CN114235990A - A screening method and application of tuberculosis serum markers - Google Patents
A screening method and application of tuberculosis serum markers Download PDFInfo
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- CN114235990A CN114235990A CN202111432830.8A CN202111432830A CN114235990A CN 114235990 A CN114235990 A CN 114235990A CN 202111432830 A CN202111432830 A CN 202111432830A CN 114235990 A CN114235990 A CN 114235990A
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Abstract
本发明公开了一种结核病血清标志物筛选方法，结核病患者血清(实验组)、健康人血清(对照组)分别进行仿生亲和或亲和层析，制备实验组仿生亲和或亲和层析抗体柱、制备对照组仿生亲和或亲和层析抗体柱；将结核杆菌培养滤液或结核杆菌胞浆蛋白分别与实验组、对照组仿生亲和或亲和层析抗体柱层析；分析与上述实验组和对照组血清抗体结合的结核杆菌蛋白，初步获得结核病检测的血清标志物；进一步方法分析上述初选结核病检测血清标志物。这种方法采用仿生亲和色谱、抗原‑抗体特异识别、质谱分析、生物信息学分析方法相结合方式，实现结核患者血清标志物的高通量筛选与评估。解决了现有技术筛选结核病血清标志物方法的局限性。
The invention discloses a method for screening tuberculosis serum markers. Tuberculosis patient serum (experimental group) and healthy human serum (control group) are respectively subjected to biomimetic affinity or affinity chromatography to prepare the biomimetic affinity or affinity chromatography of the experimental group Antibody column, preparation control group biomimetic affinity or affinity chromatography antibody column; Mycobacterium tuberculosis culture filtrate or Mycobacterium tuberculosis cytoplasmic protein were chromatographed with experimental group and control group biomimetic affinity or affinity chromatography antibody column respectively; The Mycobacterium tuberculosis proteins bound by the serum antibodies of the above experimental group and the control group were initially obtained as serum markers for tuberculosis detection; further methods were used to analyze the above-mentioned primary selection of serum markers for tuberculosis detection. This method uses a combination of biomimetic affinity chromatography, antigen-antibody specific identification, mass spectrometry analysis, and bioinformatics analysis methods to achieve high-throughput screening and evaluation of serum markers of tuberculosis patients. The limitations of the prior art methods for screening TB serum markers are solved.
Description
Technical Field
The invention relates to the field of medical diagnosis, in particular to a screening method of a tuberculosis serum marker and application of the method in the field of tuberculosis serum diagnosis.
Background
Tuberculosis (TB) is one of the infectious diseases with the highest single-disease death rate caused by the infection of organisms by tubercle bacillus (MTB), and tuberculosis patients account for 90 percent of the total number of patients. The current research on tuberculosis serum markers is a single and non-specific research mode; the following limitations exist: (1) about 50% of Mycobacterium Tuberculosis (MTB) proteins are not expressed in foreign e.coli (e.coli) host cells, resulting in the omission of such proteins in serum marker screens. (2) The expression and separation and purification processes of a large amount of MTB recombinant protein are time-consuming and labor-consuming, and most of the recombinant expressed proteins are not ideal serum markers. (3) The activity of exogenously expressed MTB protein is easily influenced by the properties of the expression host; (4) the MTB protein is identified by the total serum of a patient, and the whole protein component of the serum is very complex, so that the possibility of non-specific binding is very high, and the false positive of the detection result is easily caused.
In order to solve the limitation of the screening of the tuberculosis serum marker in the prior art, an effective screening method is needed.
Disclosure of Invention
The invention discloses a method for screening tuberculosis serum markers, which combines the bionic affinity or affinity chromatography specific adsorption of serum antibodies, antigen (tubercle bacillus antigen) -antibody specific recognition, mass spectrometry and bioinformatics analysis methods to realize high-throughput screening of the tuberculosis serum markers. Solves the limitation of the method for screening the tuberculosis serum marker in the prior art.
