Application of PCSK6 in diagnosis of sepsis

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of a biomarker in preparation of a product for diagnosing sepsis, the biomarker is proprotein convertase subtilisin 6 (PCSK6), and the biomarker has remarkably high sensitivity and specificity in the aspect of sepsis diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of PCSK6 in the preparation of products for diagnosing sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection. Bacteria, viruses and parasites can all cause infection. After infection, bacteria and toxins enter the body, activate the body's inflammatory response cells, produce and release a large number of inflammatory mediators (such as TNF-α, IL-1β, IL-6, etc.), and cause local or systemic inflammatory response. In severe sepsis, severe infection caused by immunosuppression will further aggravate the systemic inflammatory response, and the various pathological changes driven by the inflammatory response will also further deteriorate, leading to or aggravating organ failure, and even death of the patient. Sepsis not only threatens human health, but also brings a huge economic burden to medical and health care.

[0003] At present, the diagnosis of sepsis mainly relies on the detection of blood, body fluids or tissue specimens. The source of infection is determined by bacterial culture of the specimens, and sometimes imaging examinations such as ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) are also used to determine the source of infection. The time for bacterial culture of the specimens is long, usually 1-3 days. Therefore, early diagnosis of sepsis is crucial. Early prevention and early treatment can avoid the huge risks that sepsis brings to patients.

[0004] PCSK6 is a member of the proprotein convertase subtilisin (PCSKs) family, which is also known as the serine protease family and includes proteins with different functions, such as PCSK1-6, MBTPS1, etc.

[0005] PCSK1 is involved in the processing of hormones and other protein precursors at sites consisting of paired basic amino acid residues. Substrates include pro-opiomelanocortin (POMC), renin, enkephalin, dynorphin, somatostatin, insulin, and AGRP. PCSK1 activity is essential for the activation and cleavage of many peptide hormone precursors, which are related to the regulation of food intake, glucose homeostasis, and energy homeostasis (see patent CN115698336A, published on 2021-11-11).

[0006] The PCSK2 protease is packaged into and activated in dense core secretory granules and is expressed in the neuroendocrine system and brain. Single nucleotide polymorphisms in this gene may increase susceptibility to myocardial infarction and type 2 diabetes. This gene may also play a role in tumorigenesis and progression.

[0007] PCSK3 processes latent precursor proteins into their biologically active products. It is a calcium-dependent serine endoprotease that cleaves precursor proteins at their paired basic amino acid processing sites. Some of the furin substrates are: parathyroid hormone, transforming growth factor β1 precursor, proalbumin, pro-β-secretase, membrane type 1 matrix metalloproteinase, β-subunit of nerve growth factor precursor, and von Willebrand factor.

[0008] PCSK4 is present in the sperm plasma membrane acrosome and plays an important role in binding to the zona pellucida (ZP) molecules during fertilization (see patent IDP000069516B, published on May 18, 2018). PCSK5 plays a key regulatory role in the secretion and maturation of growth factors.

[0009] PCSK6, which is primarily involved in the processing of protein and peptide precursors, regulates protein transport and processing through the secretory pathway. It is expressed in many tissues, including the neuroendocrine, liver, intestine, and brain, and plays a role in tumor progression. It has also been reported that the mRNA and protein levels of PCSK6 were increased in hypoxic cardiomyocytes, thus identifying PCSK6 as a key player in cardiac remodeling after acute myocardial infarction (see non-patent literature: Secretome Analysis of Cardiomyocytes Identifies PCSK6 (Proprotein Convertase Subtilisin / Kexin Type 6) as a NovelPlayer in Cardiac Remodeling After Myocardial Infarction. Circulation. 2020; 141: 1628–1644.), in addition, it has been reported that PCSK6 may be a new antigenic target in membranous nephropathy related to the use of nonsteroidal anti-inflammatory drugs (see non-patent literature: Proprotein convertase subtilisin / kexin type 6 (PCSK6) is a likely antigenic target in membranous nephropathy and nonsteroidal anti-inflammatory drug use. Kidney International (2023) 104, 343–352).

