Serum antibody igg specifically recognizes its vaccine strain antigen spectrum and screening method and application thereof

Abstract

The application 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, serum samples of latent infection human groups without producing tuberculosis antibodies and containing anti-tubercle bacillus protein antibodies are removed, then the screened serum antibodies are subjected to biomimetic affinity and affinity chromatography, high-purity serum antibody IgG is obtained, the high-purity serum antibody IgG after biomimetic affinity and affinity chromatography is covalently coupled to a solid-phase carrier material, and different types of serum antibody columns are prepared; after affinity chromatography of different types of serum antibody columns and BCG total protein, Gly-HCl (pH1.5-3.0) can only elute the combined BCG protein on the columns but not high-abundance serum antibody IgG, LC-MS / MS can detect low, medium and high concentrations of BCG protein, and a BCG antigen spectrum specifically combined with serum antibodies of tuberculosis patients is constructed, which is used for tuberculosis protein vaccine development and / or serum protein marker library research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tuberculosis pathogen infection and immunity and its protein markers, and specifically relates to a serum antibody IgG specific recognition of a vaccine strain antigen spectrum and a screening method and application thereof. BACKGROUND

[0002] Tuberculosis is one of the human infectious diseases with the highest mortality caused by Mycobacterium tuberculosis (MTB) infection of the body; patients with pulmonary tuberculosis (PTB) account for 85% of the total number of patients, and timely diagnosis and reasonable drug use can cure 85% of the patients; according to the data of the China Center for Disease Control and Prevention, 741,000 new tuberculosis patients were added in 2023, and the tuberculosis prevention and control task is arduous; Ningxia Hui Autonomous Region is located in the western part of China and is an economically underdeveloped region. Tuberculosis can be divided into pulmonary tuberculosis and extrapulmonary tuberculosis according to the different parts of the patient infected with the pathogenic bacteria; most extrapulmonary tuberculosis patients are also infected with Mycobacterium tuberculosis in the lungs, and the pathogen further invades other tissues and organs; the latest tuberculosis diagnosis standard (WS-288-2017) defines pulmonary tuberculosis as lesions occurring in lung tissue, trachea, bronchus and pleura caused by MTB infection, which accounts for more than 80% of the total number of tuberculosis patients.

[0003] Currently, the main detection methods of tuberculosis are based on etiology, pathology, molecular biology, immunology and imaging, which have significantly improved the detection rate of tuberculosis. However, each detection method still has its limitations. In etiology, sputum acid-fast staining-microscopy and bacterial culture are the gold standard for tuberculosis detection. Sputum acid-fast staining is the most common clinical detection method for suspected pulmonary tuberculosis patients because of its convenience and non-invasive nature. However, the detection rate of sputum acid-fast staining is between 30-60%, which may lead to missed diagnosis. Sputum bacterial culture has a higher detection rate than acid-fast staining, but it takes 2-8 weeks to get the results, which is not conducive to the timely treatment of tuberculosis patients. In addition, sputum bacterial culture and sputum acid-fast staining based on etiology have the following limitations: first, about 10% of the samples are non-tuberculosis mycobacteria, which need further culture and identification; second, specific detection of non-pulmonary tuberculosis infection is difficult; third, some patients have little or no sputum; fourth, it is difficult to differentiate patients with pulmonary diseases caused by non-tuberculosis mycobacteria. Pathology is also an important way to diagnose tuberculosis, especially in the diagnosis of difficult patients such as sputum-negative pulmonary tuberculosis and extrapulmonary tuberculosis. Tuberculosis histopathology observes the formation of granulomas and the morphology of multinucleated giant cells and Langhans cells. If there are suppurative non-granulomatous tuberculosis, it will cause false negative results in histopathology. Molecular biology detection methods based on polymerase chain reaction (PCR) are easily affected by laboratory quality control differences and external homologous nucleic acid fragment cross contamination, leading to PCR amplification failure. Enzyme-linked immunospot assay (ELISPOT) based on immunology is the most sensitive method for tuberculosis detection, but it has limitations, including: first, HIV co-infected with MTB has a high mortality rate, but the detection rate of immunological methods is relatively low; second, the detection rate of old people and other patients with underlying diseases is low; third, organ inhibitors and immunosuppressant drug users limit the clinical application of this method. Imaging-based tuberculosis detection has the following limitations: first, low specificity, with the condition of "different diseases with the same image"; second, lack of judgment specificity for current infection or past infection. Therefore, the limitations of the above tuberculosis detection methods lead to missed diagnosis and misdiagnosis, which can easily further develop into active tuberculosis (ATB) patients, becoming a new source of tuberculosis transmission. Studies have shown that ATB patients infect 10-13 people per year, and super spreaders can infect more than 200 people per year. Therefore, improving the detection rate of missed and misdiagnosed tuberculosis patients is a difficult problem in tuberculosis prevention and control, and it is also a key issue that researchers and medical workers need to solve.Therefore, it is of great significance to develop new, rapid and high-sensitivity tuberculosis patient detection reagents for the prevention and control of the infectious disease.

[0004] In the prior art, a kind of tuberculosis serum marker screening method and application are disclosed in Chinese patent for invention with patent No.ZL202111432830.8, by carrying out biomimetic affinity and affinity chromatography to serum of tuberculosis patient (experimental group) and serum of healthy person (control group) respectively, prepare experimental group biomimetic affinity and affinity chromatography antibody column, prepare control group biomimetic affinity and affinity chromatography antibody column;Tubercle bacillus culture filtrate or tubercle bacillus cytoplasmic protein is chromatographed with experimental group and control group biomimetic affinity and affinity chromatography antibody column respectively;The tubercle bacillus protein combined with the serum antibody of the above-mentioned experimental group and control group is analyzed, and the serum marker for detecting tuberculosis is obtained initially;Further method analyzes the above-mentioned initial selection tuberculosis detection serum marker, the above-mentioned method adopts the combination mode of biomimetic affinity chromatography, antigen-antibody specific recognition, mass spectrometry analysis and bioinformatics analysis method, realizes the high-throughput screening and evaluation of serum marker of tuberculosis patient, solves the limitation of the prior art screening tuberculosis serum marker method;But in the above-mentioned prior art, serum antibody IgG directly adsorbed on solid carrier is used in biomimetic affinity and affinity chromatography, when eluting tubercle bacillus protein (antigen) adsorbed with serum antibody, high concentration serum antibody IgG specifically adsorbed on biomimetic affinity chromatography material can be eluted together with tubercle bacillus protein adsorbed on the antibody, so the composition in the eluent of immunochromatography is high concentration serum antibody and tubercle bacillus protein, after trypsin enzymolysis, the polypeptide fragment of this kind of sample exists the effect that low abundance tubercle bacillus protein is covered by high abundance serum antibody IgG when LC-MS / MS detection is carried out, so that low concentration protein cannot be detected, and then the number of detected proteins is small, leading to that the information of tubercle bacillus serum marker protein library is not comprehensive. SUMMARY

[0005] Therefore, it is of great significance to develop new, rapid and high-sensitivity tuberculosis patient detection reagents for the prevention and control of the infectious disease.

[0006] Therefore, it is of great significance to develop new, rapid and high-sensitivity tuberculosis patient detection reagents for the prevention and control of the infectious disease.

