An early diagnostic marker for detecting sepsis and application of protein polypeptide thereof

Abstract

The application belongs to the field of medicine, and particularly relates to an early diagnosis marker for detecting sepsis and application of a protein polypeptide thereof. It is found in the application that phosphorylation of PGK1S271 can be used as a specific detection index, which contributes to early detection of sepsis in clinic. Similarly, the current clinical drugs for the treatment of sepsis are also very limited, and the competitive inhibition protein polypeptide TAT-PGK1S271D of the PGK1S271 site is constructed, which can provide ideas and insights for the development of new drugs for the treatment of diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to an early diagnosis marker for detecting sepsis and application of a protein polypeptide thereof. BACKGROUND

[0002] The latest definition of sepsis is that the body's response to infection is out of balance, leading to life-threatening organ dysfunction. The pathophysiology of sepsis is complex, and it is a highly heterogeneous syndrome with excessive inflammation and immune suppression. Sepsis is the leading cause of morbidity and mortality in hospitalized patients. However, there is currently no effective treatment for sepsis. Nearly 50 million cases of sepsis occur worldwide each year, affecting people of all ages. Although the site of infection and pathogenic bacteria vary by geographic location and age, respiratory and gastrointestinal infections are the most common. In 2017, about 110 million patients died of sepsis, accounting for nearly 20% of all deaths worldwide. When sepsis patients need to be admitted to the intensive care unit, one-third of patients survive for less than 30 days, and the mortality rate varies by age, comorbidities, and type of organ dysfunction. Sepsis survivors have longer readmission times and higher mortality risks. Nearly half of sepsis survivors are readmitted to the hospital at least once a year, and one in six sepsis survivors eventually dies. Despite 30 years of research and more than 200 randomized controlled trials, none of our treatments can consistently save the lives of sepsis patients. The treatment of sepsis is still largely symptomatic, such as infection source control, timely use of antibiotics, resuscitation, and supportive treatment of organ dysfunction.

[0003] The pathophysiology of sepsis is complex, and it is a highly heterogeneous syndrome with excessive inflammation and immune suppression. Sepsis is the leading cause of morbidity and mortality in hospitalized patients. However, there is currently no effective treatment for sepsis. Nearly 50 million cases of sepsis occur worldwide each year, affecting people of all ages. Although the site of infection and pathogenic bacteria vary by geographic location and age, respiratory and gastrointestinal infections are the most common, although the site of infection and pathogenic microorganisms vary by geographic location and age. In 2017, about 110 million patients died of sepsis, accounting for nearly 20% of all deaths worldwide. When sepsis patients need to be admitted to the intensive care unit, one-third of patients survive for less than 30 days, and the mortality rate varies by age, comorbidities, and type of organ dysfunction. Sepsis survivors have longer readmission times and higher mortality risks. Nearly half of sepsis survivors are readmitted to the hospital at least once a year, and one in six sepsis survivors eventually dies. Despite 30 years of research and more than 200 randomized controlled trials, none of our treatments can consistently save the lives of sepsis patients. The treatment of sepsis is still largely symptomatic, such as infection source control, timely use of antibiotics, resuscitation, and supportive treatment of organ dysfunction.

[0004] At present, the method commonly used in clinic for diagnosing sepsis is that the diagnosis of sepsis mainly relies on clinical indicators, and the diagnostic criteria is "infection + SOFA≥2". SOFA score, namely Sequential Organ Failure Assessment (SOFA), is a scoring system for evaluating the severity of disease and prognosis of critically ill patients according to clinical blood biochemical detection and clinical indicators, and the higher the score, the more severe the disease. At present, the serum marker used in clinic for assisting the diagnosis of sepsis is procalcitonin (PCT). However, the increase of PCT only indicates infection, and it is difficult to distinguish between ordinary infection and sepsis.

[0005] When the external pathogenic microorganism invades, the monocyte / macrophage system first plays a role. Macrophages recognize pathogenic bacteria through the surface of the body and phagocytose and digest them, present them to T lymphocytes through antigen presentation, and produce a large number of inflammatory factors and chemotactic factors through the NF-κB and JAK-STAT pathways to regulate adaptive immune response. The produced inflammatory factors such as IL-1β and TNF-α activate the receptors on the macrophages through positive feedback to cause the activation of NF-κB. One of the causes of sepsis is the excessive activation of macrophages, which leads to a large number of inflammatory factor storms. Sepsis involves many types of cytokines, and there have been reports of IL-4, IL-10, IL-1RA related to anti-inflammatory; IL-1β, IL-6, CCL3, CCL5, MCP-1, IP-10 and TNF-α related to pro-inflammatory. The assembly and activation of NLRP3 inflammasome can cause different degrees of damage to different systems during sepsis, but in Nlrp3 knockout mice, pathogenic bacteria no longer activate NLRP3 inflammasome, the production of IL-1β is greatly reduced, and the activation and NF-κB signaling of NF-κB are weakened. Since inhibiting the NLRP3 / IL-1β pathway can reduce sepsis-mediated damage to other organs, it can be used to prevent sepsis diseases. Although there has been great progress in the bioinformatics research of sepsis, attempts have been made in clinic to weaken the inflammatory response or target cytokines for treatment, but all have ended in failure. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide an early diagnostic marker for detecting sepsis and an application of a protein polypeptide thereof.

