Application of anti-Gelsolin3 antibody related to demyelinating giline-Bar syndrome
By screening and identifying anti-Gelsolin3 antibodies in GBS patients samples, the problem of diagnosing and treating demyelinated GBS in the prior art is solved, and the early accurate diagnosis and effective treatment of the disease is achieved.
Patent Information
- Application Number
- CN202411929815.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to effectively diagnose and treat demyelinated Guillain-Barre syndrome (GBS), and the existing biomarkers and pathogenic antibodies are not specific enough, resulting in delayed diagnosis and poor treatment effect.
By screening and identifying anti-Gelsolin3 antibodies in GBS patient samples, using technical methods such as single fiber immunofluorescence and co-immunoprecipitation, the presence and specificity of the antibody is verified, and detection methods are developed to assist the diagnosis of GBS.
The anti-Gelsolin3 antibody was discovered and verified as a pathogenic antibody for demyelinated GBS, providing a new biomarker that can help improve the early diagnostic accuracy and therapeutic effect of GBS.
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Figure CN119643879A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological antibody technology, and specifically to the application of anti-Gelsolin3 antibodies related to demyelinating Guillain-Barré syndrome. Background Art
[0002] Guillain-Barré syndrome (GBS) is an autoimmune-mediated peripheral neuropathy. Its common clinical manifestations are limb weakness with or without sensory impairment. In severe cases, it can lead to respiratory muscle weakness and endanger life. The clinical diagnosis of this disease mainly relies on typical clinical manifestations, cerebrospinal fluid analysis and electrophysiological examinations. However, some patients do not show obvious symptoms in the early stage, which leads to delayed diagnosis and affects clinical recovery. The current clinical treatments for this disease include immunoglobulin and plasma exchange. Clinical data show that only 65% of patients are effective in the above treatments, about 20% of patients still have varying degrees of disability after active immunotherapy, and about 5% of patients die from serious complications such as lung infection. It is of great significance to find early biomarkers to assist in the diagnosis of GBS.
[0003] According to its pathological characteristics, GBS can be divided into two types: demyelinating and axonal. Current research at home and abroad has fully proved that the basic mechanism of GBS is that autoimmune antibodies attack the myelin sheath or axon of peripheral nerves and then induce complement deposition and the formation of membrane attack complexes. Anti-ganglioside antibodies are the most common pathogenic antibodies in GBS. Since the 1990s, a series of anti-ganglioside antibodies such as anti-GM1, anti-GQ1b, anti-GD1a, and anti-GalNAc-GD1a have been found to be closely related to the onset of axonal GBS. Among them, anti-GM1, anti-GD1a, and anti-GalNAc-GD1a antibodies are related to the onset of axonal GBS, while anti-GQ1b antibodies are related to the onset of Miller-Fisher syndrome (a variant of GBS). The pathogenicity of these antibodies has been confirmed in relevant animal models. However, the pathogenic antibodies for demyelinating GBS are still unclear.
[0004] The search for new biomarkers and pathogenic antibodies in GBS, especially pathogenic antibodies against demyelinating GBS, has always been a hot topic of research at home and abroad, and is also an urgent problem to be solved in the clinical diagnosis and treatment of GBS. In the past 20 years, Professor Jerome Devaux of France has successively detected antibodies such as anti-NF155, gliomedin, and NF186 in GBS. However, these antibodies are not specific and are also present in patients with other immune peripheral neuropathies such as chronic inflammatory demyelinating polyneuropathy. Currently, commonly used antibody detection methods include single fiber immunofluorescence, cell immunofluorescence, immunoprecipitation, Western blot, enzyme-linked immunosorbent assay, etc. Summary of the invention
[0005] The purpose of the present invention is to overcome the above technical defects, provide the application of anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome, and strive to explore new markers and pathogenic antibodies based on the GBS patient sample library. When single-fiber immunofluorescence is used to screen pathogenic antibodies, there are significant antibodies binding to the paraganglionic region of the mouse sciatic nerve in the patient's serum, and the morphology of its fluorescence distribution is significantly different from the previously discovered anti-NF155, gliomedin, NF186, CNTN1, Caspr1 and other antibodies. By using immunoprecipitation combined with mass spectrometry, 41 differential antibodies binding to sciatic nerve proteins were found in this patient, control patients and normal controls. Further cellular immunoassay confirmed that the significantly bound antibody is anti-Ge Gelsolin3 antibody. Anti-Gelsolin3 antibodies were screened in the serum of GBS patients by immunoprecipitation and Western blot, and anti-Gelsolin3 antibodies were found in patients. The serum of patients carrying the antibody was locally injected into the sciatic nerve of mice, and significant immunofluorescence binding in the paranodal ring area was observed. The antibody was not found in normal human serum, patients with neuromyelitis optica, and patients with chronic inflammatory demyelinating polyneuropathy. Since immunofluorescence localization proved that Gelsolin3 was mainly located in the paranodal ring area of the myelin sheath, and the electrophysiological manifestations of patients carrying the antibody were mainly demyelinating, it was confirmed that the anti-Gelsolin3 antibody was a pathogenic antibody of demyelinating GBS, and the antibody can be tested in GBS patients.
