Monoclonal antibodies, hybridoma cells targeting Gephyrin protein and their applications

Abstract

The present invention belongs to the field of monoclonal antibody pharmaceutical technology, specifically relating to a monoclonal antibody targeting the Gephyrin protein, hybridoma cells, and applications thereof. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2. This monoclonal antibody has a high affinity for the Gephyrin protein. At an antibody concentration of 0.1 g / L and after 121.6-fold dilution, the EC50 value is 5.31 nM.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody drugs, and particularly relates to a monoclonal antibody targeting Gephyrin protein, a hybridoma cell and applications thereof. Background Art

[0002] Existing studies have shown that gephyrin, an anchoring protein beneath the postsynaptic membrane, functions to receive inhibitory signals by clustering inhibitory glycine and γ-aminobutyric acid receptors and localizing them to the postsynaptic cytoskeleton. Abnormal gephyrin expression may contribute to cognitive impairment. GABAA receptors containing the α5 subunit (α5-GABAARs) are highly expressed in the hippocampus, with some located on the medial side of the postsynaptic cleft. They bind to gephyrin and mediate transient inhibition (i.e., miniature inhibitory postsynaptic currents, mIPSCs), which is essential for the body's sensitivity to inhaled anesthesia. Alterations in the expression of α5-GABAARs on the membrane lead to decreased learning and memory abilities in animals.

[0003] Therefore, gephyrin plays a crucial role in studying the involvement of α5-GABAR in anesthesia-induced cognitive dysfunction. Summary of the Invention

[0004] In response to the above problems, the purpose of the present invention is to provide a monoclonal antibody that can target Gephyrin protein, which has strong specificity and high affinity for Gephyrin protein, and is conducive to in-depth research on the regulatory mechanism of Gephyrin in α5-GABAAAR-mediated cognitive dysfunction and related treatment strategies.

[0005] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0006] In one aspect, the present invention provides a monoclonal antibody targeting Gephyrin protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0007] Another aspect of the present invention provides a hybridoma cell, whose deposit number is CGMCC No.46029.

[0008] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the monoclonal antibody targeting Gephyrin protein of the present invention.

[0009] In another aspect, the present invention provides a biomaterial comprising the nucleic acid molecule of the present invention. The biomaterial comprises an expression vector, an engineered bacterium or an engineered cell.

[0010] In another aspect, the present invention provides a detection reagent for detecting Gephyrin protein, comprising the monoclonal antibody targeting Gephyrin protein of the present invention.

[0011] Preferably, the above detection reagent comprises an indirect ELISA detection reagent.

[0012] In another aspect, the present invention provides a detection kit for detecting Gephyrin protein, which comprises the monoclonal antibody targeting Gephyrin protein of the present invention or the detection reagent for detecting Gephyrin protein of the present invention.

[0013] In another aspect, the present invention provides a drug for treating cognitive dysfunction caused by abnormal Gephyrin expression, comprising the monoclonal antibody targeting Gephyrin protein of the present invention.

[0014] In another aspect, the present invention provides a use of the monoclonal antibody targeting Gephyrin protein, the hybridoma cell, the isolated nucleic acid molecule, or the biomaterial of the present invention in the preparation of a detection reagent for detecting Gephyrin protein.

[0015] In another aspect, the present invention provides a use of the monoclonal antibody targeting Gephyrin protein of the present invention, the hybridoma cell of the present invention, the isolated nucleic acid molecule of the present invention, or the biomaterial of the present invention in the preparation of a drug for treating cognitive dysfunction caused by abnormal Gephyrin expression.

[0016] The hybridoma cells of the present invention were deposited on August 5, 2024 at the General Microbiology Center of the China Culture Collection Administration of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 46029.

[0017] The beneficial effects of the present invention include at least:

[0018] (1) The monoclonal antibody targeting Gephyrin protein provided by the present invention has a high affinity to Gephyrin protein. The antibody concentration is 0.1 g / l, and after 121.6-fold dilution, the EC50 value is 5.31 nM.

[0019] (2) The monoclonal antibody targeting Gephyrin protein provided by the present invention has a small molecular weight and can be obtained in large quantities. It has high specificity and low immunogenicity, and can easily enter tissues and penetrate the blood-brain barrier, thereby improving the accuracy of targeting specific molecules and avoiding adverse side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a diagram of the Gephyrin expression vector, and the red sequence is the recombinant Gephyrin sequence;

[0021] Figure 2 This marker is a DNA marker consisting of 13 double-stranded DNA fragments ranging from 250 bp to 12000 pb, of which 1500 bp and 4000 bp are indicator bands, showing bright bands.

