Use of Rv1860 protein, RV3881c protein, Rv2031c protein and Rv3803c protein in diagnosing active pulmonary tuberculosis infection

Abstract

The present invention relates to the field of disease diagnosis, and specifically to the use of Rv1860 protein, RV3881c protein, Rv2031c protein and Rv3803c protein in the preparation of diagnostics for active pulmonary tuberculosis infection. The present invention provides a product for detecting the levels of anti-Rv1860 antibodies, anti-RV3881c antibodies, anti-Rv2031c antibodies and anti-Rv3803c antibodies in serum for use in the preparation of products for identification, diagnosis, auxiliary diagnosis, screening and / or auxiliary screening of patients with active pulmonary tuberculosis infection. Experiments have shown that a biomarker combination containing four antigens can effectively screen patients with active pulmonary tuberculosis infection.

Description

Technical Field

[0001] The present invention relates to the field of disease diagnosis, and in particular to use of Rv1860 protein, RV3881c protein, Rv2031c protein and Rv3803c protein in the preparation of diagnostic kits for active pulmonary tuberculosis infection. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (MTB). It is one of the top ten leading causes of death. According to the World Health Organization's tuberculosis report, by 2019, about one-third of the world's population was infected with Mycobacterium tuberculosis, and 1.4 million people died from tuberculosis. 1 / 4-1 / 3 of people infected with Mycobacterium tuberculosis do not have any clinical symptoms, which is also called latent tuberculosis infection (LTBI). Currently, about 2 billion people suffer from LTBI, and about 10% of them will develop active tuberculosis during their lifetime. As the immune status of infected individuals changes, timely diagnosis of tuberculosis is crucial for the treatment of tuberculosis and infection control.

[0003] The lack of effective diagnostic methods for differentiating LTBI from active pulmonary tuberculosis (ATB) has hampered the prevention and control of tuberculosis. Currently, laboratory diagnosis of tuberculosis still relies on traditional methods, including acid-fast staining (AFS), nucleic acid amplification (NAA, such as gene Xpert-MTB / RIF), and sputum and other respiratory specimens for Mycobacterium tuberculosis (MTB) culture. All methods have limitations. Sputum tuberculosis culture requires 2-8 weeks to obtain culture results, and the positive rate is low, which cannot meet clinical requirements; the sputum smear method has a short cycle but poor specificity; although the gene Xpert-MTB / RIF has a fast detection speed and high sensitivity, it cannot solve the problem of detecting negative bacteria. Interferon-γ release test is also a method to assist in the diagnosis of tuberculosis, but it still cannot distinguish ATB from LTBI. Therefore, clinical diagnosis urgently needs more accurate and easier-to-operate tuberculosis diagnostic tools.

[0004] Preventing LTBI from developing ATB is one of the important strategies for tuberculosis prevention and control. Screening and identification of LTBI populations requires new biomarkers and technologies, especially for those high-burden populations, including HIV patients, diabetic patients, and immunocompromised patients. Summary of the invention

[0005] The technical problem solved by the present invention is how to screen patients with active pulmonary tuberculosis infection.

[0006] The use of a product for detecting the levels of anti-Rv1860 antibodies, anti-RV3881c antibodies, anti-Rv2031c antibodies and anti-Rv3803c antibodies in serum in the preparation of a product for the purpose of identifying, diagnosing, assisting in diagnosis, screening and / or assisting in screening patients with active pulmonary tuberculosis infection.

[0007] Optionally, the Rv1860 is a protein as follows a) or b): a) a protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; b) a protein derived from a) in which the amino acid sequence of SEQ ID NO.1 in the sequence listing is substituted and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.1;

[0008] The RV3881c is the protein of c) or d) as follows: c) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence list; d) a protein derived from c) in which the amino acid sequence of SEQ ID NO.2 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.2;

[0009] The Rv2031c is a protein of the following e) or f): e) a protein consisting of the amino acid sequence shown in SEQ ID NO.3 in the sequence list; f) a protein derived from e) in which the amino acid sequence of SEQ ID NO.3 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.3;

[0010] The Rv3803c is a protein as follows g) or h): g) a protein composed of the amino acid sequence shown in SEQ ID NO.4 in the sequence list; h) a protein derived from g) in which the amino acid sequence of SEQ ID NO.4 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.4.

[0011] The use of a product for detecting the levels of anti-Rv1860 antibodies, anti-RV3881c antibodies, anti-Rv2031c antibodies and anti-Rv3803c antibodies in serum in the preparation of a product for distinguishing patients with active pulmonary tuberculosis infection from normal people; optionally, the patient candidate to be tested is a patient with active pulmonary tuberculosis.

[0012] A marker composition for identification, diagnosis, auxiliary diagnosis, screening and / or auxiliary screening of patients with active pulmonary tuberculosis infection, consisting of anti-Rv1860 antibody, anti-RV3881c antibody, anti-Rv2031c antibody and anti-Rv3803c antibody; the Rv1860 is a protein as follows a) or b): a) a protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence table; b) a protein derived from a) in which the amino acid sequence of SEQ ID NO.1 in the sequence table is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.1;

[0013] The RV3881c is the protein of c) or d) as follows: c) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence list; d) a protein derived from c) in which the amino acid sequence of SEQ ID NO.2 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.2;

[0014] The Rv2031c is a protein of the following e) or f): e) a protein consisting of the amino acid sequence shown in SEQ ID NO.3 in the sequence list; f) a protein derived from e) in which the amino acid sequence of SEQ ID NO.3 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.3;

[0015] The Rv3803c is a protein as follows g) or h): g) a protein composed of the amino acid sequence shown in SEQ ID NO.4 in the sequence list; h) a protein derived from g) in which the amino acid sequence of SEQ ID NO.4 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.4.

