Antigen combination for auxiliary diagnosis of tuberculosis and its application and kit

Abstract

The present invention discloses a group of fusion proteins for assisting the diagnosis of tuberculosis, which include 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and Ag85B-HBHA fusion proteins. The above fusion protein combination can be used for the detection of antigen-specific tuberculosis antibodies in the body fluids of tuberculosis patients, and has the characteristics of high sensitivity, specificity and low cost for assisting the diagnosis of tuberculosis. At the same time, the new antigen combination provided by the present invention can provide a scientific reference for the diagnosis and treatment of tuberculosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antigen combination for auxiliary diagnosis of tuberculosis, an application thereof and a kit. Background Art

[0002] Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (Mtb) and remains a major threat to human health. In 2016-2019, TB was the leading cause of death from a single infectious microorganism. In addition, it is estimated that approximately 23% of the world's population has latent tuberculosis infection (LTBI) and is therefore at risk of developing active TB during their lifetime. Bacille Calmette-Guerin (BCG) is the only tuberculosis vaccine approved for clinical use, which can prevent disseminated TB in young children, but cannot provide sustained protection against TB in adolescents and adults. Early and accurate diagnosis of active TB is crucial for the prevention and control of TB, and early identification of Mtb bacteria or its components can provide direct evidence for rapid diagnosis.

[0003] Methods used to diagnose tuberculosis include tuberculin skin test (TST), sputum smear and culture test, chest X-ray (CXR), interferon-γ release assay (IGRA) and nucleic acid amplification test. TST and CXR are not specific for auxiliary diagnosis of tuberculosis. Sputum smear microscopy for identification of acid-fast bacilli remains the most commonly used test for tuberculosis, but its sensitivity is low. Although bacterial culture is the gold standard for the diagnosis of tuberculosis, it is a time-consuming, laboratory-based test. GeneXpertMTB / RIF (hereinafter referred to as Xpert) and Xpert MTB / RIF Ultra tests both diagnose active TB by simultaneously detecting Mtb complex and rifampicin resistance in patients with pulmonary tuberculosis, with high sensitivity and specificity; however, both methods are relatively expensive and require investment in equipment and technology. Serological diagnosis has its inherent advantages, such as simple and rapid operation, easy promotion, and no need for special precision instruments. A variety of purified antigens are available, especially for sputum culture-negative and extrapulmonary tuberculosis, which has important auxiliary diagnostic value. However, it is difficult to achieve sufficient sensitivity and specificity using a single antigen.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an antigen combination for auxiliary diagnosis of tuberculosis and its application and kit, so as to improve the sensitivity and specificity of auxiliary diagnosis of tuberculosis.

[0006] The present invention is achieved in that:

[0007] In the first aspect, the present invention provides a group of fusion proteins for assisting in the diagnosis of tuberculosis, which include 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and Ag85B-HBHA;

[0008] The amino acid sequence of the fusion protein 38KD-MPT32-MPT64 is shown in SEQ ID NO.1, or a sequence having more than 90% homology with SEQ ID NO.1;

[0009] The amino acid sequence of the fusion protein CFP10-Mtb81-EspC is shown in SEQ ID NO.2, or a sequence having more than 90% homology with SEQ ID NO.2;

[0010] The amino acid sequence of the fusion protein Ag85B-HBHA is shown in SEQ ID NO.3, or is a sequence having more than 90% homology with SEQ ID NO.3.

[0011] In a second aspect, the present invention provides an antigen combination for assisting in the diagnosis of tuberculosis, comprising the above-mentioned fusion proteins 38KD-MPT32-MPT64 and CFP10-Mtb81-EspC, and the antigen LAM.

[0012] In a third aspect, the present invention provides a DNA molecule encoding the above antigen combination.

[0013] In a fourth aspect, the present invention provides a recombinant expression vector containing the above-mentioned DNA molecule, which includes pET-30a-38KD-MPT32-MPT64, pET21a-CFP10-Mtb81-EspC and pET21a-Ag85B-HBHA;

[0014] The nucleotide sequence of the fusion protein 38KD-MPT32-MPT64 in the recombinant expression vector is shown in SEQ ID NO.4;

[0015] The nucleotide sequence of the fusion protein CFP10-Mtb81-EspC in the recombinant expression vector is shown in SEQ ID NO.5;

[0016] The nucleotide sequence of the fusion protein Ag85B-HBHA in the recombinant expression vector is shown in SEQ ID NO.6.

[0017] In a fifth aspect, the present invention provides a transgenic cell line or recombinant bacteria containing the above-mentioned recombinant expression vector.

[0018] In a sixth aspect, the present invention provides the use of the above-mentioned antigen combination in the preparation of a serological diagnostic reagent for tuberculosis, wherein the above-mentioned tuberculosis includes pulmonary tuberculosis, extrapulmonary tuberculosis, negative pulmonary tuberculosis and XpertMTB / RIF test negative tuberculosis.

