Serum antibody IgG specific recognition vaccine strain antigen spectrum and screening method and application thereof

Abstract

The invention provides a serum antibody IgG specific recognition vaccine strain antigen spectrum and a screening method and application thereof. Serum of an experimental group and a control group is screened in advance, and serum samples of latent infection crowds which do not generate tuberculosis antibodies and contain anti-mycobacterium tuberculosis protein antibodies are removed; then carrying out bionic affinity and affinity chromatography on the screened serum antibody to obtain a high-purity serum antibody IgG, and covalently coupling the high-purity serum antibody IgG subjected to bionic affinity and affinity chromatography to a solid-phase carrier material to prepare different types of serum antibody columns; after affinity chromatography of different types of serum antibody columns and BCG total protein, Gly-HCl (pH 1.5-3.0) can only elute BCG protein combined on the Gly-HCl instead of high-abundance serum antibody IgG, LC-MS / MS can detect low, medium and high concentration BCG protein, a BCG antigen spectrum specifically combined with the serum antibody of a tuberculosis patient is constructed, and the BCG antigen spectrum specifically combined with the serum antibody of the tuberculosis patient is established. The method is used for tuberculosis protein vaccine development and / or serum protein marker library research.

Description

Technical Field

[0001] The present invention belongs to the technical field of tuberculosis pathogen infection and immunity and protein markers thereof, and particularly relates to a serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain, and a screening method and application thereof. Background Art

[0002] Tuberculosis (TB) is one of the most lethal human infectious diseases caused by infection with Mycobacterium tuberculosis (MTB). Pulmonary tuberculosis (PTB) accounts for 85% of all cases. With timely diagnosis and appropriate medication, 85% of patients can recover. According to the notifiable infectious disease report of the Chinese Center for Disease Control and Prevention, 741,000 new cases of TB were reported in 2023, posing a daunting challenge in TB prevention and control. The Ningxia Hui Autonomous Region, located in western my country and an economically underdeveloped region, is classified as pulmonary and extrapulmonary TB, depending on the site of infection. Most extrapulmonary TB cases develop after infection with MTB in the lungs, with the pathogen further invading other tissues and organs. The latest TB diagnostic criteria (WS-288-2017) define pulmonary TB as lesions caused by MTB infection in the lungs, trachea, bronchi, and pleura, accounting for over 80% of TB cases.

[0003] Currently, tuberculosis detection methods are mainly based on methods such as etiology, pathology, molecular biology, immunology and imaging, which have significantly improved the detection rate of tuberculosis, but the various detection methods still have shortcomings in their application; in etiology, sputum acid-fast staining-microscopy and bacterial culture methods of tuberculosis patients are the gold standard for tuberculosis detection. Because sputum is easy to obtain and does not harm the tester, it has become the most commonly used clinical detection method for suspected pulmonary tuberculosis patients; however, the detection rate of sputum bacterial acid-fast staining method based on etiology is between 30-60%, which can easily lead to missed diagnosis of patients; the detection rate of sputum bacterial culture method is higher than that of acid-fast staining method, but its detection cycle is long, taking 2-8 weeks, which is not conducive to timely treatment of tuberculosis patients. In addition, sputum bacterial culture and sputum bacterial acid-fast staining of tuberculosis patients based on the etiological strategy have the following limitations: First, about 10% of the samples of sputum smear bacterial acid-fast staining are non-tuberculous mycobacteria, which require further culture and identification; second, specific detection of non-pulmonary tuberculosis infected patients is difficult; third, some patients have little or no sputum; fourth, differential diagnosis of patients with lung diseases caused by non-tuberculous mycobacteria is difficult; pathological diagnosis is also one of the important ways to confirm tuberculosis, especially in the diagnosis of difficult patients such as pulmonary tuberculosis and extrapulmonary tuberculosis with negative sputum bacteria. Tuberculosis histopathology observes granuloma formation and the morphology of multinucleated giant cells and Langerhans cells. If purulent tuberculosis without granuloma occurs, it will cause false negative results in histopathology; molecular biological detection methods based on polymerase chain reaction (PCR) are easily affected by differences in laboratory quality control and cross-contamination of external homologous nucleic acid fragments, resulting in failure of PCR amplification; enzyme-linked immunospot (ELISA) based on immunological principles is also an important way to confirm tuberculosis, especially in the diagnosis of difficult patients such as pulmonary tuberculosis and extrapulmonary tuberculosis. The ELISPOT (enzyme-linked immunosorbent assay) is the most sensitive method for tuberculosis (TB) detection, but its limitations include: first, the high mortality rate in patients with HIV and MTB co-infection, while the detection rate of immunological methods is relatively low; second, the low detection rate in the elderly and patients with other underlying diseases; third, the low detection rate in patients with organ dysfunction and those taking immunosuppressive drugs, which limits the expansion of clinical applications. Tuberculosis testing based on imaging has the following limitations: first, low specificity, resulting in the possibility of "same image for different diseases"; second, a lack of specificity in determining whether the infection is immediate or past. Consequently, these limitations lead to missed or misdiagnosed patients who are highly contagious and can develop active TB (ATB), becoming new sources of TB spread. Studies have shown that ATB patients infect an average of 10-13 people annually, and super-spreaders can infect over 200 people annually. Therefore, improving the detection rate of missed and misdiagnosed TB patients is a challenge in disease prevention and control, and a key issue that needs to be addressed by scientific researchers and medical professionals.Therefore, the development of new, rapid, and highly sensitive tuberculosis patient detection reagents is of great significance to the prevention and control of this infectious disease.

[0004] In the prior art, for example, the Chinese invention patent with patent number ZL202111432830.8 discloses a method and application for screening serum markers for tuberculosis, which comprises subjecting serum of tuberculosis patients (experimental group) and serum of healthy people (control group) to bionic affinity and affinity chromatography, respectively, to prepare bionic affinity and affinity chromatography antibody columns for the experimental group and bionic affinity and affinity chromatography antibody columns for the control group; subjecting culture filtrate of Mycobacterium tuberculosis or cytoplasmic proteins of Mycobacterium tuberculosis to chromatography on bionic affinity and affinity chromatography antibody columns for the experimental group and the control group, respectively; analyzing Mycobacterium tuberculosis proteins bound to the serum antibodies of the experimental group and the control group to preliminarily obtain serum markers for tuberculosis detection; further analyzing the above-mentioned preliminary serum markers for tuberculosis detection, and the above-mentioned method adopts a combination of bionic affinity chromatography, antigen-antibody specific recognition, mass spectrometry analysis, and bioinformatics analysis methods to achieve serum markers for tuberculosis patients. High-throughput screening and evaluation of markers solves the limitations of existing methods for screening tuberculosis serum markers. However, in the above-mentioned existing technologies, serum antibody IgG using biomimetic affinity and affinity chromatography is directly adsorbed on a solid phase carrier. When eluting the Mycobacterium tuberculosis protein (antigen) adsorbed with the serum antibody, the high-concentration serum antibody IgG specifically adsorbed on the biomimetic affinity chromatography material can be eluted together with the Mycobacterium tuberculosis protein adsorbed on the antibody. Therefore, the components of the eluate of the immunochromatography are high-concentration serum antibodies and Mycobacterium tuberculosis protein. When the polypeptide fragments of such samples after trypsin hydrolysis are subjected to LC-MS / MS detection, there is an effect that low-abundance Mycobacterium tuberculosis protein will be masked by high-abundance serum antibody IgG, making it impossible to detect low-concentration proteins, and thus the number of detected proteins is small, resulting in incomplete information in constructing the Mycobacterium tuberculosis serum marker protein library. Summary of the Invention

[0005] In view of this, the present invention provides a method for screening serum antibodies IgG that specifically recognize the antigen spectrum of its vaccine strain, so as to construct a comprehensive information library of marker proteins that are specifically recognized by serum antibodies IgG of tuberculosis patients.