[0022] Figure 3 Restriction Digestion Map, where lane 1: Plasmid; lane 2: Plasmid Digested with XbaI-XhoI; M: DNA Marker;

[0023] Figure 4 The results of Gephyrin protein purification are shown in Figure 2. M: Marker; Lane 1: Flow-through; Lanes 2-7: Purified samples.

[0024] Figure 5 Schematic diagram of the immunization process;

[0025] Figure 6 This is the EC50 fitting analysis of Gephyrin mouse monoclonal antibody-301;

[0026] Figure 7 This is the EC50 fitting analysis of Gephyrin mouse monoclonal antibody-401;

[0027] Figure 8 This is the EC50 fitting analysis of Gephyrin mouse monoclonal antibody-501;

[0028] Figure 9 This is a diagram of the ScFv heavy chain sequence of strain 301;

[0029] Figure 10 This is a diagram of the ScFv light chain sequence of strain 301;

[0030] Figure 11 This is a diagram of the three-dimensional simulated structure of the light and heavy chains of the ScFv of strain 301;

[0031] Figure 12 This is a diagram of the three-dimensional simulated structure of Gephyrin;

[0032] Figure 13 This is a graphic representation of the docking results of Gephyrin-ScFv-301-VH+VL (white) and Gephyrin molecules. DETAILED DESCRIPTION

[0033] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0034] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context clearly has a different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0035] An embodiment of the present invention provides a monoclonal antibody targeting Gephyrin protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0036] Specifically, the sequence shown in SEQ ID NO.1 is:

[0037] VQLKDSGPGLVKPSQSLSLTCTVTGMSITSDYARNWIRQFPGNKLEWMGYISYSGSTS YNPSLKSRISITRDTSKNQFFLQLNSVSTEDTATYYCANYYYGREYYFDMWGQGTTLTVSS;

[0038] The sequence shown in SEQ ID NO.2 is:

[0039] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSW.

[0040] It should be noted that Gephyrin is a protein with a multifunctional domain, including two MPT domains. In view of the characteristics of certain enzymes, the present invention uses a prokaryotic expression system to recombinantly express mouse Gephyrin, and the N-terminus of the recombinant Gephyrin is fused with a 6xHis tag for expression.

[0041] It should also be noted that the preparation method of the monoclonal antibody targeting Gephyrin protein in the present invention is as follows:

[0042] (1) Prokaryotic expression of mouse gephyrin recombinant protein: This process includes codon optimization, template synthesis and vector construction; transformation into Escherichia coli BL21 (DE3) strain, and expression identification of 3 to 5 clones; followed by protein expression amplification and affinity purification to ensure that the purity of the recombinant mouse gephyrin protein exceeds 90%.

[0043] (2) Immunization and ELISA detection: Immunization: 5 Balb / c mice were immunized and boosted according to standard procedures; Detection: ELISA detection was performed by collecting blood from the tail. When the ELISA titer reached 10^5, the next step of cell fusion experiment could be carried out.

[0044] (3) Cell fusion and screening: The spleen cells of immunized mice were collected and fused with myeloma cells. The resulting hybridoma cells were then proliferated and screened by ELISA to obtain 10 to 20 high-titer mother cell lines.

[0045] (4) Subcloning: Subcloning: Use the limiting dilution method to subclone 10 to 20 positive mother cell lines to identify 5 to 10 positive clones; provide 5 to 10 subclones, and provide 0.1 ml of culture supernatant for each subclone for ELISA cross detection.

[0046] (5) Ascites production and antibody purification: Ascites production: Three strains were selected from the selected subclones for the production of mouse ascites; the obtained ascites were purified by Protein A / G.

[0047] (6) Based on ELISA technology, a monoclonal cell line (Fv region) was selected for sequencing: Gephyrin antigen was used as the target, and after EC50 affinity identification, the quality of the obtained single-chain antibody was evaluated, and the antibody with the best affinity was selected, which was the monoclonal antibody of the present invention.

