Application of DNMT3B as a marker in preparation of diagnostic product for severe EV71 infection

Abstract

The application discloses application of DNMT3B as a marker in preparation of a severe EV71 infection diagnosis product, and belongs to the biomedical field. It is found by the application that the nucleocytolysis degree and ubiquitination level of DNA methyltransferase DNMT3B can be used for diagnosing children with severe EV71 infection. EV71 infection promotes partial transport of DNMT3B in the cell nucleus to the cytoplasm, and promotes K63 ubiquitination modification of DNMT3B by combining the 3C and 3D proteins with DNMT3B, so that DNMT3B is finally degraded by a proteasome. The reagent for detecting the nucleocytolysis degree of DNMT3B and / or the reagent for detecting the ubiquitination level of DNMT3B can be used for preparing a severe EV71 infection diagnosis product or a severe EV71 infection prognosis diagnosis product, so as to realize diagnosis of severe EV71 infection or prognosis thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of DNMT3B as a marker in the preparation of diagnostic products for severe EV71 infection. Background Technology

[0002] Enterovirus 71, belonging to the Picornaviridae family, has a genome length of approximately 7500 nt, containing a 3'-poly(a) tail and an open reading frame (ORF) encoding a polypeptide of 2940 amino acids (Yuan J, Shen L, Wu J, Zou X, Gu J, Chen J, Mao L. 2018. Enterovirus A71 Proteins: Structure and Function. Frontiers in Microbiology 9:286.). The ORF-encoded polyprotein can be cleaved into 11 mature viral proteins, of which 4 proteins are involved in the construction of the viral capsid (VP1-VP4). Non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D play important roles in viral infection, viral replication, and evasion of innate immune responses, and also mediate viral-host interactions (Xiao X, Qi J, Lei X, Wang J. 2019. Interactions Between Enteroviruses and the Inflammasome: New Insights Into Viral Pathogenesis. Frontiers in Microbiology 10:321.). The 2A and 3C proteins of EV71 possess protease activity, enabling them to cleave NLRP3 to inhibit the activation of the NLRP3 inflammasome and reduce the release of IL-1β (Wang H, Lei X, Xiao X, Yang C, Lu W, Huang Z, Leng Q, Jin Q, He B, Meng G, Wang J. 2015. Reciprocal Regulation between Enterovirus 71 and the NLRP3 Inflammasome. Cell reports 12:42-48.). The 3D protein of EV71 is an RNA-dependent RNA polymerase that forms a “3D-NLRP3-ASC” complex, which then binds to pro-caspase-1 to form caspase-1, ultimately mediating the secretion of IL-1β.

[0003] Epigenetics refers to a wide range of phenotypic genetic molecular changes independent of any alteration in the DNA sequence itself. Epigenetic modifications occur during cell proliferation to regulate transcriptional activity in normal tissues. The epigenetic state of cells plays a crucial role in determining the differentiation state and normal function of multicellular organisms. Epigenetic features include DNA methylation, histone modifications, non-coding RNA, and chromatin structure. DNA methylation is an epigenetic modification and a major epigenetic factor affecting gene expression (Moore LD, Le T, Fan G. 2013. DNA methylation and its basic function. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 38:23-38.). DNA methylation plays a crucial role in the expression of inflammatory factors. DNA methylation is catalyzed by the DNA methyltransferase (DNMT) family, which transfers a methyl group to the 5' position of the CpG dinucleotide cytosine ring, forming 5mC (Smallwood SA, Kelsey G. 2012. De novo DNA methylation: a germ cell perspective. Trends in genetics: TIG 28:33-42.). The DNMT family includes DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L, of which only DNMT1, DNMT3A, and DNMT3B possess methyltransferase activity. DNMT3 is the major de novo methyltransferase, primarily involved in constructive DNA methylation, i.e., directly synthesizing methylcytosine de novo for methylation modification without the guidance of a pre-methylated DNA template. DNMT3A and DNMT3B can establish new methylation patterns on unmodified DNA and are known as de novo methyltransferases, while DNMT1 is responsible for replicating and maintaining the methylation pattern after DNA replication (Portela A, Esteller M. 2010. Epigenetic modifications and human disease. Nature biotechnology 28:1057-1068.).