[0006] It is also necessary to provide a serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain.

[0007] It is also necessary to provide an application of serum antibody IgG to specifically recognize the antigen spectrum of its vaccine strain.

[0008] The technical solution adopted by the present invention to solve its technical problem is: A method for screening serum IgG antibodies that specifically recognize the antigen spectrum of a vaccine strain comprises the following steps: S1: Screen the serum samples of the control group and the experimental group, perform biomimetic affinity and affinity chromatography on the serum proteins of the experimental group to obtain the serum antibody IgG of the experimental group; perform biomimetic affinity and affinity chromatography on the serum proteins of the control group to obtain the serum antibody IgG of the control group; S2: The serum antibody IgG of the experimental group is fixed and coupled to the activated solid phase support material by a specific chemical covalent bond to prepare the affinity antibody column of the experimental group; the serum antibody IgG of the control group is fixed and coupled to the activated solid phase support material by a specific chemical covalent bond to prepare the affinity antibody column of the control group; S3: Prepare BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein, affinity chromatography of the BCG (strain) culture filtrate or BCG (strain) cytoplasm protein with an affinity antibody column of the experimental group, and affinity chromatography of the same BCG (strain) protein sample with an affinity antibody column of the control group, to obtain BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the control group; S4: The protein information of the BCG (strain) culture filtrate or BCG (strain) cytoplasmic proteins bound to the experimental group and the control group was analyzed by high-throughput liquid chromatography-mass spectrometry; S5: Search the BCG (strain) proteomics database to obtain basic biochemical information for each protein; analyze and compare the differences between the BCG (strain) culture filtrate or BCG (strain) cytoplasmic proteins bound to the experimental group and the BCG (strain) proteins bound to the control group, eliminate the BCG (strain) proteins bound to the control group serum antibody column, and select the BCG (strain) proteins that specifically bind to the serum of tuberculosis patients as tuberculosis protein vaccine markers and / or tuberculosis serological detection markers; S6: Perform mass spectrometry data analysis, bioinformatics analysis, and immunological analysis on the tuberculosis protein vaccine markers and / or tuberculosis serological detection markers constructed in S5 to obtain the antigen spectrum of serum antibody IgG that specifically recognizes its vaccine strain.

[0009] A serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain is obtained by the screening method for serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain as described above.

[0010] The serum antibody IgG described above specifically recognizes the antigen spectrum of the vaccine strain and is used to prepare a new marker for the development of tuberculosis protein vaccines and / or a tuberculosis serological diagnostic reagent, wherein the tuberculosis includes pulmonary tuberculosis, extrapulmonary tuberculosis, sputum bacterial culture-negative tuberculosis, negative pulmonary tuberculosis and GeneXpert MTB / RIF test-negative tuberculosis.

[0011] A kit for assisting in the detection of tuberculosis, comprising the serum antibody IgG described above that specifically recognizes the antigen spectrum of its vaccine strain.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a screening method for serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain, which comprises the following steps: screening serum samples of a control group (healthy people) and an experimental group (tuberculosis patients); subjecting the serum proteins of the experimental group and the control group to bionic affinity and affinity chromatography, respectively, to obtain serum antibody IgG of the experimental group and the control group; immobilizing and coupling the serum antibody IgG of the experimental group and the control group to an activated solid phase carrier material by means of specific chemical covalent bonds, respectively, to prepare affinity antibody columns of the experimental group and the control group; subjecting the BCG (strain) culture filtrate or the BCG (strain) cytoplasmic protein to affinity chromatography with the antibody columns of the experimental group and the control group, respectively; and analyzing the affinity between the above-mentioned experimental group and the control group. The serum antibodies of the control group were combined with the BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein to preliminarily obtain new markers for the development of tuberculosis protein vaccines and / or serum markers for tuberculosis detection; the BCG proteins specifically recognized by the above-mentioned preliminary tuberculosis serum antibody IgG were further analyzed to obtain the antigen spectrum of the vaccine strain specifically recognized by the serum antibody IgG; the above method preliminarily used ELISA to evaluate the sensitivity and specificity of the specific recognition of 5 recombinantly expressed MTB proteins and BCG whole protein with serum antibodies of tuberculosis patients, so as to eliminate serum samples that failed to produce tuberculosis antibodies due to low or abnormal immunity, and then the screened serum antibodies were used for the detection of tuberculosis. Bionic affinity and affinity chromatography were performed to obtain high-purity serum antibody IgG, and the high-purity serum antibody IgG obtained after bionic affinity and affinity chromatography was covalently coupled to a solid-phase support material to prepare a tuberculosis serum antibody IgG column (experimental group); healthy human serum samples were screened using the same method to remove serum samples of latently infected people containing anti-Mycobacterium tuberculosis protein antibodies in the serum, and the screened serum antibodies were then subjected to bionic affinity and affinity chromatography to obtain high-purity serum antibody IgG, and the high-purity serum antibody IgG obtained after bionic affinity and affinity chromatography was covalently coupled to a solid-phase support material to prepare a healthy human serum antibody IgG column (control group); After affinity chromatography between different types of serum antibody IgG columns and BCG (strain) total protein, when the BCG (strain) total protein antigen bound thereto is eluted, the high-purity serum antibody IgG that specifically binds to the BCG (strain) total protein will not fall off, and no antigen-antibody mixture will be produced. When the sample is tested by LC-MS / MS, there is no effect of low-abundance Mycobacterium tuberculosis protein being masked by high-abundance serum antibody IgG, so that both low-concentration proteins and high-concentration proteins can be detected, and the number of detected proteins is large, so that the new markers for the development of tuberculosis protein vaccines and / or the serum marker library for tuberculosis testing are more comprehensive and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The SDS-PAGE evaluation results of the recombinant MTB protein purified by nickel metal chelate chromatography.

[0014] Figure 2 These are the results of ELISA tests for five recombinant MTB proteins and BCG whole protein antigen antibodies.

[0015] Figure 3 This is the SDS-PAGE diagram of the serum antibody IgG components in affinity chromatography coupled with SPA filler.

[0016] Figure 4 Grayscale analysis of affinity ligand-coupled SPA-purified human serum antibody IgG.

[0017] Figure 5 To evaluate the adsorption performance of Sepharose-6FF-A133-IgG affinity ligand.

[0018] Figure 6 SDS-PAGE was used to evaluate the adsorption performance of the Sepharose-6FF-A133-IgG affinity ligand.

[0019] Figure 7 The results show the detection of BCG cytoplasmic protein in serum by mass spectrometry in healthy subjects and patients with pulmonary tuberculosis sputum smear (+).