[0016] The use of anti-Rv1860 antibodies, anti-RV3881c antibodies and / or anti-Rv2031c antibodies in serum as markers in the development, design and / or preparation of products for the purpose of identification, diagnosis, auxiliary diagnosis, screening and / or auxiliary screening of patients with active pulmonary tuberculosis infection;

[0017] The Rv1860 is a protein as follows a) or b): a) a protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence list; b) a protein derived from a) in which the amino acid sequence of SEQ ID NO.1 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.1;

[0018] The RV3881c is the protein of c) or d) as follows: c) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence list; d) a protein derived from c) in which the amino acid sequence of SEQ ID NO.2 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.2;

[0019] The Rv2031c is a protein of the following e) or f): e) a protein consisting of the amino acid sequence shown in SEQ ID NO.3 in the sequence list; f) a protein derived from e) in which the amino acid sequence of SEQ ID NO.3 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.3;

[0020] The Rv3803c is a protein as follows g) or h): g) a protein composed of the amino acid sequence shown in SEQ ID NO.4 in the sequence list; h) a protein derived from g) in which the amino acid sequence of SEQ ID NO.4 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.4.

[0021] A kit for identifying, diagnosing, assisting in diagnosis, screening and / or assisting in screening patients with active pulmonary tuberculosis infection, or a kit for distinguishing patients with active pulmonary tuberculosis infection from normal persons, comprising a detection chip, wherein at least Rv1860, RV3881c, Rv2031c and Rv3803c proteins are connected to the detection chip, and each of the above four proteins forms a detection point separately; preferably, the detection chip is connected to Rv1860, RV3881c, Rv2031c and Rv3803c proteins;

[0022] The Rv1860 is a protein as follows a) or b): a) a protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence list; b) a protein derived from a) in which the amino acid sequence of SEQ ID NO.1 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.1;

[0023] The RV3881c is the protein of c) or d) as follows: c) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence list; d) a protein derived from c) in which the amino acid sequence of SEQ ID NO.2 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.2;

[0024] The Rv2031c is a protein of the following e) or f): e) a protein consisting of the amino acid sequence shown in SEQ ID NO.3 in the sequence list; f) a protein derived from e) in which the amino acid sequence of SEQ ID NO.3 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.3;

[0025] The Rv3803c is a protein as follows g) or h): g) a protein composed of the amino acid sequence shown in SEQ ID NO.4 in the sequence list; h) a protein derived from g) in which the amino acid sequence of SEQ ID NO.4 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function as the protein shown in SEQ ID NO.4.

[0026] Optionally, the kit also includes reagents for use with the detection chip, the reagents including the following 1)-4): 1) pH 7.4 PBS solution, whose composition is 2mM Na2HPO4, 1mM KH2PO4, 10mM NaCl and 2mM KCl; 2) pH 7.4 PBST solution, whose composition is: PBS and Tween-20; Optionally, Tween-20 accounts for 1‰ of the total volume of the solution; 3) pH 7.4 PBS solution containing BSA; 4) fluorescently labeled anti-human secondary antibody.

[0027] The use of the above-mentioned kit in the preparation of products for the purpose of identification, diagnosis, auxiliary diagnosis, screening and / or auxiliary screening of active pulmonary tuberculosis patients.

[0028] The technical solution of the present invention has the following advantages:

[0029] 1. The present invention provides a product for detecting the levels of anti-Rv1860 antibodies, anti-RV3881c antibodies, anti-Rv2031c antibodies and anti-Rv3803c antibodies in serum for use in preparing a product for distinguishing or assisting in distinguishing active pulmonary tuberculosis patients from latent tuberculosis infection patients. The present invention selects 21 MTB proteins reported in the literature and 43 proteins of the DosR family as candidate antigens. A saccharomyces cerevisiae expression system is selected to recombinantly express 64 MTB candidate proteins. After expressing and purifying all candidate proteins, an MTB protein chip is constructed for sample screening. In the exploratory stage of biomarker development, a total of 180 serum samples were collected, including 60 ATB samples, 60 LTBI samples and 60 healthy volunteer samples, from which 5 proteins were screened, and then a model was established to obtain a biomarker combination containing 4 antigens, which can effectively distinguish ATB patients from LTBI populations. In the verification stage, the biomarker combination was further verified by ELISA method using more than 300 serum samples, and the final results showed that the chip detection results were basically consistent with the ELISA detection results.

[0030] 2. The lack of rapid laboratory technology that can effectively distinguish active tuberculosis from latent tuberculosis infection has hampered the prevention and control of infectious diseases and led to the further spread of the disease. The present invention uses gene chip technology and enzyme-linked immunosorbent assay (ELISA) method to screen and verify candidate biomarkers in serum samples, and ultimately determines a highly sensitive and specific MTB antigen combination for accurately distinguishing ATB from LTBI and healthy people.