In addition, the invention also discloses the application of the screening method, and the diagnosis accuracy of tuberculosis is improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tuberculosis serum marker screening method comprises the following steps:
a. performing biomimetic affinity or affinity chromatography on the serum protein of the experimental group to prepare an experimental group biomimetic affinity or affinity chromatography antibody column; performing biomimetic affinity or affinity chromatography on the serum protein of the control group to prepare a biomimetic affinity or affinity chromatography antibody column of the control group;
b. preparing tubercle bacillus culture filtrate protein or tubercle bacillus cytoplasm protein, carrying out chromatography with the tubercle bacillus culture filtrate or tubercle bacillus cytoplasm protein and experimental group bionic affinity or affinity antibody chromatography respectively, and carrying out chromatography with the same tubercle bacillus protein sample and control group bionic affinity chromatography column or affinity chromatography column;
c. eluting the chromatographic column after the step b is finished, and then carrying out liquid chromatography-mass spectrometry on the eluted components;
d. and (3) analyzing the tubercle bacillus culture filtrate or tubercle bacillus cytoplasmic protein combined with the serum antibody in the experimental group and the control group by liquid chromatography-mass spectrometry.
e. Searching a tubercle bacillus proteomics database to obtain the basic biochemical information of each specific protein; comparing the tubercle bacillus culture filtrate or tubercle bacillus cell plasma protein combined with the analysis experimental group with the tubercle bacillus protein combined with the control group, deducting the tubercle bacillus protein combined with the control group, and finally constructing a tubercle bacillus serum marker protein library;
f. and carrying out mass spectrum data analysis, bioinformatics analysis and immunological analysis on the proteins in the constructed tuberculosis serum marker library to obtain the tuberculosis serum marker.
Furthermore, after the proteins in the tuberculosis serum marker library obtained in step f are expressed and separated and purified through genetic engineering, an immunological method for specifically recognizing the proteins by using antigen-antibody is adopted, and the method comprises but is not limited to indirect Enzyme-Linked immunosorbent Assay (ELISA) evaluation, and the sensitivity and the specificity of the tuberculosis serum marker are verified.
The serum of the experimental group is from tuberculosis patients, and the serum of the control group is from health physical examination patients.
The biomimetic affinity material used for the affinity chromatography of the experimental group and the control group in the step a comprises but is not limited to the following small molecule compounds: l-theanine; an amino biirine; 3-aminophenol; 4-amino 1 naphthol hydrochloride; 4-2 aminoethylbenzenesulfonamide; 1-aminoanthraquinone; hexylamine; 2, 6-diaminoanthraquinone; 2, 6-diaminopyridine; 2-phenylacetamide; adenine; a hydroxysuccinimide; 2,4, 6-triaminopyrimidine; diphenylamine; a dodecylamine; diethylamine hydrochloride; dibenzylamine; a dodecylamine; any one or two of N-acetyl-L-methionine. Or the affinity ligand of the specific adsorption serum antibody is derived from Protein A (SPA) Protein obtained by the separation or the recombinant expression of A-type staphylococcus aureus, or Protein G obtained by the separation or the recombinant expression of G-type streptococcus is used as the affinity ligand and is used for preparing the affinity ligand of the specific adsorption serum antibody.
Further, the preparation of the biomimetic affinity chromatography column in the step a comprises the following steps:
a1, carrying out pretreatment, activation reaction and ligand coupling on a solid phase matrix by using an adsorbing material with Sepharose as the matrix, and constructing a bionic affinity separation material for specifically adsorbing serum antibodies;
further comprises a bionic affinity separation material which is formed by coupling the bionic affinity separation material on a sepharose solid phase carrier which takes cyanuric chloride as a spacer arm and is activated by epoxy and is used for specifically adsorbing serum antibodies;
another scheme comprises coupling Protein A (SPA) obtained by separating or recombining A-type staphylococcus aureus or Protein G obtained by separating or recombining G-type streptococcus as ligand on a sepharose solid phase carrier which is naturally or after genetic modification and is activated by epoxy with cyanuric chloride as a spacer.
a2, pretreating the serum sample, and respectively preparing the serum sample and the bionic affinity or affinity antibody column which is screened from the bionic affinity ligand library and is specifically used for adsorbing the serum antibody.
a3, analyzing the protein loading amount, purity and yield of the serum sample in different bionic affinity or affinity antibody columns.