[0020] In the picture: Figure 1 Figure A shows the soluble expression and nickel metal chelate chromatography separation and purification of the protein encoded by the recombinant Mycobacterium tuberculosis Rv0222 gene; 1: flow-through; M: Marker; 2-3: wash solution; 4-7: soluble expression protein eluate; Figure B shows the nickel metal chelate chromatography separation and purification of the recombinant Mycobacterium tuberculosis Rv1980c gene-encoded protein; M: Marker; 1: flow-through; 2-3: wash solution; 4-7: inclusion body protein eluate; Figure C shows the soluble expression and nickel metal chelate chromatography separation and purification of the protein encoded by the recombinant Mycobacterium tuberculosis Rv0577 gene Figure 2: chemiluminescence; M: Marker; 1: pET28a stock bacterial sludge; 2: protein supernatant after ultrasonic lysis; 3: soluble expression protein eluate; Figure D shows the soluble expression and nickel metal chelate chromatography separation and purification of the protein encoded by the recombinant Mycobacterium tuberculosis Rv2031c gene; M: Marker; 1: pET28a stock bacterial sludge; 2: protein supernatant after ultrasonic lysis; 3: soluble expression protein eluate; Figure E shows the nickel metal chelate chromatography separation and purification of inclusion bodies of the protein encoded by the recombinant Mycobacterium tuberculosis Rv0934 gene. Figure 2: chemiluminescence; M: Marker; 1: pET28a stock bacterial sludge; 2: protein supernatant after ultrasonic lysis; 3: inclusion body protein purification flow-through.

[0021] Figure 3Figure A shows healthy human serum after affinity chromatography on Sepharose-6FF-A73-SPA; Figure B shows healthy human serum after affinity chromatography on Sepharose-6FF-A74-SPA; Figure C shows healthy human serum after affinity chromatography on Sepharose-6FF-A75-SPA; Figure D shows healthy human serum after affinity chromatography on Sepharose-6FF-A77-SPA; Figure E shows healthy human serum after affinity chromatography on Sepharose-6FF-A78-SPA; Figure F shows healthy human serum after affinity chromatography on Sepharose-6FF-A87-SPA; Figure G shows healthy human serum after affinity chromatography on Sepharose-6FF-A133-SPA; where 1: original serum; 2: flow-through; 3-4: wash solution; 5-8: eluent; M: Marker.

[0022] Figure 5 Figure A in the middle shows the affinity chromatography of healthy human serum antibody Sepharose-6FF-A133-IgG pET28a-SPA- BL21 (DE3) ; Figure B shows a blank Sepharose-6FF matrix affinity chromatography pET28a-SPA- BL21(DE3) 1: SPA protein sample; M: Marker; 2: Flow-through; 3-4: Washing solution; 5-8: Eluent.

[0023] Figure 7 Figures A and B are Venn diagrams of protein data detected after searching the MTB and BCG databases, respectively, using serum antibody affinity chromatography on BCG cytoplasmic protein elution fractions from serum of healthy persons and patients with pulmonary tuberculosis sputum smear (+). Figures C and D are protein data detected after searching the BCG database, using serum antibody affinity chromatography on BCG cytoplasmic protein flowthrough fractions from serum of healthy persons and patients with pulmonary tuberculosis sputum smear (+). DETAILED DESCRIPTION

[0024] The technical solutions and technical effects of the embodiments of the present invention are further elaborated below in conjunction with the accompanying drawings of the present invention.

[0025] A method for screening serum IgG antibodies that specifically recognize the antigen spectrum of a vaccine strain comprises the following steps: S1: Screen the serum samples of the control group and the experimental group, perform biomimetic affinity and affinity chromatography on the serum proteins of the experimental group to obtain the serum antibody IgG of the experimental group; perform biomimetic affinity and affinity chromatography on the serum proteins of the control group to obtain the serum antibody IgG of the control group; S2: The serum antibody IgG of the experimental group is fixed and coupled to the activated solid phase support material by a specific chemical covalent bond to prepare the affinity antibody column of the experimental group; the serum antibody IgG of the control group is fixed and coupled to the activated solid phase support material by a specific chemical covalent bond to prepare the affinity antibody column of the control group; S3: Prepare BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein, affinity chromatography of the BCG (strain) culture filtrate or BCG (strain) cytoplasm protein with an affinity antibody column of the experimental group, and affinity chromatography of the same BCG (strain) protein sample with an affinity antibody column of the control group, to obtain BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the control group; S4: The protein information of the BCG (strain) culture filtrate or BCG (strain) cytoplasmic proteins bound to the experimental group and the control group was analyzed by high-throughput liquid chromatography-mass spectrometry; S5: Search the BCG (strain) proteomics database to obtain basic biochemical information for each protein; analyze and compare the differences between the BCG (strain) culture filtrate or BCG (strain) cytoplasmic proteins bound to the experimental group and the BCG (strain) proteins bound to the control group, eliminate the BCG (strain) proteins bound to the control group serum antibody column, and select the BCG (strain) proteins that specifically bind to the serum of tuberculosis patients as tuberculosis protein vaccine markers and / or tuberculosis serological detection markers; S6: Perform mass spectrometry data analysis, bioinformatics analysis, and immunological analysis on the tuberculosis protein vaccine markers and / or tuberculosis serological detection markers constructed in S5 to obtain the antigen spectrum of serum antibody IgG that specifically recognizes its vaccine strain.

[0026] Specifically, the serum antibody IgG that specifically recognizes the protein in the vaccine strain antigen spectrum library is expressed through genetic engineering and separated and purified by nickel metal chelate chromatography, and then evaluated using an immunological method of antigen-antibody specific recognition, including but not limited to indirect enzyme-linked immunosorbent assay (ELISA), to verify the sensitivity and specificity of the screened serum antibody IgG in specifically recognizing the vaccine strain antigen spectrum.

[0027] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a screening method for serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain, which comprises the following steps: screening serum samples of a control group (healthy people) and an experimental group (tuberculosis patients); subjecting the serum proteins of the experimental group and the control group to bionic affinity and affinity chromatography, respectively, to obtain serum antibody IgG of the experimental group and the control group; immobilizing and coupling the serum antibody IgG of the experimental group and the control group to an activated solid phase carrier material by means of specific chemical covalent bonds, respectively, to prepare affinity antibody columns of the experimental group and the control group; subjecting the BCG (strain) culture filtrate or the BCG (strain) cytoplasmic protein to affinity chromatography with the antibody columns of the experimental group and the control group, respectively; and analyzing the affinity of the serum antibodies of the experimental group and the control group. The method comprises the following steps: first, analyzing the BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound to the BCG, and preliminarily obtaining a novel marker for the development of tuberculosis protein vaccine and / or a serum marker for tuberculosis detection; further analyzing the BCG protein specifically recognized by the above-mentioned preliminary tuberculosis serum antibody IgG, and obtaining the antigen spectrum of the vaccine strain specifically recognized by the serum antibody IgG; the above method preliminarily uses the ELISA method to evaluate the sensitivity and specificity of the specific recognition of 5 recombinantly expressed MTB proteins and BCG whole protein with the serum antibodies of tuberculosis patients, so as to remove serum samples that fail to produce tuberculosis antibodies due to low or abnormal immunity, and then performing biomimetic affinity and affinity chromatography on the screened serum antibodies. High-purity serum antibody IgG was obtained, and the high-purity serum antibody IgG after biomimetic affinity and affinity chromatography was covalently coupled to a solid phase carrier material to prepare a tuberculosis serum antibody column IgG column (experimental group); the same method was used to screen healthy human serum samples to remove serum samples of latently infected people containing anti-Mycobacterium tuberculosis protein antibodies in the serum, and then the screened serum antibodies were subjected to biomimetic affinity and affinity chromatography to obtain high-purity serum antibody IgG, and the high-purity serum antibody IgG after biomimetic affinity and affinity chromatography was covalently coupled to a solid phase carrier material to prepare a healthy human serum antibody IgG column (control group); different types of serum antibody IgG columns were combined with BCG (bacteria After affinity chromatography of the total protein of the BCG (strain) (a mixture of equal amounts of BCG culture filtrate protein and BCG cytoplasmic protein), when the bound BCG (strain) total protein antigen is eluted, the highly pure serum antibody IgG that specifically binds to the BCG (strain) total protein will not fall off, and no antigen-antibody mixture will be produced. When the sample is tested by LC-MS / MS, there is no effect of low-abundance Mycobacterium tuberculosis protein being masked by high-abundance serum antibody IgG, so that both low-concentration proteins and high-concentration proteins can be detected, and the number of detected proteins is large, so that the information of the new markers for the development of tuberculosis protein vaccines and / or the serum marker library for tuberculosis testing is more comprehensive and accurate.