[0004] In recent years, several studies have reported epigenetic changes during viral infection. One study found that the NS1 protein of IAV suppresses the antiviral response by inhibiting histone mimics that inhibit transcriptional elongation (Marazzi I, Ho JS, Kim J, Manicassamy B, Dewell S, Albrecht RA, Seibert CW, Schaefer U, Jeffrey KL, Prinjha RK, Lee K, García-Sastre A, Roeder RG, Tarakhovsky A. 2012. Suppression of the antiviral response by an influenza histone mimic. Nature 483:428-433.). Another study showed that IAV infection directly leads to changes in promoter DNA methylation of genes encoding inflammatory proteins (Mukherjee S, Vipat VC, Chakrabarti AK. 2013. Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes. Influenza and other respiratory viruses 7:979-986.). Other studies have found that DNA methylation modification can occur in inflammatory cytokines; LPS has been reported to induce the expression of IL-6 and IL-8 in bovine endometrial cells by modifying DNA methylation (Wang J, Yan X, Nesengani LT, Ding H, Yang L, Lu W. 2018. LPS-induces IL-6 and IL-8 gene expression in bovine endometrial cells "through DNA methylation". Gene 677:266-272.). Furthermore, interleukin-32 (IL-32) is upregulated through DNA methylation modification after IAV infection.IAV infection can suppress DNMT3B expression, leading to the production of cyclooxygenase 2 and lambda-1 interferon (Fang J, Hao Q, Liu L, Li Y, Wu J, Huo X, Zhu Y. 2012. Epigenetic changes mediated by microRNA miR29 activate cyclooxygenase 2 and lambda-1 interferon production during viral infection. Journal of Virology 86:1010-1020.).

[0005] EV71 is one of the main pathogens causing hand-foot-and-mouth disease (HFMD). The virus primarily targets children under 5 years old and infants, and can cause serious neurological complications in children, such as aseptic meningitis, brainstem encephalitis, poliomyelitis-like paralysis, and neurogenic pulmonary edema. Most cases recover within a week. These children have mild symptoms, with only rashes on their hands, feet, mouth, and buttocks, and may also have cough and runny nose. However, a small number of children may develop severe illness, with persistent high fever, headache, vomiting, drowsiness and other neurological symptoms, as well as cardiopulmonary symptoms such as rapid breathing, cyanosis of the lips, and coughing up pink frothy sputum. Critically ill patients may develop coma and shock, and if not treated promptly, may die. The most severe outbreaks have occurred in the Asia-Pacific region and beyond (Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. 2010. Virology, epidemiology, pathogenesis, and control of enterovirus 71. The Lancet. Infectious diseases 10:778-790.). Currently, there is still a lack of comprehensive understanding of the pathogenesis of EV71, and it is not possible to detect severely infected children with EV71 in a timely manner in clinical practice. Therefore, it is of great significance to provide reagents for preparing diagnostic products for severe EV71 infection. Summary of the Invention

[0006] The purpose of this invention is to solve the problem that existing technologies cannot detect children with severe EV71 infection in a timely manner, and to provide an application of DNMT3B as a marker in the preparation of diagnostic products for severe EV71 infection.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Application of DNMT3B as a marker in the preparation of diagnostic products for severe EV71 infection or its prognosis. Diagnosis of severe EV71 infection or its prognosis is achieved by detecting the degree of DNMT3B nuclear export and / or the level of DNMT3B ubiquitination. Compared with children without severe EV71 infection or those who have recovered from severe EV71 infection, children with severe EV71 infection showed increased levels of DNMT3B ubiquitination and nuclear export in PBMCs.

[0009] Application of reagents for detecting the degree of DNMT3B nuclear export and / or reagents for detecting the level of DNMT3B ubiquitination in the preparation of diagnostic products for severe EV71 infection or diagnostic products for the prognosis of severe EV71 infection.

[0010] The method for detecting the degree of nucleation and ubiquitination of DNMT3B is as follows:

[0011] (1) The ubiquitination level of DNMT3B in cells of children with severe EV71 infection was detected by immunoprecipitation and immunoblotting.

[0012] (2) The degree of nucleation of DNMT3B in cells of severely EV71-infected children was detected by nucleocytoplasmic separation experiment.