[0006] The technical scheme provided by the present invention is: application of anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome, and the experimental reagents include: 4% paraformaldehyde, trypsin, acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol, iodoacetamide, ultrapure water, anti-fluorescence attenuation agent sealing, poly-lysine solution, immunoglobulin, cholera toxin B, TBS, Tween 20, goat serum, anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, MH1031 or anti-human IgG4 antibody, ECL luminescent solution, BCA protein quantitative kit, normal human serum, GBS patient and control serum, 1M Tris-HCl buffer, RIPA lysate and 0.1M glycine solution.
[0007] Furthermore, the application of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome mainly includes the following steps: collecting serum from GBS patients and control individuals, performing immunoprecipitation with sciatic nerve proteins to obtain immune complexes, separating and identifying the immune complexes using a mass spectrometer, obtaining candidate protein sequences, inserting the candidate protein encoding genes into vectors, performing immunofluorescence verification after transfection of cells, determining the localization and expression of the candidate protein, constructing candidate protein expression plasmids, expressing and purifying recombinant proteins in Escherichia coli, performing immunoprecipitation verification on the purified recombinant proteins using GBS patient serum, determining the presence of anti-Gelsolin3 IgG antibodies, further verifying the expression of anti-Gelsolin3 IgG antibodies in GBS patient serum using the Western blot method, and verifying the binding of anti-Gelsolin3 IgG antibody-positive patient serum to the sciatic nerve using a single fiber immunofluorescence method.
[0008] Preferably, the specific steps of obtaining the immune complex are:
[0009] S1. After the mice were anesthetized, the sciatic nerves were removed and RIPA lysis buffer (containing PMSF+cocktail protease inhibitor+phosphatase inhibitor) was added. The nerves were cryogenically ground and ultrasonically broken. The nerves were lysed on ice for 20 min and centrifuged at 14000 g for 30 min. The supernatant was transferred to a new 1.5 ml centrifuge tube and the protein was quantified by the BCA method.
[0010] S2. Take an appropriate amount of Protein A / G Beads and add them to a 1.5 ml centrifuge tube. Centrifuge at 1000 rpm for 1 min, discard the supernatant, and wash twice with PBS.
[0011] S3, add 10-20 μL serum (normal human serum, GBS patient serum, other control serum) to Protein A / G Beads, incubate on a shaker at 4°C for 2 h, and wash with PBS three times;
[0012] S4, add sciatic nerve protein to the above mixture, incubate overnight on a shaker at 4°C, and wash three times with PBS;
[0013] S5. Add 2X SDS loading buffer to the above reaction mixture, centrifuge at 1000 rpm for 1 min, take the supernatant and load it for SDS-PAGE, and stain with Coomassie brilliant blue;
[0014] S6, gel cutting and enzymatic hydrolysis: Cut the gel strip into pieces, decolorize the gel block with 50% acetonitrile containing 50mM ammonium bicarbonate, dehydrate the gel block with 100% acetonitrile for 5 minutes, remove the liquid phase in the system, add dithiothreitol solution with a final concentration of 10mM, incubate at 37°C for 60 minutes, incubate with 100% acetonitrile for dehydration again, remove the liquid phase, add iodoacetamide with a final concentration of 55mM, and incubate at room temperature to avoid Incubate under light for 45 minutes, then wash with ammonium bicarbonate at a final concentration of 50mM, incubate with 100% acetonitrile for dehydration, and finally resuspend the gel with 50mM ammonium bicarbonate containing 10ng / μl trypsin, incubate on ice for 1 hour, remove excess solution in the sample, and hydrolyze the gel at 37℃ overnight. The peptides after enzymatic hydrolysis are extracted from the gel with 50% acetonitrile / 5% formic acid and 100% acetonitrile in turn. The peptide solution is freeze-dried and set aside.