[0007] The technical solution adopted by the present application to solve its technical problems is:

[0008] In a first aspect, the present application protects the use of PGK1 or an active fragment, a functional fragment of PGK1 S271 phosphorylation in the preparation of products for early diagnosis, risk assessment or treatment of sepsis-related diseases.

[0009] In a second aspect, the present application protects the use of a reagent for detecting PGK1 or PGK1 S271 phosphorylation in the preparation of a product for early diagnosis, risk assessment of a sepsis-related disease.

[0010] In a specific embodiment, the reagent is an antibody or antibody fragment for detecting PGK1 S271 phosphorylation.

[0011] In a third aspect, the present application protects the use of a reagent or kit for inhibiting PGK1 S271 phosphorylation in the preparation of a product for early diagnosis, risk assessment or treatment of a sepsis-related disease.

[0012] In a specific embodiment, the reagent for inhibiting PGK1 S271 phosphorylation is a protein polypeptide TAT-PGK1 S271D.

[0013] In a more specific embodiment, the sequence of the protein polypeptide TAT-PGK1 S271D is as follows: YGRKKRRQRRR+AHX+KDLMDKAEKN.

[0014] In a fourth aspect, the present application protects a product for early diagnosis, risk assessment or treatment of a sepsis-related disease in a subject, the product containing a reagent for inhibiting PGK1 S271 phosphorylation.

[0015] In a specific embodiment, the reagent for inhibiting PGK1 S271 phosphorylation is a protein polypeptide TAT-PGK1 S271D.

[0016] In a fifth aspect, the present application protects a system for early diagnosis, risk assessment or treatment of a sepsis-related disease in a subject, the product containing the product described above.

[0017] Advantages

[0018] The present application provides a biomarker for early diagnosis of sepsis and a protein polypeptide thereof, which has the following advantages compared with the prior art:

[0019] (1) PGK1 S271 phosphorylation can earlier indicate the occurrence of sepsis infection and the degree of infection;

[0020] (2) PGK1 S271 phosphorylation has specificity and is different from other non-specific infection indicators;

[0021] (3) PGK1 S271 phosphorylation detection is convenient and can be highly expressed in serum and easily detected;

[0022] (4) The protein polypeptide TAT-PGK1 S271D can target the treatment of sepsis. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is shown that the level of p-PGK1 S271 in immortalized bone marrow macrophages and human monocytes is significantly increased under LPS stimulation.

[0024] Figure 2 It is shown that the level of p-PGK1 S271 in primary macrophages is significantly increased under LPS stimulation, and its effect is specific.

[0025] Figure 3 It is shown that the level of p-PGK1 S271 in the blood of 40 mice under sepsis model is significantly increased.

[0026] Figure 4 It is shown that the survival rate of 10 old mice under sepsis model, and the mice with PGK1 S271A point mutation significantly alleviate the development of sepsis.

[0027] Figure 5 It is shown that the competitive inhibitory polypeptide scheme TAT-PGK1 S271D made for PGK1 S271 site has the following amino acid sequence: YGRKKRRQRRR+AHX+KDLMDKAEKN.

[0028] Figure 6 It is shown that the PGK1 S271 competitive inhibitory polypeptide can effectively inhibit the activation of NLRP3 inflammasome in immortalized macrophages, and alleviate the development of sepsis.

[0029] Figure 7 It is shown that the key inhibitory effect of PGK1 S271 competitive inhibitory polypeptide on sepsis is caused by the blocking of S271 site. DETAILED DESCRIPTION

[0030] The application will be further described in detail below in combination with examples. The reagents or instruments and equipment used are not marked with the manufacturer, and are all regarded as conventional products that can be purchased in the market.

[0031] Example 1

[0032] In order to verify the phosphorylation of PGK1 S271, we constructed an antibody p-PGK1 S271 for detecting the phosphorylation of PGK1 S271 site (PGK1, NCBI mRNA accession number: NM_000291.4, NCBI Protein accession number: CCDS14438.1Specifically, for the modification of S271 site, we selected 267-276aa to synthesize a modified polypeptide C-KDLM (easy oxidation, replaced by Nle) (S-p) KDLMSKAEKN, and immunized experimental Japanese white rabbits after coupling with KLH. At the same time, we synthesized a control peptide C-KDLM (easy oxidation, replaced by Nle) SKAEKN for antibody purification and detection. After sacrificing the rabbits, we finally provided the antigen affinity purified antibody (purified using modified and non-modified polypeptides, respectively).