[0048] It should also be noted that the indirect ELISA (enzyme-linked immunosorbent assay) method was used in the present invention to determine the affinity of the monoclonal antibody targeting the Gephyrin protein; the results of the ELISA standard curve showed that the monoclonal antibody containing the targeting Gephyrin protein and its corresponding target antigen exhibited high affinity at a concentration of 2 μg / ml (100 μL antigen / well) (the antibody concentration was 0.1 g / l, and after 121.6-fold dilution, the EC50 value was 5.31 nM). Therefore, this monoclonal antibody plays an important role in exploring the molecular mechanism of Gephyrin's involvement in α5-GABAAR-mediated cognitive dysfunction and its related treatment strategies. In addition, the simulated three-dimensional single-chain antibody can also effectively bind to Gephyrin.

[0049] Another embodiment of the present invention provides a hybridoma cell, the deposit number of which is CGMCC No. 46029. Specifically, the hybridoma cell can secrete the monoclonal antibody targeting Gephyrin protein of the present invention.

[0050] Another embodiment of the present invention provides an isolated nucleic acid molecule that encodes the monoclonal antibody targeting the Gephyrin protein of the present invention. Specifically, any monoclonal antibody that can encode the monoclonal antibody targeting the Gephyrin protein of the present invention meets the requirements of the isolated nucleic acid molecule of the present invention.

[0051] Yet another embodiment of the present invention provides a biomaterial comprising the isolated nucleic acid molecule of the present invention. The biomaterial comprises an expression vector, an engineered bacterium, or an engineered cell.

[0052] It should be noted that the isolated nucleic acid molecule of the present invention can be connected to an expression vector to form a recombinant expression vector, or an engineered bacterium or an engineered cell. In addition, expression vectors, engineered bacteria (such as Escherichia coli) or engineered cells are all known in the art.

[0053] Another embodiment of the present invention provides a detection reagent for detecting Gephyrin protein, comprising the monoclonal antibody targeting Gephyrin protein of the present invention. It should be noted that the monoclonal antibody targeting Gephyrin protein of the present invention can specifically bind to Gephyrin protein and can therefore be used for quantitative or qualitative detection of Gephyrin protein.

[0054] In some specific embodiments, the above detection reagent includes an indirect ELISA detection reagent. It should be noted that the monoclonal antibody targeting Gephyrin protein in the present invention can be used as a coating antibody to construct an indirect ELISA detection system for detecting Gephyrin protein.

[0055] Another embodiment of the present invention provides a detection kit for detecting Gephyrin protein, comprising the monoclonal antibody targeting Gephyrin protein or the detection reagent for detecting Gephyrin protein of the present invention. It should be noted that the kit of the present invention is in a form known in the art, including a reagent bottle or instructions.

[0056] Yet another embodiment of the present invention provides a drug for treating cognitive dysfunction caused by abnormal Gephyrin expression, comprising the monoclonal antibody targeting Gephyrin protein of the present invention.

[0057] Another embodiment of the present invention provides a use of the monoclonal antibody targeting Gephyrin protein of the present invention, the hybridoma cell of the present invention, the isolated nucleic acid molecule of the present invention, or the biomaterial of the present invention in the preparation of a detection reagent for detecting Gephyrin protein.

[0058] Another embodiment of the present invention provides a use of the monoclonal antibody targeting Gephyrin protein of the present invention, the hybridoma cell of the present invention, the isolated nucleic acid molecule of the present invention, or the biomaterial of the present invention in the preparation of a drug for treating cognitive dysfunction caused by abnormal Gephyrin expression.

[0059] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0060] 1. Obtaining the optimized mouse gephyrin gene sequence

[0061] Gephyrin expression vectors such as Figure 1 As shown, its amino acid sequence is shown below (SEQ ID NO.3):