[0010] MBTPS1 encodes a membrane-bound enzyme called site-1 protease (SIP), which is a member of the serine proteases and acts as a proprotein convertase responsible for cleaving a variety of precursor substrates. Summary of the invention

[0011] To make up for the gaps in the prior art, the present invention uses PCSK6 as a biomarker for diagnosing sepsis, achieving higher sensitivity and specificity. Specifically, the present application screened out the biomarker of sepsis - PCSK6 through a discovery cohort, and further verified its diagnostic efficacy through a validation cohort.

[0012] A first aspect of the present invention provides a use of a biomarker or a reagent for detecting a biomarker in the preparation of a product for diagnosing sepsis, wherein the biomarker is proprotein convertase subtilisin / kexin type 6 (PCSK6).

[0013] The biomarkers are biomarkers in cells, tissues or body fluids.

[0014] Preferably, the cells may be liver cells, brain cells or intestinal cells.

[0015] Preferably, the tissue may be liver tissue, brain tissue or intestinal tissue.

[0016] Preferably, the body fluid may be blood, plasma, serum, saliva, urine, tears, amniotic fluid, cerebrospinal fluid or lymph fluid.

[0017] The sepsis includes liver damage, brain damage, intestinal damage, gastric damage, lung damage, kidney damage or heart damage caused by sepsis.

[0018] Preferably, the liver injury is caused by sepsis.

[0019] The products include test kits, test strips, chips or devices.

[0020] The product includes a reagent for detecting a biomarker. Preferably, the reagent detects the presence or absence of a biomarker or the protein expression level or concentration.

[0021] Preferably, the reagents for detecting biomarkers include reagents used by one or more of protein immunoblotting, enzyme-linked immunosorbent assay, chromatography or mass spectrometry.

[0022] The diagnosis of sepsis includes detecting the presence or absence of biomarkers or protein expression levels or concentrations. Preferably, it also includes comparing it with a threshold value, which is obtained in a previous experiment, that is, the threshold value is determined by the difference in the biomarkers between sepsis patients and healthy people through experiments and data analysis.

[0023] In a specific embodiment of the present invention, the PCSK6 expression level or concentration is different or significantly different from the threshold value (the difference is statistically significant, for example, log2(FoldChange)|(fold change)>0, P<0.05). For example: PCSK6 higher than the threshold value or significantly higher than the threshold value indicates the occurrence of sepsis, and the disease is diagnosed.

[0024] Preferably, the biomarkers are detected by one or more of protein immunoblotting, enzyme-linked immunosorbent assay, chromatography or mass spectrometry.

[0025] Further preferably, the enzyme-linked immunosorbent assay includes one or more of direct ELISA, indirect ELISA, double antibody sandwich ELISA or competitive ELISA.

[0026] Further preferably, the chromatography includes one or more of gas chromatography, liquid chromatography, capillary electrophoresis, high performance liquid chromatography or ultra high performance liquid chromatography.

[0027] Further preferably, the mass spectrometry includes one or more of electron spray ionization mass spectrometry, quadrupole mass spectrometer, ion trap mass spectrometer, matrix-assisted activation desorption / ionization time-of-flight mass spectrometer, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, MALDI quadrupole-time-of-flight mass spectrometry, electrospray ionization (ESI)-TOF mass spectrometry, ESI ion trap mass spectrometry, ESI triple quadrupole mass spectrometry or Fourier transform ion cyclotron resonance.

[0028] The second aspect of the present invention provides a use of a biomarker in the preparation of a product for diagnosing septic liver injury, wherein the biomarker is proprotein convertase subtilisin / kexin type 6 (PCSK6).

[0029] The third aspect of the present invention provides an application of a biomarker in constructing a diagnostic model of sepsis, wherein the biomarker is PCSK6.

[0030] A fourth aspect of the present invention provides a method for constructing a sepsis diagnostic model, the method comprising:

[0031] i): Collecting the biomarker expression level or concentration test results of sepsis patients and healthy controls, wherein the biomarkers include PCSK6;

[0032] ii): Build a diagnostic model using the results collected in i); or,

[0033] The construction method comprises:

[0034] 1) collecting samples from subjects and detecting the expression level or concentration of biomarkers, wherein the biomarkers include PCSK6;

[0035] 2) Conduct clinical diagnosis on the subjects and divide them into sepsis group and healthy control group;

[0036] 3) Constructing a diagnosis model based on the detection results of step 1) and the diagnosis results of step 2).