The tubercle bacillus culture filtrate or tubercle bacillus cytoplasmic protein used in the step b of the invention comprises culture filtrate protein and tubercle bacillus cell cytoplasmic protein when the tubercle bacillus is cultured. Preferably, the prepared tubercle bacillus culture filtrate or cytoplasmic protein is taken to be respectively subjected to chromatography with a test group bionic affinity or affinity antibody column and a control group bionic affinity or affinity antibody column. Furthermore, the experimental group affinity chromatographic column and the control group affinity chromatographic column are bionic affinity antibody columns or affinity antibody columns. Further, the tubercle bacillus culture filtrate or cytoplasmic protein which is not adsorbed by the affinity chromatography column is washed. Then the affinity chromatography column completed with step b is eluted in two steps. Further, obtaining the serum antibody-mycobacterium tuberculosis protein complex after the step b is completed. The tubercle bacillus protein includes, but is not limited to, tubercle bacillus culture filtrate protein or tubercle bacillus cell cytoplasm protein.
The protein concentrations of the different eluted fractions were quantified by BCA method. The concentration of all component proteins to be detected by mass spectrometry is adjusted by ultrafiltration concentration to an optimum concentration suitable for mass spectrometry, e.g., between 2mg and 10 mg/mL. The flow-through and eluted fractions were digested with trypsin and further dried for detection by mass spectrometry.
The elution of step c comprises elution using a two-step elution method. Further, the elution method comprises:
(1) the eluent used by the bionic affinity separation material is a glycine-hydrochloric acid buffer system with pH of 2.0-4.5 and salt ion concentration of 0.01-5.0moL/L, a phthalic acid-hydrochloric acid buffer system with pH of 2.0-4.5 and salt ion concentration of 0.01-5.0moL/L, and a citric acid-sodium citrate buffer system with pH of 2.0-4.5 and salt ion concentration of 0.01-5.0 moL/L. The elution system is also suitable for chromatography of affinity separation materials of specific purified serum antibodies with staphylococcus aureus Protein A (SPA) or G-group streptococcus strain-derived Protein G as ligand.
(2) The eluent used by the bionic affinity separation material can also select a Tris buffer liquid system with the pH value of 10.0-13.0 and the salt ion concentration of 0.01-5.0 moL.
Further, step e comprises the steps of:
searching tubercle bacillus proteins detected by mass spectrometry and respectively combined with an experimental group and a control group in a database (http:// www.uniprot.org /); eliminating tubercle bacillus protein adsorbed specifically to the control group serum antibody. And constructing a tuberculosis serum marker library by using the eliminated experimental group tubercle bacillus proteins.
And (f) performing bioinformatics, proteomics and immunity analysis prediction and specific serum marker screening on the screened mycobacterium tuberculosis protein, wherein the preferred method in the step f comprises the following steps:
(1) analyzing the molecular weight, isoelectric point, hydrophilicity and hydrophobicity, subcellular localization, whether the protein in the tubercle bacillus protein library is a secreted protein and a possible secretion mode.
(2) And screening out proteins with high abundance, high sequence specificity and strong immunogenicity in the protein library of the tubercle bacillus, wherein the proteins are used as candidate markers of the tuberculosis serum marker.
(3) The tuberculosis serum marker screened by the bionic affinity chromatography-mass spectrometry strategy or the affinity chromatography-mass spectrometry strategy is recombined and expressed in escherichia coli, and target protein is separated and purified to obtain high-purity recombined target protein; and finally, evaluating the detection sensitivity and specificity of the recombinant tubercle bacillus protein in the serum of the tuberculosis patient by using an indirect ELISA method, and finally screening out the tuberculosis serum marker.
Furthermore, the invention relates to a method for detecting tuberculosis, and the tuberculosis serum marker used is prepared according to the method of the invention.
Further, a kit for detecting tuberculosis, which is characterized in that: the tuberculosis serum marker used in the kit is prepared according to the method of the invention.
Compared with the prior art, the invention has the following beneficial effects:
1. purification of high purity serum antibodies: the bionic affinity chromatography or the affinity chromatography is used, so that the serum antibodies of the experimental group and the control group have high purity (> 90%), the nonspecific binding is less when the antibodies are specifically adsorbed with the tubercle bacillus antigen, the interference on the later analysis is reduced, and the screened tuberculosis serum marker has high specificity.
2. The antigen activity is high and is close to the natural state: the screening system combines the bionic affinity chromatography, antigen-antibody specific recognition, mass spectrometry and bioinformatics analysis methods; the specific combination of the protein and the serum antibody is a tubercle bacillus natural protein mixture, the biological activity of the protein is high, and the protein components and the protein abundance of the tubercle bacillus are closer to the natural growth condition of the thallus.
