Diagnostic marker for systemic lupus erythematosus and application thereof

Abstract

The invention provides a diagnostic marker for systemic lupus erythematosus and application of the diagnostic marker, and belongs to the technical field of biological medicine. The diagnostic marker is at least one protein of PDGFA (Platelet Derivative Growth Factor Amplification), APOD (Adenosine Polymerase Chain Reaction), CFHR5, AMDHD1 and SCGB3A1; the amino acid sequence of the PDGFA is as shown in SED ID NO. 1; the amino acid sequence of the APOD is as shown in SED ID NO. 2; the amino acid sequence of the CFHR5 is as shown in SED ID NO.3; the amino acid sequence of AMDHD1 is as shown in SED ID NO. 4; and the amino acid sequence of the SCGB3A1 is as shown in SED ID (Sequence of Identification) NO.5. According to the present invention, the five proteins are adopted as the SLE disease diagnosis biomarker independently or in the combination manner, such that advantages of high accuracy, high sensitivity and high specificity are provided, and the new target spot is provided for the diagnosis, the intervention improvement and the like of the SLE patient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a diagnostic marker for systemic lupus erythematosus and application thereof. BACKGROUND

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple systems and multiple organs and having multiple autoantibodies, and its pathogenesis is complex and clinical manifestations are highly heterogeneous. At present, the diagnosis of SLE relies on various clinical and laboratory markers, some of which have high specificity, such as anti-dsDNA, anti-Sm antibody, anti-nucleosome antibody (AnuA) and the like, but some patients are negative or have fluctuating titers, and the sensitivity is low. The conventional diagnostic markers are difficult to achieve high sensitivity and high specificity, and thus it is difficult to meet the needs of precise diagnosis and treatment. SUMMARY

[0003] In view of this, the present application aims to provide a diagnostic marker for systemic lupus erythematosus and application thereof, and aims to solve at least one technical problem in the background art.

[0004] The present application is implemented as follows: The present application provides, in a first aspect, a diagnostic marker for systemic lupus erythematosus, the diagnostic marker being at least one protein selected from the group consisting of PDGFA, APOD, CFHR5, AMDHD1 and SCGB3A1. The PDGFA is platelet-derived growth factor A, and its amino acid sequence is shown in SED ID NO. 1. The APOD is apolipoprotein D, and its amino acid sequence is shown in SED ID NO. 2. The CFHR5 is complement factor H-related protein 5, and its amino acid sequence is shown in SED ID NO. 3. The AMDHD1 is amide hydrolase domain-containing protein 1, and its amino acid sequence is shown in SED ID NO. 4. The SCGB3A1 is secretoglobin family 3A member 1, and its amino acid sequence is shown in SED ID NO. 5.

[0005] Preferably, the diagnostic marker is a combination of at least two proteins selected from the group consisting of PDGFA, APOD, CFHR5, AMDHD1 and SCGB3A1.

[0006] Preferably, the diagnostic marker is a combination of at least three proteins selected from the group consisting of PDGFA, APOD, CFHR5, AMDHD1 and SCGB3A1.

[0007] Preferably, the diagnostic markers are a combination of at least four of PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

[0008] Preferably, the diagnostic markers are a combination of all of PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

[0009] The second aspect of the present application provides use of a reagent for detecting the expression amount of the diagnostic markers of systemic lupus erythematosus described above in the preparation of a product for diagnosing systemic lupus erythematosus.

[0010] Preferably, the product detects the expression amount of the diagnostic markers in blood based on mass spectrometry.

[0011] Preferably, the product is a kit or a reagent.

[0012] The third aspect of the present application provides a product for diagnosing systemic lupus erythematosus, which comprises a reagent for detecting the expression amount of the diagnostic markers of systemic lupus erythematosus described above.

[0013] Preferably, the product detects the expression amount of the diagnostic markers in blood based on mass spectrometry; and the product is a kit or a reagent.