[0031] 3. Unlike most studies, the present invention applies the system biology approach to the screening of tuberculosis immunogens, rather than evaluating known immunogens and their combinations. Existing studies have shown that multiple antigens such as LAM, Ag85A, Ag85B, ESAT-6, CFP10, and MPT64 can be used to evaluate Mycobacterium tuberculosis infection, and the T-cell-based interferon gamma release assay (IGRA) is the most effective method for distinguishing MTB-infected and non-MTB-infected people, but it still cannot distinguish ATB from LTBI.

[0032] 4. Protein microarray technology has been widely used as a high-throughput technology. The purification method of the protein fixed on the chip surface will have a certain impact on the screening process. Previous reports used Escherichia coli as an expression system to produce proteins. The present invention selected Saccharomyces cerevisiae as an expression system. The proteins expressed by the yeast system have post-translational modifications and are more suitable for the screening of functional proteins. Therefore, a reasonable and scientific design was adopted. First, DosR protein and other widely reported MTB candidate antigens were prepared and positioned on the microarray, providing a unique MTB protein microarray for subsequent screening. Then a relatively small number of serum samples were used for preliminary screening. After data analysis and model establishment, a larger number of serum samples were used to verify the preliminary screening results of the ELISA microarray to ensure that the detection results of the two systems were consistent.

[0033] 5. Studies have shown that a combination of nine DosR antigens (Rv0569, Rv1996, Rv2030, Rv2031c, Rv2626, Rv2628, Rv3129, Rv3131, and Rv3133) can effectively distinguish latent infection from active tuberculosis infection, but this group contains too many proteins, thus weakening the possibility of its clinical application.

[0034] 6. The ELISA results showed that the sensitivity of the four candidate antigens in the present invention was low, ranging from 19.3% to 38.6%. However, the detection performance was significantly improved by combining the four candidate antigens into one group, with a sensitivity of 93.3% and a specificity of 97.7%. The four antigen groups performed well in distinguishing ATB from LTBI and healthy individuals, indicating that the humoral immune response of ATB patients was stronger than that of LTBI and healthy individuals.

[0035] 7. Serological biomarkers are a simple, rapid, and non-invasive disease diagnostic technology. Therefore, from the perspective of clinicians, screening new serological biomarkers for tuberculosis is of great significance. Four proteins (Rv1860, Rv2031c, Rv3881c, and Rv3803c) showed that the serum antibody levels of ATB patients were significantly higher than those of LTBI and healthy groups. In the comparison between ATB and HC and ATB and LTBI, most of the differential antibodies were IgG subtypes, indicating that the antibodies produced during MTB infection are mainly IgG subtypes.

[0036] 8. The four antigen detection panel established by the present invention has accurate detection capability when screening LTBI patients from tuberculosis patients. It not only has strong diagnostic capability but also is simple to operate and low in cost. It is a promising tool for the prevention and treatment of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 is a schematic diagram of the process of screening and identifying ATB and LTBI / healthy human serum biomarkers in Example 1 of the present invention;

[0039] Figure 2 It is the quality control of the tuberculosis-related protein microarray in Example 1 of the present invention; Figure 2 A The image on the left is a full view of a representative tuberculosis-related protein microarray detected by anti-GST signal; statistical analysis showed that the tuberculosis protein detection rate was 98.4%. There were 14 blocks on each microarray. A magnified image of one of the 14 blocks on the tuberculosis-related protein microarray is shown on the right. Figure 2 B Histograms of foreground (F) and background (B) signal intensities show that most printed spots contain a large amount of recombinant protein;

[0040] Figure 3 It is the antigen with different antibody levels in serum samples of ATB, LTBI or HC groups screened based on tuberculosis-related protein microarray in Example 1 of the present invention;

[0041] Figure A shows that there are different antigens in the antibody levels between the ATB group and the HC group;

[0042] B Volcano plot shows antigens with different antibody levels between ATB and LTBI groups; black dashed line: cutoff value (ratio>1.1, p value<0.05); the five antigens with higher antibody levels in the ATB group are marked;

[0043] C: Venn diagram, five antigens were present in the comparison between ATB and HC and ATB and LTBI; high levels of antibodies against these five antigens were detected in ATB;

[0044] D: Microarray results of 5 proteins as candidate biomarkers.

[0045] Figure 4 The results of candidate biomarker antibody detection between different groups; the upper group shows that the signal intensity of each candidate biomarker in the ATB group is significantly higher than that in the HC and LTBI control groups; the lower group shows: the ROC curve of each candidate biomarker;

[0046] Figure 5ROC curves of biomarker groups using tuberculosis-related protein microarray samples; A: comparison between ATB and healthy volunteers (HC), ATB vs HC train set refers to the comparison between ATB and HC training set, ATB vs HC validation set refers to the comparison between ATB and HC validation set; B: comparison between ATB and LTBI, ATB vs LTBI train set refers to the comparison between ATB and LTBI training set, ATB vs LTBI validation set refers to the comparison between ATB and LTBI validation set; C: comparison between ATB and LTBI plus HC, ATB vs ATB LTBI plus HC train set refers to the comparison between ATB and LTBI plus HC training set, ATB vs ATB LTBI plus HC validation set refers to the comparison between ATB and LTBI plus HC validation set; The AUC values ​​of the ROC curves in (AC) are all >0.9, and the AUC values ​​of the ROC curves of each biomarker group are higher than the AUC values ​​of any single biomarker.