[0037] The fifth aspect of the present invention provides a diagnostic model obtained by the above-mentioned construction method.

[0038] In a sixth aspect, the present invention provides a method for diagnosing sepsis, wherein the method comprises detecting the presence, expression level or concentration of PCSK6 in a sample of a subject.

[0039] The sample includes cells, tissues or body fluids.

[0040] Preferably, the cells may be liver cells, brain cells or intestinal cells.

[0041] Preferably, the tissue may be liver tissue, brain tissue or intestinal tissue.

[0042] Preferably, the body fluid may be blood, plasma, serum, saliva, urine, tears, amniotic fluid, cerebrospinal fluid or lymph fluid.

[0043] The method comprises one or more of protein immunoblotting, enzyme-linked immunosorbent assay, chromatography or mass spectrometry.

[0044] Preferably, the method comprises comparing the expression level or concentration of the detected biomarker with a threshold value, wherein the threshold value is obtained in a previous experiment, that is, the threshold value is determined by the difference between the biomarkers of sepsis patients and healthy people through experiments and data analysis. The biomarkers described in the present application are different or significantly different from the threshold value (the difference is statistically significant, for example, log2(FoldChange)|(fold change)>0, P<0.05). For example: PCSK6 is higher than the threshold value or significantly higher than the threshold value, indicating the occurrence of sepsis, and the disease is diagnosed.

[0045] The term "subject" may be a human or non-human animal, including "patients", "suspected patients" and "healthy individuals", etc. The term does not indicate a specific age or gender, and covers adult or newborn subjects and fetuses. The non-human animals may be wild animals, zoo animals, economic animals, pets, experimental animals, etc. Specifically, the non-human animals include but are not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0046] The term "diagnosis" means to find out whether a patient has a disease or condition in the past, at the time of diagnosis, or in the future, or to find out the progression or possible future progression of a disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0048] Figure 1 Shown is the Venn diagram of differential protein analysis in peripheral serum between sepsis patients and normal control group;

[0049] Figure 2 Shown is a sample correlation heatmap;

[0050] Figure 3 Shown is the principal component analysis diagram (PCA analysis);

[0051] Figure 4 Shown is a bar graph drawn based on the total number of differentially expressed proteins and the number of specific up-regulated / down-regulated proteins, with up representing up-regulation and down representing down-regulation;

[0052] Figure 5 The volcano plot of differentially expressed proteins is shown, where up represents upregulation, down represents downregulation, and nosig represents no significant difference;

[0053] Figure 6 Shown is the GO (Gene Ontology Enrichment Analysis) enrichment map;

[0054] Figure 7 Shown is the protein expression level of PCSK6 in sepsis patients and healthy subjects, **** represents p < 0.0001;

[0055] Figure 8 Shown is the receiver operating characteristic curve (ROC), where the 95% CI is 0.6738-1.000, and CI is the confidence interval;

[0056] Fig. 9 The figure shows the bar graph of the changes in the concentration of liver function indicators over time, among which, Figure A is alanine aminotransferase (ALT), Figure B is aspartate aminotransferase (AST), Figure C is total bilirubin (TBIL), Figure D is γ-glutamyl transpeptidase (γ-GT), Figure E is albumin (ALB), Figure F is alkaline phosphatase (ALP), and Figure G is total bile acid (TBA). CLP represents the sepsis model group, SHAM represents the sham operation model group, ns represents no statistically significant difference, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001;

[0057] Fig.10 Shown is the HE staining (hematoxylin-eosin staining) structure diagram, CLP represents the sepsis model group, and SHAM represents the sham operation model group;

[0058] Fig.11 The results of liver injury pathological scores at different times are shown. CLP represents the sepsis model group, SHAM represents the sham operation model group, ** represents p<0.01, **** represents p<0.0001;

