identifying, diagnosing and/or screening latent tuberculosis infection, wherein the marker composition is composed of anti-Rv0284 antibody, anti-Rv2002 antibody and anti-Rv2421c antibody; Wherein, the Rv0284 is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.1 have the same function as the protein shown in SEQ ID NO.1 after being transformed. Protein; the Rv2002 is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 have the same function as the protein shown in SEQ ID NO.2 after being transformed. Protein; the Rv2421c is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified and have the same function as the protein shown in SEQ ID NO.3. . 7. Use of the marker composition according to claim 6 in the preparation of a product for identifying, diagnosing and/or screening latent tuberculosis infection. 8. A kit for identifying, diagnosing and/or screening latent tuberculosis infection, wherein the kit comprises Rv0284 protein, Rv2002 protein and Rv2421c protein. 9. The kit according to claim 8, wherein the Rv0284 protein is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO. A protein with the same function as the protein shown in SEQ ID NO.1 after the transformation; the Rv2002 protein is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 A protein with the same function as the protein shown in SEQ ID NO.2 after being transformed; the Rv2421c protein is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 The modified protein has the same function as the protein shown in SEQ ID NO.3. 10. Use of the kit according to claim 8 or 9 in the preparation of a product for identifying, diagnosing and/or screening latent tuberculosis infection and active pulmonary tuberculosis.

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