[0033] To detect the phosphorylation level of PGK1 S271 in murine immortalized macrophages iBMDMs and human monocyte cell line THP-1, we found by immunoblotting that the phosphorylation could be detected as early as 2 hours after LPS stimulation, and the PGK1 S271 phosphorylation signal also increased with the increase of stimulation time. Similarly, after gray value analysis of the immunoblotting results by imagej software and normalization with the internal reference GAPDH, we used Graphpad software to statistically analyze the results repeated three times. The above results showed that the expression level of p-PGK1 S271 in iBMDMs and THP-1 cell line increased significantly under the induction of LPS, which had a significant difference compared with non-LPS stimulation (* represents P<0.05, ** represents P<0.01, *** represents P<0.001). It is shown that the p-PGK1 S271 site has the specificity of early detection of sepsis.

[0034] The specific steps are as follows:

[0035] 1. Cell culture and stimulation:

[0036] 1.1 Cell recovery:

[0037] (1) Take the cell cryopreservation tube to be recovered out of liquid nitrogen and place it in a 37°C water bath for rapid shaking to thaw.

[0038] (2) After disinfecting the surface of the cell cryopreservation tube with 75% alcohol, transfer it to a centrifuge tube and mix with an equal proportion of cell complete culture medium (DMEM), seal it, and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0039] (3) Discard the supernatant, add 1 mL of complete culture medium (DMEM) to resuspend the cells, then transfer the cell suspension to a cell culture flask or dish containing DMEM, and mix the cells gently.

[0040] (4) Place the cell culture flask or dish in a 37°C, 5% CO2 cell culture incubator for culture. After the cells are completely adherent, observe the cell state, and decide whether to pass or continue to culture according to the cell growth state and density.

[0041] 1.2 Cell passage:

[0042] (1) Take the cells that need to be passaged out of the incubator, discard the cell culture medium, and wash the cells twice with 1 mL of sterile 1x PBS.

[0043] (2) Add 1 mL of trypsin / EDTA solution to the cells and mix well. Incubate at room temperature for 2 min. When the cell morphology changes, the digestion is complete. Immediately add an equal volume of DMEM to terminate the trypsin digestion.

[0044] (3) After mixing the cells by blowing, transfer them to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant and resuspend the cell pellet with 1 mL of DMEM. Add the cells to the cell culture bottle or cell culture plate in proportion, and incubate in a 37°C, 5% CO2 cell culture incubator.

[0045] 1.3 Cell cryopreservation:

[0046] (1) Take the cells that need to be cryopreserved out of the incubator, discard the cell culture medium, and wash the cells twice with 1 mL of sterile 1x PBS.

[0047] (2) Add 1 mL of trypsin / EDTA solution to the cells and mix well. Incubate at room temperature for 2 min. When the cell morphology changes, the digestion is complete. Immediately add an equal volume of DMEM to terminate the trypsin digestion.

[0048] (3) After mixing the cells by blowing, transfer them to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature.

[0049] (4) Discard the supernatant, add cell cryopreservation solution and resuspend the cell pellet thoroughly, then transfer it to a cell cryopreservation tube and seal it with a sealing film.

[0050] (5) Cryopreserve the cells in a gradient, first store them at -80°C for 48 hours (h), then transfer them to liquid nitrogen for long-term storage.

[0051] 1.4 Cell stimulation:

[0052] Refer to the cell passage procedure for plating. When the cells are fully attached and the density reaches 80%-90%, add LPS (200 mg / mL) of different stimulation times to the cells, gently mix the medium, and continue to culture the cells in the incubator.

[0053] After the stimulation time, add Nigericin (10 nm) to the cell culture medium and continue to culture the cells at 37°C for 45 min

[0054] 2. Western blot

[0055] First, centrifuge the cell culture supernatant after the experiment is completed to remove suspended cells, then add Loading Buffer, and collect the proteins released by the cells outside the cells. Then add Loading Buffer directly to the cells, and collect the cell lysate. The collected protein sample needs to be placed in a dry bath oven at 95°C for 5-10 min, and stored at -20°C for standby.

[0056] Gel preparation: according to the size of the detected protein, prepare the concentrated gel and the separation gel.

[0057] Sample preparation: place the protein sample to be detected in a dry bath oven at 95°C for 5 min.