[0062] MATEGMILTNHDHQIRVGVLTVSDSCFRNLAEDRSGINLKDLVQDPSLLGGTISAYKIVPDEIEEIKETLIDWCDEKELNLILTTGGTGFAPRDVTPEATKEVIEREAPGMALAMLMGSLNVTPLGMLSRPVCGIRGKTLIINLPGSKKGSQECFQFILPALPHAIDLLRDAIVKVKEVHDELEDLPSPPPPLSPPPTTSPHKQTEDKGVQCEEEEEEKKDSGVASTEDSSSSHITAAALAAKIPDSIISRGVQVLPRDTASLSTTPSESPRAQATSRLSTASCPTPKQIRRPDESKGVASRVGSLKARLPSCSSTYSVSEVQSRCSSKENILRASHSAVDITKVARRHRMSPFPLTSMDKAFITVLEMTPVLGTEIINYRDGMGRVLAQDVYAKDNLPPFPASVKDGYAVRAADGPGDRFIIGESQAGEQPTQTVMPGQVMRVTTGAPIPCGADAVVQVEDTELIRESDDGTEELEVRILVQARPGQDIRPIGHDIKRGECVLAKGTHMGPSEIGLLATVGVTEVEVNKFPVVAVMSTGNELLNPEDDLLPGKIRDSNRSTLLATIQEHGYPTINLGIVGDNPDDLLNALNEGISRADVIITSGGVSMGEKDYLKQVLDIDLHAQIHFGRVFMKPGLPTTFATLDIDGVRKIIFALPGNPVSAVVTCNLFVVPALRKMQGILDPRPTIIKARLSCDVKLDPRPEYHRCILTWHHQEPLPWAQSTGNQMSSRLMSMRSANGLLMLPPKTEQYVELHKGEVVDVMVIGRL(Carrier information: pET28a);

[0063] The optimized gene sequence (optimized by Carmed Biotechnology (Tianjin) Co., Ltd.) is as follows (SEQ ID NO. 4):

[0064]

[0065] Target fragment synthesis and enzyme digestion identification (conditions see Table 1).

[0066] Table 1 Target fragment synthesis and enzyme digestion identification

[0067]

[0068]

[0069] 2. Mouse Gephyrin Recombinant Protein Expression

[0070] (1) Transformation of expression strains

[0071] (1) Take 100 μl of a Rosstta BL21 competent cell, place it on ice to thaw, take a 1.5 ml sterile centrifuge tube in a clean bench, and divide it equally into 50 μl competent cells;

[0072] (2) Take 20ul of the customer's plasmid and add it to the competent cells in a clean bench. Mix gently and place on ice for 30min.

[0073] (3) Heat shock at 42°C for 90 seconds, then immediately place on ice for 5 minutes. Take 30 μl of the culture and spread it on an LB plate (containing 50 μg / ml kan). Incubate at 37°C for 14 hours (overnight culture) and select a single clone for activation culture.

[0074] (2) Expression identification

[0075] (1) Pick a single clone from the LB plate in the above step, add 2 ml LB liquid medium (containing 50 μg / ml kan) to activate the cell until the OD600 is 0.6-0.8, transfer to 5 ml LB liquid medium (containing 50 μg / ml kan) and culture until the OD600 is 0.6-0.8. Then add 1 mM IPTG to induce expression at 37°C for 4 h and collect the cells;

[0076] (2) Take samples of the bacterial system under different conditions before and after induction for SDS-PAGE analysis;

[0077] (III) Amplification and affinity purification

[0078] (1) Amplification of expression: Select the BL21 strain that can express the target protein from the above expression identification, inoculate it into 1LLB medium (containing 50ug / ml kan), expand and culture at 220rpm and 37℃ until the OD600 reaches 0.6-0.8, then add IPTG with a final concentration of 1mM and induce the culture at 30℃ for 12h, and then centrifuge at 4000rpm to collect the bacteria for use;

[0079] (2) Collect the obtained bacterial sludge and resuspend 2 g of bacterial sludge in 40 ml of 20 mM PB + 150 mM NaCl, pH 7.2; ultrasonically disrupt the sludge in an ice bath at 420 W for 3 seconds, with 5 seconds intervals, for 15 minutes; centrifuge at 12000 rpm at 4°C for 20 minutes, collect the supernatant and precipitate, and set aside;

[0080] (3) Purification

[0081] The supernatant was filtered through a 0.22 μm filter membrane and then purified by Ni column affinity enrichment.

[0082] Equilibration buffer:

[0083] 20 mM PB + 150 mM NaCl, pH 8.0;

[0084] Wash Buffer:

[0085] 20 mM PB + 150 mM NaCl + 5 mM imidazole, pH 8.0;

[0086] 20 mM PB + 150 mM NaCl + 20 mM imidazole, pH 8.0;

[0087] 20 mM PB + 150 mM NaCl + 50 mM imidazole, pH 8.0;

[0088] 20 mM PB + 150 mM NaCl + 80 mM imidazole, pH 8.0;

[0089] Elution Buffer:

[0090] 20mM PB + 150mM NaCl + 300mM imidazole, pH 8.0

[0091] After flow-through and purification of the sample, the sample was boiled at 100°C after adding loading buffer and analyzed by SDS-PAGE. Figure 4 shown.