[0019] In a seventh aspect, the present invention provides a kit for assisting in the diagnosis of tuberculosis, comprising the above-mentioned antigen combination.

[0020] The present invention has the following beneficial effects:

[0021] The present invention provides a group of new fusion proteins for assisting in the diagnosis of tuberculosis, which can be used to detect antigen-specific tuberculosis antibodies in the body fluids of tuberculosis patients, and has the characteristics of high sensitivity, specificity and low cost for assisting in the diagnosis of tuberculosis. At the same time, the new antigen combination provided by the present invention can provide a scientific reference for the diagnosis and treatment of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 SDS-PAGE electrophoresis was used to identify the purity of the fusion protein. M: marker; 1: 38KD-MPT32-MPT64; 2: CFP10-Mtb81-EspC; 3: Ag85B-HBHA;

[0024] Figure 2 The results of ELISA for the detection of 38KD-MPT32-MPT64 fusion protein and single protein-specific IgG antibodies in the serum of tuberculosis patients;

[0025] Figure 3 ROC curve for evaluation of auxiliary diagnostic efficacy of 38KD-MPT32-MPT64 fusion protein and single protein;

[0026] Figure 4 A matrix diagram comparing the detection of 38KD-MPT32-MPT64 fusion protein and single protein specific IgG antibodies;

[0027] Figure 5 The results of ELISA for the detection of CFP10-Mtb81-EspC fusion protein and single protein-specific IgG antibodies in the serum of tuberculosis patients;

[0028] Figure 6ROC curve for evaluation of the auxiliary diagnostic efficacy of CFP10-Mtb81-EspC fusion protein and single protein;

[0029] Figure 7 A matrix diagram comparing the detection characteristics of CFP10-Mtb81-EspC fusion protein and single protein-specific IgG antibodies;

[0030] Figure 8 The results of ELISA for the detection of Ag85B-HBHA fusion protein-specific IgG antibodies in the serum of tuberculosis patients;

[0031] Fig. 9 The ROC curve for evaluating the diagnostic efficacy of Ag85B-HBHA fusion protein;

[0032] Fig.10 The results of ELISA for the detection of LAM polysaccharide antigen-specific IgG antibodies in the serum of tuberculosis patients;

[0033] Fig.11 ROC curve for evaluating the diagnostic efficacy of LAM polysaccharide antigen;

[0034] Fig.12 Analysis of fusion protein and LAM polysaccharide antigen-specific IgG levels for TB patients divided into subgroups based on GeneXpert MTB / RIF results;

[0035] Fig.13 Distribution characteristics of samples positive for fusion protein and LAM polysaccharide antigen-specific IgG antibodies

[0036] Fig.14 This is the ROC curve of the combined use of fusion protein and LAM polysaccharide antigen for serological auxiliary diagnosis of tuberculosis. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0038] At present, serological testing also has certain auxiliary diagnostic advantages in tuberculosis, especially in tuberculosis with negative sputum pulmonary tuberculosis, extrapulmonary tuberculosis and tuberculosis with negative GeneXpert MTB / RIF test. Compared with single antigen, fusion protein can improve the sensitivity of detection.

[0039] Fusion proteins are formed by splicing natural proteins together through polypeptide fragments, and have important application value in disease prevention, diagnosis and treatment. Fusion proteins can express several antigens at the same time, reducing the cost of antigen preparation and improving sensitivity, and have advantages in serologically assisted diagnosis of tuberculosis. In tuberculosis, the key to establishing this method is to obtain high-purity specific diagnostic antigens. Since there are many specific antigen components of Mycobacterium tuberculosis, it is necessary to screen highly sensitive antigen combinations.

[0040] Based on this, the present invention first screened 6 antigens with high sensitivity for tuberculosis antibody detection, including lipoarabinomannan (LAM) and 38KD (Rv0934), MPT32 (Rv1860), MPT64 (Rv1980c), EspC (Rv3615c), and Mtb81 (Rv1837c) proteins, combined with the reported antigens with good sensitivity CFP10 (Rv3874), Ag85B (Rv1886c), and HBHA (Rv0475), and three fusion proteins were constructed and expressed in the present invention, including 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and Ag85B-HBHA.

[0041] The preparation method of the above three fusion proteins is as follows: the fusion protein encoding gene sequence is synthesized by gene synthesis technology, the proteins are connected by polypeptides (GGGSGGGS), then restriction sites are inserted at both ends, and tags are added, and a prokaryotic expression vector is obtained after connection and transformation. After transfection, the expression of the fusion protein is induced, and the fusion protein is obtained after purification and concentration.