[0014] The present application determines that the expression amounts of PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1 in blood samples of SLE patients and healthy people change significantly, and thus proposes that the five proteins are used alone or in combination as biomarkers for the diagnosis of SLE, which has the advantages of high accuracy, high sensitivity, and high specificity, and provides a new target for the diagnosis and intervention improvement of SLE patients. The development of the diagnostic markers based on the five proteins alone or in combination into corresponding auxiliary early diagnosis reagents and kits has wide scientific research value and clinical effect, and provides great convenience for early screening, clinical diagnosis, and intervention treatment. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 FIG. 4 is a box plot of the differential expression of the five proteins in the blood of the SLE group and the HC group in the experiment; Figure 2 FIG. 5 is a ROC curve of the five proteins alone as biomarkers in the SLE group and the HC group in the experiment; Figure 3 FIG. 6 is a ROC curve of any two of the five proteins combined as biomarkers in the SLE group and the HC group in the experiment; Figure 4 FIG. 7 is a ROC curve of any three of the five proteins combined as biomarkers in the SLE group and the HC group in the experiment; Figure 5ROC curves of any four combinations of the five proteins in the experimental set as biomarkers in the SLE group and the HC group; Figure 6 This is the ROC curve of all five proteins in the experimental set as biomarkers in the SLE group and the HC group; Figure 7 This is the box plot of the differential expression of five proteins in the blood between the SLE group and the HC group in the validation set; Figure 8 To validate the ROC curves of the five proteins used as biomarkers in the SLE and HC groups; Figure 9 To validate the ROC curve of any two combinations of the five proteins in the set as biomarkers in the SLE group and the HC group; Figure 10 To validate the ROC curve of any three combinations of the five proteins in the set as biomarkers in the SLE group and the HC group; Figure 11 To validate the ROC curve of any four combinations of the five proteins in the set as biomarkers in the SLE group and the HC group; Figure 12 ROC curves of the five proteins in the validation set combined as biomarkers in the SLE and HC groups. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] 1. Experimental Subjects In the experimental set, 138 patients with systemic lupus erythematosus were selected as the SLE group, and 58 healthy subjects without systemic lupus erythematosus were selected as the HC group. In the validation set, 51 patients with systemic lupus erythematosus were selected as the SLE group, and 42 healthy subjects without systemic lupus erythematosus were selected as the HC group. The characteristic information is shown in Table 1.

[0018] Table 1

[0019] Among them, some patients with systemic lupus erythematosus lack clinical information on indicators such as urine occult blood, urine protein, anti-double-stranded DNA antibodies, antinuclear antibodies, and anti-Sm antibodies. Therefore, the calculation of the positive proportion of the above indicators is based on the total number of patients with clinical information on the indicator.

[0020] 2. Screening of differentially expressed proteins 1. Enzymatic hydrolysis (1) Put the blood sample in a refrigerated high-speed centrifuge, 4°C, 12000g centrifugation for 10 min, remove the cell debris, and transfer the supernatant to a new centrifuge tube; (2) Take 50µL of the high-speed centrifuged blood sample to the pre-washed magnetic nanomaterial (PTM-00F13303, from Hangzhou Jingjie Biological Technology Co., Ltd., PTM Bio), and incubate it in a constant-temperature mixer at 1200 rpm and 37°C for 1 hour; (3) After incubation, centrifuge at room temperature and 10000g for 5 min, discard the supernatant, and collect the magnetic beads; (4) Add 300µL of washing buffer to the magnetic bead precipitate after centrifugation, and wash the magnetic beads by oscillating at 1200g for 5 min; (5) Repeat step (4) twice; (6) Add 70μL of enzyme digestion buffer to the washed magnetic beads, mix well, and heat in a metal bath at 95°C for 10 min; (7) After heating, let the sample return to room temperature, add trypsin with a final concentration of 20ng / μL, and incubate at 37°C overnight for enzyme digestion; (8) The next day after enzyme digestion, add dithiothreitol (DTT) to a final concentration of 5mM, and reduce at 56°C for 30 min; (9) Add iodoacetamide (IAM) to a final concentration of 11mM, and incubate at room temperature in the dark for 15 min.