[0092] 3. Immunization Process

[0093] In the embodiment of the present invention, according to Figure 5 The mice were immunized according to the immunization process shown in the figure; the immunization dose was 100 μg / mouse, the immunization method was 3-4 subcutaneous multi-point injections, and one booster immunization, and the experimental animals were 5 Balb / c mice.

[0094] The specific immunization time course is shown in Table 2.

[0095] Table 2 Immunization time course

[0096]

[0097]

[0098] In addition, the results of serum test after three immunizations are shown in Table 3 below.

[0099] Table 3 Serum test results after three immunizations

[0100] 3 free 1 2 3 4 5 1 / 1k 1.521 0.835 0.958 0.692 1.623 1 / 3k 0.803 0.414 0.522 0.37 0.804 1 / 9k 0.357 0.219 0.266 0.209 0.466 1 / 27k 0.159 0.113 0.135 0.116 0.267 1 / 81k 0.082 0.054 0.068 0.076 0.213 1 / 243k 0.059 0.061 0.063 0.058 0.179 1 / 729k 0.056 0.043 0.045 0.062 0.183 diluent 0.063 0.044 0.05 0.088 0.184

[0101] The results of serum test after four immunizations are shown in Table 4 below.

[0102] Table 4 Serum test results after four immunizations

[0103] 4 free 1 2 3 4 5 1 / 1k 1.765 1.348 1.667 1.082 1.901 1 / 3k 0.829 0.603 0.713 0.531 0.954 1 / 9k 0.336 0.267 0.303 0.275 0.401 1 / 27k 0.154 0.145 0.145 0.137 0.222 1 / 81k 0.098 0.092 0.101 0.127 0.133 1 / 243k 0.089 0.093 0.082 0.13 0.143 1 / 729k 0.077 0.066 0.082 0.084 0.078 diluent 0.081 0.071 0.088 0.078 0.078

[0104] The results of serum test after five immunizations are shown in Table 5 below.

[0105] Table 5 Serum test results after five immunizations

[0106]

[0107]

[0108] IV. Fusion, Cloning, and Detection

[0109] In the embodiment of the present invention, the hybridoma is prepared as follows:

[0110] (1) Preparation of splenocytes

[0111] Balb / c mice were sacrificed by cervical dislocation and immersed in 75% alcohol for 3–5 minutes. The spleen was removed aseptically and placed in a dish containing 5 mL of incomplete culture medium. The spleen was washed three times to remove surface fat and connective tissue. The washed spleen was cut into 3–5 small pieces with scissors, then minced and passed through a cell sieve to collect the cells. The spleen cell suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed and centrifuged again using the same method. The pellet was resuspended in 10 mL of incomplete culture medium, and the number of viable cells was counted. A 108 splenic lymphocyte suspension was obtained for later use.

[0112] (2) Preparation of myeloma cells

[0113] Select a myeloma cell line and isolate myeloma cells from cells in the logarithmic growth phase cultured in vitro or from tumors growing in vivo; centrifuge the logarithmically growing myeloma cells and wash them twice with serum-free culture medium; prepare a cell suspension and count the number of viable cells; adjust the cell concentration and take a 10^7 cell suspension for later use.

[0114] (3) Cell fusion

[0115] Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5, and 20-50 mL of RPMI-1640 culture medium was added. The mixture was centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and the remaining liquid was gently aspirated with a pipette. The bottom of the centrifuge tube was gently tapped with a finger to disperse the cell pellet. The centrifuge tube was placed in a 37°C water bath. 1 mL of preheated 50% PEG was slowly added dropwise to the centrifuge tube within 30 seconds, while gently stirring. The mixture was allowed to stand for 1-5 minutes. Preheated incomplete culture medium was added dropwise along the tube wall to terminate the PEG reaction. 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 10 mL were added dropwise at 2-minute intervals, while gently rotating the centrifuge tube. The mixture was centrifuged at 800 rpm for 5-10 minutes, and the supernatant was discarded. The cell pellet was gently resuspended in HAT selection medium containing 20% ​​calf serum. Be careful not to pipette too hard to prevent the fused cells from dispersing. Add complete culture medium according to the number of 96 / 24-well plates, and then inoculate the above cells into 24-well plates containing feeder cells (1.0-1.5 mL per well) or 96-well culture plates (0.10-0.15 mL per well) (1-3 cells per well).