[0013] The reagents for detecting the degree of nucleation of DNMT3B include one or more of lymphocyte separation solutions and lysis buffers. Further, the lysis buffers include lysis buffer A and lysis buffer C (lysis buffer A contains 10 μL 1M Tris-HCl, 5 μL 1M MgCl2, 10 μL 1M NaCl, 1 μL 1M DTT, 10 μL cocktail protease inhibitor, and 964 μL ddH2O per mL; lysis buffer C contains 20 μL 1M HEPES-KOH (pH 7.9), 1.5 μL 1M MgCl2, 500 μL 1M NaCl, 1 μL 1M DTT, 0.4 μL 0.5M EDTA, 100 μL 10% NP-40, 10 μL cocktail protease inhibitor, and 367.1 μL ddH2O per mL).

[0014] The reagents for detecting DNMT3B ubiquitination levels include one or more of the following: lysis buffer, protease inhibitor, DNMT3B antibody solution, and protein dye. Further, the lysis buffer includes RIPA lysis buffer, SDS lysis buffer, and NP-40 cell lysis buffer.

[0015] A diagnostic kit for severe EV71 infection or a diagnostic kit for the prognosis of severe EV71 infection, comprising reagents for detecting the degree of nuclear export of DNMT3B and reagents for detecting the level of ubiquitination of DNMT3B.

[0016] Advantages and beneficial effects of the present invention: The present invention discovers that severe EV71 infection promotes the partial transport of DNMT3B from the cell nucleus to the cytoplasm, and through its 3C and 3D proteins, it binds to DNMT3B, promoting the ubiquitination modification of DNMT3B at the K63 position, so that it is eventually degraded by the proteasome. Therefore, the degree of DNMT3B exiting the nucleus and the ubiquitination level can be used to diagnose children with severe EV71 infection and to determine the disease outcome. Attached Figure Description

[0017] Figure 1 The EV71 infection provided in this embodiment of the invention promotes the ubiquitination and degradation of DNMT3B. RD cells were transfected with HA-DNMT3B and Myc-ubiquitin for 24 hours, then infected with EV71 or not infected with EV71 (MOI=1) for 12 hours, and then treated with MG132 (20uM) for 6 hours. The ubiquitination modification status of DNMT3B was detected by co-immunoprecipitation and Western blot analysis using HA-tagged antibodies and Myc-tagged antibodies, respectively.

[0018] Figure 2 This invention provides an example of EV71 3C and 3D detection for DNMT3B co-localization. RD cells, after overexpressing 3XHA-DNMT3B, were stimulated with 1 μL and 10 μL of EV71 (MOI=1), respectively. After 2 hours of viral stimulation, cells were treated with MG132 (10 μM). Samples were collected 12 hours after viral infection for nucleocytosis separation experiments to detect the expression of various proteins in the nucleus and cytoplasm. LaminA was used as a nuclear protein internal control, and GAPDH as a cytoplasmic protein internal control.

[0019] Figure 3 The results show the degree of DNMT3B nuclear exit (A) and ubiquitination level (B) in PBMC cells of healthy children (HI), EV71 infected patients (EIP), severe EV71 infected patients (SEIP), and rehabilitation patients (RP). Detailed Implementation

[0020] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art.

[0022] Example 1: RD cell culture and EV71 virus amplification

[0023] 1. The human rhabdomyosarcoma cell line RD used in this invention was purchased from the China Center for Type Culture Collection (CCTCC) of Wuhan University. The enterovirus 71 (EV71, C4 type) was derived from the brain of an infant who died from EV71 infection in Xiangfan City, Hubei Province. This virus was preserved and amplified in our laboratory. The culture medium MEM (Gibco) and fetal bovine serum (Gibco) used for RD cell culture were both purchased from Thermo Fisher Scientific.

[0024] 2. Under a microscope, when the RD cell growth density in the culture flask reaches 80% or higher, discard the old culture medium and add fresh serum-free culture medium, along with an appropriate amount of virus for infection. Generally, one flask of T75 cells is used for virus amplification, with 15 mL of culture medium added.

[0025] 3. Incubate virus-infected cells in a CO2 incubator for 1-3 days, observing cell status at regular intervals. When cells are nearly or completely dead, aspirate the culture medium and transfer it to sterile 15mL centrifuge tubes. The harvested culture medium, which is the virus solution, should be centrifuged at 3000rpm for 3 minutes, aliquoted, labeled, and stored at -80℃ for later use.