[0042] Among them, the fusion protein amino acid sequence and coding gene sequence are as follows:

[0043] 38KD-MPT32-MPT64 fusion protein amino acid sequence (SEQ ID NO.1);

[0044] CFP10-Mtb81-EspC fusion protein amino acid sequence (SEQ ID NO. 2);

[0045] Ag85B-HBHA fusion protein amino acid sequence (SEQ ID NO. 3);

[0046] 38KD-MPT32-MPT64 fusion protein encoding gene sequence (SEQ ID NO.4);

[0047] CFP10-Mtb81-EspC fusion protein encoding gene sequence (SEQ ID NO.5);

[0048] Ag85B-HBHA fusion protein encoding gene sequence (SEQ ID NO.6).

[0049] Specifically, the restriction sites inserted at both ends of 38KD-MPT32-MPT64 are NdeI / XhoI, the restriction sites inserted at both ends of CFP10-Mtb81-EspC are BamHI / XhoI, and the restriction sites inserted at both ends of Ag85B-HBHA are BamHI / XhoI. The tag inserted at the carboxyl terminus (C-terminus) of 38KD-MPT32-MPT64 is a His tag (LEHHHHHH), and CFP10-Mtb81-EspC and Ag85B-HBHA insert a T7 tag (MASMTGGQQMGRGS) and a His tag (LEHHHHHH) at the amino terminus (N-terminus) and the carboxyl terminus, respectively, but are not limited to T7 and His tags.

[0050] The present invention also provides expression vectors of the above-mentioned Mycobacterium tuberculosis fusion protein, including pET-30a-38KD-MPT32-MPT64, pET21a-CFP10-Mtb81-EspC and pET21a-Ag85B-HBHA.

[0051] The inventors of the present invention identified the application value of the above three fusion proteins and LAM antigens alone and in combination in serological auxiliary diagnosis of tuberculosis, and screened out a highly sensitive and specific antigen combination, which includes 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and Ag85B-HBHA fusion proteins and LAM antigens.

[0052] According to further systematic analysis, it was found that samples with elevated anti-Ag85B-HBHA antibody levels could be completely covered by the 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and LAM antigen combination. Therefore, when the antigen combination included 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and LAM, it also had high sensitivity and specificity in the auxiliary diagnosis of tuberculosis.

[0053] The present invention also provides the use of the above antigen combination in preparing a tuberculosis serological diagnostic reagent.

[0054] The above-mentioned tuberculosis is not limited to pulmonary tuberculosis, but can also be applied to extrapulmonary tuberculosis, negative pulmonary tuberculosis, and tuberculosis that is negative for GeneXpert MTB / RIF (a rapid detection product for Mycobacterium tuberculosis and rifampicin resistance).

[0055] The present invention also provides a kit for auxiliary diagnosis of tuberculosis, which comprises the above antigen combination: 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and Ag85B-HBHA fusion protein and LAM antigen, or 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and LAM antigen.

[0056] The sample tested by the kit here is serum, but it is not limited to serum and can also be applied to plasma, pleural effusion, cerebrospinal fluid and other body fluids.

[0057] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] This embodiment provides an antigen combination for assisting diagnosis of tuberculosis, which includes 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and Ag85B-HBHA fusion proteins, and LAM antigen.

[0060] Example 2

[0061] This embodiment provides an antigen combination for assisting diagnosis of tuberculosis, which includes 38KD-MPT32-MPT64 and CFP10-Mtb81-EspC fusion proteins, and LAM antigen.

[0062] Example 3

[0063] This example provides the synthesis of fusion protein encoding gene:

[0064] Each protein in the three fusion proteins in Example 1 is connected by a connecting peptide (GGGSGGGS), a His tag (LEHHHHHH) is added to the carboxyl end (C-terminus) of the 38KD-MPT32-MPT64 fusion protein, and a T7 tag (MASMTGGQQMGRGS) and a His tag (LEHHHHHH) are added to the amino end (N-terminus) and carboxyl end of the CFP10-Mtb81-EspC and Ag85B-HBHA fusion proteins, respectively. The coding genes of the three fusion proteins were obtained by gene synthesis (Shanghai Bioengineering), and restriction sites were inserted at both ends, wherein NdeI / XhoI restriction sites were inserted at both ends of 38KD-MPT32-MPT64, and BamHI / XhoI restriction sites were inserted at both ends of CFP10-Mtb81-EspC and Ag85B-HBHA. The amino acid sequence of the fusion protein and the coding gene sequence are as follows:

[0065] 38KD-MPT32-MPT64 fusion protein amino acid sequence (SEQ ID NO.1);

[0066] CFP10-Mtb81-EspC fusion protein amino acid sequence (SEQ ID NO. 2);

[0067] Ag85B-HBHA fusion protein amino acid sequence (SEQ ID NO. 3);

[0068] 38KD-MPT32-MPT64 fusion protein encoding gene sequence (SEQ ID NO.4);

[0069] CFP10-Mtb81-EspC fusion protein encoding gene sequence (SEQ ID NO.5);

[0070] Ag85B-HBHA fusion protein encoding gene sequence (SEQ ID NO.6).