[0116] After the inoculation is completed, the culture plate is placed in a 37° C., 5% CO 2 incubator for culture.

[0117] (4) Selective culture of hybridoma cells

[0118] In HAT medium, unfused myeloma cells, lacking HGPRT and TK, cannot utilize the salvage pathway to synthesize DNA and die. Unfused B lymphocytes, while possessing HGPRT and TK, cannot survive long-term in vitro and gradually die. Only hybridomas, formed by the fusion of myeloma cells with immune B cells, possess both the ability of myeloma cells to proliferate indefinitely and the gene products of HGPRT and TK from immune B cells. Therefore, they can survive and reproduce in HAT selective medium. Within one to two days of HAT selective culture, a large number of tumor cells will die. After three to four days, the tumor cells disappear, and small, round, translucent hybridoma colonies resembling myeloma cells can be observed. Hybridoma cell growth is monitored, and when they cover one-tenth of the bottom of the well, the culture supernatant can be removed and tested for specific antibodies.

[0119] (5) Hybridoma cell cloning

[0120] Only a small number of hybridoma cells grown in HAT medium secrete the predetermined specific monoclonal antibody, so screening and cloning are necessary. Cloning involves culturing and multiplying the cells in the culture well from a single cell to form a monoclonal clone, achieving uniformity in the secretion of antibodies. Gently blow dry the hybridoma cells to be cloned from the culture well and count them. Adjust the cell count to 3 to 10 × 10^3 cells / mL. Prepare a 96-well cell culture plate containing feeder cells and divide it into four groups of 24 wells each. Dilute the cell suspension by doubling to form four groups of cells: 12.5, 25, 50, and 100 cells / mL. Add 100 μL of diluted cells to each well and incubate at 37°C, 5% CO2. After 8 to 9 days, visible cell clones will form, allowing for prompt testing of antibody activity.

[0121] (6) Hybridoma cell establishment / expansion culture

[0122] The cells in the positive wells were transferred to 24-well plates for culture and testing, and the positive single cells were expanded and cultured, fixed and frozen, or used for ascites injection to prepare antibodies in vivo.

[0123] SP2 / 0 mouse myeloma cells were fused with spleen cells from a preferred mouse. After fusion, culture, observation, testing, and positive / negative control tests were performed to obtain a hybridoma cell that met the experimental requirements. Based on the results of cell line screening, the present invention summarizes the fusion results of Gephyrin-3 mice.

[0124] (1) Integration

[0125] After the mice were fused, multiple positive wells were obtained, which met the requirements of the present invention and entered the next stage of screening (Gephyrin is Gphn). The fusion plate test data are shown in Tables 6-11.

[0126] Table 6 Mouse fusion plate test results 1

[0127]

[0128] Table 7 Mouse fusion plate test results 2

[0129]

[0130] Table 8 Mouse fusion plate test results 3

[0131]

[0132] Table 9 Mouse fusion plate test results 4

[0133]

[0134]

[0135] Table 10 Mouse fusion plate test results 5

[0136]

[0137] Table 11 Mouse fusion plate test results 6

[0138]

[0139] (2) Cloning

[0140] During the cloning stage, multiple cloning wells were obtained. The detection data of the cloning stage are shown in Tables 12-16.

[0141] Table 12 Cell clone screening results (plate 1)

[0142]

[0143]

[0144] Table 13 Cell clone screening results (plate 2)

[0145]

[0146] Table 14 Cell clone screening results (plate 3)

[0147]

[0148] Table 15 Cell clone screening results (plate 4)

[0149]

[0150] Table 16 Cell clone screening results (plate 5)

[0151]

[0152]

[0153] (3) Planting

[0154] Through mouse fusion, primary cloning by limiting dilution, and supernatant analysis, multiple cell lines were generated. Five cell lines were selected for subsequent ascites fluid preparation. Data from expanded cell supernatant analysis are shown in the table (K: thousand, W: ten thousand, X: multiple). Raw cell supernatant analysis data are shown in Tables 17-18.