[0058] Loading: prepare 1x Tris-Glycine electrophoresis buffer (TGS) in advance, then check for leaks by loading the gel plate. According to the experimental conditions, select the appropriate loading volume, and add Protein Ladder (protein marker), sample, and loading buffer (Loading Buffer) to each well. First, electrophorese at 80V for 40 min, and then electrophorese at 120V until the separation is complete when the protein marker band migrates to the separation gel.

[0059] Membrane transfer: prepare 1x transfer buffer in advance and cool it at 4°C. According to the number of loading wells, cut a PVDF membrane of the corresponding size. Stack the filter paper-gel-film-filter paper in order, and remove the air bubbles. Place the membrane transfer clamp in the membrane transfer tank, pour in the transfer buffer, cover the electrophoresis cover, connect the power supply, and place the membrane transfer tank in an ice-water mixture for membrane transfer. According to the molecular weight size, select the transfer time and conditions (230mA constant current or 95V constant voltage transfer for 80-120 min).

[0060] Blocking: prepare 5% skimmed milk using 1x PBST (containing 0.1% Tween 20) buffer in advance. After membrane transfer is completed, place the PVDF membrane clamp in the blocking box and pour in an appropriate amount of milk. Block at room temperature for 1h at 20rpm.

[0061] Primary antibody: after blocking is completed, wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer, then place it in the incubation box, add PGK1 S271 phosphorylated antibody, and incubate at 4°C overnight.

[0062] Secondary antibody: after the primary antibody incubation is completed, recover the primary antibody. Then wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer, add the corresponding secondary antibody, and incubate on a shaker at room temperature for 1h. After incubation, wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer for development.

[0063] Development: Mix A and B of ECL developing solution at a ratio of 1:1. Place the membrane on the flat plate of the developing instrument, drop the developing solution, and develop the membrane.

[0064] Example 2

[0065] To verify that PGK1 S271 phosphorylation can also be detected in primary bone marrow-derived macrophages. We used wild type (WT) and PGK1 S271A point mutant mice, respectively, to extract the femur and tibia, and to extract macrophages from the bone marrow. Under the stimulation of LPS and Nig, the phosphorylation level of PGK1 S271 was detected by immunoblotting, and the repeated three experiments were statistically analyzed by Graphpad. The results showed that the phosphorylation of PGK1 S271 in WT primary macrophages was significantly enhanced under the induction of LPS, while the phosphorylation of PGK1 S271 in PGK1 S271A primary macrophages could not be detected. The gray value analysis of the immunoblotting results was performed by imagej software, and the homogenization with the internal reference GAPDH was used for statistical analysis of three independent experiments by Graphpad software, which indicated the specificity of p-PGK1271 antibody detection (*P<0.05, ***P<0.001). Similarly, we detected the phosphorylation of PGK1 S271 in bone marrow-derived primary macrophages by immunofluorescence, and the statistical analysis showed that the results were consistent with those of immunoblotting (**P<0.01).

[0066] The specific steps are as follows:

[0067] 1. Preparation of mouse bone marrow primary macrophages

[0068] (1) The mice were sacrificed by cervical dislocation, and the four limbs of the mice were fixed on the operation board with a fixed needle for subsequent use.

[0069] (2) The eye scissors and forceps and the fur on the abdomen of the mouse were disinfected with 75% alcohol. The leg skin was cut open with eye scissors, the leg bone was exposed, the residual muscle tissue was removed, and the tibia and femur were cut short and placed in alcohol for 15 s, and then placed in sterile pre-cooled PBS.

[0070] (3) Ignite the alcohol lamp, take out the separated leg bone and wash it with 75% alcohol and PBS buffer. Hold the femur (or tibia) with surgical forceps and cut off the two ends of the epiphysis. Use a 5 mL syringe needle to pierce the end of the leg bone.

[0071] (4) Use a 5 mL syringe to aspirate 1640 culture medium to repeatedly flush the bone marrow cavity until the bone color is white. Collect the flushed bone marrow solution in a 15 mL centrifuge tube.

[0072] (5) Collect the cells by centrifugation at 1000 rpm for 5 min, resuspend the L929 cell supernatant with the DMEM medium at a ratio of 1:9 and place in a 10 cm culture dish.

[0073] (6) Place the cell culture plate in a 37°C incubator with 5% CO2 and appropriate humidity, and replace the medium every other day. After one week, mouse primary bone marrow macrophages can be obtained.

[0074] 2. Cell stimulation

[0075] Referring to the cell passage step, when the cells are completely adherent and the density reaches 80%-90%, add LPS (200 mg / mL) to the cells, mix the medium gently, and continue to culture the cells in the incubator.