[0071] Example 4

[0072] This example is fusion protein expression and purification

[0073] (1) Construction of fusion protein expression vector

[0074] The synthetic gene was cloned into the expression vector to form pET-30a-38KD-MPT32-MPT64, pET21a-CFP1-Mtb81-EspC and pET21a-Ag85B-HBHA recombinant expression vectors. After the sequencing was correct, the competent cells were transfected to express the fusion protein.

[0075] (2) Fusion protein expression

[0076] ① Transform competent cells with recombinant plasmid. Take 1 μl of the recombinant vector to transform Rosetta (DE3), heat shock at 42°C for 90 seconds, let stand on ice for 2 minutes, then spread on LB solid plate containing 30 μg / mL kanamycin and 34 μg / mL chloramphenicol at a final concentration, and culture at 37°C overnight.

[0077] ②Select the best induction conditions for small-scale culture. Pick a single colony of the expression strain Rosetta (DE3) and place it in a conical flask containing 10ml LB medium. Add kanamycin and chloramphenicol at a final concentration of 30μg / mL and 34μg / mL, and culture overnight at 37℃ and 220rpm. Inoculate the overnight cultured bacteria into 10mL LB medium at a ratio of 1:100, add kanamycin and 34μg / mL chloramphenicol at a final concentration of 30μg / mL, and culture at 37℃ and 220rpm. When the OD value reaches 0.6, add IPTG at a final concentration of 0.5mM, 220rpm, and induce at 20℃ overnight; induce at 37℃ for 4h, and the one without IPTG inducer is used as a negative control. The cells were collected by centrifugation at 4000rpm for 10min, and the supernatant was discarded. The cells were suspended with 500μL PBS (PH7.4) buffer, ultrasonically disrupted for 6min, with an ultrasonic interval of 0.5s and a rest of 1.5s. The supernatant and precipitate were collected by centrifugation respectively. The precipitate was dissolved with 500μL inclusion body dissolution solution (8M Urea, 50mM Tris-HCl, 300mM NaCl, pH8.0), and 40μL of sample and 10μL 5×protein loading buffer were mixed and boiled in a water bath for 10min. For SDS-PAGE detection, 12% SDS-PAGE, Tris-Gly electrophoresis buffer (Tris 3.0g, glycine 14.4g, SDS 1.0g, fixed to 1L) were prepared, and the sample volume was 10μL. The concentrated gel was 80V for 20min, and the separation gel was 120V for 60min. After the gel electrophoresis, the gel was stained for 20min and decolorized. After identification, it was determined that the best induction condition was 0.5 mM IPTG, 20°C, and 220 rpm overnight induction.

[0078] ③ Induce the expression of fusion protein in large quantities. Inoculate the cultured bacteria into 3L LB liquid medium at a ratio of 1:100, add 50μg / mL ampicillin and 34μg / mL chloramphenicol at a final concentration, culture at 37°C and 220rpm. When the OD value reaches 0.6, add 0.5mM IPTG at a final concentration, 20°C and 220rpm, induce overnight, and collect the bacterial cells by centrifugation.

[0079] (3) Fusion protein purification and identification

[0080] ① Ultrasonic disruption of bacteria. Dissolve the collected bacteria in disruption buffer (8M Urea, 50mM Tris, 300mM NaCl, 0.1% Triton X-114, pH8.0), and ultrasonically disrupt the bacteria in an ice bath at a power of 400W for 20min (ultrasonication for 2S, pause for 6S as one cycle). After ultrasonication, centrifuge at 12000rpm, 4℃ for 20min, and collect the supernatant for the next step of purification.

[0081] ② Nickel agarose affinity chromatography

[0082] Clean the equilibrated column: Take 5 mL of Ni-NTA and clean the equilibrated column with 5 times the column bed volume of Binding buffer at a flow rate of 5 mL / min;

[0083] Upper column: flow rate is 2 mL / min, and the flow-through liquid is collected;

[0084] Wash 1: Wash the column with 20 times the column volume of Binding buffer to remove endotoxin at a flow rate of 5 mL / min.

[0085] Wash 2: Wash with wash buffer at a flow rate of 5 mL / min;

[0086] Elution: Elution with elution buffer at a flow rate of 2 mL / min, and collect the eluate.