[0026] Example 2: Construction of Recombinant Plasmids

[0027] The gene sequences of DNMT3B and ubiquitin were retrieved from the NCBI website. Suitable polymerase chain reaction (PCR) primers were designed using Snapgene software, and the target fragments were obtained through PCR amplification. Using the pcDNA3.1 / Myc-His plasmid as an expression vector, restriction endonucleases were used for digestion, followed by ligation of the fragments using T4 ligase. The ligation products were transformed into *E. coli*, and the cells were screened and expanded using resistant culture medium. Finally, endotoxin-free overexpression plasmids of DNMT3B and ubiquitin, suitable for cell transfection, were obtained using an endotoxin-free plasmid extraction kit from Omega, and named pcDNA-DNMT3B and myc-ubiquitin, respectively. Using pcDNA-DNMT3B as a template, the DNMT3B fragment was subcloned and inserted into the pKH3 plasmid (containing a 3xHA tag) to obtain the 3xHA-DNMT3B plasmid.

[0028] Example 3: Protein immunoprecipitation and Western blot analysis confirmed that EV71 infection promotes the ubiquitination and degradation of DNMT3B.

[0029] 1. RD cells were transfected with 3xHA-DNMT3B and Myc-ubiquitin for 24 hours, then infected with EV71 or uninfected cells (MOI=1) for 12 hours, followed by treatment with MG132 (20uM) for 6 hours. The ubiquitination status of DNMT3B was detected by co-immunoprecipitation and Western blotting analysis using HA-tagged antibodies and Myc-tagged antibodies, respectively. Myc (M4439) and hemagglutination (HA) antibodies were purchased from MBL International. MG132 was purchased from TargetMol.

[0030] 2. Protein Immunoprecipitation Assay. Take a 10cm culture dish, discard the culture medium, add 1mL of pre-chilled PBS, and scrape the cells from the bottom of the dish into a pre-chilled EP tube using a cell scraper. After collecting the sample, centrifuge at 3000rpm for 3min, discard the supernatant, wash the cells again with PBS, leaving the cells, add 792μL of RIPA cell lysis buffer and 8μL of 100X protease inhibitor, mix the cell pellet well, sonicate each sample for 5 seconds, with a 3-second interval, repeat 3 times to lyse the cells, centrifuge at 12000rpm for 15min at 4℃ in a refrigerated centrifuge, collect the supernatant into a new EP tube, add 10μL of protein A / G pre-wash to remove non-specifically bound proteins, and place on a 4℃ rotary shaker for 1 hour. Remove the sample, centrifuge at 3000rpm for 3min at 4℃, aspirate the supernatant into a new EP tube, add 0.6μL of DNMT3B antibody, and continue to place on a 4℃ shaker for 4 hours. Remove the sample, add 18 μL of protein A / G precipitate to the protein complex, and continue shaking at 4°C for 4 hours. Remove the sample, centrifuge at 3000 rpm for 2 min at 4°C, discard the supernatant, leaving the lower protein A / G precipitate, add 800 μL of wash buffer (equal volume of RIPA lysis buffer and 1M NaCl solution), centrifuge at 3000 rpm for 2 min, wash 4 times or more, and centrifuge at 12000 rpm for 1 min on the last wash, discard the supernatant, add 28 μL of SDS lysis buffer and 5 μL of protein loading buffer, boil in boiling water for 5 min, centrifuge at 12000 rpm for 5 min after boiling, and obtain the results by Western blot.

[0031] 3. Western blot analysis of proteins. HA-tagged antibody was used for immunoblotting (IP), and Myc-tagged antibody was used for immunoblotting (IB) to detect the ubiquitination status of DNMT3B.

[0032] (1) Aspirate the supernatant of the cells to be tested, add 1 mL of PBS to wash once, remove the PBS, add 500 μL of trypsin, place in a 37℃ incubator to digest for 3-5 minutes, then add serum-containing culture medium and blow the cells apart with a pipette.

[0033] (2) Transfer the cell mixture to a 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add 1 mL of PBS to resuspend and wash the cells, and centrifuge at 1000 rpm for 5 minutes.