[0007] Therefore, it is of great significance to develop new, rapid and high-sensitivity tuberculosis patient detection reagents for the prevention and control of the infectious disease.

[0008] The technical scheme adopted by the present application to solve its technical problems is:

[0009] A screening method for serum antibody IgG specific recognition of vaccine strain antigen spectrum, comprising the following steps:

[0010] 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 serum antibodies IgG of the experimental group; perform biomimetic affinity and affinity chromatography on the serum proteins of the control group to obtain serum antibodies IgG of the control group;

[0011] S2: fix and couple the serum antibodies IgG of the experimental group to the activated solid-phase carrier material in a specific chemical covalent bond manner to prepare an affinity antibody column of the experimental group; fix and couple the serum antibodies IgG of the control group to the activated solid-phase carrier material in a specific chemical covalent bond manner to prepare an affinity antibody column of the control group;

[0012] S3: prepare BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein, perform affinity chromatography on the BCG (strain) culture filtrate or BCG (strain) cell plasma 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) cell plasma protein combined with serum antibodies of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein combined with serum antibodies of the control group;

[0013] S4: analyze the protein information of the BCG (strain) culture filtrate or BCG (strain) cell plasma protein combined by the experimental group and the control group by high-throughput liquid chromatography-mass spectrometry;

[0014] S5: search a BCG (strain) proteomics database to obtain basic biochemical information of each protein; analyze and compare the differences between the BCG (strain) culture filtrate or BCG (strain) cell plasma protein combined by the experimental group and the BCG (strain) protein combined by the control group, eliminate the BCG (strain) proteins combined with the serum antibody column of the control group, and select the BCG (strain) proteins specifically combined with the serum of tuberculosis patients as tuberculosis protein vaccine markers and / or tuberculosis serological detection markers;

[0015] S6: perform mass spectrometry data analysis, bioinformatics analysis, and immunology analysis on the tuberculosis protein vaccine markers and / or tuberculosis serological detection markers constructed in S5 to obtain a serum antibody IgG specific recognition of its vaccine strain antigen spectrum.

[0016] A serum antibody IgG specific recognition of its vaccine strain antigen spectrum is obtained by the screening method of the serum antibody IgG specific recognition of its vaccine strain antigen spectrum as described above.

[0017] Use of serum antibody IgG specific to recognize its vaccine strain antigen profile as described above for the preparation of new markers for the development of tuberculosis protein vaccine and / or in the serological diagnostic reagents for tuberculosis, including pulmonary tuberculosis, extrapulmonary tuberculosis, tuberculosis with negative sputum bacterial culture, bacteriological negative pulmonary tuberculosis and tuberculosis with negative GeneXpert MTB / RIF test.

[0018] A kit for aiding in the detection of tuberculosis, comprising serum antibody IgG specific to recognize its vaccine strain antigen profile as described above.

[0019] The advantageous effects of the present application compared to the prior art are:

[0020] The application provides a screening method for serum antibody IgG specific recognition of antigen spectrum of a vaccine strain, screening of serum samples of a control group (healthy people) and an experimental group (tuberculosis patients), biomimetic affinity and affinity chromatography of serum proteins of the experimental group and the control group, respectively, to obtain serum antibody IgG of the experimental group and the control group; the serum antibody IgG of the experimental group and the control group is respectively fixed and coupled to an activated solid carrier material in a specific chemical covalent bond mode to prepare an affinity antibody column of the experimental group and the control group; BCG (strain) culture filtrate or BCG (strain) cytoplasmic protein is subjected to affinity chromatography of the antibody column of the experimental group and the control group; the BCG (strain) culture filtrate protein or the BCG (strain) cytoplasmic protein combined with the serum antibody of the above experimental group and the control group is analyzed to preliminarily obtain a new marker for tuberculosis protein vaccine development and / or a serum marker for tuberculosis detection; further analysis of the BCG protein specifically recognized by the serum antibody IgG of the above preliminary tuberculosis serum antibody IgG is performed to obtain serum antibody IgG specific recognition of antigen spectrum of a vaccine strain; the above method is used to evaluate the sensitivity and specificity of specific recognition of five kinds of recombinant MTB proteins and BCG whole protein to serum antibody of tuberculosis patients by ELISA method in advance, so as to remove serum samples that fail to produce tuberculosis antibodies due to low immunity or abnormality, then the screened serum antibody is 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 is covalently coupled to a solid carrier material to prepare a tuberculosis serum antibody column IgG column (experimental group); the serum samples of healthy people are screened by the same method to remove serum samples of latent infection population containing anti-tubercle bacillus protein antibodies in serum, then the screened serum antibody is 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 is covalently coupled to a solid carrier material to prepare a healthy person serum antibody IgG column (control group); after affinity chromatography of different types of serum antibody IgG columns and BCG (strain) total protein, the high-purity serum antibody IgG specifically combined with the BCG (strain) total protein antigen does not fall off, and an antigen-antibody mixture is not generated, when sample LC-MS / MS detection is performed, the effect that low-abundance tubercle bacillus proteins are covered by high-abundance serum antibody IgG does not exist, so that low-concentration proteins and high-concentration proteins can be detected, the number of detected proteins is large, and the information of a new marker for tuberculosis protein vaccine development and / or a serum marker for tuberculosis detection is more comprehensive and accurate. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 SDS-PAGE evaluation results of recombinant MTB protein nickel metal chelate chromatography separation and purification.

[0022] Figure 2The results of ELISA detection of antibodies against five recombinant MTB proteins and BCG whole protein antigens.

[0023] Figure 3 SDS-PAGE of serum antibody IgG fractions purified by SPA with affinity ligand coupling.

[0024] Figure 4 Gray scale analysis of human serum antibody IgG purified by SPA with affinity ligand coupling.

[0025] Figure 5 Evaluation of adsorption performance of Sepharose-6FF-A133-IgG affinity ligand.

[0026] Figure 6 SDS-PAGE of Sepharose-6FF-A133-IgG affinity ligand adsorption performance evaluation.

[0027] Figure 7 Mass spectrometric detection of serum from healthy people and patients with positive sputum smear for tuberculosis of BCG cytoplasmic protein.

[0028] In the figure: Figure 1 Figure A is the soluble expression and nickel metal chelate chromatography separation and purification of the recombinant Mycobacterium tuberculosis Rv0222 gene encoded protein; 1: flow through; M: Marker; 2-3: wash waste; 4-7: soluble expression protein eluent; Figure B is the inclusion body nickel metal chelate chromatography separation and purification of the recombinant Mycobacterium tuberculosis Rv1980c gene encoded protein; M: Marker; 1: flow through; 2-3: wash waste; 4-7: inclusion body protein eluent; Figure C is the soluble expression and nickel metal chelate chromatography separation and purification of the recombinant Mycobacterium tuberculosis Rv0577 gene encoded protein; M: Marker; 1: pET28a original bacterial slurry; 2: protein supernatant after ultrasonic bacterial disruption; 3: soluble expression protein eluent; Figure D is the soluble expression and nickel metal chelate chromatography separation and purification of the recombinant Mycobacterium tuberculosis Rv2031c gene encoded protein; M: Marker; 1: pET28a original bacterial slurry; 2: protein supernatant after ultrasonic bacterial disruption; 3: soluble expression protein eluent; Figure E is the nickel metal chelate chromatography separation and purification of the recombinant Mycobacterium tuberculosis Rv0934 gene encoded protein inclusion body M: Marker; 1: pET28a original bacterial slurry; 2: protein slurry after ultrasonic bacterial disruption; 3: inclusion body protein purification flow through.