[0087] Dialysis: The eluate fractions were dialyzed into: 4M Urea, 50mM Tris, 300mM NaCl, pH8.0 (38KD-MPT32-MPT64) or 1×PBS, pH7.4 (CFP10-Mtb81-EspC) or 50mM Tris, 300mM NaCl, 0.2% SKL, pH8.0 (Ag85B-HBHA). Dialysis was performed overnight, and then concentrated with PEG20000, filtered through a 0.45μm filter membrane, and aliquoted and stored at -80°C.

[0088] Among them, Binding buffer (8M Urea, 50mM Tris, 300mM NaCl, 0.1% Triton X-114, pH8.0);

[0089] Wash buffer (8M Urea, 50mM Tris, 300mM NaCl, 20 / 50mM Imidazole, pH8.0);

[0090] Elution buffer (8M Urea, 50mM Tris, 300mM NaCl, 500mM Imidazole, pH8.0).

[0091] ③SDS-PAGE electrophoresis to identify fusion protein

[0092] The molecular weight of the antibody was predicted using a protein molecular weight calculator, and the purity and molecular weight of the antibody were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0093] SDS-PAGE electrophoresis detection process: Take 5 μg of purified fusion protein, add protein electrophoresis loading buffer (add β-mercaptoethanol), boil for 5 minutes, use 10% SDS-PAGE gel for electrophoresis separation, 150V, electrophoresis for about 1 hour. After the electrophoresis, stain with Coomassie Brilliant Blue dye and incubate at 4°C overnight. On the second day, decolorize with decolorizing solution until the bands are clear and the background is clean, and then take pictures with a camera to record the staining results.

[0094] SDS-PAG test results ( Figure 1 ): 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and Ag85B-HBHA three fusion proteins contain 867, 981 and 514 amino acids, respectively, and the predicted molecular weights are 89kDa, 105kDa and 55kDa, respectively. SDS-PAGE electrophoresis identified that the protein molecular weight is close to the predicted molecular weight, and the purity is greater than 90%. In the figure, M: marker; 1: 38KD-MPT32-MPT64; 2: CFP10-Mtb81-EspC; 3: Ag85B-HBHA.

[0095] Example 5

[0096] This example is an identification of the distribution characteristics of 38KD-MPT32-MPT64 fusion protein and single protein-specific IgG antibodies in the serum of tuberculosis patients.

[0097] (1) Detection of tuberculosis antigen-specific IgG antibody levels in tuberculosis patients

[0098] Fifty active tuberculosis patients were enrolled, and 17 healthy subjects were included as controls. Peripheral blood was collected from patients, serum was separated, and stored in aliquots at -80°C for later use. ELISA was used to detect the tuberculosis antigen-specific IgG antibody level in the serum of tuberculosis patients.

[0099] ELISA detection process: The coating solution was coated with 5μg / ml 38KD, MPT32, MPT64 and 38KD-MPT32-MPT64 fusion proteins at 4°C overnight. The next day, the plate was washed three times with Wash Buffer working solution, and 5% skim milk powder was added to block non-specific binding sites, 200μl / well, and reacted at 37°C for 2 hours. After washing three times with a plate washer, sera from tuberculosis patients and healthy controls diluted 1:50 with 5% skim milk powder were added and incubated at 4°C overnight. After washing five times, 1:60000 diluted horseradish peroxidase-labeled goat anti-human IgG (H+L) was added and reacted at 37°C for 1h. After washing five times, TMB substrate solution was added, 100μl / well, and reacted at 37°C for 30min. Stop solution was added, 50μl / well. OD was measured using an enzyme reader 450 The test results were analyzed by Graphpad software.

[0100] Among them, the main reagent sources and preparation:

[0101] Coating buffer (BD OptEIA Coating Buffer, 51-2713KC) pH 9.5 0.1 M sodium carbonate

[0102] Wash buffer (BD OptEIA Wash Buffer, 51-9003739) 20× concentrated wash buffer should be diluted with deionized or distilled water to a 1× working solution.

[0103] The color developing solution A (BD OptEIA Substrate Reagent A, 51-2606KZ) is a buffer solution containing hydrogen peroxide.

[0104] The color developing solution B (BD OptEIA Substrate Reagent B, 51-2607KZ) contains an organic solvent of 3,3',5,5'-tetramethylbenzidine (TMB).

[0105] Stop solution (BD OptEIA Stop Solution, 51-2608KZ) 1. Sulfuric acid.

[0106] 5% milk: Add 5 g of milk to 100 ml of 1× PBS solution.

[0107] ELISA test results ( Figure 2 ): In tuberculosis patients, the levels of all antigen-specific IgG antibodies were significantly higher than those in healthy controls, and the difference was statistically significant (p<0.05). Taking the (OD450 mean + 2*standard deviation) of the healthy control group as the detection critical value, the detection rates of 38KD, MPT32, MPT64 and 38KD-MPT32-MPT64 specific IgG antibodies in the serum of tuberculosis patients were 36% (18 / 50), 36% (18 / 50), 24% (12 / 50) and 54% (27 / 50), respectively, and the specificities were 94.1% (1 / 17), 94.1% (1 / 17), 100% (0 / 17) and 94.1% (1 / 17), respectively. The results showed that the 38KD-MPT32-MPT64 fusion protein had good specificity, and the sensitivity was significantly improved compared with the use of protein antigens alone.