[0034] (3) Discard the supernatant, add 200 μL of NP-40 cell lysis buffer and 4 μL of protein inhibitor 50×cocktail to resuspend the cells, mix well, and place the centrifuge tube on ice for half an hour for lysis. Then centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the supernatant to a new 1.5 mL centrifuge tube, being careful not to aspirate the cell pellet at the bottom, and label it.

[0035] (4) Before determining the protein concentration, prepare a 2 μg / μL BSA protein solution as a standard. Take the corresponding volume of EP tubes, add 1 mL of 1×BioRad protein dye, then add 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of BSA standard, and 2 μL of the sample to be tested, respectively. Mix well, and add 200 μL from each tube to the assay plate. Set up one well as a blank control, place the plate in a microplate reader, and read the value at a wavelength of 595 nm. Record the results and calculate the protein concentration based on the BSA standard curve.

[0036] (5) Using the lowest protein concentration group as a reference, adjust the concentration of each sample group to the same state, and dilute and make up the difference with NP 40 cell lysis buffer. Then, according to the sample volume, add the corresponding amount of 5× protein loading buffer, mix well, boil for 10 minutes to denature the protein. Centrifuge at 12000 rpm for 1-3 minutes and set aside.

[0037] (6) Prepare the SDS-PAGE gel in advance. The concentration of the stacking gel is 5%, and the concentration of the separating gel is 12%. The gel concentration can be adjusted according to the size of the protein to be tested. Place the gel in the electrophoresis tank, pour in the electrophoresis buffer, shake the electrophoresis tank to remove air bubbles at the bottom, and observe whether the sample wells on the gel are normal. If they are skewed, adjust them to be straight. Add the protein marker and sample in sequence.

[0038] (7) Turn on the power and run the gel at 90V. Observe the situation through the marker. When the protein enters the separating gel, switch to 110V and continue running the gel for 1-3 hours. When the protein to be tested reaches the appropriate position, turn off the power and prepare for transfer.

[0039] (8) Prepare 1L of transfer buffer and cut the membrane and filter paper according to the size of the gel. The PVDF membrane used in this experiment needs to be soaked in methanol for 15s before transfer, then soaked in double-distilled water for 2min, and finally soaked in the prepared transfer buffer for more than 5min.

[0040] (9) Remove the protein gel, cut off the stacking gel portion, gently rinse away any surface impurities, remembering the front and back sides, and immerse it in transfer buffer. Open the transfer plate and place the gels from the negative electrode in the following order: soft support, three layers of filter paper, protein gel, PVDF membrane, three layers of filter paper, soft support. Ensure there are no air bubbles between the gel and membrane; these can be gently removed with a glass rod. After loading the transfer plate, align the positive and negative electrodes and place it in the transfer apparatus. Turn on the power and transfer at a constant current of 200mA for 2 hours. Keep the transfer apparatus on ice or in a refrigerator at 4°C throughout the process.

[0041] (10) Prepare 10% skim milk solution using PBS. Take out the membrane, mark the side closest to the gel, and place it in the milk solution for sealing. Shake on a shaker at a suitable speed for 1-2 hours.

[0042] (11) Discard the milk and rinse several times with PBS, being careful not to rinse directly onto the membrane. After rinsing with milk, place the membrane in a DNMT3B antibody solution prepared with PBS (the specific dilution ratio depends on the antibody) and shake overnight at 4°C or incubate at room temperature for 2-4 hours.

[0043] (12) After the primary antibody incubation is complete, aspirate the membrane and store it at 4°C. Wash the membrane with TBST, rinsing it three times on a shaker with a 10-minute interval between each rinse.

[0044] (13) Prepare 3% skim milk with PBS, dilute goat anti-rabbit IgG (generally at a ratio of 1:5000), place it in a membrane, and incubate on a shaker at room temperature for 40 min-1 h.

[0045] (14) Discard the secondary antibody, wash the membrane with TBST, and rinse on a shaker 5-6 times, with a 10-minute interval between each rinse.