[0076] After the stimulation time, add nigericin (10 nm) to the cell culture medium and continue to culture the cells at 37°C for 45 min

[0077] 2. Western blot

[0078] First, centrifuge the cell culture supernatant after the experimental treatment to remove suspended cells, then add Loading Buffer, and collect the proteins released by the cells into the extracellular space. Then add Loading Buffer directly to the cells and collect the cell lysate. The collected protein samples need to be boiled in a dry oven at 95°C for 5-10 min and stored at -20°C for later use.

[0079] Gel preparation: Prepare concentrated and separating gels according to the size of the proteins to be detected.

[0080] Sample preparation: Place the protein samples to be detected in a dry oven at 95°C for 5 min.

[0081] Loading: Prepare 1x Tris-Glycine electrophoresis buffer (TGS) in advance, then check for leaks by loading the gel plate. Choose the appropriate loading volume according to the experimental conditions, and add Protein Ladder (protein marker), sample, and loading buffer (Loading Buffer) to each well. First, electrophorese at 80V for 40 min, then electrophorese at 120V until the separation is complete when the protein marker band migrates to the separating gel.

[0082] Transfer: Prepare 1x transfer buffer in advance and place it in 4℃ to cool down. Cut the PVDF membrane to the appropriate size according to the number of wells. Stack the filter paper-gel-membrane-filter paper in order and remove the air bubbles. Place the transfer clamp in the transfer tank, pour in the transfer buffer, cover the electrophoresis cover, connect the power supply, and place the transfer tank in the ice water mixture for transfer. Select the transfer time and conditions according to the molecular weight (230mA constant current or 95V constant voltage transfer for 80-120min).

[0083] Blocking: Prepare 5% skimmed milk in 1x PBST (containing 0.1% Tween 20) buffer in advance. After transfer, place the PVDF membrane clamp in the blocking box and pour in the appropriate amount of milk. Block at room temperature for 1h at 20rpm.

[0084] Primary antibody: After blocking, wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer, then place it in the incubation box, add PGK1 S271 phosphorylated antibody, and incubate at 4℃ overnight.

[0085] Secondary antibody: After incubation of the primary antibody, recover the primary antibody. Then wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer, add the corresponding secondary antibody, and incubate on the shaker at room temperature for 1h. After incubation, wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer for development.

[0086] Development: Mix the ECL developing solution by mixing A and B liquids in a ratio of 1:1. Place the membrane on the flat plate of the developer, add the developing solution, and develop the color.

[0087] 3. Immunofluorescence:

[0088] (1) Select the appropriate cell well plate and place sterile cell slides in it, then evenly inoculate the cells in the cell well plate, and wait for the density to grow to 60-80%.

[0089] (2) Remove the culture medium in the culture dish containing the cells of interest, and wash 3 times with PBS.

[0090] (3) Add fixing solution (4% paraformaldehyde) and fix for 15min.

[0091] (4) Wash 3 times with PBS, add 0.02% NP40 to break the membrane for 10min, and wash 3 times with PBS.

[0092] (5) Block 200μL of blocking solution per well for 15min, recover the blocking solution, and wash 3 times with PBS.

[0093] (6) After incubation at 4℃ overnight, wash 3 times with PBST.

[0094] (7) Incubate the secondary antibody in dark room temperature for 60 min, wash 3 times with PBST.

[0095] (8) Stain the nucleus with DAPI for 10 min in dark, wash 4 times with PBST.

[0096] Example 3

[0097] To show that the p-PGK1 S271 site has specificity for the detection of sepsis, we verified the phosphorylation level of PGK1 S271 in 40 C57BL / 6 mice fed in the Experimental Animal Center of Nanjing Medical University. We constructed a sepsis mouse model by intraperitoneal injection of LPS, and then extracted whole blood for immunoblotting to detect the phosphorylation level of PGK1 S271 in serum. The results showed that the phosphorylation level of PGK1 S271 in the serum of sepsis mice increased significantly, while this change could not be detected in normal mice. This indicates that the development of sepsis is closely related to the level of PGK1 S271 phosphorylation. The results of immunoblotting were analyzed by imagej software for gray value, and after normalization with the internal reference GAPDH, the ROC curve was drawn. The ROC curve shows that AUC = 0.9642, P < 0.0001, indicating that p-PGK1 S271 has good predictive value.

[0098] The specific steps are as follows:

[0099] A sepsis model was constructed by intraperitoneal injection of a lethal dose of LPS (20 mg / kg), and whole blood was extracted 12 h later.