[0108] (2) Draw the ROC curve to evaluate the efficacy of 38KD-MPT32-MPT64 fusion protein and single protein-specific IgG antibody detection in the serum of tuberculosis patients in assisting the diagnosis of tuberculosis.

[0109] ROC curve of 38KD-MPT32-MPT64 fusion protein and single protein for assisting diagnosis of tuberculosis ( Figure 3 ): The areas under the curve of 38KD, MPT32, MPT64 and 38KD-MPT32-MPT64 were 0.7788, 0.7729, 0.7006 and 0.8588 respectively. The results showed that 38KD-MPT32-MPT64 fusion protein had better auxiliary diagnosis efficacy than protein antigen alone.

[0110] (3) Draw a matrix diagram to compare the consistency of the detection results of 38KD-MPT32-MPT64 fusion protein and single protein specific IgG antibodies.

[0111] Each antigen was detected with the (OD450 mean + 2*standard deviation) of the healthy control group as the detection critical value, and the serum IgG antibody test results of 50 tuberculosis patients were qualitatively classified (1 and 0). 1 represents a positive antigen-specific antibody test, and 0 represents a negative antigen-specific antibody test. The test results of 38KD-MPT32-MPT64 fusion protein and individual proteins were displayed in a matrix diagram using Prism7.0 software (black: 1 / positive; white: 0 / negative).

[0112] result( Figure 4 ): The test results of 38KD-MPT32-MPT64 fusion protein were highly consistent with the test results of 38KD, MPT32, and MPT64 single antigens. 38KD-MPT32-MPT64 can detect 96.3% (26 / 27) of 38KD positive samples, 88.9% (16 / 18) of MPT32 positive samples, and 83.3% (10 / 12) of MPT64 positive samples. When (38KD+MPT32+MPT64) was combined, as long as any one of the antigen-specific IgG antibodies tested positive, the sample was judged to be positive, and 58% (29 / 50) of the patients could detect antigen-specific IgG antibodies. The 38KD-MPT32-MPT64 fusion protein was compared with the combined (38KD+MPT32+MPT64) results in tuberculosis patients with a consistency rate of 88% (44 / 50), a missed detection rate of 8% (4 / 50), and a new detection rate of 4% (2 / 50). The missed detection may be due to the interference of certain epitope-specific antibody detection caused by the fusion protein; the new detection may be related to the superposition of low-level different antigen-specific antibody detection results. In short, the fusion protein detection results are highly consistent with the combined analysis results of antigen detection alone, but the detection method is significantly optimized.

[0113] Example 6

[0114] This example is an identification of the distribution characteristics of CFP10-Mtb81-EspC fusion protein and single protein-specific IgG antibodies in the serum of tuberculosis patients.

[0115] (1) ELISA detection process: 5 μg / ml CFP10, Mtb81, EspC and CFP10-Mtb81-EspC fusion protein were coated with coating solution at 4°C overnight. Other detection processes were the same as the above ELISA detection process.

[0116] ELISA test results ( Figure 5 ): In tuberculosis patients, the levels of CFP10, Mtb81 and CFP10-Mtb81-EspC antigen-specific IgG antibodies were significantly higher than those in healthy controls, and the differences were statistically significant (p<0.05); no statistical difference was found in the level of EspC-specific IgG antibodies compared with healthy controls. Taking the (OD450 mean + 2*standard deviation) of the healthy control group as the detection cutoff value, the detection rates of CFP10, Mtb81, EspC and CFP10-Mtb81-EspC-specific IgG antibodies in the serum of tuberculosis patients were 26% (10 / 50), 26% (13 / 50), 26% (13 / 50) and 40% (20 / 50), respectively, and the specificities were 94.1% (1 / 17), 94.1% (1 / 17), 100% (0 / 17) and 100% (0 / 17), respectively. The results showed that the 38KD-MPT32-MPT64 fusion protein had good specificity and its sensitivity was significantly improved compared with the use of protein antigen alone.

[0117] (2) Draw the ROC curve to evaluate the efficacy of CFP10-Mtb81-EspC fusion protein and single protein-specific IgG antibody detection in the serum of tuberculosis patients in assisting the diagnosis of tuberculosis.

[0118] ROC curves of CFP10-Mtb81-EspC fusion protein and single protein for assisted diagnosis of tuberculosis ( Figure 6 ): The areas under the curve of CFP10, Mtb81, EspC and CFP10-Mtb81-EspC were 0.6747, 0.6900, 0.5853 and 0.7118 respectively. The results showed that CFP10-Mtb81-EspC fusion protein had better auxiliary diagnosis efficacy than protein antigen alone.