[0015] Furthermore, the specific steps of obtaining the candidate protein sequence are:
[0016] K1, the peptide was dissolved in liquid chromatography mobile phase A and separated using the EASY-nLC1000 ultra-high performance liquid phase system, mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile, the liquid phase gradient was set as follows: 0-16min, 9%-25% B phase, 16-22min, 25%-40% B phase, 22-26min, 40%-80% B phase; 26-30min, 80% B phase, the flow rate was maintained at 450n / min;
[0017] K2, peptides were separated by ultra-high performance liquid phase system and injected into NSI ion source for ionization and then analyzed by ThermoScientificTMQ Exactive mass spectrometer. The ion source voltage was set to 2.2kV. The peptide parent ions and their secondary fragments were detected and analyzed by high-resolution Orbitrap. The primary mass spectrometer scanning range was set to 350-1800m / z, and the scanning resolution was set to 70,000; the secondary scanning resolution was set to 17,500, and the data acquisition mode used the data-dependent scanning (DDA) program, that is, after the primary scan, the top 20 peptide parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometer analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 5e4, the signal value was set to 1e4ions / s, the maximum injection time was set to 100ms, and the dynamic exclusion time of the tandem mass spectrometer scanning was set to 15.0s to reduce the repeated scanning of the parent ion.
[0018] K3. Data search: The secondary mass spectrometry data were searched using Proteome Discoverer 1.3, and the search parameters were set as follows: the database was set to a combined search of Homo Sapiens (SwissProt, 20,366 sequences) and Musculus (SwissProt, 17,045 sequences), the enzyme cleavage method was set to Trypsin / P, the missed cleavage site was set to 2, the primary parent ion mass error tolerance was set to 10 ppm, the secondary fragment ion mass error tolerance was set to 0.02 Da, the fixed modification was set to cysteine alkylation, the variable modification was set to methionine oxidation, protein N-terminal acetylation, the peptide ion score was required to be higher than 20, and the identification result peptide confidence was set to High.
[0019] Preferably, the specific steps of the immunofluorescence verification are: Gelsolin3 is selected as a candidate protein according to the localization of the protein, the full-length cDNA encoding Gelsolin3 is inserted into a pcDNA3.1(+) vector containing an enhanced green fluorescent protein (EGFP) tag at the C-terminus, human embryonic kidney 293 cells are seeded in a 24-well plate together with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and incubated for 24 hours, and the cells are transfected with a plasmid expressing gelsolin3-EGFP using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen), and after 48 hours, the transfected cells are fixed with 4% paraformaldehyde for 10 minutes and stained with 0.5% Triton at room temperature. X-100 and 5% normal goat serum for 20 min of permeabilization / blocking. After washing three times with PBS, the cells were incubated with diluted serum (1:100) and anti-gelsolin antibody (1:250) at 4°C overnight. The cells were subsequently washed with PBS and incubated with Alexa Fluor 647-conjugated goat anti-human IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG for 1 h at room temperature. After washing, the cells were mounted with antifade mounting medium containing DAPI, and images were captured using an Axio Imager Z2 microscope (Zeiss, Jena, Germany).
[0020] Preferably, the specific steps of purifying the recombinant protein are: first constructing a Gelsolin3 expression plasmid, extracting total RNA from the sciatic nerve of C56BL / 6J mice using TRIzol reagent (Invitrogen, CA, USA), and using PrimeScript TMII First-strand cDNA synthesis kit (Takara, Beijing, China) was used to synthesize complementary DNA (cDNA). The full-length Gelsolin3 gene was amplified from the cDNA by PCR and subsequently cloned into the pET-28a(+) vector (Novagen), which provided a 6×His tag for the recombinant protein at the C-terminus. The recombinant vector was then transformed into the host Escherichia coli BL21(DE3) for protein expression. The culture conditions were optimized and the cells were fermented at 37°C to an OD600 of 0.8 and then induced with 0.5 mM isopropyl-β-D-thiogalactoside at 16°C for 20 h. The cells were harvested and suspended in 20 mM PBS at pH 7.5. The cells were lysed by sonication at 200 W for 30 min (1 s on, 2 s off) in a buffer containing Tris and 500 mM NaCl. The lysates were centrifuged at 14,000 g for 20 min at 4 °C. The supernatant was purified using a nickel chelate column (TransGen, Beijing). The proteins were eluted with lysis buffer containing 500 mM imidazole. The protein samples were extensively dialyzed against a buffer containing 20 mM Tris, 100 mM NaCl, and 2 mM EDTA at pH 7.5. The proteins were identified by SDS-PAGE and Western blot.