[0046] (15) After washing the membrane, remove it, place it on filter paper and gently press it dry. Add a 1:1 mixture of horseradish peroxidase chromogenic substrate to the membrane, incubate for 5 minutes, and then place it in a chemiluminescence imaging analyzer for color development. The exposure time can be adjusted as needed. Save the results and analyze them. The results are as follows: Figure 1 As shown, EV71 infection promotes the ubiquitination and degradation of DNMT3B.

[0047] Example 4: EV71 infection promotes the translocation of DNMT3B from the nucleus to the cytoplasm.

[0048] 1. RD cells were stimulated with 1 μL and 10 μL of EV71 (MOI=1) after overexpressing DNMT3B protein. After 2 h of viral stimulation, MG132 (10 μM) was added to treat the cells. The cells were collected 12 h after viral infection.

[0049] 2. Perform nucleocytoplasmic separation experiments. Wash with PBS, centrifuge to remove the supernatant, add 10 times the volume of lysis buffer A, and lyse on ice for 30 minutes, gently vortexing or pipetting the cells every 15 minutes. Then add 1 / 20 volume of 10% NP-40 detergent, vortex for 5 seconds, place on ice for 5 minutes, and vortex again for 5 seconds. Then centrifuge at 3000 rpm at 4°C for 20 minutes. Transfer the extracted supernatant to a new 1.5 mL EP tube, centrifuge at 12000 rpm for 30 minutes, and transfer the supernatant to a new EP tube again; this is the cytoplasmic protein solution. Meanwhile, resuspend the precipitate with 5 times the volume of lysis buffer A, stand on ice for 10-20 minutes, centrifuge at 4°C and 3000 rpm for 5 minutes, remove the supernatant, add 10 times the volume of lysis buffer C to the precipitate, sonicate for 15 minutes, sonicate again, and then centrifuge at 4°C and 12000 rpm for 30 minutes. The resulting supernatant is the nucleoprotein solution. The lysis buffer A consists of 10 μL 1M Tris-HCl, 5 μL 1M MgCl2, 10 μL 1M NaCl, 1 μL 1M DTT, 10 μL cocktail protease inhibitor, and 964 μL ddH2O per mL. The lysis buffer C consists of 20 μL 1M HEPES-KOH (pH 7.9), 1.5 μL 1M MgCl2, 500 μL 1M NaCl, 1 μL 1M DTT, 0.4 μL 0.5M EDTA, 100 μL 10% NP-40, 10 μL cocktail protease inhibitor, and 367.1 μL ddH2O per mL.

[0050] The expression of various proteins in the cell nucleus and cytoplasm was detected. LaminA was used as a nuclear protein internal control, and GAPDH as a cytoplasmic protein internal control. Western blot analysis was performed, following the methods described in Example 3. Results are as follows: Figure 2 As shown, endogenous DNMT3B is localized throughout the cell before EV71 infection, but after infection, some nuclear DNMT3B translocates from the nucleus to the cytoplasm.

[0051] Example 5: Detection of the degree of nuclear export and ubiquitination of DNMT3B in relevant cells of healthy children, children with ordinary EV71 infection, and children with severe EV71 infection.

[0052] Blood samples from healthy children were collected from Wuhan University Hospital. Patients were collected from the ICU of Zhongnan Hospital of Wuhan University and had experienced the entire process of EV71 infection, severe illness, and recovery. Patient presentation: age less than 3 years, EV71 positive, body temperature (axillary temperature) greater than 39℃, poor response to conventional antipyretics, abnormal respiration, and peripheral blood WBC count exceeding 15*10. 9The patient presented with stress-induced hyperglycemia, with blood glucose levels exceeding 8.3 mmol / L. Lymphocyte separation medium was purchased from Tianjin Haoyang Biotechnology Co., Ltd., and RPMI-1640 cell culture medium was purchased from Thermo Fisher Scientific.