[0059] Fig.12 The figure shows the relative protein expression of PCSK6 in hepatocytes of sepsis model mice and sham operation model mice, ** represents p < 0.01, CLP represents sepsis model group, SHAM represents sham operation model group;

[0060] Fig.13 The graph shows the protein concentration of PCSK6 in the serum of sepsis model mice and sham operation model mice, **** represents p < 0.0001, CLP represents the sepsis model group, and SHAM represents the sham operation model group;

[0061] Fig.14 Shown is the model mouse operating characteristic curve (ROC), wherein the 95% CI is 0.9420-1.000, and CI is the confidence interval. DETAILED DESCRIPTION

[0062] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0063] It should be noted that the methods used in the present invention are all conventional methods unless otherwise specified, and the reagents used in the present invention are all commercially available products unless otherwise specified.

[0064] Animals used in the examples: C57BL / 6J mice, weighing about 20 g, male, healthy and free of special pathogens, Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0008. Feed with complete nutritional feed, room temperature 22-25°C, humidity 30%-50%.

[0065] The instruments used in the examples are: DYY-7 protein electrophoresis instrument (Beijing Liuyi Instrument Factory), ImageQuant LAS4000 chemiluminescence imaging analyzer (GE General Electric Company), Spectra MR multifunctional microplate reader (DYNEX Technologies Company), Vanquish Neo chromatograph (Thermo Company), Astral liquid phase tandem mass spectrometer (Thermo Company).

[0066] All data in the examples of the present application are expressed as x±s, and the inter-group comparisons were performed by one-way analysis of variance. P<0.05 was considered statistically significant, and SPSS25.0 software package was used for statistical analysis.

[0067] Example 1 Screening for biomarkers for diagnosis of sepsis

[0068] 1. Discovery Queue

[0069] Participants were recruited and whole blood samples were collected from the PLA General Hospital. The diagnosis of the disease population was based on the Sepsis 3.0 definition and SOFA score. Exclusion criteria were malignant tumors, chronic respiratory diseases, and any confounding factors of anticoagulants. Normal physical examinations of the normal population were normal and did not have the above diseases. Based on the above diagnostic criteria, the composition of the discovery cohort included 11 patients with confirmed sepsis and 10 healthy controls.

[0070] 2. Detection of protein in samples

[0071] The human whole blood samples were allowed to stand to obtain serum. High-abundance proteins were removed using a filter column, and the protein concentration was determined using the BCA method. The samples were then subjected to protein denaturation, reduction, alkylation, and enzymatic hydrolysis. The samples were desalted using a Waters-C18 SPE column, and then eluted, concentrated, and re-dissolved. The supernatant was tested on an instrument and analyzed by Astral-DIA proteomics.

[0072] 3. Results

[0073] Astral-DIA proteomic analysis detected a total of 4910 proteins, of which 4794 proteins were shared between the sepsis group and the healthy control group ( Figure 1 ). There are significant differences between the two groups of samples, and the correlation between samples within the group is strong, and the data reliability is high ( Figure 2 , Figure 3 ). According to the set threshold |log2(FoldChange)|(fold change)>0, P<0.05, cluster analysis was performed on the differentially expressed proteins between the groups ( Figure 4 , Figure 5), 20 differentially expressed proteins with significantly different serum protein expression levels in sepsis patients compared with the healthy control group were screened, including CD14, PRH1, WFDC2, PGLYRP1, REG1B, RETN, GXYLT1, CELA2A, ATG, MYL3, PCSK6, etc., and further enriched by GO pathway analysis ( Figure 6 ), PCSK6 was selected as a diagnostic marker for sepsis. PCSK6 expression was significantly upregulated in patients with sepsis ( Figure 7 ), the difference is significant.

[0074] Example 2 Validation of PCSK6 in sepsis diagnosis

[0075] In order to further verify the diagnostic efficacy of PCSK6 in sepsis, this example again collected 20 new sepsis patients and 10 healthy controls from the PLA General Hospital to form a validation cohort. The patient inclusion criteria were consistent with the discovery cohort in Example 1 used for omics screening, and whole blood samples were collected from sepsis patients and normal people.