[0021] Furthermore, the specific steps of the immunoprecipitation validation are as follows: the purified gelsolin 3 (GSN3) protein is subjected to protein immunoprecipitation (IP) analysis using patient serum, and the negative control group is normal serum without the target antibody, which is used to evaluate nonspecific binding. First, the purified protein and serum are incubated at 4 degrees for 4 hours, and the Protein A / G magnetic beads (Smart-Lifesciences Biotechnology Co., Ltd, SA032005) are washed three times with PBS-T. Then, the antigen-antibody complex is added to the Protein A / G magnetic beads, incubated overnight at 4 degrees, and after washing the magnetic beads 5 times with PBS-T, the immunoprecipitated protein is eluted with 2×SDS loading buffer, separated with 7.5% SDS-page, transferred to a PVDF membrane, and washed with 5% The membrane was blocked with BSA for 2 hours, then incubated with primary antibody GSN (11644-2-AP, Proteintech, China) at 4 degrees overnight, washed, incubated with enzyme-labeled goat anti-rabbit IgG (H+L) (AS014, Abclonal) at room temperature for 1 hour, and developed with ECL luminescent solution (BKLS0100, Millipore).
[0022] Preferably, the specific steps of further verifying the expression of anti-Gelsolin3 IgG antibodies in GBS patient serum using the Western blot method are as follows: taking 40 μg of purified GSN3 protein, diluting it with 5x loading buffer and denaturing it at 95°C for 5 minutes, loading it into a 7.5% polyacrylamide gel for electrophoresis at a constant voltage of 80V, and then transferring the protein to a PVDF membrane at a constant current of 200 mA for 100 minutes, blocking the membrane with 5% skim milk powder in TBS-T for 2 hours, diluting the patient serum (1:50) with protein-free blocking buffer and incubating it at 4°C overnight, washing it, and incubating the membrane with anti-human IgG antibody ( BA1070, Boster, Wuhan, China), anti-human IgG1 antibody (A10648, Invitrogen, USA), anti-human IgG2 antibody (MH1722, Invitrogen, USA), anti-human IgG3 antibody (MA5-42730, Invitrogen, USA), MH1031 or anti-human IgG4 antibody (A-10654, Invitrogen, USA) were incubated at room temperature for 1 h, and finally, ECL luminescent solution (Millipore, BKLS0100) was used for color development. As shown in the figure below, the expression of anti-GSN3 IgG antibody was detected in GBS patients.
[0023] Furthermore, the specific steps of using the single fiber immunofluorescence method to verify the binding of anti-Gelsolin3IgG antibody positive patient serum to the sciatic nerve are as follows: the sciatic nerve was dissected from an adult C57BL / 6J mouse and fixed with 4% paraformaldehyde at room temperature for 40 minutes. Then, the sciatic nerve fibers on the slide were combed with a fine needle, and the slide was air-dried overnight at room temperature and kept at -20°C until use. For this test, the combed fibers were blocked with PBS containing 10% normal goat serum and 0.3% TritonX-100 at room temperature for 2 hours, and then incubated with diluted patient serum (1:200) and rabbit anti-Caspr1 (1:300) in PBS at 4°C overnight. The slide was washed and incubated with DyLight 650-conjugated goat anti-human IgG (1:500, Abcam, USA) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000, Thermo Fisher Scientific, USA) at room temperature. Fisher) for 2 hours, the slides were mounted with anti-fluorescence attenuation mounting medium (Solarbio), and images were taken using a laser confocal microscope. Significant fluorescence binding was observed in the paranodal ring region of the myelin sheath of sciatic nerve fibers.
[0024] Preferably, the sciatic nerve protein is derived from mice;
[0025] Furthermore, the immunofluorescence validation was performed using human embryonic kidney 293 cells for transfection and validation;
[0026] Preferably, the Western blot method is validated using anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody and anti-human IgG4 antibody;
[0027] Preferably, the anti-Gelsolin3 IgG antibody is specifically expressed in patients with Guillain-Barré syndrome (GBS).