[0028] Furthermore, the screening of serum samples from the control group and the experimental group in step S1 is specifically as follows: BCG cytoplasmic protein and the prepared MTB recombinant protein antigen are coated on a solid phase carrier, the serum sample to be tested is added, and the level of serum antibodies is detected by enzyme-labeled color development, and the serum samples from the control group and the experimental group are screened to remove serum samples in the experimental group that do not produce tuberculosis antibodies and serum samples of latent tuberculosis infected persons containing anti-Mycobacterium tuberculosis antibodies in the control group, thereby improving the accuracy of serum antibody IgG in the experimental and control group samples.

[0029] Furthermore, in step S1, serum proteins are subjected to bionic affinity and affinity chromatography to obtain serum antibody IgG; specifically, the serum proteins are purified by bionic affinity and affinity chromatography respectively, and then the serum purified by bionic affinity and affinity chromatography is mixed in equal mass, and the mixed serum is pretreated to obtain serum antibody IgG with high diversity and high purity, and the purity of serum antibody IgG reaches more than 85%.

[0030] In the prior art, in the Chinese invention patent with patent number ZL202111432830.8, specific antibody adsorption is performed during serum antibody purification, and then the bionic affinity or affinity chromatography column adsorbed with the antibody is directly subjected to immunochromatography with tuberculosis antigens. Therefore, this method cannot elute serum antibody IgG from the chromatography column, and the antibody-antigen specific adsorption experiment is performed directly on the bionic affinity chromatography column; therefore, the antibodies of the bionic affinity filler and the affinity filler can only be specifically adsorbed with tuberculosis antigens respectively. This patent purifies the serum antibodies first, that is, elutes them from the chromatography column, then pre-treats them, and finally covalently couples the mixed antibodies to agarose gel, so the serum antibody IgG has high diversity and high purity.

[0031] Furthermore, in the step S2, the activated solid phase support material is prepared by the following method: the activated solid phase support material is synthesized by epoxy activation reaction, amination reaction and cyanuric chloride substitution reaction.

[0032] Furthermore, before the S2 step, it is necessary to screen affinity materials that specifically bind to serum IgG to obtain the optimal affinity material for specific adsorption of serum IgG. Specifically, seven affinity materials activated with different spacer arms (epoxy activation-amination-cyanuric chloride substitution reaction) are coupled to Staphylococcus aureus protein A (SPA), and different adsorption effects are obtained by affinity chromatography on healthy human serum IgG collected clinically. After verification by SDS-PAGE gel electrophoresis, grayscale analysis of protein purity differences is performed to screen out the optimal affinity material for specific adsorption of serum IgG.

[0033] Furthermore, the S3 step also includes eluting the BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound to the serum antibodies of the experimental group and the BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound to the serum antibodies of the control group, so as to wash off the bound BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein respectively.

[0034] Furthermore, in step S3, the BCG (strain) culture filtrate or BCG (strain) cytoplasmic protein used includes culture filtrate protein from BCG (strain) culture or BCG (strain) cell cytoplasmic protein. The prepared BCG (strain) culture filtrate or cytoplasmic protein is chromatographed on an affinity antibody column for the experimental group and an antibody column for the control group, respectively. Furthermore, the BCG (strain) culture filtrate or cytoplasmic protein not adsorbed by the affinity chromatography column is washed, and the protein concentration of the different eluted fractions is quantitatively analyzed using the BCA assay. Ultrafiltration is used to concentrate all protein components to be analyzed by mass spectrometry to an optimal concentration suitable for mass spectrometry, such as between 2 mg and 10 mg / mL. The flow-through and eluted fractions are digested with trypsin and then dried for mass spectrometry analysis.

[0035] Furthermore, the eluent is PBS or glycine-HCl. In one embodiment, the eluent is Gly-HCl with a pH of 1.5-3.0.

[0036] Furthermore, the S5 includes the following steps: searching the database (http: / / www.uniprot.org / ) for BCG (strain) proteins detected by mass spectrometry that bind to the experimental group and the control group, respectively; removing BCG (strain) proteins that are specifically adsorbed by serum antibodies of the control group, and using the removed BCG (strain) proteins of the experimental group to construct a serum antibody IgG library that specifically recognizes the vaccine strain antigen; Furthermore, the S6 includes the following steps: analyzing the molecular weight, isoelectric point, hydrophilicity, subcellular localization, whether it is a secretory protein and the possible secretion mode of the proteins in the BCG (strain) protein library; screening out proteins with high abundance, strong sequence specificity and strong immunogenicity in the BCG (strain) protein library as candidate markers for the serum antibody IgG of the present invention to specifically recognize the antigen spectrum of its vaccine strain; the serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain screened by the above-mentioned bionic affinity chromatography-mass spectrometry strategy or the affinity chromatography-mass spectrometry strategy is recombinantly expressed in Escherichia coli, and the target protein is separated and purified by nickel metal chelate chromatography to obtain a high-purity recombinant target protein; finally, the indirect ELISA method is used to evaluate the detection sensitivity and specificity of the recombinant tuberculosis protein in the serum of tuberculosis patients, and finally the serum antibody IgG of the present invention that specifically recognizes the antigen spectrum of its vaccine strain is screened out.

[0037] A serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain is obtained by the screening method for serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain as described above.

[0038] The serum antibody IgG described above specifically recognizes the antigen spectrum of the vaccine strain and is used for preparing a new marker for the development of tuberculosis protein vaccines and / or a tuberculosis serological diagnostic reagent, characterized in that the tuberculosis includes pulmonary tuberculosis, extrapulmonary tuberculosis, tuberculosis with negative sputum bacterial culture, negative pulmonary tuberculosis and GeneXpert MTB / RIF test-negative tuberculosis.

[0039] Furthermore, the pulmonary tuberculosis is extrapulmonary tuberculosis and sputum bacterial culture-negative tuberculosis.