[0076] Then, serum from the whole blood samples was taken for ELISA assay, and the results showed that PCSK6 was specifically highly expressed in the serum of sepsis patients compared with healthy controls, and was used to draw the receiver operating characteristic (ROC) curve, see Figure 8 , where AUC = 0.85, cutoff value is 0.75, sensitivity is 95%, and specificity is 80%.

[0077] Example 3 Expression of PCSK6 in a mouse sepsis model

[0078] 1. Experimental design

[0079] Twenty C57BL / 6J male mice were randomly divided into two groups, with 10 mice in each group. In one group, the mouse sepsis model was prepared by cecal ligation and puncture. The mice were anesthetized conventionally and then opened the abdomen. The cecum was found and bluntly separated. The cecum was ligated 1.0-1.5 cm away from the distal end of the cecum. The cecum was then punctured with a needle. Sesame-sized feces were squeezed out from the puncture hole. The cecum was returned to the abdominal cavity, and the abdominal wall muscles and skin were sutured layer by layer. The sham operation model of mice was prepared by the sham operation method in the other group. The mice were anesthetized conventionally and then opened the abdomen. The cecum was bluntly separated to avoid bleeding and damage of mesenteric vessels. The abdominal wall muscles and skin were then sutured layer by layer.

[0080] 2. Protein expression detection

[0081] 1) Detection of PCSK6 protein expression in mouse hepatocytes

[0082] 24 hours after surgery, the mice in both groups were anesthetized by intraperitoneal injection of 3% sodium pentobarbital, 0.2 ml / 100 g, and the liver was perfused. The perfused liver was removed and primary hepatocytes were isolated and extracted by density centrifugation. Approximately 2×10 cells were collected from each group. 6 After extracting total cell protein, the protein expression of PCSK6 in cells was detected by Western blotting. The primary antibodies were rabbit anti-mouse PCSK6 polyclonal antibody and mouse anti-mouse β-actin monoclonal antibody (both at a dilution ratio of 1:1000); the secondary antibodies were HRP (horseradish peroxidase)-labeled goat anti-rabbit IgG monoclonal antibody and HRP-labeled goat anti-mouse IgG monoclonal antibody (both at a dilution ratio of 1:5000). The ultrasensitive chemiluminescence kit was used for development and imaging. After observation by chemiluminescence imaging analyzer, the gray value of the target protein was analyzed by ImageJ software (National Institutes of Health, USA). β-actin was used as an internal reference to calculate the relative expression of the target protein.

[0083] 2) Detection of PCSK6 protein expression in mouse peripheral serum

[0084] 24 hours after surgery, whole blood samples were collected from the two groups of mice by enucleation, and then the whole blood was placed in a centrifuge at 3000r / min for 10 minutes to obtain serum. The ELISA test kit provided by Shanghai Aimeng Youning Biotechnology Co., Ltd. was used to detect the PCSK6 level in the obtained serum. The ELISA experimental operation was strictly carried out and calculated according to the operating instructions of the kit to minimize human errors.

[0085] 3. Identification of liver damage

[0086] The liver function indicators ALT, AST, TBIL, γ-GT, ALP, ALB and TBA of mice in each group were detected at different time points, and the liver damage was evaluated by combining HE staining and liver injury pathological scoring.

[0087] 4. Experimental results

[0088] 1) Liver injury in sepsis model

[0089] The levels of ALT, AST, TBIL, γ-GT, ALP, and TBA in the liver tissues of mice increased first and then decreased in a time-dependent manner, with the most obvious increase at 12h and 24h after CLP and a decrease at 48h. The ALB level was the lowest at 24h after CLP and rebounded at 48h. Fig. 9 HE staining ( Fig.10 ) and liver injury pathological score ( Fig.11) The results showed that compared with the Sham group, the liver tissues of mice in the 6, 12, and 24h groups after CLP surgery showed varying degrees of damage and progressively worsened over time, mainly manifested as disordered hepatic cord structure, inflammatory cell infiltration, and hepatocyte edema and degeneration. However, the above pathological damage of the liver tissue in the 48h group after CLP modeling was significantly alleviated compared with the CLP-24h group. This and the liver function indicators together suggested that in the sepsis state, liver tissue damage increased in a time-dependent manner, was most significant at 24h of sepsis, and decreased at 48h.

