Serum protein markers for identifying systemic lupus erythematosus and application thereof

Abstract

The invention discloses a group of serum protein markers for identifying systemic lupus erythematosus. The serum protein markers comprise MCP-3, ST1A1, TNF, TGF-alpha, CSF-1, AXIN1, CXCL9, IL-15RA, IL18, CCL3, IL10, 4E-BP1, PD-L1, SIRT2, CCL11, CDCP1, IL-18R1, FGF-23, CD40, STAMBP, IL6, IL-17C, CD8A, CXCL10, CXCL5, DNER, FGF-19, LAP TGF-beta-1, CCL28, CASP-8, IL8, CCL23, CCL20, CCL19, MCP-1, VEGFA, uPA, OPG, OSM, various novel protein markers which are expected to be used for screening and identifying systemic lupus erythematosus are identified.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteomics or systemic lupus erythematosus diagnostic markers, and in particular relates to a group of serum protein markers for identifying systemic lupus erythematosus and applications thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by inflammation and immune-mediated damage to multiple organ systems, including the mucocutaneous system, musculoskeletal system, blood system, and renal system. However, SLE often affects multiple tissues and organs throughout the body, resulting in high heterogeneity in clinical manifestations and difficulty in clinical diagnosis. Currently, the diagnosis of SLE in clinical practice mainly relies on the evaluation of clinical symptoms combined with the detection of autoantibodies. These traditional diagnostic methods are limited by defects such as low sensitivity or specificity, invasiveness, and delayed diagnosis, which greatly delays the best diagnosis and treatment period for patients.

[0003] Proteomics testing is a qualitative and quantitative analysis of proteins in biological samples, which is widely used in biopharmaceutical development, clinical diagnosis and basic biological research. As an important functional molecule in organisms, proteins are responsible for the regulation and execution of life activities. However, the current work on SLE and proteomics is mostly based on mass spectrometry methods, which cannot target and identify low-abundance proteins. In terms of sensitivity, compared with other technologies, Olink technology has significant advantages, which can achieve a detection sensitivity of fg / ml, and the detection range can span 10 orders of magnitude. Not only can the required detection sample volume reach the uL level, but many low-abundance proteins can also be accurately detected. At present, there are relatively few studies on olink proteomics and SLE. Therefore, the development of SLE-specific serum protein biomarkers based on olink technology can provide new ideas and possibilities for the prevention, screening and treatment of SLE, so as to truly transfer it from the laboratory to the clinic.

[0004] Purpose of the Invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art, provide a group of serum protein markers for identifying systemic lupus erythematosus and their applications, and provide a useful tool for identifying systemic lupus erythematosus.

[0006] On the one hand, the present invention provides a group of serum protein markers for identifying systemic lupus erythematosus, wherein the serum protein markers include MCP-3, ST1A1, TNF, TGF-alpha, CSF-1, AXIN1, CXCL9, IL-15RA, IL18, CCL3, IL10, 4E-BP1, PD-L1, SIRT2, CCL11, CDCP1, IL-18R1, FGF-23, CD40, STAMBP, IL6, IL-17C, CD8A, CXCL10, CXCL5, DNER, FGF-19, LAP TGF-beta-1, CCL28, CASP-8, IL8, CCL23, CCL20, CCL19, MCP-1, VEGFA, uPA, OPG, OSM, LIF, and IFN-gamma.

[0007] Preferably, the method for selecting serum protein markers comprises the following steps:

[0008] S1. Collect serum samples: Collect serum samples from healthy subjects HC and patients with systemic lupus erythematosus SLE;

[0009] S2, perform olink96 Proteomics 96 inflammation panel protein sequencing on the serum samples collected in S1 to screen for differentially expressed proteins;

[0010] S3. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of differentially expressed proteins in S2 for systemic lupus erythematosus.

[0011] On the other hand, the present invention provides a kit for identifying systemic lupus erythematosus, wherein the kit comprises the serum protein marker or a detection reagent thereof.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. The present invention provides a group of serum protein markers for identifying systemic lupus erythematosus, which can effectively identify systemic lupus erythematosus;

[0014] 2. The present invention provides a kit for identifying systemic lupus erythematosus, which provides a new idea for the targeted treatment of systemic lupus erythematosus from the perspective of serum protein.

[0015] Description of the Figures and Schedules

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0017] Figure 1The number and distribution of differentially expressed proteins between the healthy subjects group (HC) and the systemic lupus erythematosus group (SLE).

[0018] Figure 2 The heat map of differential protein clustering between the healthy subjects group (HC) and the systemic lupus erythematosus group (SLE).

[0019] Figure 3 This is a differential analysis of the top 4 differentially expressed proteins between the healthy subjects group (HC) and the systemic lupus erythematosus group (SLE).