[0029] Figure 3Figure A is Sepharose-6FF-A73-SPA affinity chromatography of healthy human serum; Figure B is Sepharose-6FF-A74-SPA affinity chromatography of healthy human serum; Figure C is Sepharose-6FF-A75-SPA affinity chromatography of healthy human serum; Figure D is Sepharose-6FF-A77-SPA affinity chromatography of healthy human serum; Figure E is Sepharose-6FF-A78-SPA affinity chromatography of healthy human serum; Figure F is Sepharose-6FF-A87-SPA affinity chromatography of healthy human serum; Figure G is Sepharose-6FF-A133-SPA affinity chromatography of healthy human serum; wherein 1: serum as original; 2: flow-through; 3-4: washing impurities; 5-8: eluent; M: Marker.

[0030] Figure 5 Figure A is Sepharose-6FF-A133-IgG affinity chromatography of healthy human serum antibody; Figure B is Sepharose-6FF-A133-IgG affinity chromatography of healthy human serum antibody; Figure C is Sepharose-6FF-A133-IgG affinity chromatography of healthy human serum antibody; Figure D is Sepharose-6FF-A133-IgG affinity chromatography of healthy human serum antibody; Figure E is Sepharose-6FF-A133-IgG affinity chromatography of healthy human serum antibody; Figure F is Sepharose-6FF-A133-IgG affinity chromatography of healthy human serum antibody; Figure G is Sepharose-6FF-A133-IgG affinity chromatography of healthy human serum antibody; wherein 1: serum as original; 2: flow-through; 3-4: washing impurities; 5-8: eluent; M: Marker. BL21 (DE3) Figure B is Sepharose-6FF-A133-IgG affinity chromatography of pET28a-SPA-BCG cytoplasmic protein; Figure C is Sepharose-6FF-A133-IgG affinity chromatography of pET28a-SPA-BCG cytoplasmic protein; Figure D is Sepharose-6FF-A133-IgG affinity chromatography of pET28a-SPA-BCG cytoplasmic protein; Figure E is Sepharose-6FF-A133-IgG affinity chromatography of pET28a-SPA-BCG cytoplasmic protein; Figure F is Sepharose-6FF-A133-IgG affinity chromatography of pET28a-SPA-BCG cytoplasmic protein; Figure G is Sepharose-6FF-A133-IgG affinity chromatography of pET28a-SPA-BCG cytoplasmic protein; wherein 1: serum as original; 2: flow-through; 3-4: washing impurities; 5-8: eluent; M: Marker. BL21(DE3) 1: SPA protein as original; M: Marker; 2: flow-through; 3-4: washing impurities; 5-8: eluent.

[0031] Figure 7 Figures A and B are mass spectrometry detection of BCG cytoplasmic protein elution components of healthy human and pulmonary tuberculosis smear (+) patient serum antibody column affinity chromatography, respectively searching MTB and BCG databases to detect protein data; Figures C and D are mass spectrometry detection of flow-through components of healthy human and pulmonary tuberculosis smear (+) patient serum antibody column affinity chromatography of BCG cytoplasmic protein, searching BCG database to detect protein data. DETAILED DESCRIPTION

[0032] The technical solutions and technical effects of the embodiments of the present application are further described in detail below in combination with the accompanying drawings of the present application.

[0033] A screening method for serum antibody IgG specific recognition of vaccine strain antigen spectrum, comprising the following steps:

[0034] S1: screening control group and experimental group serum samples, performing biomimetic affinity and affinity chromatography on serum proteins of the experimental group to obtain serum antibody IgG of the experimental group; performing biomimetic affinity and affinity chromatography on serum proteins of the control group to obtain serum antibody IgG of the control group;

[0035] S2: Serum antibody IgG of the experimental group is fixed and coupled to the activated solid carrier material by specific chemical covalent bond to prepare an affinity antibody column of the experimental group; serum antibody IgG of the control group is fixed and coupled to the activated solid carrier material by specific chemical covalent bond to prepare an affinity antibody column of the control group;

[0036] S3: Prepare BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein, and perform affinity chromatography of the BCG (strain) culture filtrate or the BCG (strain) cell plasma protein with the affinity antibody column of the experimental group, and perform affinity chromatography of 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) cell plasma protein combined with serum antibody of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein combined with serum antibody of the control group;

[0037] S4: Analyze the protein information of the BCG (strain) culture filtrate or BCG (strain) cell plasma protein combined by the experimental group and the control group by high-throughput liquid chromatography-mass spectrometry;

[0038] S5: Retrieve basic biochemical information of each protein from a BCG (strain) proteomics database; analyze and compare the differences between the BCG (strain) culture filtrate or BCG (strain) cell plasma protein combined by the experimental group and the BCG (strain) protein combined by the control group, eliminate the BCG (strain) protein combined in the control group serum antibody column, and select the BCG (strain) protein specifically combined with the serum of the tuberculosis patient as a tuberculosis protein vaccine marker and / or a tuberculosis serological detection marker;

[0039] S6: Perform mass spectrometry data analysis, bioinformatics analysis and immunology analysis on the tuberculosis protein vaccine marker and / or the tuberculosis serological detection marker constructed in S5, to obtain the vaccine strain antigen spectrum specifically recognized by serum antibody IgG.