[0021] 2. Desalination (1) Acidify the enzyme-digested peptide fragments to pH=2~3 with 10% trifluoroacetic acid (TFA), centrifuge at room temperature and 12000g for 10 min, and transfer the supernatant to a new ep tube; (2) Activation: Add 50μL of activation solution to the StageTip, and centrifuge at 1500g for 1 min; (3) Equilibrium: Add 50μL of desalination solution to the StageTip, and centrifuge at 1500g for 1 min; (4) Repeat step (3) once; (5) Loading: Discard the waste liquid in the ep tube, and add the acidified peptide fragments to the StageTip, centrifuge at 1500g for 1 min; (6) Desalination: Add 50μL of desalination solution to the StageTip, and centrifuge at 1500g for 1 min; (7) Repeat step (6) once.

[0022] (8) Elution: Load the StageTip into a new ep tube, add 20μL of elution solution to the StageTip, and centrifuge at 750g for 1 min; (9) Repeat step (8) once, combine the two elution solutions, freeze and spin dry, and store for later use.

[0023] 3. Liquid chromatography-mass spectrometry analysis The peptide segments were dissolved in the mobile phase of liquid chromatography and then separated using a Vanquish Neo ultra-high performance liquid system. The mobile phase A was a water solution containing 0.1% formic acid; the mobile phase B was a water solution containing 0.1% formic acid and 80% acetonitrile. The liquid phase gradient was set as follows: 0 min~1.6 min, 4%B~22.5%B; 1.6 min~2.0 min, 22.5%B~35%B; 2.0 min~2.6 min, 35%B~55%B; 2.6 min~2.7 min, 55%B~99%B; 2.7 min~6.8 min, 99%B; 6.8 min~7.6 min, 99%B, and the flow rate was maintained at 300 nl / min.

[0024] After being separated by the ultra-high performance liquid system, the peptide segments were injected into the NSI ion source for ionization and then entered the Orbitrap Astral mass spectrometer for analysis. The ion source voltage was set to 1900 V, the peptide segment parent ions were detected and analyzed using the Orbitrap detector, and the secondary fragment ions were detected and analyzed using the Astral detector. The primary mass spectrometry scan range was set to 380 m / z~980 m / z, and the scan resolution was set to 240000; the secondary mass spectrometry scan range was fixed to start at 150 m / z, and the secondary scan resolution was set to 80000. The data acquisition mode used the data-independent scanning (DIA) program, i.e., after the primary scanning, the peptide segment ions in multiple consecutive windows entered the HCD collision cell, and were fragmented using 25% fragmentation energy for secondary mass spectrometry analysis. In order to improve the effective utilization rate of mass spectrometry, the automatic gain control (AGC) was set to 500%, and the maximum injection time was set to 3 ms.

[0025] 4. Database search The DIA data was searched using the DIA-NN (v 1.8) search engine with the software default parameters. The database was Homo_sapiens_9606_SP_20230103.fasta (20389 sequences), the enzyme cutting mode was set to Trypsin / P, and the maximum number of missed cuts was set to 1. The fixed modification was set to N-term M excision and C carbamidomethylation. A deep learning algorithm was used to construct a theoretical spectrum library, and a reverse library was added to calculate the false positive rate (FDR) caused by random matching; the FDR of precursor identification was set to 1%.

[0026] III. Verification of diagnostic efficiency Figure 1 and Figure 7The comparison chart of the expression of five proteins in the blood of SLE and HC groups in the experimental set and the validation set respectively, according to the relationship between the difference of the expression of five proteins in the blood of SLE and HC experimental objects and the clinical parameters, the evaluation is carried out through statistical test and regression analysis. The diagnostic efficiency is evaluated by receiver operating characteristic curve (ROC curve). When the area-under-the-curve (AUC) of the ROC curve is greater than or equal to 0.9, the diagnostic index is considered to be “highly accurate”, when 0.8≤AUC<0.9, the diagnostic index is considered to be “accurate”, when 0.7≤AUC<0.8, the diagnostic index is considered to be “moderately accurate”.

[0027] The five proteins are PDGFA, APOD, CFHR5, AMDHD1 and SCGB3A1. The PDGFA is platelet-derived growth factor A, the amino acid sequence of which is shown as SED ID NO. 1. The APOD is apolipoprotein D, the amino acid sequence of which is shown as SED ID NO. 2. The CFHR5 is complement factor H-related protein 5, the amino acid sequence of which is shown as SED ID NO. 3. The AMDHD1 is amide hydrolase domain 1, the amino acid sequence of which is shown as SED ID NO. 4. The SCGB3A1 is secretoglobin family 3A member 1, the amino acid sequence of which is shown as SED ID NO. 5.