[0040] A kit for assisting in the detection of tuberculosis, comprising the serum antibody IgG described above that specifically recognizes the antigen spectrum of its vaccine strain.

[0041] The use of the present invention is described by way of the following examples. Example 1:

[0042] S1: Screen the serum samples of the control group and the experimental group, perform biomimetic affinity and affinity chromatography on the serum proteins of the experimental group to obtain a rich variety of serum antibodies IgG of the experimental group; perform biomimetic affinity and affinity chromatography on the serum proteins of the control group to obtain a rich variety of serum antibodies IgG of the control group; 1. Screening of serum samples from the control group and experimental group: Five homemade recombinant MTB proteins and BCG cytoplasmic proteins were used as antigens. ELISA was used to screen patient serum samples that showed strong recognition of all of the above proteins as the experimental group. Healthy human serum that did not recognize the five recombinant MTB proteins and BCG cytoplasmic proteins served as the control group and was used for subsequent experiments and the preparation of the control group antibody column. (1) Coating: Recombinant Mycobacterium tuberculosis proteins MPT64 (Rv1980c), echA1 (Rv0222), CFP32 (Rv0577), HspX (Rv2031c), pstS1 (Rv0934), and BCG cytoplasmic complete protein were diluted to 5 μg / mL in coating buffer and 100 μL was added to each well at 4°C overnight. The 96-well plate was washed three times with 1× PBST buffer for 3 min each time. (2) Blocking: 1× PBS with 5% skim milk powder, add 250 μL to each well, and incubate at 37°C for 2 h. Wash the plate three times with 1× PBST buffer, each time for 3 min. (3) Sample addition: Dilute the serum sample to be tested with 5% skim milk powder (set up a blank control group at the same time), add 100 μL of the diluted serum sample to each well, and incubate at 37°C for 1 h. Wash the 96-well plate three times with 1× PBST buffer, each time for 3 min. (4) Enzyme-labeled secondary antibody: Add HRP-labeled goat anti-human antibody to a skim milk powder solution prepared in 1× PBS at a dilution of 1:5000; add 100 μL of secondary antibody to each well and incubate at 37°C for 1 h. Wash the plate three times with 1× PBST buffer, each time for 3 min. (5) Color development: Add 50 μL of TMB color development solution (solution A, solution B) to each well, protect from light, and incubate at 37°C for 10 min.

[0043] (6) Termination of reaction: Add 50 μL of 2 mol / L sulfuric acid solution to each well to terminate the reaction.

[0044] (7) Result determination: Place the ELISA plate on an ELISA reader and read the result at 450 nm.

[0045] The serum samples of 87 tuberculosis patients and 81 healthy people were analyzed by ELISA method (1)-(7) above. The ELISA results were analyzed by GraphpadPrism to screen out 12 serum samples of control group (healthy people) and 12 serum samples of experimental group (tuberculosis patients); The SDS-PAGE results of the recombinant MTB protein nickel metal chelate chromatography separation and purification experiment are as follows Figure 1 The results of ELISA tests on five recombinant MTB proteins and BCG whole protein antigen antibodies are shown in Figure 2 As shown; ELISA results were analyzed by GraphpadPrism to screen out serum samples from healthy individuals and tuberculosis patients.

[0046] 2. Preparation of high-purity serum antibody IgG: Twelve serum antibodies from tuberculosis patients with the highest sensitivity to five recombinant MTB proteins and BCG cytoplasmic proteins (highest OD450 values ​​measured by microplate reader) screened by ELISA were mixed at a ratio of 0.5 mL per person to form a 6 mL tuberculosis patient serum antibody group (experimental group); twelve serum antibodies from healthy individuals with the lowest OD450 values ​​measured by microplate reader were mixed at a ratio of 0.5 mL per person to form a 6 mL healthy individual serum antibody group (control group); The serum proteins of the experimental group and the control group were purified by biomimetic affinity and affinity chromatography, respectively (the biomimetic affinity purification was the same as that in the paper (1) Guorong Ma, Ruiqiang Xu, Wang Pei, Yang Zhang, Rang Yang, Zhihao Zhao, Yating Li, Chenjie Feng, Dongwu Jin, Wei Ma, Yumei Ma, Zhongren Ma, Screening and validation of an alkaline-tolerant biomimetic affinity chromatography A5-87 resin for purification with the discarded bovine serum Immunoglobulin G, [J] Journal of Chromatography A, 1714 (2023) 464580, https: / / doi.org / 10.1016 / j.chroma. 2023.464580). Then, the serum purified by biomimetic affinity and affinity chromatography was mixed in equal mass, and the mixed serum was pretreated to obtain serum antibody IgG with high diversity and purity. The pretreatment was as follows: (1) SDS-PAGE gel electrophoresis (qualitative) was used to evaluate the purity of serum antibody IgG in biomimetic affinity and affinity chromatography samples; then grayscale scanning software was used to evaluate the purity of serum antibody IgG after purification by the two methods; (2) serum antibody IgG purified by the two methods were transferred into dialysis bags respectively, and dialyzed in 0.5 mol pH 8.5 Na2SO4 solution at 4-10 ° C for 4-6 h; (3) BCA protein quantification method was used to detect the concentration of serum antibody IgG after dialysis by biomimetic affinity chromatography and affinity chromatography respectively; (4) serum antibody IgG samples purified by the two methods were mixed in a 1:1 ratio.

[0047] The purity of serum IgG antibody in the experimental group reached more than 85%, and the purity of serum IgG antibody in the control group reached more than 85%.

[0048] S2: The serum antibody IgG of the experimental group is fixed and coupled to the activated solid phase support material by a specific chemical covalent bond to prepare the affinity antibody column of the experimental group; the serum antibody IgG of the control group is fixed and coupled to the activated solid phase support material by a specific chemical covalent bond to prepare the affinity antibody column of the control group; 1. Screening of the best affinity material: Seven types of Sepharose-6FF-SPA affinity fillers with different lengths of spacer arms were synthesized; the prepared affinity materials with different spacer arms were covalently coupled to the alkali-resistant recombinant SPA on agarose microspheres after activation (epoxy activation-amination-cyanuric chloride substitution reaction) of different spacer arms. The 12 serum samples with the lowest OD values ​​screened by ELISA were mixed in equal amounts and affinity chromatography was performed on healthy human serum IgG to screen out affinity materials that could specifically adsorb serum antibody IgG. Figure 3 As shown, 12% SDS-PAGE gel electrophoresis verification showed that Sepharose-6FF-A133-SPA (Figure G) was the best affinity purification material for serum antibodies.