[0119] (3) Draw a matrix diagram to compare the consistency of the detection results of CFP10-Mtb81-EspC fusion protein and single protein-specific IgG antibodies.

[0120] Each antigen was detected with the (OD450 mean + 2*standard deviation) of the healthy control group as the detection critical value, and the serum IgG antibody test results of 50 tuberculosis patients were qualitatively classified (1 and 0). 1 represents a positive antigen-specific antibody test, and 0 represents a negative antigen-specific antibody test. The test results of CFP10-Mtb81-EspC fusion protein and individual proteins were displayed in a matrix diagram using Prism7.0 software (black: 1 / positive; white: 0 / negative).

[0121] result( Figure 7 ): The test results of CFP10-Mtb81-EspC fusion protein are highly consistent with the test results of CFP10, Mtb81, and EspC single antigens. CFP10-Mtb81-EspC fusion protein can detect 90% (9 / 10) of CFP10 positive samples, 92.3% (12 / 13) of Mtb81 positive samples, and 61.5% (8 / 13) of EspC positive samples. When (CFP10+Mtb81+EspC) is combined, as long as any one of the antigen-specific IgG antibodies is tested positive, the sample is judged to be positive, and 50% (25 / 50) of patients can detect antigen-specific IgG antibodies. The CFP10-Mtb81-EspC fusion protein can detect 72% (18 / 25) of the (CFP10+Mtb81+EspC) combined analysis positive samples, with a consistency rate of 82% (41 / 50) in tuberculosis patients, a missed detection rate of 14% (7 / 50), and a new detection rate of 4% (2 / 50). Among the missed samples, 1 was a CFP10-positive sample, 1 was a Mtb81-positive sample, and 5 were EspC-positive samples. Considering that the fusion protein mainly affects certain epitopes of EspC, its detection is partially interfered. The new detection may be related to the superposition of low-level detection results of different antigen-specific antibodies.

[0122] Example 7

[0123] This example is an identification of the distribution characteristics of Ag85B-HBHA fusion protein-specific IgG antibodies in the serum of tuberculosis patients.

[0124] (1) ELISA detection process: 5 μg / ml Ag85B-HBHA fusion protein was coated with the coating solution at 4°C overnight. Other detection processes were the same as the above ELISA detection process.

[0125] ELISA test results ( Figure 8): The level of Ag85B-HBHA antigen-specific IgG antibody in tuberculosis patients was significantly higher than that in healthy people, and the difference was statistically significant (p<0.05). Taking the (OD450 mean + 2*standard deviation) of the healthy control group as the detection cutoff value, the detection rate of Ag85B-HBHA-specific IgG antibody in the serum of tuberculosis patients was 30% (15 / 50) and the specificity was 94.1% (1 / 17).

[0126] (2) Draw the ROC curve to evaluate the diagnostic efficacy of Ag85B-HBHA fusion protein and single protein-specific IgG antibodies in the serum of tuberculosis patients.

[0127] ROC curve of Ag85B-HBHA fusion protein for assisted diagnosis of tuberculosis ( Fig. 9 ):The areas under the curve of Ag85B-HBHA are 0.7000 respectively.

[0128] Example 8

[0129] This example is an identification of the distribution characteristics of LAM polysaccharide antigen-specific IgG antibodies in the serum of tuberculosis patients.

[0130] (1) ELISA test process: The coating solution was coated with 1 μg / ml of Mycobacterium tuberculosis, Strain H37Rv, Purified Lipoarabinomannan (LAM); from BEI Resources, Catalog No.: NR-14848) overnight at 37°C. The serum of tuberculosis patients and healthy controls was diluted 1:200. Other test processes were the same as the above ELISA test process.

[0131] ELISA test results ( Fig.10 ): The levels of LAM antigen-specific IgG antibodies in tuberculosis patients were significantly higher than those in healthy people, and the difference was statistically significant (p<0.05). Taking the (OD450 mean + 2*standard deviation) of the healthy control group as the detection cutoff value, the detection rate of LAM-specific IgG antibodies in the serum of tuberculosis patients was 28% (14 / 50) and the specificity was 94.1% (1 / 17).

[0132] (2) Draw a ROC curve to evaluate the efficacy of LAM polysaccharide antigen-specific IgG antibody detection in the sera of tuberculosis patients in assisting the diagnosis of tuberculosis.

[0133] ROC curve of LAM polysaccharide antigen-assisted diagnosis of tuberculosis ( Fig.11 ): The area under the curve of LAM antigen was 0.8294.