[0020] Figure 4 This is a differential analysis of the top 4 differentially expressed proteins between the healthy subjects group (HC) and the systemic lupus erythematosus group (SLE).

[0021] Figure 5 This is a differential analysis of the top 4 differentially expressed proteins between the healthy subjects group (HC) and the systemic lupus erythematosus group (SLE).

[0022] Figure 6 This is a differential analysis of the top 4 differentially expressed proteins between the healthy subjects group (HC) and the systemic lupus erythematosus group (SLE).

[0023] Figure 7 This is an integrated differential analysis of the top 4 differentially expressed proteins between the healthy subjects group (HC) and the systemic lupus erythematosus group (SLE). DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the embodiments are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0025] Example 1: Sequencing screening of differential serum protein markers

[0026] 1.1 Collection of serum samples:

[0027] A total of 51 patients diagnosed with systemic lupus erythematosus (SLE) by the Department of Rheumatology and Immunology of Drum Tower Hospital Affiliated to Nanjing University Medical School were collected, and fresh serum specimens (without any anticoagulant) were collected for routine testing. The diagnosis was based on the revised criteria for systemic lupus erythematosus established by the American College of Rheumatology (ACR) in 1997. At the same time, fresh serum specimens of 22 healthy volunteers HC from the Physical Examination Center of Drum Tower Hospital Affiliated to Nanjing University Medical School were collected and stored in a -80℃ refrigerator for future use.

[0028] 1.2 Serum Olink Proteomics Sequencing:

[0029] 1.2.1 Sample randomization: Use the RAND function in the Excel table to output the numerical value corresponding to each sample, and then arrange the values ​​from small to large so that the samples are randomly distributed in the 96-well plate.

[0030] 1.2.2 Sample sampling: According to the randomized sample distribution table, take 20 μL of each sample into a 96-well plate as an external control; add 5 μL of mixed plasma sample to wells A12 and B12, add 5 μL of Negative Control (NC) to wells C12, D12, and E12, and add 5 μL of Interplate Control (IPC) to wells F12, G12, and H12, and mix well.

[0031] 1.2.3 Hybridization incubation: Prepare hybridization mixture first, take out the reagent and thaw it at room temperature, vortex and centrifuge, take a nuclease-free 1.5ml centrifuge tube, add 280μL of Incubation Solution, 40μL of IncubationStabilizer, 40μL of A-probes, and 40μL of B-probes in sequence, vortex and centrifuge. Take out an 8-tube strip and add 47μL of hybridization mixture to each well. Take out a 96-well plate, mark it as an incubation plate, use an 8-channel pipette to add 3μL of hybridization mixture to each well, and then add 1μL of sample to each well one by one. After sticking the sealing film, centrifuge at 400g for 1 minute, put it in the PCR instrument, set it to 4℃ infinity, record the time of putting it into the PCR instrument, and the incubation time must meet 16-24 hours.

[0032] 1.2.4 Extension and amplification: Set the PCR program to 50℃ for 20 minutes; 95℃ for 5 minutes; 95℃ for 30 seconds, 54℃ for 1 minute, and 60℃ for 1 minute for 17 cycles; maintain at 10℃. After the settings are completed, run the PCR instrument and press the pause button to maintain the temperature of the PCR instrument at 50℃. Prepare the extension and amplification mixture. Take a 15mL centrifuge tube and add 9385μL of High Purity Water, 1100μL of PEA Solution, 55μL of PEA Enzyme, and 22μL of PCR Polymerase in sequence, vortex and centrifuge. Take out the incubation plate from the PCR instrument and centrifuge. Use an 8-channel pipette to add 96μL of extension and amplification mixture to each well. After sealing the film, vortex and centrifuge, place it on the PCR instrument preheated at 50℃, and then resume the running program.

[0033] 1.2.5 Chip pretreatment: Unpack and take out the chip, check whether the chip and syringe are damaged. Take out the syringe, press down the piston hole, and push the liquid in the syringe. Click the instrument Target 96 option, the instrument pushes out the sample stage, puts in the chip and cover, and automatically returns to the sample stage. Click Start, and the chip pretreatment begins.

[0034] 1.2.6 Operation on the machine: Take out a 1.5ml centrifuge tube, add 550μL of Detection Solution, 230μL of High Purity Water, 7.8μL of Detection Enzyme, and 3.1μL of PCR Polymerase in sequence, vortex and centrifuge. Take out another strip tube and dispense 95μL per well. Take out a 96-well plate, add 7.2μL of the mixture to each well, and after the extension amplification program is completed, add 2.8μL of the product, vortex and mix well for use. Prepare the diluted sample plate, primer plate and pre-treated chip, add 5μL of primers to the left side of the chip, and add 5μL of diluted product to the right side in sequence to avoid bubbles. After the sample is added, put it into the sample loading station, set the run name, and click Run.