[0047] Figure 6 It is the ROC curve of the biomarker panel using Elisa samples in Example 1 of the present invention; a: comparison between ATB and HC; ATB vs HC train set refers to the comparison between ATB and HC training set, and ATB vs HC validation set refers to the comparison between ATB and HC validation set; b: comparison between ATB and LTBI, ATB vs LTBI train set refers to the comparison between ATB and LTBI training set, and ATB vs LTBI validation set refers to the comparison between ATB and LTBI validation set; c: comparison between ATB and LTBI plus HC, ATB vs ATB LTBI plus HC train set refers to the comparison between ATB and LTBI plus HC training set, and ATB vs ATB LTBI plus HC validation set refers to the comparison between ATB and LTBI plus HC validation set; in each different comparison, the AUC value of the ROC curve of each biomarker group is higher than that of any single biomarker. DETAILED DESCRIPTION

[0048] Example 1

[0049] 1.1 Research Materials

[0050] Methods: 160 serum samples were collected from patients with active tuberculosis (ATB), latent tuberculosis infection (LTBI) and healthy volunteers. The patients ranged in age from 16 to 93 years. In the training phase, three groups of 180 serum samples (60 samples in each group) were used for microarray detection; in the validation phase, three groups of 300 serum samples (100 samples in each group) were further tested by ELISA. All samples were collected at Jiangxi Chest Hospital from December 2019 to September 2020, and sample collection was approved by the Ethics Committee of Jiangxi Chest Hospital (No.: (2019)50). Participants were enrolled after obtaining written consent and were conducted in accordance with the Declaration of Helsinki (revised in 2013).

[0051] ATB patients were diagnosed based on clinical symptoms, acid-fast bacilli (AFB) sputum smear, bacterial culture results and other criteria. The inclusion criteria for the ATB group were: tuberculosis-specific clinical symptoms, sputum AFB positive and / or bacterial culture positive. The inclusion criteria for the LTBI group were: patients without tuberculosis-specific clinical symptoms, sputum AFB negative and IGRA positive (X.DOT-TB, Foshan Tuberculosis Medical Center, China). The inclusion criteria for the HC group were healthy volunteers with negative IGRA (X.DOT-TB, Foshan Tuberculosis Medical Center, China). All three groups of patients were collected before treatment. All patients had no other immune-related diseases. The clinical data of all patients are shown in Table 1.

[0052] 1.2 Construction of MTB antigen microarray

[0053] MTB antigen comes from the Institute of Biophysics, Chinese Academy of Sciences. After sequencing verification, the gene was introduced into the target vector pEGH-A27 through the Gateway-LR reaction to construct an expression vector. The constructed vector can express the recombinant protein tagged with N-terminal GST, which is convenient for subsequent purification.

[0054] The MTB microarray candidate antigens were expressed using the Saccharomyces cerevisiae Y258 strain and purified by a high-throughput strategy as follows. The strain was first inoculated in a 12-well plate and cultured in SC-URA / D-Raffinose medium until the OD600 reached 1.0, and then galactose was added to a final concentration of 20% (w / v) to induce protein expression. After induction, cells were collected when the OD600 reached 2.0. The harvested cells were mixed with 200 μL of lysis buffer and transferred to a 96 × 0.5 mL culture dish (Sangguang Biotech, Shanghai, China) containing 100 μL of frozen glass beads. Cell lysis was performed at 4 °C and immediately applied to glutathione beads. GST-fusion proteins were purified by GST affinity chromatography and purified according to standard methods. Purified antigens were labeled on microarray slides after immunoblotting analysis using anti-GST antibodies (Sangon Biotech, Shanghai, China).

[0055] The MTB protein microarray was constructed by Guangzhou Bochong Biotechnology Co., Ltd. (Foshan, China) and contains 64 recombinant MTB (H37Rv) proteins (including 43 DosR proteins and 21 proteins reported in the literature, see Table 1). Human IgG (Sigma, USA) and IgM (Rockland, USA) were used as positive controls, bovine serum albumin (BSA) was used as negative control, and the microarray was incubated with anti-GST antibody (CWBIO, Beijing, China) for quality evaluation. All purified MTB proteins and controls were repeated on one slide. The quality of the protein microarray was evaluated by probing with anti-GST antibody ( Figure 2 ). The MTB protein microarray was stored at -80°C before use.

[0056] Table 1

[0057]

[0058]

[0059]

[0060] #: indicates unsuccessful recombinant expression of protein.

[0061] 1.3 MTB protein microarray testing of serum samples

[0062] First, the MTB protein microarray was preheated from -80°C to room temperature for half an hour and then incubated in blocking buffer (containing 3% (w / v) BSA and 0.1% (v / v) Tween 20 in PBS buffer) at 37°C for 1 h.

[0063] Secondly, serum samples were diluted with blocking buffer at a volume ratio of 1:1000 and incubated for 1 h at 37°C for MTB protein microarray measurement. After washing three times with PBST, the microarray was incubated with Alexa647-conjugated goat anti-human IgG in the dark for 1 h.

[0064] Finally, after washing three times with PBST solution (pH 7.4), the microarray was rinsed with double distilled water and dried. The microarray was scanned with a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA) and analyzed using GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA).