[0134] Example 9

[0135] This example shows the serum IgG antibody levels of tuberculosis patients stratified by GeneXpert MTB / RIF test results.

[0136] (1) Based on the results of Xpert MTB / RIF testing, tuberculosis patients were divided into two groups: Xpert positive group (Xpert+, n=42) and Xpert negative group (Xpert-, n=8). The IgG antibody levels in the serum of the two groups were analyzed and compared.

[0137] Serum IgG antibody levels in tuberculosis patients stratified by Xpert test results ( Fig.12 ): In the Xpert+ group, the levels of four antigen-specific IgG antibodies were significantly higher than those of healthy people. In the Xpert- group, the level of 38KD-MPT32-MPT64-specific IgG antibodies was significantly higher than that of healthy people, indicating that this fusion protein has a certain significance for the serological diagnosis of Xpert-negative tuberculosis patients. The level of LAM antigen-specific IgG antibodies in the Xpert+ group was significantly higher than that in the Xpert- group, and there was a certain correlation between LAM antibody positivity and positive molecular biological detection of Mycobacterium tuberculosis.

[0138] Example 10

[0139] Combined analysis of fusion protein and polysaccharide antigen-specific IgG antibodies in sera of tuberculosis patients

[0140] Through the above examples, it can be concluded that the sensitivity of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, Ag85B-HBHA and LAM are 54.0% (27 / 50), 42.0% (21 / 50), 30.0% (15 / 50)) and 28.0% (14 / 50), respectively, and the specificity is 94.1% (1 / 17), 100.0% (0 / 17), 94.1% (1 / 17) and 94.1% (1 / 17), respectively. For the same patient, as long as any one antigen-specific IgG antibody is detected positive, the patient is defined as tuberculosis antibody positive. Using this method, 37 patients were identified as TB-specific Ab positive (TB-Ab+), and 13 patients were identified as TB-specific Ab negative (TB-Ab-). The detection rates of fusion protein and polysaccharide antigen-specific IgG in tuberculosis patients and healthy subjects, as well as the detection sensitivity of tuberculosis subgroups (TB-Xpert+ group and TB-Xpert- group) were further analyzed (Table 1). When the antigens were analyzed separately, the detection sensitivity of antigen-specific IgG antibodies in the TB-Xpert+ group was higher than that in the TB-Xpert- group. When the combined analysis was performed, the detection sensitivity of the TB-Xpert+ group and the TB-Xpert- group was similar due to the significant improvement in the detection sensitivity.

[0141] Table 1. Detection sensitivity of specific IgG antibodies in sera of tuberculosis patients by antigen analysis alone and in combination

[0142]

[0143] Embodiment 11

[0144] This example shows the recognition characteristics of each antigen-specific IgG antibody in tuberculosis patients

[0145] In Example 10, 37 tuberculosis antibody positive samples were detected, and a Venn diagram was further drawn to show the recognition characteristics of each antigen-related IgG in all TB-Ab+ samples.

[0146] Venn Diagram Results( Fig.13 ): Among the 37 TB-Ab+ patients, the number of patients with IgG antibodies reactive to any one, two, three, and four antigens was 16, 7, 10, and 4, respectively. Samples with elevated anti-Ag85B-HBHA antibody levels could be fully covered by the combination of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and LAM antigens. The sensitivity of this combination was 74.0% (37 / 50) and the specificity was 88.2% (2 / 17) (Table 1).

[0147] Example 12

[0148] This example is a ROC curve analysis of the performance of tuberculosis antigen combination in assisting diagnosis of tuberculosis

[0149] The ROC curves of the three fusion proteins and LAM combinations were drawn by using the binary logistic regression analysis method combined with ROC curve analysis ( Fig.14 ).

[0150] The area under the curve (AUC) value of the combination of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, Ag85B-HBHA fusion protein and LAM polysaccharide antigen was 0.8965. The AUCs of the aforementioned 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, Ag85B-HBHA and LAM antigens alone were 0.8588, 0.7118, 0.7000 and 0.8294, respectively. When the optimal cutoff value was applied (Youden index obtained the maximum cutoff value; Youden index = sensitivity + specificity - 1), the sensitivity of IgG antibodies to 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, Ag85B-HBHA, LAM and the combination of four antigens was 82.0%, 50.0%, 58.0%, 82.0% and 84.0%, respectively, and the specificity was 82.4%, 94.1%, 82.4%, 76.47% and 82.4%, respectively. Compared with the combination of four antigens, the combination of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC and LAM had the same AUC (0.8965), sensitivity (84.0%) and specificity (82.4%).

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of 38KD-MPT32-MPT64 in the preparation of a kit for assisting in the diagnosis of tuberculosis with negative GeneXpert MTB / RIF test; The amino acid sequence of the fusion protein 38KD-MPT32-MPT64 is shown in SEQ ID NO.1.

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