[0040] Specifically, the protein in the vaccine strain antigen spectrum library specifically recognized by serum antibody IgG is expressed by genetic engineering, and then separated and purified by nickel metal chelate chromatography, followed by an immunological method of specific recognition of antigens and antibodies, which includes but is not limited to indirect enzyme-linked immunosorbent assay (ELISA) evaluation, to verify the sensitivity and specificity of the serum antibody IgG specifically recognizing the vaccine strain antigen spectrum.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] The application provides a screening method for serum antibody IgG specific recognition of vaccine strain antigen spectrum, screening of serum samples of a control group (healthy people) and an experimental group (tuberculosis patients), biomimetic affinity and affinity chromatography are respectively performed on serum proteins of the experimental group and the control group, and serum antibody IgG of the experimental group and the control group is obtained; the serum antibody IgG of the experimental group and the control group is respectively fixed and coupled to an activated solid carrier material in a specific chemical covalent bond mode, and an affinity antibody column of the experimental group and the control group is prepared; BCG (strain) culture filtrate or BCG (strain) cytoplasmic protein is subjected to affinity chromatography with the antibody column of the experimental group and the control group; the BCG (strain) culture filtrate protein or the BCG (strain) cytoplasmic protein combined with the serum antibody of the above experimental group and the control group is analyzed, and a new marker for tuberculosis protein vaccine development and / or a serum marker for tuberculosis detection is preliminarily obtained; further analysis of the BCG protein specifically recognized by the serum antibody IgG of the above preliminary tuberculosis serum antibody IgG is performed, and the serum antibody IgG specifically recognizing the vaccine strain antigen spectrum is obtained; the above method is used to evaluate the sensitivity and specificity of five kinds of recombinant expression MTB proteins and BCG whole proteins and tuberculosis patient serum antibody specific recognition by using an ELISA method in advance, so that serum samples that cannot produce tuberculosis antibodies due to low immunity or abnormality are removed, then the screened serum antibody is subjected to biomimetic affinity and affinity chromatography, high-purity serum antibody IgG is obtained, the high-purity serum antibody IgG after biomimetic affinity and affinity chromatography is covalently coupled to a solid carrier material, and a tuberculosis serum antibody column IgG column (experimental group) is prepared; the serum samples of healthy people are screened in the same way, so that the serum samples of latent infection people containing anti-mycobacterium tuberculosis protein antibodies in the serum are removed, then the screened serum antibody is subjected to biomimetic affinity and affinity chromatography, high-purity serum antibody IgG is obtained, the high-purity serum antibody IgG after biomimetic affinity and affinity chromatography is covalently coupled to a solid carrier material, and a healthy person serum antibody IgG column (control group) is prepared; after affinity chromatography of different types of serum antibody IgG columns and BCG (strain) total protein (BCG culture filtrate protein and BCG cytoplasmic protein are mixed in equal amounts), the high-purity serum antibody IgG specifically combined with the BCG (strain) total protein antigen does not fall off, and an antigen and antibody mixture is not generated, when sample LC-MS / MS detection is performed, the effect that low-abundance mycobacterium tuberculosis proteins are covered by high-abundance serum antibody IgG does not exist, so that low-concentration proteins and high-concentration proteins can be detected, the number of detected proteins is large, and the information of a new marker for tuberculosis protein vaccine development and / or a serum marker for tuberculosis detection is more comprehensive and accurate.

[0043] Further, the serum samples of the control group and the experimental group screened in the S1 step are as follows: BCG cytoplasmic proteins and prepared MTB recombinant protein antigens are coated on a solid carrier, the serum sample to be detected is added, the level of serum antibody is detected by enzyme-labeled substance coloration, and the serum samples of the control group and the experimental group are screened to remove serum samples without producing tuberculosis antibodies in the experimental group, serum samples of latent tuberculosis infection containing anti-mycobacterium tuberculosis antibodies in the control group, and improve the accuracy of serum antibody IgG of the experimental group and the control group.

[0044] Further, the serum protein is subjected to biomimetic affinity and affinity chromatography in the S1 step to obtain serum antibody IgG; specifically, the serum protein is subjected to biomimetic affinity and affinity chromatography purification respectively, then the serum subjected to biomimetic affinity and affinity chromatography purification is mixed at equal quality, the mixed serum is pretreated, and serum antibody IgG with high diversity and high purity is obtained, and the purity of the serum antibody IgG reaches more than 85%.

[0045] In the Chinese invention patent with the patent number ZL202111432830.8 in the prior art, specific antibodies are adsorbed during serum antibody purification, then the biomimetic affinity or affinity chromatography column adsorbed with the antibodies is directly used in immunochromatography with tubercle bacillus antigens, so that the method cannot elute serum antibody IgG from the chromatography column, and is directly used in antibody-antigen specific adsorption experiments on the biomimetic affinity chromatography column. Therefore, the antibodies of the biomimetic affinity filler and the affinity filler can only be specifically adsorbed with tubercle bacillus antigens respectively. In the present patent, serum antibodies are first purified, that is, eluted from the chromatography column, then pretreated, and finally covalently coupled to agarose gel after mixing, so that the serum antibody IgG has high diversity and high purity.

[0046] Further, in the S2 step, the activated solid carrier material is prepared by the following method: the activated solid carrier material is synthesized through an epoxy activation reaction, an aminoization reaction and a tricyanogen chloride substitution reaction.

[0047] Further, before the S2 step, the screening of affinity materials specifically binding serum IgG is needed to obtain the best affinity material specifically adsorbing serum IgG; specifically, 7 kinds of affinity materials activated by different spacer arms (epoxy activation-aminoization-tricyanogen chloride substitution reaction) are coupled with Staphylococal Protein A (SPA), different adsorption effects of affinity chromatography of collected healthy human serum IgG are obtained, the differences in protein purity are analyzed by gray scale analysis after verification by SDS-PAGE gel electrophoresis, and the best affinity material specifically adsorbing serum IgG is screened.

[0048] Further, the S3 step further comprises eluting the BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein bound by the serum antibodies of the experimental group and the serum antibodies of the control group, respectively, to wash down the bound BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein.

[0049] Further, the BCG (strain) culture filtrate or BCG (strain) cell plasma protein used in the S3 step comprises culture filtrate protein and BCG (strain) cell plasma protein during the culture of the BCG (strain); the prepared BCG (strain) culture filtrate or cell plasma protein is subjected to affinity chromatography with the experimental group antibody column and the control group antibody column, respectively; further, the BCG (strain) culture filtrate or cell plasma protein not adsorbed by the affinity chromatography column is washed; the protein concentration of different elution components is quantitatively analyzed by BCA method; the concentration of all component proteins to be detected by mass spectrometry is treated to an optimal concentration for mass spectrometry detection, such as 2 mg-10 mg / mL, by ultrafiltration concentration method; after the flow-through and elution components are digested by trypsin, further drying treatment is performed for mass spectrometry analysis and detection.

[0050] Further, the eluent is PBS or glycine hydrochloride; in an embodiment, the eluent is Gly-HCl with pH of 1.5-3.0.

[0051] Further, the S5 comprises the following steps: searching the BCG (strain) proteins bound by the experimental group and the control group, respectively, detected by mass spectrometry in a database (http: / / www.uniprot.org / ); eliminating the BCG (strain) proteins specifically adsorbed by the serum antibodies of the control group; and constructing a serum antibody IgG specific recognition vaccine strain antigen spectrum library using the eliminated BCG (strain) proteins of the experimental group.

[0052] Further, the S6 comprises the following steps: analyzing the molecular weight, isoelectric point, hydrophilic / hydrophobic property, subcellular localization, whether it is a secretory protein and possible secretion mode of the proteins in the BCG (strain) protein library; screening the proteins with high abundance, large sequence specificity and strong immunogenicity in the BCG (strain) protein library as candidate markers of the serum antibody IgG specific recognition vaccine strain antigen spectrum of the application; recombinantly expressing the serum antibody IgG specific recognition vaccine strain antigen spectrum screened by the above-mentioned biomimetic affinity chromatography-mass spectrometry strategy or affinity chromatography-mass spectrometry strategy in Escherichia coli and isolating and purifying the target protein by nickel metal chelate chromatography to obtain high-purity recombinant target protein; and finally evaluating the detection sensitivity and specificity of the recombinant Mycobacterium tuberculosis protein in the serum of tuberculosis patients by indirect ELISA method, and finally screening the serum antibody IgG specific recognition vaccine strain antigen spectrum of the application.

[0053] A serum antibody IgG specifically recognizes the antigen profile of the vaccine strain, which is obtained by the screening method of the serum antibody IgG specifically recognizing the antigen profile of the vaccine strain as described above.