3. The reaction conditions are mild: the antigen-antibody affinity chromatography process under the system is in a mild state (10mM, pH 7.4 including but not limited to a PBS buffer system), the binding amount ratio of the tubercle bacillus antigen and the patient serum antibody is closer to the in vivo environment, and the tuberculosis serum marker screened by the method has high sensitivity and accuracy in detecting tuberculosis.
Drawings
FIG. 1 is a BiAC-grouped SDS-PAGE graph of human serum Immunoglobulin G (IgG); m is a protein marker, T0 and H0 are respectively the mixed original samples of the serum of the tuberculosis patient and the serum of the healthy control group, T1-T4 and H1-H4 are respectively the samples of the serum of the tuberculosis patient and the serum of the healthy control group flowing through in the chromatography process, and T5 and T6 are respectively SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) graphs of the samples of the serum of the tuberculosis patient and the serum of the healthy control group eluted by 0.05moL/L Gly-HCl solution with the pH value of 2.5 in the chromatography process.
FIG. 2 is a flow chart of the present invention.
FIG. 3 is SDS-PAGE picture of recombinant protein obtained by recombinant expression of tubercle bacillus protein in E.coli system and separation and purification by Ni + filler and molecular exclusion technology.
FIG. 4 ELISA method to evaluate the sensitivity and specificity of recombinant proteins in the serum of tuberculosis patients.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Preparation example 1 construction of biomimetic affinity ligand library
The synthetic process of the bionic affinity ligand and the prior patent disclosed by the inventor: "antibody affinity purification materials and their uses, 2014.11.19, china, CN 104148018A" are similarly described.
The bionic affinity small molecule structure specifically adsorbed to the human serum antibody constructed at this time comprises but is not limited to the following 19 types: l-theanine; aminopyrine; 3-aminophenol; 4-amino 1 naphthol hydrochloride; 4-2 aminoethylbenzenesulfonamide; 1-aminoanthraquinone; hexylamine; 2, 6-diaminoanthraquinone; 2, 6-diaminopyridine; 2-phenylacetamide; adenine; (ii) a hydroxy succinimide; 2,4, 6-triaminopyrimidine; diphenylamine; a dodecylamine; diethylamine hydrochloride; dibenzylamine; a dodecylamine; N-acetyl-L-methionine.
Taking the data of the mixed serum sample of the bionic affinity medium A5-87(5:4-2 amino ethyl benzene sulfonamide, 87: ethylenediamine) separated and purified healthy control group as an example.
Replacing 5g of system with 10mM PBS, coupling micromolecule compounds 5:4-2 amino ethyl benzene sulfonamide and 87: bionic affinity ligand medium A5-87 of ethylenediamine, mixing with 10.5mL of mixed healthy human serum with the concentration of 12.7mg/mL in a 50mL centrifuge tube, shaking and uniformly mixing for 30min at room temperature, standing for 10min, loading the mixture and bionic affinity medium into a gravity column, flushing the medium A5-87 with 20 times volume of PBS, directly eluting target protein combined on the medium with 0.05mol Gly-HClH, quantifying BCA protein, and analyzing the purification effect of the affinity protein through gray-scale scanning combined BCA protein quantification data after SDS-PAGE electrophoresis. The result shows that the bionic affinity medium is used for adsorbing an antibody in a mode of 16.3mg/mL, the purity of the target protein is 90.21%, and the overall yield of the protein is 87.21%.
Preparation example 2 sample pretreatment:
experimental groups: taking 100 mu L of serum samples of 20 human tuberculosis patients to obtain 2mL of serum samples, centrifuging and mixing at 2000rpm, removing precipitate, and diluting the serum samples with 8 times of 0.005-0.5mol of PBS buffer solution;
control group: the treatment method was the same as the above experimental group in 20 healthy human serum samples.
Balancing the bionic affinity chromatography column: the biomimetic affinity separation matrix gravity column was washed with 10mL of 0.1M acetic acid, 10mL of double distilled water, 10mL of 0.1M NaOH, and 10mL of double distilled water, respectively, and finally all the chromatography columns were equilibrated with 10mL of PBS (10mM, pH 7.4), respectively.
