CN115993458A - Peripheral blood protein marker kit for diagnosis of chronic obstructive pulmonary disease - Google Patents

Abstract

The invention provides a peripheral blood protein marker kit for diagnosing chronic obstructive pulmonary disease, which belongs to the biomedical field, and is characterized by comprising differential proteins alpha-1-acid glycoprotein 1, histine-rich glycoprotein, heparin cofactor 2, protein AMBP and protein Z-dependent protease inhibitor.

Description

Peripheral blood protein marker kit for diagnosis of chronic obstructive pulmonary disease

Technical Field

The invention relates to the biomedical field, in particular to a peripheral blood protein marker kit for diagnosing chronic obstructive pulmonary disease.

Background

Chronic obstructive pulmonary disease refers to a common, preventable and therapeutic chronic tracheal disease that is the third most common chronic disease in our country, with significant exposure to toxic particles or gases leading to airway and/or alveoli abnormalities. The disease has slower onset and insignificant initial symptoms, and most patients delay the good opportunity for treatment. Early diagnosis and treatment of slow obstructive pulmonary disease is therefore a key step in reducing the incidence and mortality of slow obstructive pulmonary disease. The gold standard for diagnosing COPD in clinic is a measure of lung function, but early lung function of patients is often normal, and early diagnosis and intervention are difficult. In order to establish a diagnosis method with good convenience, rapidness, sensitivity and specificity for COPD, it is important to find serum biomarkers for COPD diagnosis.

At present, a plurality of researchers use proteomics to screen differential proteins related to the chronic obstructive pulmonary disease, and prove that indexes such as Fetuin-B (FETUB), MKK3 and the like are related to the reflection of chronic obstructive pulmonary disease acute phase inflammation. Based on proteomics analysis and bioinformatics analysis of isotope labeling relative and absolute quantitative technology, differential proteins in serum of chronic obstructive pulmonary disease patients and healthy people are analyzed based on proteomics, and Differential Expression Proteins (DEPs) between tobacco induced chronic obstructive pulmonary disease patients and normal smokers are screened to find a diagnosis marker of chronic obstructive pulmonary disease towards gas.

Disclosure of Invention

Aiming at the problems, the invention provides a kit for diagnosing chronic obstructive pulmonary disease with higher accuracy. Five slow-resistance lung-related differential proteins are successfully screened out by using iTRAQ proteomics and biological analysis, wherein the AGP-1 and AMBP expression is up-regulated, the HRG, HC II and ZPI expression is down-regulated, and the results obtained by ELISA verification are consistent, so that the protein can be used as a candidate serum biomarker for diagnosis.

In order to achieve the above purpose, the present invention provides the following technical solutions.

The invention provides application of a reagent for detecting a protein marker in preparing a kit for diagnostic screening and grading evaluation of chronic obstructive pulmonary disease, which is characterized in that the protein marker comprises one or a combination of more of the following components: AGP-1, AMBP, HRG, HC II, ZPI.

Further, the kit detects the level of protein markers in the sample using ELISA techniques.

Preferably, the sample is serum.

Further, the kit for diagnosis, screening and grading evaluation of the chronic obstructive pulmonary disease takes one or a combination of more of AGP-1, AMBP, HRG, HC II or ZPI as a marker for diagnosis, screening and grading evaluation.

Preferably, the diagnosis and screening kit for chronic obstructive pulmonary disease detects the level of AGP-1, AMBP, HRG, HC II or ZPI in the sample by ELISA technique, wherein the levels of AGP1 and AMBP are positively correlated with chronic obstructive pulmonary disease; levels of HRG, HC ii and ZPI are inversely related to chronic obstructive pulmonary disease.

Preferably, the kit for the graded assessment of chronic obstructive pulmonary disease is for detecting the level of AGP-1, AMBP, HRG, HC II or ZPI in a sample by ELISA technique, wherein the levels of AGP1 and AMBP are positively correlated with the grade of chronic obstructive pulmonary disease; levels of HRG, HC ii and ZPI are inversely related to chronic obstructive pulmonary disease progression.

Compared with the prior art, the invention has the beneficial effects.

Compared with the traditional method for judging whether the airflow limitation is increased or not by checking the lung function in clinic, the method for diagnosing the slow-release lung has better early diagnosis value by taking AGP1, HRG, HC II, AMBP and ZPI as candidate serum protein markers, can avoid missed diagnosis of the slow-release lung due to slower onset of the disease of patients, relatively insignificant early symptoms of the disease and insufficient knowledge of partial patients on clinical symptoms such as cough, expectoration and the like, and delays the optimal opportunity of treatment.