[0054] The serum antibody IgG specifically recognizing the antigen profile of the vaccine strain as described above is used for preparing a new marker for tuberculosis protein vaccine development and / or a tuberculosis serological diagnostic reagent, and the tuberculosis includes pulmonary tuberculosis, extrapulmonary tuberculosis, tuberculosis with negative sputum bacterial culture, bacterium-negative pulmonary tuberculosis and GeneXpert MTB / RIF detection-negative tuberculosis.

[0055] Further, the pulmonary tuberculosis is extrapulmonary tuberculosis and tuberculosis with negative sputum bacterial culture.

[0056] A kit for assisting in detecting tuberculosis includes the serum antibody IgG specifically recognizing the antigen profile of the vaccine strain as described above.

[0057] The use of the present application is introduced through the following examples. Example 1:

[0058] S1: The serum samples of the control group and the experimental group are screened, the serum proteins of the experimental group are subjected to biomimetic affinity and affinity chromatography to obtain a variety of serum antibody IgG of the experimental group; the serum proteins of the control group are subjected to biomimetic affinity and affinity chromatography to obtain a variety of serum antibody IgG of the control group;

[0059] 1. Screening of serum samples of the control group and the experimental group:

[0060] The patient serum sample with strong recognition effect on all the above proteins is selected as the experimental group by using the self-made 5 kinds of recombinant MTB proteins and BCG cytoplasmic proteins as antigens by ELISA method; and the serum of healthy people who cannot recognize the above 5 kinds of recombinant MTB proteins and BCG cytoplasmic proteins is used as the control group for the preparation of the antibody column of the control group in the subsequent experiment;

[0061] (1) Coating: the recombinant Mycobacterium tuberculosis proteins MPT64 (Rv1980c), echA1 (Rv0222), CFP32 (Rv0577), HspX (Rv2031c), pstS1 (Rv0934) and BCG cytoplasmic whole protein are diluted to 5 μg / mL with a coating buffer, 100 μL is added to each well, and it is incubated at 4℃ overnight. The 96-well plate is washed with 1×PBST buffer for 3 times, 3 min each time;

[0062] (2) Blocking: 5% skimmed milk powder is prepared with 1×PBS, 250 μL is added to each well, and it is incubated at 37℃ for 2 h. The plate is washed with 1×PBST buffer for 3 times, 3 min each time;

[0063] (3) Sample loading: dilute the serum sample to be tested with 5% skimmed milk powder (also set up a blank control group), add 100 μL of diluted serum sample to each well, incubate at 37°C for 1 h. Wash the 96-well plate with 1x PBST buffer for 3 times, 3 min each time;

[0064] (4) Enzyme-labeled secondary antibody: add HRP-labeled goat anti-human antibody to the 1x PBS prepared skimmed milk powder solution at a ratio of 1:5000; add 100 μL of secondary antibody to each well, incubate at 37°C for 1 h. Wash the plate with 1x PBST buffer for 3 times, 3 min each time;

[0065] (5) Color development: add 50 μL of TMB color developing solution (A and B) to each well, avoid light, incubate at 37°C for 10 min.

[0066] (6) Reaction termination: add 50 μL of 2 mol / L sulfuric acid solution to each well to terminate the reaction.

[0067] (7) Result determination: place the enzyme-labeled plate into an enzyme-labeled instrument and read at 450 nm.

[0068] The obtained 87 serum samples of tuberculosis patients and 81 serum samples of healthy people were determined by ELISA method (1)-(7) above, and 12 serum samples of the control group (healthy people) and 12 serum samples of the experimental group (tuberculosis patients) were screened out by Graphpad Prism analysis of ELISA results;

[0069] The SDS-PAGE results of the nickel metal chelate chromatography separation and purification experiment of the recombinant MTB protein are shown in Figure 1 The ELISA detection results of the five kinds of recombinant MTB protein and BCG whole protein antigen antibody are shown in Figure 2 The ELISA results of the healthy people and tuberculosis patients serum samples were screened out by Graphpad Prism analysis.

[0070] 2. Preparation of high-purity serum antibody IgG:

[0071] The 12 tuberculosis patient serum antibodies with the highest sensitivity (highest OD450 value measured by enzyme-labeled instrument) to the five kinds of recombinant MTB protein and BCG cytoplasmic protein screened out by ELISA method were mixed at a ratio of 0.5 mL per person to form a 6 mL tuberculosis patient serum antibody group (experimental group); the 12 health serum antibodies with the lowest OD450 value measured by enzyme-labeled instrument were mixed at a ratio of 0.5 mL per person to form a 6 mL health serum antibody group (control group);

[0072] The serum proteins of the experimental group and the control group were respectively subjected to biomimetic affinity and affinity chromatography purification (the biomimetic affinity purification was the same as 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), and then the serum after biomimetic affinity and affinity chromatography purification was mixed in equal quality, and the mixed serum was pretreated to obtain serum antibodies IgG with high diversity and high purity;

[0073] The pretreatment was: (1) SDS-PAGE gel electrophoresis (qualitative) to evaluate the purity of serum antibodies IgG in the biomimetic affinity and affinity chromatography samples; then the purity of serum antibodies IgG after purification by the two methods was evaluated by gray scale scanning software; (2) serum antibodies IgG purified by the two methods were respectively moved into dialysis bags, and dialyzed in 0.5 moL pH8.5 Na2SO4 solution at 4-10°C for 4-6h; (3) the concentrations of serum antibodies IgG after dialysis by biomimetic affinity chromatography and affinity chromatography were respectively detected by BCA protein quantification method; (4) the serum antibodies IgG samples purified by the two methods were mixed in a ratio of 1:1.

[0074] The purity of serum antibodies IgG in the experimental group reached more than 85%, and the purity of serum antibodies IgG in the control group reached more than 85%.

[0075] S2: The serum antibodies IgG of the experimental group were fixed and coupled to the activated solid carrier material by specific chemical covalent bond to prepare the experimental group affinity antibody column; the serum antibodies IgG of the control group were fixed and coupled to the activated solid carrier material by specific chemical covalent bond to prepare the control group affinity antibody column;

[0076] 1. Screening of the best affinity material:

[0077] Seven kinds of different length spacer arm Sepharose-6FF-SPA affinity filler were synthesized; the alkali-resistant recombinant SPA was covalently coupled to the activated (epoxy activation-amino activation-cyanuric chloride substitution reaction) Sepharose microspheres with different spacer arms, and the 12 serum samples with the lowest OD value were mixed in the same quality, and the IgG in the healthy human serum was affinity chromatographed, and the affinity material capable of specifically adsorbing serum antibody IgG was screened. Figure 3 As shown in the 12% SDS-PAGE gel electrophoresis, the results showed that Sepharose-6FF-A133-SPA (Figure G) was the best affinity purification material for serum antibody.