Bionic affinity chromatography of the pretreated serum sample: and (3) respectively taking 5mL of diluted serum sample solutions of the experimental group and the control group, washing the equilibrium chromatographic column by using excessive PBS (phosphate buffer solution) with the volume about 20 times of the column volume, collecting penetration liquid, and preparing to obtain the experimental group affinity chromatographic column and the control group affinity chromatographic column.
SDS-PAGE analysis of human serum IgG Biomimetic affinity chromatography (BiAC) cohort fractions:
and eluting the adsorbed proteins on the experimental group affinity chromatographic column and the control group affinity chromatographic column by using 10mL of 50mM Gly-HCl (pH 2.5) and 0.05mol of NaOH solution, and detecting the adsorption effect of the bionic affinity chromatographic column on the IgG antibodies in the serum of the experimental group and the control group by using 12.5% SDS-PAG gel electrophoresis. Adding a protein sample solution to be detected into a5 xSDS-PAGE loading buffer according to a ratio of 4:1(v/v), carrying out water bath at 95 ℃ for 10min, centrifuging for 5min at 12,000g, cooling, and carrying out electrophoresis detection on a supernatant. The effect is shown in figure 3.
Column washing and preservation: and respectively washing proteins bound on the experimental group affinity chromatographic column and the control group affinity chromatographic column by using 10-20 mL of 0.5mol NaOH solution, washing the chromatographic columns by using 20mL of double distilled water, finally washing the bionic affinity chromatographic column by using 10mL of 20% ethanol solution, and storing the experimental group affinity chromatographic column and the control group affinity chromatographic column by using 20% ethanol solution.
Example 1
The flow of the present invention is shown in fig. 2. And (3) taking the prepared tubercle bacillus culture filtrate or tubercle bacillus cell cytoplasmic protein to respectively carry out chromatography with the experimental group bionic affinity chromatography column and the control group bionic affinity chromatography column. Wherein the column chromatography materials of the experimental group bionic affinity column and the control group bionic affinity column are A5-87.
Liquid chromatography-mass spectrometry analysis of the eluted fractions:
collecting all eluted control group and experimental group protein components adsorbed on the bionic affinity chromatographic column A5-87 by a bionic-immunoaffinity method; and all eluted fractions were treated with 25mM NH4HCO3And (4) replacing the buffer solution system. Then, all samples are respectively enzymolyzed by Trypsin, the concentration of the components is adjusted to be 0.01mg-10mg/mL after the enzymolyzed polypeptide is desalted by Ziptip, and then LC-MS/MS analysis is carried out. And injecting the detected mass spectrum peak into an LTQ-VELOS mass spectrometer on line for ion identification of the polypeptide fragment. Eluting components flowing out of the reversed phase chromatographic column are sprayed out from a nanoliter-grade electric spray nozzle (the voltage is 1.9Kv), the temperature of an ion transfer capillary is 200 ℃, the Mass spectrum adopts full scanning (Mass ranges, m/z: 350-1500), and twenty fragment patterns are collected from Mass spectrum fragment patterns after full scanning (full scan) to carry out secondary Mass spectrum scanning (MS2 scan).
Mass spectrometry data analysis and standard setting of candidate proteins:
database search for MS/MS (Secondary Mass Spectroscopy) by BioWorksTM3.1.6 software (default parameters) search proteomics database to get the raw file transformed to mgf file, and the grouped components in this study were searched separately for protein sequences in the tubercle bacillus complex in the UNIPROT database. The amino acid fixed modification of the peptide segment sequence was carbamidomethyl (C), the variable modification was oxidation (M), and the mass deviation was 20 ppm. The tryptic fragment was allowed 2 deletions. The data filtering parameters are set as: XCorr is more than or equal to 1.9 and + 1; XCorr is more than or equal to 2.5 and + 2; XCorr is more than or equal to 3.75, + 3; delta Cn is more than or equal to 0.1.