[0155] Table 1724 well supernatant results 1

[0156]

[0157] Table 18 24-well supernatant results 2

[0158] Gphn-1 13 14 15 16 17 18 19 1 2.188 2.193 2.244 0.753 0.395 1.574 0.509 3 2.131 2.107 2.185 0.696 0.27 1.572 0.463 9 1.511 1.547 2.245 0.651 0.136 1.452 0.31 27 0.813 0.806 1.824 0.537 0.07 1.042 0.149 81 0.343 0.4 1.335 0.425 0.043 0.768 0.062 243 0.155 0.174 1.104 0.329 0.028 0.459 0.033 729 0.086 0.103 0.869 0.211 0.018 0.336 0.02 Negative 0.015 0.012 0.015 0.017 0.013 0.013 0.016

[0159] (IV) Ascites antibody test

[0160] The present invention performed ascites (antibody) preparation on all the obtained cell lines, screened out 3 ascites samples (301, 401 and 501), and the test data are shown in Table 19.

[0161] Table 19 Ascites ELISA test results

[0162] Dilution multiple 301 401 501 1000 2.11 1.681 1.389 3000 1.848 1.376 1.085 9000 1.594 1.082 0.898 27000 1.308 0.855 0.473 81000 0.87 0.493 0.234 243000 0.809 0.291 0.133 729000 0.619 0.165 0.071 Negative 0.042 0.054 0.015

[0163] 5. Antibody EC50 Detection

[0164] (1) Preparation of 96-well plate

[0165] (1) On the first day, dilute the antigen to 2 μg / ml using antigen dilution buffer, then add 100 μl of antigen to each well. Seal the wells and incubate at 4°C overnight.

[0166] (2) The next day, discard the supernatant. Add 200 μl of blocking buffer to each well and incubate at 37°C for 1 hour. Discard the supernatant before use.

[0167] (2) EC50 titration

[0168] (1) Dilute the antibody protein into 16 gradients using the 5-fold gradient dilution method. Add 100 μl of sample to the appropriate wells, seal the wells, and incubate at 37°C for 1 hour.

[0169] (2) Discard the supernatant and wash 5 times with washing buffer.

[0170] (3) Add 100 μl of HRP-labeled goat anti-mouse (1:3000 dilution) to each well and incubate at 37°C for 1 h.

[0171] (4) Discard the supernatant and wash 5 times with washing buffer.

[0172] (5) Add 100 μl of TMB (mixture of Solution A and Solution B) one-step substrate reagent to each well and incubate at room temperature in a dark room for 15 min with gentle shaking.

[0173] (6) Add 50 μl of stop solution to each well and read the sample immediately at a wavelength of 450 nm.

[0174] The EC50 test results of three ScFv antibodies are as follows Figure 6 、 Figure 7 and Figure 8As shown, the results showed that the EC50 fitting analysis of Gephyrin mouse monoclonal antibody -301 was 5.31nM when the antibody concentration was 0.1g / l and diluted 121.6 times; the EC50 fitting analysis of Gephyrin mouse monoclonal antibody -401 was 201.61nM when the antibody concentration was 0.1g / l and diluted 3.2 times; the EC50 fitting analysis of Gephyrin mouse monoclonal antibody -501 was 55.62nM when the antibody concentration was 0.05g / l and diluted 5.8 times.

[0175] 6. Sequencing

[0176] Amplify the mouse VH and VL genes from hybridoma cell lysate and ligate the target genes into the T vector as follows:

[0177] (1) Primers

[0178] The primer sequences are shown in Table 20.

[0179] Table 20 Amplification primers

[0180] Primer Name Sequence (5' to 3') Vk-s MSCVK-1 5'-AYATCCAGCTGACTCAGC-3' Vk-B MSCJK-12 5'-TTTKATTTCCAGYTTGGTCCC-3' Vλ-S MSCVL-1 5'ATGCTGTTGTGACTCAGGAATC-3' Vλ-B MSCJL-B 5'GCCTAGGACAGTCAGTTTGG-3' VH5'Sense MSCVH1 5'GTRMAGCTTCAGGAGTC-3' Vh3'-R MSCGlab-B 5'CAGATGGGGSTGTYGTTTTGGC-3'

[0181] (2) RNA extraction (QIAGEN, RNeasy Mini Kit)

[0182] (1) Resuscitate T2 hybridoma cells, transfer to a 50 ml centrifuge tube, add 20 ml DMEM, and centrifuge at 300 g for 10 min.