[0028] The advantages of the present invention compared with the prior art are:
[0029] Based on immunoprecipitation, mass spectrometry, cell transfection and immunofluorescence, a new anti-Gelsolin3 IgG antibody was identified from a GBS patient. Then, using a single fiber immunofluorescence and Western blot double verification method, 17 out of 415 GBS patients were detected to be positive for anti-Gelsolin3 IgG antibodies, and the electrophysiological typing of the patients with positive antibodies was mainly demyelinating. However, the antibody was not found in 415 normal controls and 113 patients with chronic inflammatory demyelinating polyneuropathy. Therefore, the present invention discloses that the anti-Gelsolin3 IgG antibody is a newly discovered antibody expressed in demyelinating GBS patients, and provides a detection method for the antibody. In particular, in the single fiber immunofluorescence method, significant fluorescence binding can be observed in the paranodal ring area of the myelin sheath of sciatic nerve fibers, providing intuitive evidence for the diagnosis of GBS. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 It is a schematic diagram showing a single fiber immunofluorescence assay for the application of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome of the present invention;
[0031] Figure 2 Schematic diagram of the application of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome of the present invention for co-immunoprecipitation to identify differential protein binding between GBS173, GBS225 (control) and normal control serum;
[0032] Figure 3 It is a schematic diagram of 41 differentially expressed proteins identified between GBS173 and the control serum group using the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome of the present invention;
[0033] Figure 4It is a schematic diagram of the application of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome of the present invention, wherein HEK293 cells transfected with a pcDNA3.1(+) vector containing a full-length GSN3 cDNA and an enhanced green fluorescent protein (EGFP) tag are incubated with GBS173 serum;
[0034] Figure 5 Schematic diagram of the application of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome of the present invention, wherein the frozen sections of sciatic nerve of C57BL / 6 mice are incubated with GBS173 serum and commercial rabbit IgG anti-gelsolin antibody;
[0035] Figure 6 A schematic diagram of the application of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome of the present invention in screening 415 GBS patients using single fiber immunofluorescence;
[0036] Figure 7 In the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome of the present invention, we further verified the expression of the antibody and the schematic diagram of IgG subtype by Western blot for positive patients.
[0037] in, Figure 1 GBS173 patient IgG has a novel and typical binding in the paraganglionic loop of Ranvier of the sciatic nerve;
[0038] Figure 2 Total proteins extracted from sciatic nerves of C57BL / 6 mice were incubated with different sera and then subjected to 10% SDS-PAGE electrophoresis followed by staining with Coomassie Brilliant Blue. The gel located in the white box was collected for detection of target antigens by mass spectrometry;
[0039] Figure 3 Gelsolin 3 (GSN3), which is localized in the paraganglionic region of Ranvier peripheral nerves, was selected for further validation using a cell-based assay;
[0040] Figure 4 Immunostaining showed that IgG in GBS173 serum clearly bound to HEK293 cells expressing GSN3;
[0041] Figure 5 Immunostaining shows consistent binding sites in the delusional region for GBS173 IgG (green) and commercial IgG anti-gelsolin antibody (red). Immunostaining for Caspr1 (purple) was used to localize IgG binding;
[0042] Figure 6 Shown in the figure are antibody-positive patients;
[0043] Figure 7 Shown in the figure is a patient who was positive for total IgG and IgG1 subtypes. DETAILED DESCRIPTION
[0044] The essential contents of the present invention are specifically introduced below in conjunction with the embodiments. Due to space limitations, the description of the experimental process cannot be very detailed, and all parts not described in detail in the experiments are routine operations well known to those skilled in the art.
[0045] Example: Figure 1-Figure 7 As shown, in this embodiment, the experimental reagents include: 4% paraformaldehyde (Beijing Solebow Technology Co., Ltd., P1110), trypsin (Promega), acetonitrile (Fisher Chemical), formic acid (Fluka), ammonium bicarbonate (Sigma), dithiothreitol (Sigma), iodoacetamide (Sigma), ultrapure water, anti-fluorescence attenuation agent sealing (Abcam, USA, ab104139), poly-lysine solution (Sigma, USA, P8920); immunoglobulin (IVIg, China Hualan Biotechnology Co., Ltd.); cholera toxin B (Sigma, USA, C1655); TBS (), Tween 20 (), goat serum (), anti-human IgG antibody (BA1070, Boster, Wuhan, China), anti-human IgG1 antibody (A10648, Invitrogen, USA), anti-human IgG2 antibody (MH1722, Invitrogen, USA), anti-human IgG3 antibody (MA5-42730, Invitrogen, USA), MH1031 or anti-human IgG4 antibody (A-10654, Invitrogen, USA), ECL luminescent solution (Millipore, BKLS0100, USA), BCA protein quantification kit (Jiangsu Biyuntian), normal human serum, GBS patient and control serum, 1M Tris-HCl buffer (PH = 9.0): Beijing Solebow Technology Co., Ltd. (#T1160); RIPA lysis buffer: Beijing Solebow Technology Co., Ltd. (#R0020); 0.1M glycine solution (pH 2.7): Zeye Biotechnology (#ZY61990G).