And (3) analyzing and predicting the basic biological functions of the detected tubercle bacillus protein:
the information of the tubercle bacillus proteolysis polypeptide fragments detected by mass spectrometry can be compared with the tubercle bacillus whole protein polypeptide fragments subjected to virtual enzyme digestion; and finally obtaining basic information such as the amino acid sequence, the molecular weight, the isoelectric point and the like of the protein from a UNIPROT database (http:// www.uniprot.org /). For further study to determine the protein properties, SignalP v5.0(http:// www.cbs.dtu.dk/services/SignalP-5.0/) was used to analyze whether it had a signal peptide; lipop v1.0 was used to analyze whether it was lipoprotein; whether the protein is secreted to the external environment in a TAT model is analyzed by using TatP v1.0 (http:// www.cbs.dtu.dk/services/TatP /); GRAVY (http:// www.bioinformatics.org/sms 2/protein _ GRAVY. html) evaluates the overall hydrophilicity and hydrophobicity; TMHMM (http:// www.cbs.dtu.dk/services/TMHMM /) software analyzes whether the detected protein is a transmembrane protein, and the specific number of transmembrane proteins; psortb v3.0 (http:// www.psort.org/Psortb /) analyzed for its subcellular location; functional groups (http:// genetic. pateur. fr/Tubercullist /) analyze the Functional type of the protein.
The recognition specificity between the serum antibody (healthy, tubercle disease) and the target antigen (tubercle bacillus culture filtrate protein or tubercle bacillus cell plasma protein) specifically adsorbed on the bionic affinity or avidity antibody column is influenced by the following factors: (1) the specific recognition between the low-concentration antibody and the tubercle bacillus antigen in the healthy body caused by the inoculation of the bacillus calmette-guerin (bovine tubercle bacillus attenuated strain live vaccine); (2) non-specific recognition of antibodies and tubercle bacillus proteins generated by other non-pathogenic bacilli which have certain homology with tubercle bacillus in the environment and stimulate organisms; (3) non-specific combination of antibodies in human serum and a small part of tubercle bacillus protein; (4) the bionic affinity filler is non-specifically combined with the tubercle bacillus protein. The probability that this theoretically occurs depends on the choice of the final detection tool. If ELISA or immunoblotting (Western-blot) is used, the non-specific protein may not be obtained effectively; the detection rate of the above-mentioned nonspecific proteins is enhanced if a high-sensitivity mass spectrometric detection is used. Therefore, reasonable screening standards are set, and judgment of interference factors on experimental results can be reduced to a certain extent.
And (3) carrying out mass spectrometry on the proteome information of tubercle bacillus proteins recognized by serum antibodies of tuberculosis patients or serum antibodies of healthy control groups. The tubercle bacillus proteomics database and the human proteomics database (evaluation of the adsorption effect of the bionic affinity or affinity filler on the antibody) are respectively searched for the tubercle bacillus antigen-serum antibody compound. The following screening criteria were set for tuberculin information obtained by searching the tubercle bacillus proteomics database: (1) only one or more than one peptide fragment sequence specifically combined with the serum antibody of the tuberculosis patient determines the tubercle bacillus compound protein and is initially included in the range of candidate biomarkers; (2) whether the tubercle bacillus antigen combined with the serum antibody of the tuberculosis patient and the healthy human serum antibody is a candidate serum marker or not is judged; depending on the results of mass spectrometric detection of the same protein detected in the experimental group and the control group, if the number of peptide fragments of the same protein detected by mass spectrometry in the two components is equal to or has no obvious difference from the coverage rate of the peptide fragment sequence, the protein cannot be used as a component of the tuberculosis serum marker antigen library; otherwise, the tubercle bacillus protein is taken as a serum marker for detecting candidate tuberculosis.
Based on the above criteria, 237 tubercle bacillus proteins shown in table I are finally detected and used as the primary tuberculosis serum markers; a large number of candidate proteins have been used as serological tests and vaccine studies for tuberculosis: PstSI (38kda), FbpB, FbpC, FbpA, Hspx, BfrB, EsxA, EsxB Mtp63, Mpt64, etc. were screened.
Table one: tuberculosis patient serum antibody identified tuberculin list
The tubercle bacillus protein which is screened based on the bionic-immunoaffinity chromatography-mass spectrometry strategy and is specifically identified with the serum of a tuberculosis patient is partially removed according to the following standards by means of bioinformatics and immunology knowledge, so that the cost is saved, and the biomarker verification efficiency is improved. The screening method comprises the following steps:
(1) selecting a high abundance of tubercle bacillus proteins that bind to serum from a tuberculous patient, whereby the preferred proteins are characterized by a mass spectrometric detection of a number of protein peptides greater than 2 or more, including but not limited to all tubercle bacillus proteins listed in table one.