[0065] 1.4 Protein microarray data analysis

[0066] For the MTB protein microarray, the median foreground and background intensities of each point on the array were measured using GenePix-pro6.0 software. The ratio of the foreground signal to the background signal of each point was used as the signal value of the point, and then the average signal value of each pair of repetitions was used as the signal value of the protein. The signal value cutoff was set to 2.0 to identify positive signals. SAM (Significance Analysis of Chips, R Software (v3.6.1)) was used for differential protein identification. The t-test was selected to evaluate the difference significance of each protein between each group based on the signal value. Tuberculosis-specific candidate autoantigens were screened using p value <0.05 and differential fold change>1.1. In addition, the receiver operating curve (ROC) was used to distinguish between active tuberculosis and non-(active) tuberculosis. The present invention defines the disease discrimination ability of each candidate biomarker (discrimination ability = (sensitivity + specificity) / 2). In order to further improve the sensitivity and specificity of clinical detection of active pulmonary tuberculosis group, the best candidate marker protein for model construction was screened from the above-mentioned multiple candidate marker proteins.

[0067] 1.5 ELISA analysis

[0068] MTB antigen was diluted to 1 μg / mL with coating buffer (carbonate buffer, pH 9.6), incubated overnight at 4°C, and coated on 96-well plates. The plates were washed three times with PBST and blocked with blocking buffer (PBS, 3% (w / v) BSA, 0.1% (v / v) tween20) for 3 h at room temperature. 100 μL of serum sample was diluted 1:100 with PBST buffer, added to the coated plates, and then incubated at room temperature for 30 minutes. After washing the plates five times with PBST, anti-human IgG antibody (CW Biotech, Beijing, China) was diluted with PBST at a volume ratio of 1:10000 with PBST, added and incubated at room temperature for 30 minutes. After washing five more times with PBST, the plates were stained with TMB substrate (InnoRegents, Zhejiang, China) at 37°C in the dark for 10 minutes. The reaction was stopped with 2M sulfuric acid solution. The optical density was measured at 450 nm / 620 nm using a microplate reader (Perlong, Beijing, China).

[0069] 1.6 Statistical methods

[0070] The Kolmogorov-Smirnov test was used to test the normality of data distribution. Differences between groups were analyzed using Fisher's exact test or Pearson's chi-square test (categorical data) and t-test or Mann-Whitney test (continuous data). The diagnostic value of potential biomarkers was evaluated by ROC analysis and calculation of the area under the curve (AUC). Random forest was used to build a model for the tuberculosis biomarker panel. In the random forest analysis, 1000 trees were constructed using the R package randomForest (version 4.6.14) and 10 cross-validations were performed with 100 replications. The critical value for statistical significance was set at p value < 0.05. All data analyses were performed using R statistical software (version 3.6.1) and related software packages.

[0071] 2. Results

[0072] 2.1 Study design

[0073] A two-stage strategy was used to identify new biomarkers for ATB ( Figure 1). In the exploration phase, serum samples from 60 ATB patients, 60 LTBI patients, and 60 healthy volunteers were analyzed on a protein microarray containing 64M. Potential candidate proteins were screened based on sensitivity and specificity for ATB and LTBI. The SAM (significance analysis of microarray) method was used to screen a group of four proteins with the best sensitivity and specificity. In the subsequent validation phase, an additional 300 independent serum samples were evaluated in separate ELISA experiments (100 serum samples from ATB, 100 for LTBI patients, and 100 for HC patients Figure 1 ).

[0074] 2.2 Antigen purification and identification

[0075] In this embodiment, MTB antigens were expressed in Saccharomyces cerevisiae. The N-terminus of each antigen was labeled with GST to facilitate purification and identification of the antigen. The candidate protein was purified by GST affinity chromatography and identified with anti-GST antibodies.

[0076] 2.3 Protein microarray to analyze test reliability

[0077] Purified recombinant proteins and controls (elution buffer, GST, BSA, and histones) were spotted in duplicate on slides and the quality of the microarrays was assessed by probing with anti-GST antibodies ( Figure 2 A). Microarray consisting of 64 MTB H37Rv proteins, including 43 DosR proteins ( Figure 2 A) and 21 MTB proteins reported in the literature (see Table 1). Analysis of the foreground intensity (F) and background intensity (B) of the fluorescence signal showed that the protein microarray had a low background signal (average signal intensity value of 100) and a high foreground fluorescence signal intensity (average signal intensity value of 13796). The foreground intensity curve (Figure 2B) and the background intensity curve were almost completely separated, indicating that the proteins on the microarray can be used for subsequent serum sample testing.

[0078] To evaluate the stability and repeatability between experiments, the same mixed serum samples (QC samples) were measured on a total of 7 independent arrays. The average correlation coefficient between arrays was 0.98, and the Pearson correlation coefficient (R2) for each pair of replicate protein spots was greater than 0.99 (HC, 0.99; LTBI, 0.99, ATB, 1), indicating that the results obtained from the microarray are stable and repeatable. HC is a healthy person.