[0046] Experimental animals: C57BL / 6 mice aged 6-8 weeks.
[0047] 1. Immunoprecipitation of patient serum and sciatic nerve protein:
[0048] S1. After the mice were anesthetized, the sciatic nerves were removed and RIPA lysis buffer (containing PMSF+cocktail protease inhibitor+phosphatase inhibitor) was added. The nerves were cryogenically ground and ultrasonically broken, and lysis was continued on ice for 20 min. The nerves were centrifuged at 14000 g for 30 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube for protein quantification using the BCA method.
[0049] S2. Take an appropriate amount of ProteinA / GBeads and add it to a 1.5 ml centrifuge tube. Centrifuge at 1000 rpm for 1 min, discard the supernatant, and wash twice with PBS.
[0050] S3, add 10-20 μL serum (normal human serum, GBS patient serum, other control serum) to ProteinA / GBeads, incubate on a shaker at 4°C for 2 h, and wash with PBS three times;
[0051] S4, add sciatic nerve protein to the above mixture, incubate overnight on a shaker at 4°C, and wash three times with PBS;
[0052] S5, add 2X SDS loading buffer to the above reaction, centrifuge at 1000 rpm for 1 min, take the supernatant and load it for SDS-PAGE, and stain with Coomassie brilliant blue;
[0053] S6, gel cutting and enzymatic hydrolysis: Cut the gel strip into pieces, decolorize the gel block with 50% acetonitrile containing 50mM ammonium bicarbonate, dehydrate the gel block with 100% acetonitrile for 5 minutes, remove the liquid phase in the system, add dithiothreitol solution with a final concentration of 10mM, incubate at 37°C for 60 minutes, incubate with 100% acetonitrile for dehydration again, remove the liquid phase, add iodoacetamide with a final concentration of 55mM, and incubate at room temperature to avoid Incubate under light for 45 minutes; then wash with ammonium bicarbonate at a final concentration of 50mM, incubate with 100% acetonitrile for dehydration, and finally resuspend the gel with 50mM ammonium bicarbonate containing 10ng / μl trypsin, incubate on ice for 1 hour, remove excess solution in the sample, and hydrolyze the gel at 37°C overnight. The peptides after enzymatic hydrolysis are extracted from the gel using 50% acetonitrile / 5% formic acid and 100% acetonitrile in turn. The peptide solution is freeze-dried and set aside.
[0054] 2. Mass spectrometry identification:
[0055] ThermoScientific TMO Exactive mass spectrometer was used for analysis. The specific process is as follows:
[0056] K1, the peptide was dissolved in liquid chromatography mobile phase A and separated using the EASY-nLC1000 ultra-high performance liquid phase system, mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile, the liquid phase gradient was set as follows: 0-16min, 9% to 25% B phase; 16-22min, 25% to 40% B phase; 22-26min, 40% to 80% B phase; 26-30min, 80% B phase, the flow rate was maintained at 450n / min;
[0057] The peptide fragments were separated by ultra-high performance liquid phase system and injected into the NSI ion source for ionization.
[0058] ThermoScientificTMQExactive mass spectrometer was used for analysis, the ion source voltage was set to 2.2kV, the peptide parent ions and their secondary fragments were detected and analyzed using high-resolution Orbitrap, the primary mass spectrometer scanning range was set to 350-1800m / z, the scanning resolution was set to 70,000; the secondary scanning resolution was set to 17,500, and the data acquisition mode used the data-dependent scanning (DDA) program, that is, after the primary scan, the top 20 peptide parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometer analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 5e4, the signal value was set to 1e4ions / s, the maximum injection time was set to 100ms, and the dynamic exclusion time of the tandem mass spectrometer scanning was set to 15.0s to reduce repeated scanning of the parent ions;
[0059] K3, data search; secondary mass spectrometry data were searched using ProteomeDiscoverer1.3, and the search parameters were set as follows: the database was set to a combined search of HomoSapiens (SwissProt, 20366 sequences) and Musculus (SwissProt, 17045 sequences); the enzyme cleavage method was set to Trypsin / P; the missed cleavage site was set to 2; the primary parent ion mass error tolerance was set to 10ppm; the secondary fragment ion mass error tolerance was set to 0.02Da; the fixed modification was set to cysteine alkylation; the variable modification was set to methionine oxidation and protein N-terminal acetylation; the peptide ion score was required to be higher than 20, and the identification result peptideconfidence was set to High.