According to the invention, through proteomics analysis and bioinformatics analysis by isotope labeling relative and absolute quantitative technology, based on proteomics analysis of differential proteins in serum of chronic obstructive pulmonary disease patients and healthy people, differential Expression Proteins (DEPs) between tobacco induced chronic obstructive pulmonary disease patients and normal smokers are screened to find early diagnosis markers of chronic obstructive pulmonary disease. The method and the kit have the outstanding advantages of high flux, high specificity, rapid and accurate detection and the like, and have important clinical value for diagnosing chronic obstructive pulmonary diseases. The invention is suitable for early screening and diagnosis of chronic obstructive pulmonary disease, and has important significance for improving the diagnosis rate of chronic obstructive pulmonary disease, realizing early intervention treatment and reducing the death rate of chronic obstructive pulmonary disease.

Drawings

FIG. 1 shows the results of an iTRAQ-based proteomic analysis.

FIG. 2 shows GO notes and KEGG path associations corresponding to functional characterizations of DEPs.

FIG. 3 shows the results of ELISA quantitative analysis of the screened differential proteins.

Detailed Description

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.

Examples

1. A peripheral blood protein marker kit for use in the diagnosis of chronic obstructive pulmonary disease comprising the following components:

an early diagnosis marker of chronic obstructive pulmonary disease, wherein the early diagnosis marker of chronic obstructive pulmonary disease comprises differential protein alpha-1-acid glycoprotein 1 (AGP-1), histidine-rich glycoprotein (HRG), heparin cofactor 2 (HC II), protein AMBP and protein Z-dependent protease inhibitor (ZPI).

2. The screening method comprises the following steps:

study object: experimental group 6 male subjects (n=3, smokers not suffering from COPD; n=3, smokers suffering from COPD), experimental group 68 male subjects (n=26, smokers not suffering from COPD; n=8, global obstructive pulmonary disease initiative GOLD i phase; n=12, GOLD ii phase; n=14, GOLD iii phase; n=8, GOLD iv phase, smokers suffering from COPD) first group tested potential serum biomarker candidates by iTRAQ-based proteomics; the second group validated serum biomarker candidates by ELISA. Patient enrollment criteria for COPD were: (1) male patients, ages 50-70 years; (2) The patient is diagnosed according to the GOLD guidelines and must be in stationary phase; (3) Smoking history is 10 years or more, and smoking stopping time is 5 years or more. The exclusion criteria were: (1) Patients with unstable cardiovascular and cerebrovascular diseases, obvious damage to liver and kidney functions and mental diseases; (2) Other lung diseases such as asthma, tuberculosis, pneumonia and the like are diagnosed within 2 months; (3) Patients taking prescribed immunosuppression drugs include hormonal drugs (e.g., methylprednisolone), cytotoxic drugs (e.g., azathioprine), calmodulin inhibitors (e.g., cyclosporine), biological agents (e.g., anti-lymphoglobulin), and new generation monoclonal antibodies. In addition, selecting male smokers with ages of 50-70 and without COPD as a control group;

(II) sample collection: plasma samples were collected after fasting for each subject. Centrifuging the sample at room temperature for 30 minutes, and temporarily storing at-80 ℃ for use;

(III) iTRAQ quantitative proteomics experiments:

1. protein extraction: serum high abundance protein removal was performed using a Proteo miner kit to extract protein. DTT was added at a final concentration of 10mM, and the mixture was subjected to a water bath at 56℃for 1 hour. IAM was added at a final concentration of 55mM and left in the dark for 45min. Finally, 5 times of cold acetone is added, and the mixture is kept stand at-20 ℃ for precipitation overnight. Centrifuging at 30000 rpm at 4deg.C for 15min, discarding supernatant, and precipitating to obtain enriched protein. The precipitate was simply air dried, residual acetone was removed, TEAB (0.5M) was added for reconstitution, and ice bath sonicated for 5min (2 sec/3 sec). Centrifuging again at 4deg.C and 30000 rpm for 15min, and collecting supernatant for quantification;

2. protein concentration measurement (Bradford quantification): a standard curve of BSA standard was prepared, and the standard amounts (0.2. Mu.g/. Mu.L BSA) of 9 tubes were 0,2,4,6,8, 10, 12, 14, 16, 18. Mu.L in this order, and pure water was added to each tube to give a final volume of 20. Mu.L. Tube 1 is a reference without protein and the other tubes contain 0.4, 0.8, 1.2, 1.6, 2, 2.4, 2.8, 3.2, 3.6 μg protein, respectively. 180 mu L protein assay reagent was added to each tube, mixed and incubated at room temperature for 10min. Measuring absorbance at 595nm with an enzyme-labeled instrument, and reading the absorbance of each sample with the tube 1 as a reference;