[0035] 1.2.7 Data analysis and export: Export the run name file after the instrument is shut down.

[0036] Example 2: Clinical application of serum differential protein markers

[0037] 2.1 Identification and analysis of olink sequencing results:

[0038] The differential expression protein quantitative analysis was performed with 1.5-fold as the threshold of differential expression change and t-test p-value < 0.05 as the significance threshold. Figure 1 The results showed that compared with the healthy subjects, 37 proteins were significantly upregulated in patients with systemic lupus erythematosus, and 13 proteins were significantly downregulated in patients with systemic lupus erythematosus.

[0039] Quantitative proteomics analysis was used to determine the expression of the above differential proteins in 51 SLE group and 22 HC group samples. The clustering heat map analysis of differential proteins between the different groups is shown in Figure 2. Figure 2 shown.

[0040] 2.2 Analysis of the diagnostic efficacy of SLE differential proteins:

[0041] The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. The AUC of a completely worthless diagnostic test is 0.5, and the AUC of an ideal diagnostic test is 1. It is generally believed that an AUC between 0.7 and 0.9 has a certain diagnostic value.

[0042] The areas under the AUC curves of the MCP-3, ST1A1, TNF, TGF-alpha, CSF-1, AXIN1, CXCL9, IL-15RA, IL18, CCL3, IL10, 4E-BP1, PD-L1, SIRT2, CCL11, CDCP1, IL-18R1, FGF-23, CD40, STAMBP, IL6, IL-17C, CD8A, CXCL10, CXCL5, DNER, FGF-19, LAP TGF-beta-1, CCL28, CASP-8, IL8, CCL23, CCL20, CCL19, MCP-1, VEGFA, uPA, OPG, OSM, LIF, and IFN-gamma proteins provided by the present invention are all greater than 0.7, indicating that the above markers can well distinguish HC from SLE. The specific results are shown in Table 1. The top four proteins were MCP-3, ST1A1, TNF, and TGF-alpha, with p values ​​all < 0.000. The differential protein analysis between the groups was as follows Figure 3-6 As shown in Figure 2, the integrated difference analysis is as follows Figure 7 As shown, the areas under the AUC curve were 0.9875, 0.9639, 1.0, and 0.9828, respectively, indicating that the above markers have good sensitivity and specificity.

[0043] Table 1 Summary of differentially expressed proteins with an area under the AUC curve greater than 0.7

[0044] Protein name Area under the AUC curve Protein name Area under the AUC curve MCP-3 0.97 IL-17C 0.82 ST1A1 0.96 CD8A 0.80 TNF 0.94 CXCL10 0.78 TGF-alpha 0.92 CXCL5 0.78 CSF-1 0.92 DNER 0.76 AXIN1 0.91 FGF-19 0.75 CXCL9 0.90 LAP TGF-beta-1 0.75 IL-15RA 0.90 CCL28 0.74 IL18 0.89 CASP-8 0.73 CCL3 0.88 IL8 0.73 IL10 0.88 CCL23 0.73 4E-BP1 0.88 CCL20 0.73 PD-L1 0.86 CCL19 0.72 SIRT2 0.85 MCP-1 0.72 CCL11 0.85 VEGFA 0.71 CDCP1 0.84 uPA 0.71 IL-18R1 0.84 OPG 0.71 FGF-23 0.83 OSM 0.70 CD40 0.83 LIF 0.70 STAMBP 0.83 IFN-gamma 0.70 IL6 0.82

[0045] The present invention provides a screening idea and method for serum differential protein markers for SLE diagnosis. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A group of serum protein markers for identifying systemic lupus erythematosus, characterized in that: The serum protein markers include one or more of MCP-3, ST1A1, TNF, TGF-alpha, CSF-1, AXIN1, CXCL9, IL-15RA, IL18, CCL3, IL10, 4E-BP1, PD-L1, SIRT2, CCL11, CDCP1, IL-18R1, FGF-23, CD40, STAMBP, IL6, IL-17C, CD8A, CXCL10, CXCL5, DNER, FGF-19, LAP TGF-beta-1, CCL28, CASP-8, IL8, CCL23, CCL20, CCL19, MCP-1, VEGFA, uPA, OPG, OSM, LIF, and IFN-gamma.

2. A set of serum protein markers for identifying systemic lupus erythematosus according to claim 1, characterized in that: The method for selecting the serum protein marker comprises the following steps: S1. Collect serum samples: Collect serum samples from healthy subjects HC and patients with systemic lupus erythematosus SLE; S2, perform olink96 Proteomics 96 inflammation panel protein sequencing on the serum samples collected in S1 to screen for differentially expressed proteins; S3. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of differentially expressed proteins in S2 for systemic lupus erythematosus.

3. A kit for identifying systemic lupus erythematosus, characterized in that: The kit comprises the serum protein marker or a detection reagent thereof according to claim 1.

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