[0100] Treatment of blood samples and extraction of cells:

[0101] After mixing the blood sample, centrifuging at 3000 rpm for 10 min at 4°C, it was found that the blood sample was layered, the upper layer was light yellow serum, and the lower layer was red cell sediment;

[0102] The serum was aspirated and stored separately, and 2-3 times the volume of red blood cell lysis solution was added to the cell sediment, which was gently blown evenly and then allowed to stand for 10 min, and mixed again every 2-3 min;

[0103] After standing, PBS was added to the sample to terminate cell lysis, and after centrifugation at 3000 rpm for 5 min at 4°C, if the lysis was complete, the cell sediment at the bottom would appear white, if the lysis was not complete, the upper liquid was aspirated and the lysis step was repeated;

[0104] Discard the supernatant, resuspend the cell pellet with PBS to wash away the residual red blood cell lysate, centrifuge at 3000 rpm for 5 min at 4℃, discard the PBS, add an appropriate amount of 2x Loading Buffer, mix and blow evenly, collect the protein sample, cook the sample in a dry oven at 95℃ for 5-10 min, store at -20℃ for later use, and then detect by western blot method.

[0105] Western blot

[0106] Gel preparation: prepare concentrated gel and separation gel according to the size of the protein to be detected.

[0107] Sample preparation: place the protein sample to be detected in a dry oven at 95℃ for 5 min.

[0108] Loading: prepare 1x Tris-Glycine electrophoresis buffer (TGS) in advance, then assemble the gel plate to detect the leakage. According to the experimental conditions, select the appropriate loading volume, add Protein Ladder (protein marker), sample, and loading buffer (Loading Buffer) in each well. First, electrophorese at 80V for 40 min, then electrophorese at 120V until separation is complete when the protein marker band migrates to the separation gel.

[0109] Membrane transfer: prepare 1x transfer buffer in advance and cool it at 4℃. According to the number of loading wells, cut the corresponding size of PVDF membrane. Stack them in the order of filter paper-gel-membrane-filter paper, and remove air bubbles. Place the membrane transfer clamp in the membrane transfer tank, pour in the transfer buffer, cover the electrophoresis cover, connect the power supply, and place the membrane transfer tank in an ice-water mixture for membrane transfer. According to the molecular weight, select the transfer time and conditions (230mA constant current or 95V constant voltage transfer for 80-120 min).

[0110] Blocking: prepare 5% skimmed milk with 1x PBST (containing 0.1% Tween 20) buffer in advance. After membrane transfer, place the PVDF membrane clamp in the blocking box and pour in an appropriate amount of milk. Block at room temperature for 1h at 20rpm.

[0111] Primary antibody: after blocking, wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer, then place it in the incubation box, add the corresponding primary antibody, and incubate at 4℃ overnight.

[0112] Secondary antibody: after primary antibody incubation, recover the primary antibody. Then wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer, add the corresponding secondary antibody, and incubate on a shaker at room temperature for 1h. After incubation, wash 3 times with 1x PBST (containing 0.1% Tween 20) buffer for development.

[0113] Development: Mix ECL developing solution A and B at a ratio of 1:1. Place the membrane on the flat plate of the developing instrument, add developing solution dropwise, and develop.

[0114] Example 4

[0115] To verify that the PGK1 S271 site can inhibit sepsis, we commissioned the Nanjing Medical University Experimental Animal Center to construct PGK1 S271A mice through Crisper-cas12a technology. All experimental mice were genotyped before the experiment, and the control group used the same batch of mice. All experimental mice were C57BL / 6 background, 6-8 weeks old. Mice were raised in a specific pathogen-free environment at Nanjing Medical University Experimental Animal Center and were fed by animal base staff. After adapting to the required experimental environment for seven days, the study was conducted, and all experimental procedures met the standards of the Animal Welfare Ethics Committee and relevant regulations. The sepsis model was constructed by intraperitoneal injection of a lethal dose of LPS (20 mg / kg), and the survival of the mice was monitored. The study found that PGK1 S271A mice showed low mortality and low incidence of sepsis (* indicates P<0.05). The results showed that PGK1 S271A rescued mice from sepsis.

[0116] The specific steps are as follows:

[0117] WT and PGK1 S271A mice of the same batch were injected intraperitoneally with a lethal dose of LPS (20 mg / kg, sigma, Cat#L2630), and their survival rates were observed within 72 hours. The survival rate curve was plotted using Graphpad.

[0118] Example 5:

[0119] After proving that inhibiting PGK1 S271 phosphorylation has a controlling effect on the occurrence and development of sepsis, we further developed a drug protein peptide PGK110AA-S271D for PGK1 S271, with the amino acid sequence as follows: (YGRKKRRQRRR (SEQ ID NO: 1) + AHX (aminoacetic acid) + KDLMDKAEKN (SEQ ID NO: 2)) (structure as shown in Figure 5 , in the hope of treating sepsis by competitively inhibiting the phosphorylation of PGK1 S271.