[0049] The grayscale analysis of the eluted fractions was performed using Image Lab. The purity of the serum antibody IgG eluted fractions of Sepharose-6FF-A73-SPA affinity chromatography was 49.1%, 56.9%, 51.9%, and 42.2%; the purity of the serum antibody IgG eluted fractions of Sepharose-6FF-A74-SPA affinity chromatography was 49.1%, 56.9%, 51.9%, and 42.2%; The purities of the serum antibody IgG elution fractions by affinity chromatography were 68.2%, 47.2%, 40%, and 31.3%; the purities of the serum antibody IgG elution fractions by Sepharose-6FF-A75-SPA affinity purification were 13.4%, 8.2%, 6.8%, and 8.1%; the purities of the serum antibody IgG elution fractions by Sepharose-6FF-A77-SPA affinity purification were 9.6% and 26.9%; the purities of the serum antibody IgG elution fractions by Sepharose-6FF-A78-SPA affinity purification were 57.8%, 59.4%, 27.1%, and 15.2%; the purities of the serum antibody IgG elution fractions by Sepharose-6FF-A87-SPA affinity chromatography were 41.4%, 31.6%, 17.9%, and 15.4%; and the purities of the serum antibody IgG elution fractions by Sepharose-6FF-A133-SPA affinity purification were 90%, 93.8%, 89.6%, and 85.1%. After affinity chromatography of human serum antibody IgG on Sepharose-6FF-A133-SPA, Sepharose-6FF-A73-SPA, Sepharose-6FF-A75-SPA, and Sepharose-6FF-A77-SPA, the grayscale analysis of the SDS-PAGE elution fractions was performed between the two groups. The t-test was performed, and P < 0.05, indicating that the affinity chromatography of serum IgG on Sepharose-6FF-A133-SPA was statistically significant with each group, and the purity of the grayscale analysis of the SDS-PAGE elution fractions of human serum antibody IgG on Sepharose-6FF-A133-SPA affinity chromatography was the highest. The results are shown as follows Figure 4As shown in the figure, Sepharose-6FF-A133-SPA is the affinity material with the best specific adsorption of serum IgG in affinity chromatography.

[0050] 2. Preparation of activated solid support material: (1) Epoxy activation reaction: Weigh 210 g of Sepharose 6FF agarose, wash it 10 times with ultrapure water, remove the drained Sepharose 6FF agarose, and add 441 mL (VDMSO:VECH = 6:4) epoxy activation solution. Shake the epoxy activation solution in the reaction system, add 8.0 g of NaOH pellets, react for 1 hour, and measure the pH to 10.8. Add 8.0 g of NaOH pellets, and measure the pH to 10.5 after 1.5 hours. Add 8.0 g of NaOH and continue the reaction, and measure the pH to 10.2. The reaction is continued for a total of 4 hours. Cleaning: Wash 10 times with 10 times the volume of ultrapure water, ultrafilter to dry the water, and store at 4°C for later use.

[0051] (2) Amination reaction: Weigh 175 g of epoxy-activated Sepharose 6FF and divide it into 7 groups of 25 g each. The amination reaction solution is composed of 20% of different amino compounds and 80% ultrapure water. Add 52.5 mL of amination solution to each group. The amination reaction conditions are: 60°C, 170 rpm, and 16 h. Washing: Wash 10 times with 10 times the volume of ultrapure water, drain the water by ultrafiltration, and store at 4°C until use.

[0052] (3) Cyanuric chloride substitution reaction: Add 0.16 M cyanuric chloride in acetone solution to the Sepharose-6FF after the amination reaction. The cyanuric chloride in acetone solution has been pre-frozen for 4 h (-20°C). Mix thoroughly and add Na2CO3 particles. Adjust the pH to 7-8. Incubate on an ultra-low temperature shaker at 4°C for 4 h. Wash: Wash 10 times with 10 times the volume of ultrapure water, then drain the water by ultrafiltration and set aside.

[0053] 2. Preparation of affinity antibody columns for experimental and control groups: The serum IgG elution fractions from different groups of serum were collected by Sepharose-6FF-A133-SPA affinity chromatography, and equal masses of serum IgG from tuberculosis patients were mixed and dialyzed against 1 moL of Na2SO4 (pH 8.5). The mixed fractions were then mixed with the agarose microspheres activated (epoxy activation-amination-cyanuric chloride substitution reaction) as described above, and the mixtures were placed in a constant temperature shaker at 25°C and 170 rpm for 24 h to prepare a serum antibody IgG chromatography column (experimental group). During the reaction, 100 μL of the reaction solution was collected at 0 h, 2 h, 12 h, and 24 h for BCA protein quantification. A healthy human serum antibody IgG chromatography column (control group) was prepared using the same method.

[0054] The specific adsorption purity of Sepharose-6FF-A133-SPA for serum IgG antibodies from tuberculosis patients and healthy subjects was 70% and 86%, respectively.

[0055] 3. Verify whether the preparation of different groups of antibodies is successful.

[0056] SPA specifically binds to the Fc segment of IgG antibodies in human serum. Different groups of Sepharose-6FF-A133-IgG affinity ligands and blank Sepharose-6FF control group were used for affinity chromatography of pET28a-SPA- BL21(DE3) Protein, evaluate the affinity ligand adsorption performance, and verify by 12% SDS-PAGE gel electrophoresis, such as Figure 5 shown.

[0057] Different groups of Sepharose-6FF-A133-IgG affinity chromatography recombinant SPA, 6-8 as the purified eluent, the experimental results were analyzed by Image Lab, the purity was 55%, 78.5%, 79.6%, and 81.4% respectively; the purity of the 5-8 eluents of SPA protein purified by blank Sepharose-6FF matrix was 1.6%, 3.0%, and 7.2% respectively. The grayscale analysis of the two groups of eluents was performed by t-test, P < 0.0001, as shown in Figure 5. Figure 6 shown.

[0058] The results showed significant differences in adsorption between the Sepharose-6FFA133-IgG affinity material and the blank Sepharose-6FF affinity chromatography recombinant SPA. The results also demonstrated that high-purity serum antibodies from different groups were covalently bound to activated agarose microspheres (epoxy activation-amination-cyanuric chloride substitution reaction) using a chemical coupling method, successfully preparing serum antibody columns from different groups.

[0059] S3: preparing BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein, affinity chromatography of the BCG (strain) culture filtrate or BCG (strain) cytoplasm protein with an affinity antibody column of the experimental group, and affinity chromatography of the same BCG (strain) protein sample with an affinity antibody column of the control group, obtaining BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the control group, and eluting the BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the control group; Prepared BCG (strain) culture filtrate or BCG (strain) cell cytoplasmic proteins were chromatographed on an experimental affinity antibody column or a control affinity antibody column, respectively, followed by washing with Gly-HCl at pH 3.0. The experimental affinity antibody column was a column prepared from IgG antibodies from tuberculosis patient serum, while the control affinity antibody column was a column prepared from IgG antibodies from healthy human serum.

[0060] Steps S4, S5, and S6 are as follows: Liquid chromatography-tandem mass spectrometry analysis of eluted fractions: Bacillus Calmette-Guérin (BCG) strain protein fractions adsorbed by serum antibody IgG were collected from the experimental and control groups. All samples were enzymatically digested with Trypsin. After Ziptip desalting, the peptides were adjusted to a concentration of 0.01 mg / mL to 10 mg / mL and analyzed by LC-MS / MS (Thermo Fusion Lumos). The mass spectrometer was connected to an Easy-NLC1200 via a spray device. A 2 μm reversed-phase C18 resin (PepMap RSLC) was used on a 15 cm column with an inner diameter of 0.075 mm. A linear gradient separation was performed at a flow rate of 300 nL / min, from 95% solvent A (0.1% formic acid, 2% acetonitrile, 98% water) to 28% solvent B (0.1% formic acid, 80% acetonitrile) over 60 min. The spray voltage was 2.1 kV, the ion transfer capillary temperature was 275°C, and the radiofrequency lens was 60%. The mass spectrometer was operated in positive ion mode, with automatic switching between MS and MS / MS in data-dependent mode using the TUNE and Xcalbur 4.0.27.19 software packages. A full MS scan was acquired from 350 to 1500 m / z at a high resolution of R = 60,000 (defined at m / z = 400). MS / MS fragmentation analysis was performed for ions with charge states 2-7 and a collision energy of 30%, yielding the 20 most abundant multiply charged ion fragments.