[0053] 1. PBMC cell isolation

[0054] Peripheral blood was collected from the child. The collected blood or cells should be kept away from freezing and refrigeration during collection, processing, and transportation. A sterile centrifuge tube was removed, and lymphocyte separation medium was added. Simultaneously, the obtained peripheral blood sample was diluted with an equal volume of PBS and thoroughly mixed to form a peripheral blood diluent. This diluent was then added to the lymphocyte separation medium, slowly and evenly, ensuring that the blood diluent and lymphocyte separation medium formed distinct layers (the upper layer being the blood diluent, and the lower layer being the lymphocyte separation medium). The tube was centrifuged at low speed (500×g) for 20 minutes at room temperature. After centrifugation, distinct layers were observed in the centrifuge tube, from top to bottom: a plasma-PBS mixture layer, a mononuclear cell white membrane layer, a human lymphocyte separation medium layer, and a red blood cell layer. Carefully aspirate the white membrane layer using a Pasteur tube or sterile pipette tip and place it in a new centrifuge tube, being careful not to aspirate the lymphocyte separation medium. Add an appropriate amount of RPMI-1640 cell culture medium or buffer, and mix thoroughly by pipetting. Centrifuge at low speed at room temperature for 10 minutes, discard the supernatant, and retain the cell pellet. Add an appropriate amount of cell culture medium or buffer, gently and thoroughly mix, and then centrifuge to wash the cells: centrifuge at low speed at 4°C for 5 minutes. Discard the supernatant, retain the cell pellet, centrifuge at low speed at 4°C for 5 minutes, discard the supernatant to obtain PBMC cells, and seed the PBMC cells into culture flasks at a certain density for further culture.

[0055] 2. The degree of DNMT3B nuclear export and ubiquitination level in PBMC cells were detected according to the methods in Examples 3 and 4. The results showed that the degree of DNMT3B nuclear export and ubiquitination level in cells of severely EV71-infected children were higher than those in non-severely EV71-infected children, recovered children, and healthy children. Figure 3 The results show the degree of DNMT3B nuclear exit and ubiquitination level in PBMC cells of some patients.

Claims

1. The application of a reagent for detecting the marker DNMT3B in the preparation of diagnostic products for severe EV71 infection or diagnostic products for the prognosis of severe EV71 infection, characterized in that, The application includes detecting indicators mediated by the binding of EV71's 3C and / or 3D proteins to DNMT3B, the indicators being selected from one or more of the K63 ubiquitination level of DNMT3B and the degree of nucleocytoplasmic translocation of DNMT3B.

2. The application according to claim 1, characterized in that, The reagents for detecting the K63 ubiquitination level of DNMT3B include one or more of the following: DNMT3B-specific recognition antibody, K63 ubiquitination-specific recognition antibody, and proteasome inhibitor.

3. The application according to claim 1, characterized in that, The reagents for detecting the degree of nucleocytoplasmic translocation of the DNMT3B include cytoplasmic lysis buffer and nuclear lysis buffer used for nucleocytoplasmic separation.

4. The application according to claim 3, characterized in that, The cytoplasmic lysis buffer contains lysis buffer A, and the nuclear lysis buffer contains lysis buffer C; The lysis buffer A consists of the following components per 1 mL: 10 μL 1M Tris-HCl, 5 μL 1M MgCl2, 10 μL 1M NaCl, 1 μL 1M DTT, 10 μL cocktail protease inhibitor, and 964 μL ddH2O. The lysis buffer C consists of the following components per 1 mL: 20 μL 1M HEPES-KOH (pH 7.9), 1.5 μL 1M MgCl2, 500 μL 1M NaCl, 1 μL 1M DTT, 0.4 μL 0.5M EDTA, 100 μL 10% NP-40, 10 μL cocktail protease inhibitor, and 367.1 μL ddH2O.

5. The application according to any one of claims 1-4, characterized in that, The applications include the detection of peripheral blood mononuclear cells in subjects.

6. The application according to claim 5, characterized in that, The diagnostic product for severe EV71 infection compares the level of ubiquitination at the K63 position of DNMT3B and / or the degree of nuclear exit in peripheral blood mononuclear cells of the subjects with corresponding indicators in non-severe EV71 infected subjects or healthy children.

7. The application according to claim 5, characterized in that, The diagnostic product for the prognosis of severe EV71 infection compares the level of K63 ubiquitination and / or degree of nuclear exit of DNMT3B in peripheral blood mononuclear cells of severely ill children after treatment with corresponding indicators before treatment or in recovered children.

8. The application according to any one of claims 1-4, characterized in that, The diagnostic product for severe EV71 infection is a reagent kit.

9. The application according to any one of claims 1-4, characterized in that, The diagnostic product for the prognosis of severe EV71 infection is a reagent kit.

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