3. SDS electrophoresis: 12% SDS polyacrylamide gel was prepared. Each sample was mixed with a 2 Xloading buffer and heated at 95℃for 5min. The loading of each sample was 30. Mu.g and the Marker loading was 10. Mu.g. 120V constant pressure electrophoresis for 120min. After electrophoresis, the sample is dyed for 2 hours by using a dye solution, and decolorized by using a decolorizing solution for 3 to 5 times. Each time for 30min;

4. and (3) protein enzymolysis: 100. Mu.g of protein was precisely removed for each sample. The preparation method comprises the following steps of: the enzyme=20:1 ratio was added to the Trypsin and the enzyme was digested for 4h at 37 ℃. Adding Trypsin again according to the proportion, and continuing enzymolysis at 37 ℃;

5. iTRAQ marker: after trypsin digestion, the peptide fragments were pumped dry using a vacuum centrifugal pump. The peptide was reconstituted with 0.5M TEAB and iTRAQ-labeled according to the manual. Each set of peptide fragments was labeled with a different iTRAQ tag and incubated at room temperature. Mixing the labeled peptide fragments, and performing liquid phase separation by using an SCX column;

6. SCX separation: the sample is subjected to liquid phase separation by using an Shimadzu LC-20AB liquid phase system and an UltremexSCX column with the model of 4.6X250 mm. The labeled and drained mixed peptide was reconstituted with 4ml buffera (25mM NaH2PO4 in 25%CAN,pH2.7). Gradient elution was performed at a rate of 1 mL/min after column entry: eluting with bufferA for 10min, and gradually mixing with 5-35% bufferB (25 mM NaH) ₂ PO ₄ 1M KCl in 25% CAN, pH 2.7) for 11min, and finally eluting with 35-80% bufferB for 1min. The whole elution process is monitored under the absorbance of 214nm, and 20 components are obtained through screening. Desalting each component by using a StrataX desalting column, and then freezing and draining;

7. LC-ESI-MSMS analysis based on Triple TOF 5600: the liquid phase system associated with the mass spectrometer was nanoacquality (wat5.2 ers) and included both Symmetry C18 column (5 μm gauge, 180umx mm) and BEH 130C 18 column (1.7 μm gauge, 100umx mm). The symmetry C18 column was used for peptide fragment adsorption and desalting and the BEH 130C 18 column was used for separation. To both the mobile phases used, liquid a (water: acetonitrile: formic acid=98:2:0.1) and liquid B (water: acetonitrile: formic acid=2:98:0.1), a proportion of correction liquid (Thermo FisherScientific) was added. Each loading was 2.25. Mu.g (9. Mu.L), and the solution A was eluted at a flow rate of 2. Mu.L/min for 15min, followed by peptide adsorption and desalting. The elution was then started with a mobile phase containing 5% B solution at a flow rate of 300nL/min for 1min, starting to establish the elution gradient: the gradient line type of the B liquid is increased from 5% to 35% within 40min, then from 35% to 80% within 5min, then 80% is continuously eluted for 5min, and finally the column is recovered within 2 min. The resulting protein was then retrieved in the homosapiens database using Proteome Discoverer 1.4.1 (Thermo Fisher Scientific) and Mascot version 2.3 (Matrix Science, london, UK);

(IV) bioinformatic analysis: functional characterization of the labeled DEPs was determined by Gene Ontology (GO) annotation analysis using the UniProt-GO database (http:// www.ebi.ac.uk/GOA /). The Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, by which the most predominant biochemical metabolic pathways and signal transduction pathways involved in proteins were determined. Classifying the DEPs into three functional categories, namely Biological Process (BP), cellular Component (CC) and Molecular Function (MF) based on GO enrichment analysis of the DEPs;

(V) enzyme-linked reaction adsorption assay (ELISA): determining the level of the differential protein in the sample by using a double-antibody sandwich enzyme-labeled immunoassay method, verifying the amplified sample size of the screened differential expression protein by using an ELISA technology, and verifying the screened differential protein in the late stage slow-resistant lung by using the ELISA technology;

and (six) statistical analysis: the counting data is checked, and the counting data is described as + -S. The sensitivity and specificity of protein markers in early diagnosis of chronic obstructive pulmonary was assessed using the subject working profile (Receiver operating characteristic CUINC, ROC). All experimental data are statistically analyzed by adopting SPSS 20.0 software, and the difference is statistically significant when P < 0.05;