[0060] 3. Verify candidate proteins by cell immunofluorescence;
[0061] Gelsolin3 was selected as a candidate protein based on the localization of the protein, and the full-length cDNA encoding gelsolin3 was inserted into the pcDNA3.1(+) vector containing an enhanced green fluorescent protein (EGFP) tag at the C-terminus. Human embryonic kidney 293 cells were seeded in 24-well plates with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution and incubated for 24 h. The cells were transfected with the plasmid expressing gelsolin3-EGFP using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). After 48 h, the transfected cells were fixed with 4% paraformaldehyde for 10 min and permeabilized / blocked with 0.5% TritonX-100 and 5% normal goat serum for 20 min at room temperature. After washing three times with PBS, the cells were incubated with diluted serum (1:100) and anti-gelsolin antibody (1:250) at 4°C overnight. The cells were subsequently washed with PBS and incubated with AlexaFluor647-conjugated goat anti-human IgG and AlexaFluor568-conjugated goat anti-rabbit IgG for 1 h at room temperature. After washing, the cells were mounted with anti-fading mounting medium containing DAPI, and images were captured using an AxioImagerZ2 microscope (Zeiss, Jena, Germany).
[0062] 4. Expression and purification of recombinant Gelsolin3:
[0063] First, Gelsolin3 expression plasmid was constructed, and total RNA was extracted from the sciatic nerve of C56BL / 6J mice using TRIzol reagent (Invitrogen, CA, USA). TMII First-strand cDNA synthesis kit (Takara, Beijing, China) was used to synthesize complementary DNA (cDNA). The full-length Gelsolin3 gene was amplified from the cDNA by PCR and subsequently cloned into the pET-28a(+) vector (Novagen), which provided a 6×His tag for the recombinant protein at the C-terminus. The recombinant vector was then transformed into the host Escherichia coli BL21(DE3) for protein expression. The culture conditions were optimized and fermented at 37°C to an OD600 of 0.8. The cells were then induced with 0.5 mM isopropyl-β-D-thiogalactoside at 16°C for 20 h. The cells were harvested and cultured. It was suspended in a buffer containing 20 mM Tris and 500 mM NaCl at pH 7.5 and lysed by 200 W ultrasonic treatment for 30 min (1 s on, 2 s off). The lysate was centrifuged at 14,000 g for 20 min at 4°C. The supernatant was purified using a nickel chelate column (TransGen, Beijing). The protein was eluted with a lysis buffer containing 500 mM imidazole. The protein sample was extensively dialyzed with a buffer containing 20 mM Tris, 100 mM NaCl and 2 mM EDTA at pH 7.5. The protein was identified by SDS-PAGE and Western blot.
[0064] 5. Immunoprecipitation to verify the expression of anti-Gelsolin3 IgG antibodies in the serum of GBS patients:
[0065] The purified gelsolin3 (GSN3) protein was subjected to protein immunoprecipitation (IP) analysis using patient serum. The negative control group was normal serum without the target antibody, which was used to evaluate nonspecific binding. First, the purified protein and serum were incubated at 4 degrees for 4 hours; the ProteinA / G magnetic beads (Smart-Lifesciences Biotechnology Co., Ltd, SA032005) were washed three times with PBS-T, and then the antigen-antibody complex was added to the ProteinA / G magnetic beads and incubated overnight at 4 degrees. After washing the magnetic beads 5 times with PBS-T, the immunoprecipitated protein was eluted with 2×SDS loading buffer, separated with 7.5% SDS-page, transferred to a PVDF membrane, and the membrane was blocked with 5% BSA for 2 hours. Then, the primary antibody GSN (11644-2-AP, Proteintech, China) was incubated overnight at 4 degrees. After washing, the protein was stained with enzyme-labeled goat anti-rabbit IgG.
[0066] IgG (H+L) (AS014, Abclonal) was incubated at room temperature for 1 hour and then developed with ECL luminescent solution (BKLS0100, Millipore).