[0061] Database search and data analysis LC-MS / MS ion spectra were processed, mass spectrometrically detected, and database searched using BioInformation Solutions. The Mycobacterium bovis (strain BCG / Pasteur 1173P2, 3877 proteins) library was searched against the UniProt protein database to retrieve the BCG cytoplasmic whole protein from serum antibody affinity chromatography of healthy individuals and tuberculosis patients. Specific and differential protein species were screened in the BCG whole protein from serum antibody affinity chromatography of patients with positive tuberculosis sputum smears. A spectrum of BCG protein antigens specifically recognized by tuberculosis serum was constructed, such as Figure 7 shown.

[0062] Using the proteolytic peptide information from the UniProt protein database for Mycobacterium bovis (strain BCG / Pasteur 1173P2, 3877), we searched and compared BCG proteolytic peptide fragments detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) that were adsorbed by serum IgG to identify the corresponding BCG protein. Finally, we analyzed and predicted the basic biochemical properties and functions of the detected BCG protein.

[0063] The recognition specificity between serum antibodies (healthy people, tuberculosis patients) specifically adsorbed on the antibody column and BCG proteins / antigens is affected by the following factors: first, the specific recognition between low-concentration antibodies in healthy people caused by BCG (live attenuated bovine tuberculosis vaccine) vaccination and Mycobacterium tuberculosis antigens; second, the specific recognition of antibodies against pathogen antigens produced by healthy people with normal immunity when infected by aerosols containing Mycobacterium tuberculosis complex bacteria in the air; third, the non-specific binding of antibodies in human serum to individual BCG proteins; (4) the non-specific binding between affinity chromatography columns and BCG proteins. The theoretical probability of this situation occurring depends on the choice of the final detection tool. If the immunoblotting (Western-blot, WB) method is used, the above-mentioned non-specific proteins may not be obtained; if high-sensitivity LC-MS / MS detection is used, the detection rate of the above-mentioned non-specific proteins will be enhanced. Therefore, setting reasonable screening criteria can reduce the interference factors in the judgment of experimental results to a certain extent.

[0064] The LC-MS / MS analysis of BCG protein information recognized by tuberculosis patient serum antibodies or healthy control group serum antibodies sets the following screening criteria: (1) BCG protein is identified by one or more peptide sequences that specifically bind to tuberculosis patient serum antibodies and is initially included in the candidate protein range; (2) Whether BCG protein that binds to both tuberculosis patient serum antibodies and healthy person serum antibodies is listed as a candidate serum marker depends on the mass spectrometry detection data results of the same protein detected in the experimental group and the control group. If the number of peptides identified by mass spectrometry in the two components is equal to the coverage of the peptide sequence or the difference is not significant, the protein cannot be used as a component of the vaccine strain antigen spectrum antigen library specifically recognized by serum antibody IgG; otherwise, the BCG protein is listed as a candidate pathogen protein spectrum for tuberculosis detection. Based on the above criteria, we finally queried the protein Uniprot database and found a total of 3877 proteins in the Mycobacterium bovis (strain BCG / Pasteur1173P2) database. LC-MS / MS detected 2649 and 3281 proteins in the BCG protein elution and flow-through fractions of healthy subjects and sputum smears of patients with tuberculosis (+) by Sepharose-6FF-A133-IgG affinity chromatography, respectively. Figure 7 As shown in the figure, the analysis data showed that the protein detection rates were 68% and 85%, respectively. The BCG proteome database was searched to screen out high-abundance proteins that were specifically adsorbed by BCG on Sepharose-6FF-A133-IgG affinity chromatography in the serum of patients with pulmonary tuberculosis sputum smears (+).

[0065] LC-MS / MS detection data analysis, search the BCG protein database, and draw a Venn diagram, such as Figure 7 As shown in the figure, 52 proteins were specifically adsorbed to the BCG cytoplasmic total protein eluate after Sepharose-6FF-A133-IgG affinity chromatography of serum from patients with positive pulmonary tuberculosis sputum smears. Data comparison revealed one protein with a coverage greater than 10%, 14 with a coverage of 5%-10%, and the remaining proteins with a coverage below 5%. Bioinformatics analysis revealed 19 enzyme proteins, 8 unidentified proteins, 7 domain-containing proteins, 6 family proteins, 6 membrane proteins, and 2 transcriptional regulatory proteins. The remaining proteins included iron uptake proteins, conserved lipoproteins, phosphomannose proteins, and proteins of the UvrABC system.

[0066] LC-MS / MS analysis was performed to identify differentially expressed proteins in the BCG cytoplasmic protein eluate fractions from different groups of serum samples after Sepharose-6FF-A133-IgG affinity chromatography. A total of 103 significantly upregulated proteins were identified in the intersecting serum samples from healthy individuals and patients with positive pulmonary tuberculosis sputum smears. Proteins were found to be differentially expressed in the BCG cytoplasmic protein eluate fractions from different serum groups after Sepharose-6FF-A133-IgG affinity chromatography. Of these 103 proteins, 22 were screened for differential expression with a ratio greater than 1.5. Bioinformatics analysis revealed 5 membrane proteins, 5 enzyme proteins, 3 domain-containing proteins, and 2 unidentified proteins. The remaining proteins included ubiquitinated proteins, transcriptional regulatory proteins, conserved secretory proteins, antitoxin proteins, serine-rich proteins, tuberculin-related peptide proteins, and inv proteins.

[0067] Therefore, sera from tuberculosis patients and healthy controls were subjected to chromatography on the affinity material Sepharose-6FF-A133-IgG, followed by enzymatic digestion of BCG cytoplasmic protein fractions and LC-MS / MS analysis. A total of 2,649 BCG proteins were detected, and 74 differentially expressed proteins were screened (Table 1). The specific BCG proteins recognized by serum IgG from patients with positive sputum smears and the differentially expressed proteins were screened, thereby constructing a repertoire of BCG antigens specifically recognized by serum antibodies from tuberculosis patients.