[0183] (2) Discard the supernatant, add 30 ml of PBS to a 50 ml centrifuge tube, centrifuge at 300 g for 10 min, discard the supernatant, add 700 μl of RLT to the cell pellet, and pipette thoroughly to completely lyse the cells.

[0184] (3) Pipette 350 μl of lymphocyte sample from the above lysis solution, blow thoroughly, homogenize, and oscillate to completely lyse.

[0185] (4) Add 350 μl of 70% ethanol and mix thoroughly by pipetting back and forth.

[0186] (5) Add 700 μl of liquid to the center of the column, centrifuge at room temperature for 15 seconds, and discard the waste liquid.

[0187] (6) Add 700 μl of Buffer RW1 to the column, centrifuge at room temperature for 15 seconds, and discard the waste liquid and collection tube.

[0188] (7) Place the column in a new collection tube, add 500 μl of Buffer RPE (add 4 times the volume of ethanol to Buffer RPE for the first use), centrifuge at room temperature for 15 seconds, and discard the waste liquid.

[0189] (8) Add 500 μl of Buffer RPE and centrifuge at room temperature for 2 minutes.

[0190] (9) Leave the tube in the air for 1 minute and discard the collection tube.

[0191] (10) Elute with 50ul DEPC water.

[0192] (III) cDNA synthesis (20ul system)

[0193] The first-strand synthesis kit (Transcriptor First Strand cDNA Synthesis Kit) of Roche was used; the amplification system and reaction procedure are shown in Table 21.

[0194] Table 21cDNA synthesis amplification system and reaction procedure

[0195]

[0196] (IV) PCR amplification of antibody genes

[0197] (1) Using 2 μg-20 μg of total RNA reverse transcribed into cDNA as a template, the PCR reaction system was configured according to Table 22 below to amplify the antibody light chain and heavy chain genes (30 total systems, 27 reaction systems PCR).

[0198] Table 22 PCR amplification system and reaction procedure

[0199]

[0200] (2) The PCR products were subjected to 1.5% agarose gel electrophoresis. VH, VL and VK should all be bands of approximately 330 bp. After recovery and quantification, the next round of PCR was performed or frozen at -20°C for future use.

[0201] (3) The gel-recovered products were connected to the T vector and prepared for sequencing; the sequencing work was undertaken by Tianjin Kamed Biotechnology Co., Ltd.; the light and heavy chain sequences of the ScFv of strain 301 were as follows: Figure 9 and Figure 10 In addition, the three-dimensional simulated structure of the light and heavy chains of ScFv of strain 301 is shown in Figure 11 As shown, the three-dimensional simulation structure of Gephyrin is shown in Figure 12 As shown, the docking results of Gephyrin-ScFv-301-VH+VL (white) and Gephyrin molecules are shown in Figure 2. Figure 13 shown.

[0202] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A monoclonal antibody targeting Gephyrin protein, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. A hybridoma cell, characterized in that Its deposit number is CGMCC No.46029.

3. An isolated nucleic acid molecule, characterized in that Used to encode the monoclonal antibody targeting Gephyrin protein according to claim 1.

4. Biomaterial, characterized in that The nucleic acid molecule according to claim 3 is included, and the biological material includes an expression vector, an engineered bacterium or an engineered cell.

5. A detection reagent for detecting Gephyrin protein, characterized in that: The invention comprises the monoclonal antibody targeting Gephyrin protein according to claim 1.

6. The detection reagent for detecting Gephyrin protein according to claim 5, characterized in that Detection reagents include indirect ELISA detection reagents.

7. A detection kit for detecting Gephyrin protein, characterized in that: The invention comprises the monoclonal antibody targeting Gephyrin protein according to claim 1 or the detection reagent for detecting Gephyrin protein according to claim 5 or 6.

8. Use of the monoclonal antibody targeting Gephyrin protein according to claim 1, the hybridoma cell according to claim 2, the isolated nucleic acid molecule according to claim 3, or the biomaterial according to claim 4 in the preparation of a detection reagent for detecting Gephyrin protein.

Images