[0079] 2.4 Differential antibodies identified by MTB protein microarray

[0080] As described above, the MTB protein microarray was used in the exploratory phase to analyze a relatively small number of serum samples (60 samples from ATB patients, 60 samples from LTBI patients, and 60 samples from healthy volunteers). Each serum sample was incubated on the MTB protein microarray, and antibodies against the proteins on the microarray were identified by developing an array with Cy3-labeled anti-human IgG antibodies and Cy5-labeled anti-human IgM antibodies. The data obtained from the microarray were first processed using the Significance Analysis of Microarrays (SAM) algorithm. The signal intensity was normalized according to the median of the microarray and compared between the two groups to determine differential antibodies, with the threshold set at p value <0.05 and differential fold change >1.1. Using these criteria, 19 and 12 differential proteins were identified in the comparison of ATB with HC and ATB with LTBI, respectively ( Figure 3 A, 3B). The results of the Venn diagram comparison analysis showed that five proteins (Rv1860, Rv2031c, Rv3881c, Rv3803c, and Rv0526) were significantly different between ATB and HC and between ATB and LTBI ( Figure 3 C). The fluorescence signal results of protein microarray showed that these five proteins could distinguish the ATB group from the HC and LTBI groups ( Figure 3 D).

[0081] 2.5 ROC analysis and derivation of ATB diagnostic marker combinations

[0082] To identify potential serum biomarkers for the diagnosis of active pulmonary tuberculosis, receiver operating characteristic (ROC) curves of the five proteins were plotted and the areas under the curves (AUCs) were calculated. Box plot analysis was also performed to compare the differences among the five antigen groups. The antibodies to the five proteins in serum were significantly different between the ATB, LTBI, and HC groups (p value < 0.05) ( Figure 4 The sensitivity and specificity of each protein were calculated. The area under the ROC curve was used to evaluate the diagnostic value of the five proteins. The AUC values ​​of the five proteins ranged from 0.599 to 0.725 ( Figure 4 ), with sensitivities ranging from 19.3% to 38.6%. Although a range of ATB-related antibodies were found in the serum, no single antibody was particularly effective in distinguishing ATB from LTBI and HC individuals.

[0083] Through the analysis and calculation of different combinations of candidate antigens, four proteins (Rv1860, Rv3881c, Rv2031c and Rv3803c) were finally screened for model construction. First, the data were randomly divided into two groups, one as a training set (70% samples) and the other as a test set (30% samples). Then, a random forest was used to build a model to obtain a panel with good performance for distinguishing ATB from LTBI / HC individuals. In the training set, the areas under the ROC curves of ATB and HC, ATB and LTBI, and LTBI and HC reached 0.973, 0.981, and 0.964, respectively. Figure 5 The optimized four-protein combination test had a sensitivity of 86% and a specificity of 97.6% for distinguishing ATB patients from healthy subjects, and a sensitivity of 93.3% and a specificity of 97.7% for distinguishing ATB patients from LTBI patients. In the test set, the area under the ROC curve was similar (>0.9) ( Figure 5 ), indicating the stability of the panel in distinguishing ATB from LTBI individuals and healthy individuals. The results showed that the marker combination of four proteins (rv1860, Rv3881c, Rv2031c and Rv3803c) has good diagnostic performance and can be used for the diagnosis of active and latent tuberculosis infection.

[0084] 2.6 Validation of ATB biomarker panel using ELISA

[0085] In order to evaluate the application value of the identified antigens, these biomarkers were validated by ELISA method, and 300 independent sera were added for ELISA experiments. Four candidate antigens (Rv1860, Rv3881c, Rv2031c and Rv3803c) were purified and used for ELISA detection. The results were consistent with the results of the discovery phase using protein microarray. The four proteins showed significantly higher signals in the ATB group than in the HC or LTBI groups. The team effectively distinguished ATB from LTBI in both the training and test sets of the ELISA method ( Figure 6 ). The results of the ELISA experiment were also used to evaluate the performance of the random forest model biomarker panel. The areas under the ROC curve were 0.976, 0.971, and 0.972 for the ATB group vs. HC group, ATB group vs. LTBI group, and LTBI group vs. LTBI / HC group in the training set, respectively. The sensitivity and specificity for distinguishing ATB from HC were 91.2% and 98.8%, respectively. The sensitivity and specificity for distinguishing ATB from LTBI were 93.3% and 97.7%, respectively.