[0068] Table 1 List of BCG proteins specifically recognized by serum antibodies from tuberculosis patients (partial proteins)

[0069] Bacillus Calmette-Guerin (BCG) is the only vaccine approved by the World Health Organization (WHO) for the prevention of tuberculosis. The genetic identity between the standard strain of Mycobacterium tuberculosis and the BCG strain is as high as 98%. Both belong to the same strain of the Mycobacterium tuberculosis complex. BCG is derived from bovine tuberculosis bacteria that have been cultured for more than 230 generations in a medium containing ox bile, reducing the virulence of the pathogen and producing a live vaccine that is harmless to humans and can produce immunity. Compared to the M. bovis strain, BCG lacks five regions encoding a total of 38 open reading frames. The attenuated M. bovis strain, BCG, lacks 16 regions compared to the MTB-H37Rv strain and has a total of 129 open reading frames. The complete genome of BCG Pasteur strain 1173P2 has been sequenced. It measures 4,374,522 base pairs (bp) and contains 3,954 protein-encoding genes. Although BCG is a strain attenuated by passage from Mycobacterium bovis, its genome is still 30 kilobases larger than that of Mycobacterium bovis AF2122 / 97, primarily due to the presence of two independent, tandemly repeated segments, DU1 and DU2. BCG can enhance protection against miliary tuberculosis and tuberculous meningitis in children, with a protective effect of up to 86%. Due to limitations in tuberculosis culture facilities and equipment, as well as biosafety, the present invention does not use the complete proteins (cytoplasmic proteins and culture filtrate proteins) of the standard strain of Mycobacterium tuberculosis H37Rv as the antigen mixture for affinity adsorption with tuberculosis patient serum antibody IgG. Instead, the vaccine strain Mycobacterium bovis (strain BCG / Pasteur1173P2), which has good biosafety and is highly homologous (with a genetic homology of up to 98%), is selected as the antigen mixture for affinity adsorption with tuberculosis patient serum antibody IgG.

[0070] Compared with the existing patent No. ZL202111432830.8, the present invention screens out a large number of BCG (strain) proteins, and both low-abundance Mycobacterium tuberculosis proteins and high-abundance Mycobacterium tuberculosis proteins can be detected. The constructed tuberculosis patient serum antibody IgG specific adsorption BCG protein marker protein library information is more comprehensive and accurate. The reasons are as follows: First, the number of BCG proteins detected by mass spectrometry in this study is greater than the number of proteins in the existing invention (ZL202111432830.8); Second, during the LC-MS / MS detection of complex protein mixtures, high-abundance proteins will have a shielding / masking effect on the spectrum detection signals of low-abundance proteins in the components; In the present invention, the serum of patients or healthy people is covalently coupled to the activated Sepharose-6FF matrix, and the serum antibodies coupled thereto are not eluted, while the serum antibody IgG in the patent information of the existing patent number ZL202111432830.8 is eluted from the biomimetic affinity material BiAC-A115-94 together with the Mycobacterium tuberculosis antigen adsorbed thereon.

[0071] The above disclosure is only a preferred embodiment of the present invention, and it is certainly not intended to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiment and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for screening serum antibodies IgG that specifically recognize the antigen spectrum of vaccine strains, characterized in that: The following steps are involved: S1: Screen the serum samples of the control group and the experimental group, perform bionic affinity and affinity chromatography on the serum proteins of the experimental group to obtain the serum antibody IgG of the experimental group; perform bionic affinity and affinity chromatography on the serum proteins of the control group to obtain the serum antibody IgG of the control group; S2: The serum antibody IgG of the experimental group is fixed and coupled to the activated solid phase carrier material by a specific chemical covalent bond to prepare an affinity antibody column of the experimental group; the serum antibody IgG of the control group is fixed and coupled to the activated solid phase carrier material by a specific chemical covalent bond to prepare an affinity antibody column of the control group; S3: Prepare BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein, and perform affinity chromatography on the BCG (strain) culture filtrate or BCG (strain) cytoplasm protein with the affinity antibody column of the experimental group, and perform affinity chromatography on the same BCG (strain) protein sample with the affinity antibody column of the control group, to obtain BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by serum antibodies of the control group; S4: The protein information of the BCG (strain) culture filtrate or BCG (strain) cytoplasmic protein bound to the experimental group and the control group was analyzed by high-throughput liquid chromatography-mass spectrometry; S5: Search the BCG (strain) proteomics database to obtain the basic biochemical information of each protein; analyze and compare the differences between the BCG (strain) culture filtrate or BCG (strain) cytoplasm protein bound to the experimental group and the BCG (strain) protein bound to the control group, eliminate the BCG (strain) protein bound to the control group serum antibody column, and select the BCG (strain) protein that specifically binds to the serum of tuberculosis patients as a tuberculosis protein vaccine marker and / or a tuberculosis serological detection marker; S6: Perform mass spectrometry data analysis, bioinformatics analysis and immunological analysis on the tuberculosis protein vaccine markers and / or tuberculosis serological detection markers constructed in S5 to obtain the antigen spectrum of serum antibody IgG that specifically recognizes its vaccine strain.

2. The method for screening serum antibody IgG that specifically recognizes the antigen spectrum of the vaccine strain according to claim 1, characterized in that: The screening of the serum samples of the control group and the experimental group in step S1 is specifically as follows: BCG cytoplasmic protein and the prepared MTB recombinant protein antigen are coated on a solid phase carrier, the serum sample to be tested is added, and the level of serum antibodies is detected by enzyme labeling, and the serum samples of the control group and the experimental group are screened to remove the serum samples in the experimental group that do not produce tuberculosis antibodies and the serum samples of tuberculosis latently infected persons that contain anti-Mycobacterium tuberculosis antibodies in the control group.

3. The screening method for serum antibody IgG that specifically recognizes the antigen spectrum of the vaccine strain according to claim 2, characterized in that: In the step S2, the activated solid phase carrier material is prepared by the following method: the activated solid phase carrier material is synthesized by epoxy activation reaction, amination reaction and cyanuric chloride substitution reaction.

4. The method for screening serum antibody IgG that specifically recognizes the antigen spectrum of the vaccine strain according to claim 2, characterized in that: Before the step S2, it is necessary to screen the affinity material that specifically binds to serum IgG to obtain the best affinity material that specifically adsorbs serum IgG.

5. The method for screening serum antibody IgG that specifically recognizes the antigen spectrum of the vaccine strain according to any one of claims 1 to 4, characterized in that: The S3 step also includes eluting the BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by the serum antibodies of the experimental group and the BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein bound by the serum antibodies of the control group, so as to wash off the bound BCG (strain) culture filtrate protein or BCG (strain) cytoplasm protein respectively.

6. The method for screening serum antibody IgG that specifically recognizes the antigen spectrum of the vaccine strain according to claim 5, characterized in that: The eluent is PBS or glycine hydrochloride.

7. A serum antibody IgG that specifically recognizes the antigen spectrum of its vaccine strain, characterized in that: The method is obtained by the screening method of serum antibody IgG that specifically recognizes the antigen spectrum of the vaccine strain as described in any one of claims 1 to 6.

8. The use of the serum antibody IgG according to any one of claims 1 to 7 that specifically recognizes the antigen spectrum of the vaccine strain for the preparation of a new marker for the development of tuberculosis protein vaccines and / or a tuberculosis serological diagnostic reagent, characterized in that: The tuberculosis includes pulmonary tuberculosis, extrapulmonary tuberculosis, sputum bacterial culture-negative tuberculosis, sputum-negative tuberculosis and GeneXpert MTB / RIF test-negative tuberculosis.

9. The use of the serum antibody IgG as claimed in claim 8 that specifically recognizes the antigen spectrum of the vaccine strain for preparing a new marker for the development of tuberculosis protein vaccine and / or a tuberculosis serological diagnostic reagent, characterized in that: The pulmonary tuberculosis is extrapulmonary tuberculosis and tuberculosis with negative sputum bacterial culture.

10. A kit for assisting in the detection of tuberculosis, characterized in that: The method comprises the serum antibody IgG as described in any one of claims 1 to 7 which specifically recognizes the antigen spectrum of the vaccine strain.

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