[0078] The elution components were analyzed by Image Lab, and the purity of the elution components of Sepharose-6FF-A73-SPA affinity chromatography serum antibody IgG was 49.1%, 56.9%, 51.9%, and 42.2%; the purity of the elution components of Sepharose-6FF-A74-SPA affinity chromatography serum antibody IgG was 68.2%, 47.2%, 40%, and 31.3%; the purity of the elution components of Sepharose-6FF-A75-SPA affinity chromatography serum antibody IgG was 13.4%, 8.2%, 6.8%, and 8.1%; the purity of the elution components of Sepharose-6FF-A77-SPA affinity chromatography serum antibody IgG was 9.6% and 26.9%; the purity of the elution components of Sepharose-6FF-A78-SPA affinity chromatography serum antibody IgG was 57.8%, 59.4%, 27.1%, and 15.2%; the purity of the elution components of Sepharose-6FF-A87-SPA affinity chromatography serum antibody IgG was 41.4%, 31.6%, 17.9%, and 15.4%; the purity of the elution components of Sepharose-6FF-A133-SPA affinity chromatography serum antibody IgG was 90%, 93.8%, 89.6%, and 85.1%. The gray scale analysis of the elution components of SDS-PAGE after Sepharose-6FF-A133-SPA and Sepharose-6FF-A73-SPA, Sepharose-6FF-A75-SPA, and Sepharose-6FF-A77-SPA affinity chromatography of human serum antibody IgG was performed by t test between the two groups, P<0.05, indicating that the Sepharose-6FF-A133-SPA affinity chromatography of serum IgG had statistical significance with each group, and the gray scale analysis of the elution components of SDS-PAGE after Sepharose-6FF-A133-SPA affinity chromatography of human serum antibody IgG had the highest purity. The results are shown in Figure 4As shown, Sepharose-6FF-A133-SPA is the best specific adsorption affinity material for affinity chromatography of serum IgG.

[0079] 2. Preparation of activated solid carrier material:

[0080] (1) Epoxy activation reaction: 210 g of Sepharose 6FF agarose was washed with ultrapure water for 10 times, and the drained Sepharose 6FF agarose was taken out and added with 441 mL of epoxy activation solution (VDMSO:VECH=6:4). The epoxy activation solution in the reaction system was shaken, 8.0 g of NaOH particles were added, and the reaction was carried out for 1 hour. The pH was measured to be 10.8, 8.0 g of NaOH particles were added, and the pH was measured to be 10.5 after 1.5 hours. 8.0 g of NaOH was added for continuous reaction, and the pH was measured to be 10.2. The total reaction time was 4 hours. Washing: washed with 10 times the volume of ultrapure water, ultrafiltration to drain water, and stored at 4℃ for standby.

[0081] (2) Amino reaction: 175 g of epoxy activated Sepharose 6FF was divided into 7 groups, each group of 25 g. The amino reaction solution was composed of 20% different amino compounds and 80% ultrapure water. Each group was added with 52.5 mL of amino reaction solution, and the amino reaction conditions were: 60℃, 170 r / min, 16h. Washing: washed with 10 times the volume of ultrapure water for 10 times, ultrafiltration to drain water, and stored at 4℃ for standby.

[0082] (3) Cyanuric chloride substitution reaction: the amino reaction Sepharose-6FF was added with 0.16 M cyanuric chloride acetone solution, the cyanuric chloride acetone solution was pre-frozen for 4 hours (-20℃), Na2CO3 particles were fully mixed and added, the pH was adjusted to 7-8, and the reaction was carried out at 4℃ in an ultra-low temperature shaker for 4 hours. Washing: washed with 10 times the volume of ultrapure water for 10 times, ultrafiltration to drain water for standby.

[0083] 2. Preparation of affinity antibody columns for experimental group and control group:

[0084] The Sepharose-6FF-A133-SPA affinity chromatography serum IgG elution components of different groups were collected, mixed in equal mass, and dialyzed with 1 moL of Na2SO4 (pH8.5). After mixing in equal mass, the serum IgG of the patients with pulmonary tuberculosis was mixed with the above activated (epoxy activation-amino reaction-cyanuric chloride substitution reaction) agarose microspheres, and then placed in a constant temperature shaker at 25℃, 170 r / min for 24 hours to prepare a serum antibody IgG chromatography column (experimental group). During the reaction, 100 μL of reaction solution was taken at 0h, 2h, 12h, and 24h for BCA protein quantification. The same method was used to prepare a serum antibody IgG chromatography column (control group).

[0085] The specific adsorption purity of Sepharose-6FF-A133-SPA for serum antibody IgG of tuberculosis patients and healthy people is 70% and 86%, respectively.

[0086] 3. Verify whether the preparation of different groups of antibodies is successful.

[0087] SPA specifically binds to the Fc fragment of IgG class antibodies in human serum. Different groups of Sepharose-6FF-A133-IgG affinity ligands and blank Sepharose-6FF control groups are used for affinity chromatography of pET28a-SPA protein. BL21(DE3) The adsorption performance of the affinity ligand is evaluated, and 12% SDS-PAGE gel electrophoresis is used for verification, as shown in Figure 5 .

[0088] Different groups of Sepharose-6FF-A133-IgG affinity chromatography of recombinant SPA, 6-8 are purification eluates, and the experimental results are analyzed by Image Lab, with purities of 55%, 78.5%, 79.6%, and 81.4%, respectively. The purities of the eluates of the blank Sepharose-6FF matrix purified SPA protein 5-8 are 1.6%, 3.0%, and 7.2%, respectively. The gray scale analysis of the eluates of the two groups shows that the t-test between the two groups is P<0.0001, as shown in Figure 6 .

[0089] The results show that there is a significant difference in the adsorption of recombinant SPA by Sepharose-6FF A133-IgG affinity material and blank Sepharose-6FF affinity chromatography. The results show that high-purity serum antibodies of different groups are covalently bound to activated (epoxy activation-amination-cyanuric chloride substitution reaction) agarose microspheres by chemical coupling method, and different groups of serum antibody columns are successfully prepared.

[0090] S3: Prepare BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein, and perform affinity chromatography of BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein with experimental group affinity antibody column, and perform affinity chromatography of the same BCG (strain) protein sample with control group affinity antibody column. Obtain BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein combined with serum antibodies of the experimental group and BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein combined with serum antibodies of the control group, and elute the BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein combined with serum antibodies of the experimental group and the BCG (strain) culture filtrate protein or BCG (strain) cell plasma protein combined with serum antibodies of the control group.

[0091] The prepared BCG (strain) culture filtrate or BCG (strain) cell cytoplasmic protein was respectively subjected to chromatography with an experimental group affinity antibody column and a control group affinity antibody column, and then washed with Gly-HCl with a pH of 3.0. The experimental group affinity antibody column was a tuberculosis patient serum antibody IgG chromatography column, and the control group affinity antibody column was a healthy person serum antibody IgG chromatography column.