[0120] Thus we used solid-phase peptide synthesis method to synthesize PGK1266-276 small peptides with TAT active sequence (TAT), in which S271 site is permanently phosphorylated. TAT, a special peptide sequence in HIV-1 reverse transcription activator, is a cell-penetrating peptide with cell penetration. It can quickly enter cells in a non-specific manner and deliver its connected substances into cells.

[0121] Example 6

[0122] The PGK1 S271D short peptide was infected into ibmdm cells for 12 hours and 4 hours, respectively, and the cells were stimulated with LPS and Nig to activate NLRP3 inflammasome. By detecting the activation level of NLRP3 inflammasome downstream factors by Western blot, we found that the addition of PGK1 S271D short peptide significantly inhibited the cleavage of GSDMD and IL-1β band, thereby inhibiting the activation of NLRP3 inflammasome, and the short peptide added for 4 hours had inhibitory effect on NLRP3 inflammasome.

[0123] The specific steps are as follows:

[0124] Cell culture and stimulation:

[0125] 1.1 Cell recovery:

[0126] (1) Take the cell cryopreservation tube to be recovered out of liquid nitrogen and place it in a 37°C water bath for quick shaking to thaw.

[0127] (2) After sterilizing the cell cryopreservation tube surface with 75% alcohol, transfer it to a centrifuge tube and mix with an equal proportion of complete cell culture medium (DMEM). Seal and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0128] (3) Discard the supernatant, add 1 mL of complete medium (DMEM) to resuspend the cells, then transfer the cell suspension to a cell culture bottle or dish containing DMEM, and mix the cells gently.

[0129] (4) Place the cell culture bottle or dish in a 37°C, 5% CO2 cell incubator for culture. After the cells are fully attached, observe the cell state and decide whether to pass or continue culture according to the cell growth state and density.

[0130] 1.2 Cell passage:

[0131] (1) Take the cells to be passaged out of the incubator, discard the cell culture medium, and wash the cells twice with 1 mL of sterile 1×PBS.

[0132] (2) Add 1 mL trypsin / EDTA solution to the cells and mix well, stand at room temperature for 2 min, and immediately add an equal volume of DMEM to terminate the trypsin digestion after the cell morphology changes.

[0133] (3) After mixing the cells by blowing, transfer them to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant, resuspend the cell pellet with 1 mL DMEM, and add it to the cell culture bottle or cell culture plate in proportion, and place it in a 37°C, 5% CO2 cell incubator for culture.

[0134] 1.3 Cell cryopreservation:

[0135] (1) Take the cells to be cryopreserved out of the incubator, discard the cell culture medium, and wash the cells twice with 1 mL sterile 1x PBS.

[0136] (2) Add 1 mL trypsin (EDTA) solution to the cells and mix well, stand at room temperature for 2 min, and immediately add an equal volume of DMEM to terminate the trypsin digestion after the cell morphology changes.

[0137] (3) After mixing the cells by blowing, transfer them to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature.

[0138] (4) Discard the supernatant, add cell cryopreservation solution and resuspend the cell pellet, then transfer it to a cell cryopreservation tube and seal it with a sealing film.

[0139] (5) Cryopreserve the cells in a gradient, first store them at -80°C for 48 hours (h), then transfer them to liquid nitrogen for long-term storage.

[0140] 1.4 Cell stimulation:

[0141] Refer to the cell passage step to plate, and when the cells are fully attached and the density reaches 80%-90%, add different concentrations of PGK1 S271D short peptide to the cells, along with LPS (200 mg / mL), gently mix the medium, and place the cells in the incubator for continued culture for 4 hours;

[0142] After 4 hours, add Nigericin (10 nm) to the cell culture medium and continue to culture the cells at 37°C for 45 min;

[0143] Western blot

[0144] Firstly, centrifuge the cell culture supernatant to remove suspended cells, then add Loading Buffer to collect the proteins released by cells. Then add Loading Buffer directly to the cells to collect cell lysate. The collected protein samples need to be boiled in a dry oven at 95°C for 5-10 min and stored at -20°C for later use.

[0145] The protein expression level was detected by western blot according to the method described in Example 3.

[0146] Example 7

[0147] To rule out the possibility of TAT transmembrane peptide inhibiting the activation of NLRP3 inflammasome, and to detect whether PGK1 WT (266-276) also has the ability to regulate the activation of NLRP3 inflammasome, we continue to construct TAT short peptide and TAT-PGK1 S271D amino acid sequence as follows: (YGRKKRRQRRR+AHX+KDLMDKAEKN), TAT-PGK1 WT amino acid sequence as follows: (YGRKKRRQRRR+AHX+KDLMSKAEKN), TAT short peptide amino acid sequence as follows: (YGRKKRRQRRR+AHX). Add the three short peptides to the cell culture medium respectively, and use LPS and nigericin to induce the activation of NLRP3 inflammasome in ibmdm cells. It was found that TAT short peptide had no effect on the activation of NLRP3 inflammasome in macrophages, while PGK1 266-276 could also inhibit the activation of NLRP3 inflammasome, but its inhibitory effect was far less than that of PGK1 S271D short peptide.