[0086] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention. Sequence Listing <110> Jiangxi Chest Hospital <120> Use of Rv1860 protein, RV3881c protein, Rv2031c protein and Rv3803c protein in diagnosing active pulmonary tuberculosis infection <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 325 <212> PRT <213> Artificial sequence <400> 1 Met His Gln Val Asp Pro Asn Leu Thr Arg Arg Lys Gly Arg Leu Ala 1 5 10 15 Ala Leu Ala Ile Ala Ala Met Ala Ser Ala Ser Leu Val Thr Val Ala 20 25 30 Val Pro Ala Thr Ala Asn Ala Asp Pro Glu Pro Ala Pro Pro Val Pro 35 40 45 Thr Thr Ala Ala Ser Pro Pro Ser Thr Ala Ala Ala Pro Pro Ala Pro 50 55 60 Ala Thr Pro Val Ala Pro Pro Pro Pro Ala Ala Ala Asn Thr Pro Asn 65 70 75 80 Ala Gln Pro Gly Asp Pro Asn Ala Ala Pro Pro Pro Ala Asp Pro Asn 85 90 95 Ala Pro Pro Pro Pro Val Ile Ala Pro Asn Ala Pro Gln Pro Val Arg 100 105 110 Ile Asp Asn Pro Val Gly Gly Phe Ser Phe Ala Leu Pro Ala Gly Trp 115 120 125 Val Glu Ser Asp Ala Ala His Phe Asp Tyr Gly Ser Ala Leu Leu Ser 130 135 140 Lys Thr Thr Gly Asp Pro Pro Phe Pro Gly Gln Pro Pro Pro Val Ala 145 150 155 160 Asn Asp Thr Arg Ile Val Leu Gly Arg Leu Asp Gln Lys Leu Tyr Ala 165 170 175 Ser Ala Glu Ala Thr Asp Ser Lys Ala Ala Ala Arg Leu Gly Ser Asp 180 185 190 Met Gly Glu Phe Tyr Met Pro Tyr Pro Gly Thr Arg Ile Asn Gln Glu 195 200 205 Thr Val Ser Leu Asp Ala Asn Gly Val Ser Gly Ser Ala Ser Tyr Tyr 210 215 220 Glu Val Lys Phe Ser Asp Pro Ser Lys Pro Asn Gly Gln Ile Trp Thr 225 230 235 240 Gly Val Ile Gly Ser Pro Ala Ala Asn Ala Pro Asp Ala Gly Pro Pro 245 250 255 Gln Arg Trp Phe Val Val Trp Leu Gly Thr Ala Asn Asn Pro Val Asp 260 265 270 Lys Gly Ala Ala Lys Ala Leu Ala Glu Ser Ile Arg Pro Leu Val Ala 275 280 285 Pro Pro Pro Ala Pro Ala Pro Ala Pro Ala Glu Pro Ala Pro Ala Pro 290 295 300 Ala Pro Ala Gly Glu Val Ala Pro Thr Pro Thr Thr Pro Thr Pro Gln 305 310 315 320 Arg Thr Leu Pro Ala 325 <210> 2 <211> 460 <212> PRT <213> artificial sequence <400> 2 Met Thr Gln Ser Gln Thr Val Thr Val Asp Gln Gln Glu Ile Leu Asn 1 5 10 15 Arg Ala Asn Glu Val Glu Ala Pro Met Ala Asp Pro Pro Thr Asp Val 20 25 30 Pro Ile Thr Pro Cys Glu Leu Thr Ala Ala Lys Asn Ala Ala Gln Gln 35 40 45 Leu Val Leu Ser Ala Asp Asn Met Arg Glu Tyr Leu Ala Ala Gly Ala 50 55 60 Lys Glu Arg Gln Arg Leu Ala Thr Ser Leu Arg Asn Ala Ala Lys Ala 65 70 75 80 Tyr Gly Glu Val Asp Glu Glu Ala Ala Thr Ala Leu Asp Asn Asp Gly 85 90 95 Glu Gly Thr Val Gln Ala Glu Ser Ala Gly Ala Val Gly Gly Asp Ser 100 105 110 Ser Ala Glu Leu Thr Asp Thr Pro Arg Val Ala Thr Ala Gly Glu Pro 115 120 125 Asn Phe Met Asp Leu Lys Glu Ala Ala Arg Lys Leu Glu Thr Gly Asp 130 135 140 Gln Gly Ala Ser Leu Ala His Phe Ala Asp Gly Trp Asn Thr Phe Asn 145 150 155 160 Leu Thr Leu Gln Gly Asp Val Lys Arg Phe Arg Gly Phe Asp Asn Trp 165 170 175 Glu Gly Asp Ala Ala Thr Ala Cys Glu Ala Ser Leu Asp Gln Gln Arg 180 185 190 Gln Trp Ile Leu His Met Ala Lys Leu Ser Ala Ala Met Ala Lys Gln 195 200 205 Ala Gln Tyr Val Ala Gln Leu His Val Trp Ala Arg Arg Glu His Pro 210 215 220 Thr Tyr Glu Asp Ile Val Gly Leu Glu Arg Leu Tyr Ala Glu Asn Pro 225 230 235 240 Ser Ala Arg Asp Gln Ile Leu Pro Val Tyr Ala Glu Tyr Gln Gln Arg 245 250 255 Ser Glu Lys Val Leu Thr Glu Tyr Asn Asn Lys Ala Ala Leu Glu Pro 260 265 270 Val Asn Pro Pro Lys Pro Pro Pro Ala Ile Lys Ile Asp Pro Pro Pro 275 280 285 Pro Pro Gln Glu Gln Gly Leu Ile Pro Gly Phe Leu Met Pro Pro Ser 290 295 300 Asp Gly Ser Gly Val Thr Pro Gly Thr Gly Met Pro Ala Ala Pro Met 305 310 315 320 Val Pro Pro Thr Gly Ser Pro Gly Gly Gly Leu Pro Ala Asp Thr Ala 325 330 335 Ala Gln Leu Thr Ser Ala Gly Arg Glu Ala Ala Ala Leu Ser Gly Asp 340 345 350 Val Ala Val Lys Ala Ala Ser Leu Gly Gly Gly Gly Gly Gly Gly Val 355 360 365 Pro Ser Ala Pro Leu Gly Ser Ala Ile Gly Gly Ala Glu Ser Val Arg 370 375 380 Pro Ala Gly Ala Gly Asp Ile Ala Gly Leu Gly Gln Gly Arg Ala Gly 385 390 395 400 Gly Gly Ala Ala Leu Gly Gly Gly Gly Met Gly Met Pro Met Gly Ala 405 410 415 Ala His Gln Gly Gln Gly Gly Ala Lys Ser Lys Gly Ser Gln Gln Glu 420 425 430 Asp Glu Ala Leu Tyr Thr Glu Asp Arg Ala Trp Thr Glu Ala Val Ile 435 440 445 Gly Asn Arg Arg Arg Gln Asp Ser Lys Glu Ser Lys 450 455 460 <210> 3 <211> 144 <212> PRT <213> Artificial Sequence <400> 3 Met Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro 1 5 10 15 Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu Arg 20 25 30 Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu 35 40 45 Gly Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys 50 55 60 Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala Glu 65 70 75 80 Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly 85 90 95 Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp Asp 100 105 110 Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val 115 120 125 Ser Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn 130 135 140 <210> 4 <211> 299 <212> PRT <213> Artificial Sequence <400> 4 Met Lys Gly Arg Ser Ala Leu Leu Arg Ala Leu Trp Ile Ala Ala Leu 1 5 10 15 Ser Phe Gly Leu Gly Gly Val Ala Val Ala Ala Glu Pro Thr Ala Lys 20 25 30 Ala Ala Pro Tyr Glu Asn Leu Met Val Pro Ser Pro Ser Met Gly Arg 35 40 45 Asp Ile Pro Val Ala Phe Leu Ala Gly Gly Pro His Ala Val Tyr Leu 50 55 60 Leu Asp Ala Phe Asn Ala Gly Pro Asp Val Ser Asn Trp Val Thr Ala 65 70 75 80 Gly Asn Ala Met Asn Thr Leu Ala Gly Lys Gly Ile Ser Val Val Ala 85 90 95 Pro Ala Gly Gly Ala Tyr Ser Met Tyr Thr Asn Trp Glu Gln Asp Gly 100 105 110 Ser Lys Gln Trp Asp Thr Phe Leu Ser Ala Glu Leu Pro Asp Trp Leu 115 120 125 Ala Ala Asn Arg Gly Leu Ala Pro Gly Gly His Ala Ala Val Gly Ala 130 135 140 Ala Gln Gly Gly Tyr Gly Ala Met Ala Leu Ala Ala Phe His Pro Asp 145 150 155 160 Arg Phe Gly Phe Ala Gly Ser Met Ser Gly Phe Leu Tyr Pro Ser Asn 165 170 175 Thr Thr Thr Asn Gly Ala Ile Ala Ala Gly Met Gln Gln Phe Gly Gly 180 185 190 Val Asp Thr Asn Gly Met Trp Gly Ala Pro Gln Leu Gly Arg Trp Lys 195 200 205 Trp His Asp Pro Trp Val His Ala Ser Leu Leu Ala Gln Asn Asn Thr 210 215 220 Arg Val Trp Val Trp Ser Pro Thr Asn Pro Gly Ala Ser Asp Pro Ala 225 230 235 240 Ala Met Ile Gly Gln Ala Ala Glu Ala Met Gly Asn Ser Arg Met Phe 245 250 255 Tyr Asn Gln Tyr Arg Ser Val Gly Gly His Asn Gly His Phe Asp Phe 260 265 270 Pro Ala Ser Gly Asp Asn Gly Trp Gly Ser Trp Ala Pro Gln Leu Gly 275 280 285 Ala Met Ser Gly Asp Ile Val Gly Ala Ile Arg 290 295