[0092] The S4, S5 and S6 steps are as follows:

[0093] Liquid chromatography-tandem mass spectrometry analysis of eluted components:

[0094] The BCG (strain) protein components adsorbed by serum antibodies IgG of the experimental group and the control group were collected respectively; all samples were subjected to Trypsin enzymolysis, and the enzymolysis polypeptides were desalted by Ziptip, and then the component concentration was adjusted to 0.01 mg-10 mg / mL, and then LC-MS / MS (Thermo Fusion Lumos) analysis was performed. The mass spectrometer is connected with Easy-NLC1200 through a spraying device, adopts a 2 μm reversed-phase resin (PepMap RSLC) with a C18, a 15 cm chromatographic column with an inner diameter of 0.075 mm, and a flow rate of 300 nL / min, and is subjected to linear gradient separation for 60 min from 95% A solvent (0.1% formic acid, 2% acetonitrile, 98% water) to 28% B solvent (0.1% formic acid, 80% acetonitrile). The spraying voltage is 2.1 KV, the ion transfer capillary temperature is 275℃, and the radio frequency lens is 60%. The mass spectrometer is operated in a positive ion mode, and automatically switches between MS and MS / MS in a data-dependent mode using TUNE and Xcalbur 4.0.27.19 software package. One complete MS scan is obtained from 350 to 1500 m / z under high resolution R=60,000 (defined at m / z=400), MS / MS fragment analysis is performed for ions with a charge state of 2-7 and a collision energy of 30%, and 20 most abundant polycharged ion fragments are obtained.

[0095] Database retrieval and data analysis

[0096] LC-MS / MS ion spectra were processed, mass spectrometry detected and database searched using Bio Information Atics Solutions. According to UniProt protein database search Mycobacterium bovis (strain BCG / Pasteur 1173P2, 3877 species of protein) library search BCG cytoplasm whole protein of serum antibody column affinity chromatography of healthy people and tuberculosis patients, screening specific and differential protein species in BCG whole protein of serum antibody column affinity chromatography of pulmonary tuberculosis sputum smear (+) patients, constructing pulmonary tuberculosis serum specific recognition BCG protein antigen spectrum, such as Figure 7

[0097] According to the UniProt protein database Mycobacterium bovis (strain BCG / Pasteur 1173P2, 3877) proteolytic polypeptide information, search and compare the serum antibody IgG adsorbed BCG proteolytic polypeptide fragments detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and determine the corresponding BCG protein information. Finally, the basic biochemical properties and functions of the detected BCG proteins were analyzed and predicted.

[0098] The recognition specificity between serum antibodies (healthy people, tuberculosis patients) specifically adsorbed on the antibody column and BCG proteins / antigens can be affected by the following factors: 1. Specific recognition between low concentration of antibodies in healthy people and Mycobacterium tuberculosis antigens caused by BCG vaccination; 2. Specific recognition of antibodies produced by patients in response to pathogenic antigens and BCG antigens caused by infection of healthy people with normal immunity by air containing Mycobacterium tuberculosis complex bacteria aerosol; 3. Non-specific binding of antibodies in human serum to individual BCG proteins; (4) Non-specific binding between affinity chromatography column and BCG proteins. The probability of this situation theoretically occurring depends on the choice of the final detection tool. For example, if the Western-blot (WB) method is used, the above non-specific proteins may not be obtained; if high sensitivity LC-MS / MS detection is used, the detection rate of the above non-specific proteins will be enhanced. Therefore, setting reasonable screening standards can to some extent reduce the interference factors on the determination of experimental results.

[0099] ​LC-MS / MS analysis of the information of the BCG protein recognized by the serum antibody of the tuberculosis patient or the serum antibody of the healthy control group is set as follows: (1) one or more peptide sequences specifically combined with the serum antibody of the tuberculosis patient determines the BCG protein, which is preliminarily included in the candidate protein range; (2) whether the BCG protein combined with the serum antibody of the tuberculosis patient and the serum antibody of the healthy person is listed as a candidate serum marker depends on the mass spectrometry data results of the same protein detected in the experimental group and the control group. If the number of peptides and the coverage of peptide sequences of the same protein detected by mass spectrometry in the two groups are equal or have no significant difference, the protein cannot be used as a vaccine strain antigen profile antigen library component specifically recognized by the serum antibody IgG. Otherwise, the BCG protein is listed as a candidate pathogen protein profile for tuberculosis detection. Based on the above criteria, finally, by querying the protein Uniprot database, the Mycobacterium bovis (strain BCG / Pasteur1173P2) database protein is 3877, and the number of LC-MS / MS detected in the serum of the healthy person and the sputum smear (+) patient with pulmonary tuberculosis Sepharose-6FF-A133-IgG affinity chromatography BCG protein elution and flow-through component is 2649 and 3281, respectively, as shown in Figure 7 , the analysis data results show that the protein detection rate is 68% and 85%, respectively. Search the BCG protein database, and screen out the high-abundance proteins specifically adsorbed by the serum of the sputum smear (+) patient with pulmonary tuberculosis Sepharose-6FF-A133-IgG affinity chromatography BCG.

[0100] LC-MS / MS detection data analysis, search BCG protein database, draw a Venn diagram, as shown in Figure 7 , it can be seen from the figure that there are 52 specific adsorption proteins in the Sepharose-6FF-A133-IgG affinity chromatography BCG cytoplasmic whole protein eluate of the serum of the sputum smear (+) patient with pulmonary tuberculosis. There is one protein with a coverage of more than 10%, and there are 14 proteins with a coverage of 5%-10%. The coverage of the remaining proteins is less than 5%. Bioinformatics analysis includes 19 kinds of various enzyme proteins, 8 kinds of unidentified proteins, 7 kinds of structure domain proteins, 6 kinds of family proteins, 6 kinds of membrane proteins, 2 kinds of transcriptional regulation proteins, and the remaining proteins include iron uptake proteins, conserved lipid proteins, phosphomannose proteins, and UvrABC system proteins.

[0101] The LC-MS / MS was used to detect the different groups of Sepharose-6FF-A133-IgG affinity chromatography BCG cytoplasmic protein elution components of the difference protein, the intersection of the two groups of samples of healthy people and pulmonary tuberculosis sputum smear (+) patients serum significantly up-regulated protein was 103 kinds. The protein in the serum of different groups of Sepharose-6FF-A133-IgG affinity chromatography BCG cytoplasmic protein elution sample was different. From the 103 kinds of proteins, 22 kinds of proteins with a difference ratio greater than 1.5 were screened, bioinformatics analysis, including 5 kinds of membrane proteins and 5 kinds of enzyme proteins, 3 kinds of domain-containing proteins, 2 kinds of unidentified proteins, the remaining proteins including ubiquitination protein, transcription regulation protein, conserved secretory protein, antitoxin protein, serine-rich protein, tuberculin-related peptide protein, inv protein.

[0102] Therefore, the LC-MS / MS analysis of the tuberculosis patients and healthy people serum antibody affinity material Sepharose-6FF-A133-IgG chromatography BCG cytoplasmic protein components after enzymolysis, a total of 2649 kinds of BCG proteins were detected, and 74 kinds of differential proteins were screened (as shown in Table 1). The types of BCG proteins specifically recognized by the serum IgG of pulmonary tuberculosis sputum smear (+) patients and the types of differential proteins were screened, and the antigen spectrum of the serum antibody of pulmonary tuberculosis patients specifically recognizing BCG was constructed.