(seventh) identification of serum differential proteins: by iTRAQ quantitative proteomics experiments, we identified 144 DEPs between COPD patients and control group, 80 of which expressed up-regulated differential proteins and 64 of which expressed down-regulated differential proteins;

(eight) GO enrichment analysis and path enrichment analysis of differential proteins: functional characterization of DEPs by bioinformatics analysis revealed corresponding GO notes and KEGG path associations (FIGS. 2B-D). Wherein the GO project is enriched (CC, BP and MF) are P, P02647, P0 9, P01009, P10909, P02671, P01031, P08, P, Q14520, B4E344, P02763, P08603, F6KPG5, B4DUV1, B2R950, P00744, A8K5J8, P01023, P01011, P, A0M8Q6, P Q53H26, P, O, Q, P02675, P, K7ERI9, O, P0C0L4, P27169, P02743, P01871, K7ER74, P13671, D3JV41, P02760, P, Q16610, P, Q14515, P00739, Q5NV84, P68871, Q5VY30, Q8TDL5, P10645, C9J6H 2P, Q9UK55, Q5NV90, P24593, Q, P02647, P10909, P01031, P08, P, Q, P02763, P08603, P00134, A8K5J8, P01023, P, A0M8Q6, P, B7Z550, P, B2R6W1, P0C0L4, P01871, P13671, A8K8Z4, D3JV41, P02760, Q16610, P, Q5NV84, P, Q5NV90, Q, P02647, P01009, B7Z1F8, P01031, B2R950, P01023, P01011, P, Q5UGI6, B4DR57, P0C0L4, K7ER74, P02760, P, B4DPP8, B2RMS9, Q9K 55, B8. According to selection principle, at least 2-3 of CC, BP or MF are enriched, several proteins of the complement system or that have been reported in COPD are removed, and the following proteins are temporarily screened: alpha-1-acid glycoprotein 1 (AGP-1), histine-rich glycoprotein (HRG), heparin cofactor 2 (HC II), protein AMBP, protein Z-dependent protease inhibitor (ZPI);

(nine) differential protein validation of ELISA: the results of ELISA quantitative analysis of the above-screened differential proteins were shown in FIG. 3 by ELISA measurement of the peripheral blood serum proteins of the validation group. Among them, AGP1 is significantly elevated in COPD patient serum and is associated with higher grade, the higher the level (as in fig. 3A). HRG was significantly reduced in COPD patient serum and correlated with higher grade, lower level (as in fig. 3B). HCII is significantly reduced in COPD patient serum and is associated with higher grade, lower level (as in fig. 3C). AMBP is significantly elevated in COPD patient serum and is associated with higher grade, the higher the level (as in fig. 3D). ZPI is significantly reduced in COPD patient serum and is associated with higher grade, lower level (as in fig. 3E).

The invention mainly screens the Differential Expression Proteins (DEPs) between the patients with the tobacco induced chronic obstructive pulmonary disease and normal smokers through proteomics analysis and bioinformatics analysis of isotope labeling relative and absolute quantitative technology, and verifies that the five screened differential expression proteins can be used as candidate serum biomarkers for early diagnosis of the chronic obstructive pulmonary disease by ELISA technology.

Claims (6)

1. Use of a reagent for detecting a protein marker in the preparation of a kit for diagnostic screening, fractionation assessment of chronic obstructive pulmonary disease, characterized in that the protein marker comprises one or a combination of several of the following: AGP-1, AMBP, HRG, HC II, ZPI.

2. The use according to claim 1, wherein the kit detects the level of protein markers in the sample using ELISA techniques.

3. The use according to claim 2, wherein the sample is serum.

4. The use according to claim 1, wherein the kit for diagnostic screening and grading evaluation of chronic obstructive pulmonary disease uses AGP-1, AMBP, HRG, HC ii or ZPI as diagnostic screening and grading evaluation marker.

5. The use according to claim 4, wherein the diagnostic screening kit for chronic obstructive pulmonary disease detects the level of AGP-1, AMBP, HRG, HC ii or ZPI in the sample by ELISA, the levels of AGP1 and AMBP being positively correlated with chronic obstructive pulmonary disease; levels of HRG, HC ii and ZPI are inversely related to chronic obstructive pulmonary disease.

6. The use according to claim 4, wherein the kit for the graded assessment of chronic obstructive pulmonary disease is for detecting the level of AGP-1, AMBP, HRG, HC ii or ZPI in a sample by ELISA technique, the levels of AGP1 and AMBP being positively correlated with the grade of chronic obstructive pulmonary disease; levels of HRG, HC ii and ZPI are inversely related to chronic obstructive pulmonary disease progression.

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