[0148] The specific steps are as follows:

[0149] 1. Cell culture and stimulation:

[0150] 1.1 Cell recovery:

[0151] (1) Take the cell cryopreservation tube to be recovered out of liquid nitrogen and place it in a 37°C water bath for quick thawing.

[0152] (2) After disinfecting the surface of the cell cryopreservation tube with 75% alcohol, transfer it to a centrifuge tube and mix with an equal amount of complete cell culture medium (DMEM). Seal it and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0153] (3) Discard the supernatant, add 1 mL of complete culture medium (DMEM) to resuspend the cells, then transfer the cell suspension to a cell culture bottle or dish containing DMEM and mix the cells gently.

[0154] (4) Put the cell culture bottle or dish in the cell culture box at 37°C, 5% CO2, and culture until the cells are completely adherent. Observe the cell state, and determine whether to pass or continue to culture according to the cell growth state and density.

[0155] 1.2 Cell passage:

[0156] (1) Take the cells that need to be passed out of the incubator, discard the cell culture medium, and wash the cells twice with 1 mL of sterile 1x PBS.

[0157] (2) Add 1 mL of trypsin / EDTA solution to the cells and mix well. Incubate at room temperature for 2 min until the cells are digested. Then add an equal volume of DMEM to terminate the trypsin digestion.

[0158] (3) After mixing the cells by blowing, transfer them to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant, resuspend the cell pellet with 1 mL of DMEM, and add it to the cell culture bottle or cell culture plate in proportion. Place it in a 37°C, 5% CO2 cell culture box for culture.

[0159] 1.3 Cell freezing:

[0160] (1) Take the cells that need to be frozen out of the incubator, discard the cell culture medium, and wash the cells twice with 1 mL of sterile 1x PBS.

[0161] (2) Add 1 mL of trypsin (EDTA) solution to the cells and mix well. Incubate at room temperature for 2 min until the cells are digested. Then add an equal volume of DMEM to terminate the trypsin digestion.

[0162] (3) After mixing the cells by blowing, transfer them to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature.

[0163] (4) Discard the supernatant, add cell freezing solution and resuspend the cell pellet thoroughly, then transfer it to a cell freezing tube and seal it with a sealing film.

[0164] (5) Freeze the cells in a gradient, first store them at -80°C for 48 hours (h), then transfer them to liquid nitrogen for long-term storage.

[0165] 1.4 Cell stimulation:

[0166] Refer to the cell passage procedure for plating. When the cells are completely adherent and the density reaches 80%-90%, add different concentrations of PGK1 S271D short peptide to the cells, along with LPS (200 mg / mL), mix the medium gently, and place the cells in the incubator for continued culture for 4 hours.

[0167] 4 hours later, Nigericin (10 nm) was added into the cell culture medium and the cells were incubated for another 45 min at 37°C

[0168] 2. Western Blot

[0169] The cell culture supernatant was first centrifuged to remove the suspended cells, and then the Loading Buffer was added to collect the proteins released from the cells. The Loading Buffer was directly added to the cells to collect the cell lysate. The collected protein sample was boiled in a dry oven at 95°C for 5-10 min and stored at -20°C for later use. The protein expression level was detected according to the western blot method described in Example 1.

[0170] The protection scope of the present application is not limited to the above examples. Any changes and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. Use of a reagent for detecting PGK1 S271 phosphorylation in the manufacture of a product for early diagnosis, risk assessment of sepsis-related diseases.

2. Use according to claim 1, characterized in that, The reagent is an antibody or antibody fragment for detecting PGK1 S271 phosphorylation.

3. Use of an agent or a kit inhibiting the phosphorylation of PGK1 S271 in the manufacture of a product for the treatment of a sepsis-related disease, characterized in that, The reagent is a protein polypeptide TAT-PGK1 S271D, the sequence of which is as follows: YGRKKRRQRRR+AHX+KDLMDKAEKN.

4. A product for use in the treatment of a sepsis-related disease in a subject, characterized in that, The product contains a reagent for inhibiting PGK1 S271 phosphorylation, which is a protein polypeptide TAT-PGK1 S271D, the sequence of which is as follows: YGRKKRRQRRR+AHX+KDLMDKAEKN.

5. A system for the treatment of sepsis-related diseases in a subject, characterized in that, The system contains the product of claim 4.

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