[0103] Table 1 List of BCG proteins specifically recognized by the serum antibody of tuberculosis patients (part of the proteins)

[0104]

[0105] Bacillus Calmette-Guerin Vaccine (BCG) is the only vaccine approved by the World Health Organization (WHO) for tuberculosis prevention; the gene level homology between Mycobacterium tuberculosis standard strain and BCG strain is as high as 98%. Both belong to the Mycobacterium tuberculosis complex strain system. BCG is derived from bovine tuberculosis bacteria cultured in bovine bile-containing medium for more than 230 generations, and the virulence of the pathogenic bacteria is reduced, forming a live vaccine harmless to the human body but capable of producing immunity. Compared with the bovine Mycobacterium M. bovis strain, 5 regions are deleted, a total of 38 open reading frames. The attenuated strain of bovine Mycobacterium-BCG lacks 16 regions compared with the MTB-H37Rv strain, a total of 129 open reading frames. The whole genome sequencing of BCG Pasteur 1173P2 has been completed, and the full length is 4374522 bp, containing 3954 genes encoding proteins; although BCG is a strain attenuated by passage of bovine Mycobacterium, the full length of its genome is still 30 kb larger than that of bovine Mycobacterium AF2122 / 97 strain, mainly because BCG genome contains two independent tandem repeat fragments DU1 and DU2. BCG can enhance children's resistance to miliary tuberculosis and tuberculous meningitis infection, and the protection can reach 86%. Based on the conditions of tuberculosis bacillus culture facility equipment and biosafety, etc., the invention does not use Mycobacterium tuberculosis H37Rv strain protein whole protein (cytoplasmic protein and culture filtrate protein) as the antigen mixture for affinity adsorption of serum antibody IgG of tuberculosis patients, but selects Mycobacterium bovis (strain BCG / Pasteur 1173P2) with high biosafety and high homology (gene level homology as high as 98%) as the antigen mixture for affinity adsorption of serum antibody IgG of tuberculosis patients.

[0106] Compared with the existing patent with the patent number ZL202111432830.8, the number of BCG (strain) protein screened by the present application is more, and both the low-abundance Mycobacterium tuberculosis protein and the high-abundance Mycobacterium tuberculosis protein can be detected, and the constructed serum antibody IgG specificity adsorption BCG protein marker protein library information of tuberculosis patients is more comprehensive and accurate. The reasons are as follows: first, the number of BCG protein detected by mass spectrometry in the present application is more than that in the existing invention (ZL202111432830.8); second, in the process of LC-MS / MS detection of complex protein mixture, high-abundance proteins can shield / cover the mass spectrometry signal of low-abundance proteins in the component; and in the present application, the serum of patients or healthy people is covalently coupled to the activated Sepharose-6FF matrix, and the serum antibodies coupled thereon are not eluted, while in the existing patent information with the patent number ZL202111432830.8, the serum antibody IgG is eluted from the biomimetic affinity material BiAC-A115-94 together with the Mycobacterium tuberculosis antigen adsorbed thereon.

[0107] The above only discloses the preferred embodiments of the present application, and of course cannot limit the scope of the rights of the present application, and those skilled in the art can understand that all or part of the processes of the above embodiments are implemented, and equivalent changes made according to the claims of the present application still belong to the scope covered by the present application.

Claims

1. A screening method for serum antibody IgG specific recognition of its vaccine strain antigen profile, characterized by, Comprise the following steps: S1: screening out the serum samples of control group and experimental group, carrying out biomimetic affinity and affinity chromatography on the serum proteins of experimental group to obtain serum antibody IgG of experimental group; carrying out biomimetic affinity and affinity chromatography on the serum proteins of control group to obtain serum antibody IgG of control group; S2: coupling the serum antibody IgG of experimental group to the activated solid carrier material by specific chemical covalent bond to prepare an affinity antibody column of experimental group; coupling the serum antibody IgG of control group to the activated solid carrier material by specific chemical covalent bond to prepare an affinity antibody column of control group; S3: preparing BCG strain culture filtrate protein or BCG strain cytoplasm protein, and carrying out affinity chromatography on the BCG strain culture filtrate protein or BCG strain cytoplasm protein with the affinity antibody column of experimental group and the affinity antibody column of control group respectively, to obtain BCG strain culture filtrate protein or BCG strain cytoplasm protein combined with serum antibody of experimental group and BCG strain culture filtrate protein or BCG strain cytoplasm protein combined with serum antibody of control group; S4: analyzing the protein information of the BCG strain culture filtrate protein or BCG strain cytoplasm protein combined by the experimental group and the control group by high-throughput liquid chromatography-mass spectrometry; S5: searching the BCG strain proteomics database to obtain the basic biochemical information of each protein; analyzing and comparing the differences between the BCG strain culture filtrate protein or BCG strain cytoplasm protein combined by the experimental group and the BCG strain culture filtrate protein or BCG strain cytoplasm protein combined by the control group, eliminating the BCG strain proteins combined with the serum antibody column of the control group, and selecting the BCG strain proteins specifically combined with the serum of tuberculosis patients as the tuberculosis protein vaccine markers and / or tuberculosis serological detection markers; S6: performing mass spectrometry data analysis, bioinformatics analysis and immunology analysis on the tuberculosis protein vaccine markers and / or tuberculosis serological detection markers constructed in S5 to obtain the vaccine strain antigen spectrum specifically recognized by serum antibody IgG.

2. The method of screening for serum antibody IgG specific recognition of antigenic profile of vaccine strain as claimed in claim 1 wherein, In the S1 step, the serum samples of control group and experimental group are screened out by coating BCG cytoplasmic protein and prepared MTB recombinant protein antigens on a solid carrier, adding the serum samples to be detected, detecting the level of serum antibody by enzyme-labeled material coloration, and screening out the serum samples of control group and experimental group to remove serum samples without producing tuberculosis antibodies in the experimental group and serum samples of tuberculosis latent infection patients containing anti-mycobacterium tuberculosis antibodies in the control group.

3. The method of screening for serum antibody IgG specific recognition of antigenic profile of vaccine strain as claimed in claim 2, wherein, In the S2 step, the activated solid carrier material is prepared by the following methods: synthesizing the activated solid carrier material by epoxy activation reaction, amination reaction and cyanuric chloride substitution reaction.

4. The method of screening for serum antibody IgG specific recognition of antigenic profile of vaccine strain as claimed in claim 2, wherein, Before the S2 step, the screening of affinity materials specifically binding serum IgG is required to obtain the best affinity material for specifically adsorbing serum IgG. ​ 5. The method for screening of serum antibody IgG specific recognition of antigen profile of vaccine strain according to any one of claims 1-4, characterized in that, The S3 step further comprises eluting the BCG strain culture filtrate protein or BCG strain cytoplasmic protein bound by the serum antibodies of the experimental group and the serum antibodies of the control group, respectively, to wash down the bound BCG strain culture filtrate protein or BCG strain cytoplasmic protein.

6. The method of screening for serum antibody IgG specific recognition of antigenic profile of vaccine strain as claimed in claim 5 wherein, The eluent is PBS or glycine hydrochloride.

7. Serum antibodies IgG specifically recognizing the antigenic profile of its vaccine strain, characterized by, The serum antibodies IgG are obtained by the screening method of any one of claims 1-6.

8. Use of serum antibody IgG specific to recognize its vaccine strain antigen profile of claim 7 in screening novel markers for development of tuberculosis protein vaccine and / or in preparing serodiagnostic reagents for tuberculosis, characterized by, The tuberculosis includes pulmonary tuberculosis and extrapulmonary tuberculosis.

9. A kit for aiding in the detection of tuberculosis, characterized in that, The serum antibodies IgG are obtained by the screening method of claim 7.

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