Claims

1. Use of a product for detecting the levels of anti-Rv1860 antibodies, anti-RV3881c antibodies, anti-Rv2031c antibodies and anti-Rv3803c antibodies in serum in the preparation of a product for identification, diagnosis, auxiliary diagnosis, screening and / or auxiliary screening of patients with active pulmonary tuberculosis infection, characterized in that: The Rv1860 is a protein consisting of the amino acid sequence shown in SEQ ID NO.1; The RV3881c is a protein consisting of the amino acid sequence shown in SEQ ID NO.2; The Rv2031c is a protein consisting of the amino acid sequence shown in SEQ ID NO.3; The Rv3803c is a protein consisting of the amino acid sequence shown in SEQ ID NO.

4.

2. Use of a product for detecting the levels of anti-Rv1860 antibodies, anti-RV3881c antibodies, anti-Rv2031c antibodies and anti-Rv3803c antibodies in serum in the preparation of a product for distinguishing active tuberculosis infection patients from normal people, characterized in that: The Rv1860 is a protein consisting of the amino acid sequence shown in SEQ ID NO.1; The RV3881c is a protein consisting of the amino acid sequence shown in SEQ ID NO.2; The Rv2031c is a protein consisting of the amino acid sequence shown in SEQ ID NO.3; The Rv3803c is a protein consisting of the amino acid sequence shown in SEQ ID NO.

4.

3. Use of anti-Rv1860 antibodies, anti-RV3881c antibodies, anti-Rv2031c antibodies and anti-Rv3803c antibodies in serum as markers in the development, design and / or preparation of products for identification, diagnosis, auxiliary diagnosis, screening and / or auxiliary screening of patients with active pulmonary tuberculosis infection, characterized in that: The Rv1860 is a protein consisting of the amino acid sequence shown in SEQ ID NO.1; The RV3881c is a protein consisting of the amino acid sequence shown in SEQ ID NO.2; The Rv2031c is a protein consisting of the amino acid sequence shown in SEQ ID NO.3; The Rv3803c is a protein consisting of the amino acid sequence shown in SEQ ID NO.4.

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