(2) According to the bioinformatics prediction of biochemical properties such as Mw, pI, GRAVY, TMDs and the like, proteins which are difficult to express in Escherichia coli are removed, and the proteins mainly mean that the proteins have strong protein hydrophobicity (GRAVY > 0.6); or an MTB protein of extreme size (Mw <8kDa, or Mw >80kDa), containing a transmembrane domain (TMDs > 1). In addition to the two main screening criteria described above, several of the following criteria may be used as references for further optimized tuberculosis serum marker screening strips.
(3) Preferably only tuberculin proteins with pathogenic mycobacteria (RD Region) are present; thus preferred proteins include, but are not limited to, tubercle bacillus proteins encoded by the Rv3874, Rv3875, Rv1980, Rv3117, Rv1770 and Rv1771 genes. (4) secretory proteins that bind preferentially to the serum of tuberculosis patients and proteins that have been identified as virulence factors; thus preferred proteins include, but are not limited to, the tubercle bacillus virulence factor proteins identified in bioinformatic analyses and published articles.
(5) Preferred subcellular localization assay results: selecting in order secreted protein > cell wall protein > cell membrane protein > cell plasma protein; thus secreted proteins and cell wall proteins are preferred.
(6) The peptide fragment sequence Alignment of the final selected tubercle bacillus protein with the human proteomic database protein is carried out by means of Basic Local Alignment Search Tool (BLAST), and the tubercle bacillus protein with large difference is preferably used as a potential tuberculosis serological detection biomarker.
(7) Coli, a tuberculin protein that is easily expressed in e.coli; such proteins should have Mw values of 10-60kDa, pI values of 4.5-10, proteins without transmembrane domains, and tubercle bacillus proteins with signal peptides which have the signal peptide sequence removed upon recombinant expression.
Based on the above criteria, 111 tubercle bacillus proteins are screened out as candidate proteins (according to gene numbers) of tuberculosis serum markers: rv0009, Rv0066, Rv0118, Rv0129, Rv0144, Rv0147, Rv0164, Rv0172, Rv0211, Rv0234, Rv0242, Rv0243, Rv0315, Rv0350, Rv0363, Rv0440, Rv0462, Rv0467, Rv0475, Rv0524, Rv0569, Rv0577, Rv0685, Rv 0333, Rv0761, Rv0798, Rv0814, Rv 0811, Rv0860, Rv0864, Rv0871, Rv0896, Rv0932, Rv0934, Rv0984, Rv1071, Rv 1071074, Rv1094, Rv1133, Rv1177, Rv1198, Rv 081267, Rv 081261310 1310, 1310, Rv0984, Rv1071, Rv1074, Rv 1443, Rv 353807, Rv 739, Rv 3247, Rv 3260, Rv 7389, Rv 7376, Rv 324776, Rv 32479, Rv 324776, Rv 327389, Rv 3260, Rv 324776, Rv 32479, Rv 3260, Rv 32479, Rv 324776, Rv 32479, Rv 324776, Rv 32479, Rv 3260, Rv 32479, Rv 3260, Rv 32479, Rv 7389, Rv 32479, Rv 3260, Rv 7389, Rv 3260, Rv 32479, Rv 3260, Rv 7389, Rv 739, Rv 3260, Rv 739, Rv 32479, Rv 739, Rv 7389, Rv 739.
However, the work load of expressing and isolating more than a hundred tubercle bacillus proteins in E.coli is very large; the invention randomly selects 20 tubercle bacillus proteins, uses escherichia coli as host bacteria to carry out prokaryotic expression, uses nickel filler to separate and purify recombinant protein, and finally evaluates the sensitivity and specificity in the serum (experimental group) of tuberculosis patients and the serum (control group) of healthy people.
Prokaryotic expression and separation purification of candidate protein
1. Construction of recombinant expression vectors
(1) Designing and synthesizing a primer, namely searching a gene sequence corresponding to the MTB protein in an NCBI database according to the MTB protein serial Number (Access Number) specifically identified by the serum of the tuberculosis patient. Then, a bioinformatics method is used for analyzing whether the protein has a signal peptide or not, whether the protein is a transmembrane protein or not, and whether the hydrophobicity of the protein is strong or weak. The final primer design removes the signal peptide of the protein with the signal peptide sequence, removes the protein with transmembrane structural domain and the protein with larger and smaller molecular weight (Mw >70kDa or Mw <10 kDa); rejecting a strongly hydrophobic protein comprising a transmembrane domain; randomly screened 20 target genes. And finally, obtaining the target gene according to the gene sequence thereof by conventional primer design and Polymerase Chain Reaction (PCR).