[0090] 2) PCSK6 protein is specifically highly expressed in the sepsis mouse model. Specifically:

[0091] Hepatocytes: 24 hours after the mice underwent cecal ligation and puncture, primary hepatocytes were isolated and extracted, and total cell protein was extracted for protein immunoblotting. The results are as follows Fig.12 As shown in the figure, compared with the sham operation model group, the expression of PCSK6 in the hepatocytes of mice in the sepsis model group was significantly increased, and the difference was significant.

[0092] Serum: 24 hours after the mice underwent cecal ligation and puncture, blood was collected from the eyeballs and serum was separated. The expression of PCSK6 in the serum of mice was detected using an ELISA kit. The results are as follows: Fig.13 As shown in the figure, compared with the sham operation model group, the PCSK6 level in the peripheral serum of mice in the sepsis model group was significantly increased, and the difference was significant.

[0093] Example 4: Diagnostic Validation in a Mouse Sepsis Model

[0094] Another 40 mice were randomly divided into groups according to the random number method. 20 mice were randomly selected as the control group for sham surgery, and the other 20 mice were randomly selected as the CLP group for cecal ligation and puncture (the preparation of sham surgery model and CLP model refers to the method in Example 3). 24 hours after the operation, hepatocytes were separated and extracted, fully ground and lysed, and then centrifuged for 5 minutes. The supernatant was used for ELISA experiment to detect the PCSK6 protein content in liver tissue, and a ROC curve was formed to predict the diagnostic value.

[0095] The CLP model constructed showed liver damage, which was consistent with the results of Example 3. The expression level of PCSK6 protein in the hepatocytes of the CLP model group was significantly higher than that in the sham operation group, and the ROC results were as follows: Fig.14 As shown, AUC = 0.975, the cutoff value is 0.85, the sensitivity is 100%, and the specificity is 85%.

Claims

1. Use of a biomarker in the preparation of a product for diagnosing sepsis, characterized in that: The biomarker is proprotein convertase subtilisin / kexin type 6 (PCSK6).

2. The use according to claim 1, characterized in that: The biomarkers are biomarkers in cells, tissues or body fluids; Preferably, the cells are liver cells, brain cells or intestinal cells; Preferably, the tissue is liver tissue, brain tissue or intestinal tissue; Preferably, the body fluid is selected from blood, plasma, serum, saliva, urine, tears, amniotic fluid, cerebrospinal fluid or lymph.

3. The use according to claim 1 or 2, characterized in that: The sepsis includes liver damage, brain damage, intestinal damage, gastric damage, lung damage, kidney damage or heart damage caused by sepsis.

4. The use according to claim 1, characterized in that: The products include test kits, test strips, chips or devices.

5. The use according to claim 1, characterized in that: The product includes a reagent for detecting a biomarker, and the reagent detects the presence or absence of a biomarker or the protein expression level or concentration.

6. The use according to claim 1, characterized in that: The diagnosis of sepsis includes detecting the presence or absence of biomarkers or the protein expression level or concentration.

7. The use according to claim 5 or 6, characterized in that: The biomarkers are detected by one or more of protein immunoblotting, enzyme-linked immunosorbent assay, chromatography or mass spectrometry.

8. Use of a biomarker in the preparation of a product for diagnosing septic liver injury, characterized in that: The biomarker is proprotein convertase subtilisin / kexin type 6 (PCSK6).

9. A method for constructing a sepsis diagnostic model, characterized in that: The construction method comprises: i): Collecting the biomarker expression level or concentration test results of sepsis patients and healthy controls, wherein the biomarkers include PCSK6; ii): Build a diagnostic model using the results collected in i); or, The construction method comprises: 1) collecting samples from subjects and detecting the expression level or concentration of biomarkers, wherein the biomarkers include PCSK6; 2) Conduct clinical diagnosis on the subjects and divide them into sepsis group and healthy control group; 3) Constructing a diagnosis model based on the detection results of step 1) and the diagnosis results of step 2).

10. A sepsis diagnostic model obtained by the construction method according to claim 9.

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