[0028] The above-mentioned five proteins are used alone or in combination as serum diagnostic markers for distinguishing SLE group from HC group. The ROC curve results of SLE and HC in the experimental set are shown in Figures 2 to 6 , the ROC curve results of SLE and HC in the validation set are shown in Figures 8 to 12 , the AUC, sensitivity and specificity of the experimental set and the validation set are shown in Tables 2 to 6.

[0029] Table 2

[0030] Table 3

[0031] Table 4

[0032] Table 5

[0033] Table 6

[0034] from Figure 1 andFigure 7 It can be seen that the expression levels of APOD and SCGB3A1 in the blood of systemic lupus erythematosus patients (SLE) are higher than those in healthy controls (HC), and the differences are very significant (p less than 0.001); the expression levels of PDGFA, CFHR5 and AMDHD1 in the blood of SLE patients are lower than those in healthy controls HC, and the differences are very significant (p less than 0.001).

[0035] Figure 2 、 Figure 8 The results in Table 2 show that the above differentially expressed proteins can effectively distinguish SLE from HC alone (AUC>0.8), but the sensitivity and specificity need to be improved. The diagnostic sensitivity or specificity of some proteins is less than 70%.

[0036] like Figures 3 to 6 、 Figures 9 to 12 As shown in Tables 3 to 6, any combination of two, three, four, or all of the five proteins can effectively distinguish SLE from HC as biomarkers, and the AUC, sensitivity, or specificity are significantly better than those of a single protein. The advantages of combinations of three or more are even more obvious, with significantly improved AUC, sensitivity, or specificity.

[0037] Experimental results demonstrate that the five proteins can serve as blood diagnostic biomarkers to distinguish healthy individuals from those with SLE, and can be used to develop diagnostic products for systemic lupus erythematosus, such as kits and reagents. This biomarker combination demonstrates enhanced sensitivity, specificity, and accuracy, providing important evidence for further clinical research and offering new insights into the diagnosis and treatment of SLE.

[0038] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A diagnostic marker for systemic lupus erythematosus, characterized in that: The diagnostic marker is at least one protein selected from PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1; The PDGFA is platelet-derived growth factor A, and its amino acid sequence is shown in SED ID NO.1; The APOD is apolipoprotein D, and its amino acid sequence is shown in SED ID NO.2; The CFHR5 is complement factor H-related protein 5, and its amino acid sequence is shown in SED ID NO.3; The AMDHD1 is an amide hydrolase domain 1, and its amino acid sequence is shown in SED ID NO.4; The SCGB3A1 is secretoglobulin family 3A member 1, and its amino acid sequence is shown in SED ID NO.

5.

2. The diagnostic marker for systemic lupus erythematosus according to claim 1, characterized in that The diagnostic marker is a combination of at least two proteins among PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

3. The diagnostic marker for systemic lupus erythematosus according to claim 2, characterized in that The diagnostic marker is a combination of at least three proteins among PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

4. The diagnostic marker for systemic lupus erythematosus according to claim 3, characterized in that The diagnostic marker is a combination of at least four proteins among PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

5. The diagnostic marker for systemic lupus erythematosus according to claim 4, characterized in that The diagnostic markers are all combinations of PDGFA, APOD, CFHR5, AMDHD1 and SCGB3A1.

6. Use of a reagent for detecting the expression level of the diagnostic marker for systemic lupus erythematosus according to any one of claims 1 to 5 in the preparation of a product for diagnosing systemic lupus erythematosus.

7. The use according to claim 6, characterized in that The product detects the expression level of the diagnostic marker in the blood based on mass spectrometry.

8. The use according to claim 6, characterized in that The product is a kit or a reagent.

9. A product for diagnosing systemic lupus erythematosus, characterized in that: The product comprises a reagent for detecting the expression level of the diagnostic marker for systemic lupus erythematosus according to any one of claims 1 to 5.

10. The product for diagnosing systemic lupus erythematosus according to claim 9, characterized in that: The product detects the expression level of the diagnostic marker in the blood based on mass spectrometry; the product is a kit or reagent.

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