(2) Constructing and expressing a recombinant expression vector: and respectively constructing target genes on an expression vector PET28a by adopting a classical restriction endonuclease cutting and T4 DNase connection mode for the target genes and a target vector PET28a obtained by PCR, wherein an Rv3875 gene expression protein is constructed on an expression vector PET32a containing Trx protein due to less than 10kDa, and sequencing to verify whether a target gene sequence is successfully and correctly constructed. And finally, transforming the verified recombinant expression vector into an escherichia coli BL21(DE3) host cell for classical recombinant protein expression and verification. Finally, the genes are successfully recombined and expressed in the escherichia coli: recombinant proteins encoded by Rv0475, Rv0577, Rv0871, Rv0934, Rv1980, Rv2031, Rv2462, Rv2831, Rv2986, Rv3048c, Rv3248c, Rv3417c, Rv3457, Rv3763 and Rv3875 genes;
2. separation and purification of the recombinant expression protein:
each 1g of the cells were thoroughly resuspended in 15-30 mL of 50mM Tris-HCl (pH8.0) buffer containing 20mM imidazole, the cells were sonicated, centrifuged at 12,000rpm for 30min at 4 ℃ and the supernatant collected, and the particulate matter was removed by filtration through a filter having a pore size of 0.45. mu.m.
The recombinant protein exists in the host cell in the form of inclusion body, then the thallus is collected, after the ultrasonication, the thallus is centrifuged at 12,000rpm for 10min to collect protein precipitate, 8M urea is used for redissolving the recombinant protein, the recombinant protein is centrifuged at 12,000rpm for 30min, and the precipitate is discarded. The supernatant was filled into 3kDa dialysis bags and renatured by dialysis with 7M, 6M, 5M,4M,3M and 2Mol/L urea solutions, respectively. And finally, purifying the recombinant MTB protein containing His-tag by using a Ni + metal chelating chromatographic column. The recombinant protein is further desalted and purified by a size exclusion chromatography (Superdex TM G75, GE), and the sensitivity and specificity of the recombinant protein in the serum of a tuberculosis patient are detected by an ELISA method.
ELISA method for evaluating sensitivity and specificity of recombinant protein in serum of tuberculosis patient
The evaluation experiments of sensitivity and specificity were carried out in 198 serum of tuberculosis patients and 60 serum of healthy examiners: diluting the purified recombinant protein to 1-5 μ g/mL with 0.05mmol/L carbonate buffer (pH 9.6), coating a 96-well plate with 100 μ L per well, overnight at 4 ℃, washing 5 times with 1 XPBST (10mM PBS solution containing 0.5% Tween 20) washing buffer by daily use on a plate washing machine; adding 300 μ L of sealing solution containing 3% skimmed milk powder into each well, sealing at 37 deg.C for 2 hr, washing the plate with PBST for 6 times, and drying; adding 100 mu L of confining liquid into each hole of a lining gun, and adding 3-8 mu L of tuberculosis patients and control human serum samples into corresponding holes respectively. Incubating for 1h at 37 ℃, and washing a 96-well plate for 6 times; adding pre-prepared HRP-labeled goat anti-human IgG polyclonal antibody 100 mu L diluted by PBST according to 5,000-10,000 times into each hole, incubating for 1h at 37 ℃, and washing a 96-hole plate for 6 times; adding 100 mu L of TMB substrate buffer solution into each hole, and incubating for 10-30 min at 37 ℃; 50 μ L of 2mol/L H was added to each well2SO4Solution, and reaction is ended; measuring Abs value at 450nm wavelength of microplate reader, and determining the experimental result (determining the sample to be tested as positive if OD450 value of the sample is greater than the sum of the negative control sample OD450 average value and 3 times of standard deviation, and determining the sample as negative if the value is less than the sum)
The results show that: as shown in FIG. 4, the sensitivity of the above-mentioned selected tuberculin in patient serum is between 37.12 + -1.31 (Rv3763 gene-encoded protein) and 56.96 + -2.83 (Rv0577 gene-encoded protein); the specificity is between 85.83 + -1.44 (Rv3048c gene coding protein) and 96.67 + -1.77 (Rv3248c gene coding protein).
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