[0067] VI. WB verification of the expression of anti-Gelsolin3 IgG antibodies in the serum of GBS patients:
[0068] 40 μg of purified GSN3 protein was diluted with 5x loading buffer and denatured at 95°C for 5 minutes, loaded into 7.5% polyacrylamide gel for electrophoresis at 80V constant voltage, and then transferred to PVDF membrane at 200mA constant current for 100 minutes; the membrane was blocked in TBS-T with 5% skim milk powder for 2 hours; the patient serum was diluted with protein-free blocking buffer (1:50) and incubated at 4°C overnight. After washing, the membrane was incubated with anti-human IgG antibody (BA1070, Boster, Wuhan, China) and anti-human IgG1 antibody (A10648, The cells were incubated with anti-human IgG2 antibody (MH1722, Invitrogen, USA), anti-human IgG3 antibody (MA5-42730, Invitrogen, USA), MH1031 or anti-human IgG4 antibody (A-10654, Invitrogen, USA) at room temperature for 1 hour, and finally, ECL luminescent solution (Millipore, BKLS0100) was used for color development. As shown in the figure below, the expression of anti-GSN3 IgG antibody can be detected in GBS patients.
[0069] VII. Single fiber immunofluorescence verification of the binding of anti-Gelsolin3 IgG antibody positive patient serum to the sciatic nerve:
[0070] Sciatic nerves were dissected from adult C57BL / 6J mice and fixed with 4% paraformaldehyde for 40 min at room temperature. Then, the sciatic nerve fibers were teased on slides with a fine needle. The slides were air-dried overnight at room temperature and kept at -20°C until use. For this experiment, the teased fibers were blocked with PBS containing 10% normal goat serum and 0.3% TritonX-100 for 2 h at room temperature and then incubated with diluted patient serum (1:200) and rabbit anti-Caspr1 (1:300) in PBS at 4°C overnight. The slides were washed and incubated with DyLight650-conjugated goat anti-human IgG (1:500, Abcam, USA) and AlexaFluor488-conjugated goat anti-rabbit IgG (1:1000, ThermoFisher, USA) for 2 h at room temperature. The slides were mounted with anti-fluorescence attenuation mounting medium (Solarbio) and images were taken using a laser confocal microscope. There was significant fluorescence binding in the paranodal ring region of the myelin sheath of sciatic nerve fibers.
[0071] The present invention and its embodiments are described above, which is not restrictive. The drawings are only one of the embodiments of the present invention. In short, if ordinary technicians in the field are inspired by it and design embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.
Claims
1. The use of anti-Gelsolin3 antibodies related to demyelinating Guillain-Barré syndrome, characterized in that: The method mainly includes the following steps: collecting serum from GBS patients and control individuals, co-immunoprecipitating with sciatic nerve proteins to obtain immune complexes, separating and identifying the immune complexes using a mass spectrometer, obtaining candidate protein sequences, inserting the candidate protein encoding gene into a vector, transfecting cells and performing immunofluorescence verification, determining the localization and expression of the candidate protein, constructing a candidate protein expression plasmid, expressing and purifying the recombinant protein in Escherichia coli, using GBS patient serum to perform immunoprecipitation verification on the purified recombinant protein, determining the presence of anti-Gelsolin3 IgG antibodies, further verifying the expression of anti-Gelsolin3 IgG antibodies in GBS patient serum using the Western blot method, and verifying the binding of anti-Gelsolin3 IgG antibody-positive patient serum to the sciatic nerve using a single fiber immunofluorescence method.
2. The use of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome according to claim 1, characterized in that: The sciatic nerve protein is derived from mouse.
3. The use of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome according to claim 1, characterized in that: The mass spectrometer is a Thermo Scientific TMO Exactive mass spectrometer.
4. The use of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome according to claim 1, characterized in that: The immunofluorescence validation was performed using human embryonic kidney 293 cells for transfection and validation.
5. The use of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome according to claim 1, characterized in that: The Western blot method was validated using anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody and anti-human IgG4 antibody.
6. The use of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome according to claim 1, characterized in that: The anti-Gelsolin3 IgG antibody is specifically expressed in patients with Guillain-Barré syndrome (GBS).
7. The use of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome according to claim 1, characterized in that: The single fiber immunofluorescence method was imaged and verified using a laser confocal microscope.
8. An experimental reagent, characterized in that: The experimental reagent is used for the application of anti-Gelsolin3 antibodies related to demyelinating Guillain-Barré syndrome. The experimental reagent includes 4% paraformaldehyde, trypsin, acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol, iodoacetamide, ultrapure water, anti-fluorescence attenuation agent sealing, poly-lysine solution, immunoglobulin, cholera toxin B, TBS, Tween 20, goat serum, anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, MH1031 or anti-human IgG4 antibody, ECL luminescent solution, BCA protein quantification kit, normal human serum, GBS patient and control serum, 1M Tris-HCl buffer, RIPA lysis buffer and 0.1M glycine solution.