Monoclonal antibody targeting Gephyrin protein, hybridoma cell and application

Abstract

The invention belongs to the technical field of monoclonal antibody drugs, and particularly relates to a monoclonal antibody targeting Gephyrin protein, a hybridoma cell and application. The amino acid sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.2. The monoclonal antibody has high affinity with Gephyrin protein, the antibody concentration is 0.1 g / l, and the EC50 value is 5.31 nM after the monoclonal antibody is diluted by 121.6 times.

Description

Technical Field

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

[0002] Existing studies have shown that Gephyrin, an anchoring protein under the postsynaptic membrane, achieves the function of inhibiting signal reception by aggregating inhibitory glycine and γ-aminobutyric acid receptors and positioning them on the cytoskeleton of the postsynaptic membrane. Abnormal expression of Gephyrin may lead to cognitive dysfunction. GABAA receptors containing α5 subunits (α5-GABAARs) are highly expressed in the hippocampus, and some are distributed on the inner side of the gap of the postsynaptic membrane. 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. Changes in 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 view of 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 helpful for in-depth research on the regulatory mechanism of Gephyrin in α5-GABAAR-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, wherein 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] Another aspect of the present invention provides an isolated nucleic acid molecule for encoding the monoclonal antibody targeting Gephyrin protein in the present invention.

[0009] In another aspect, the present invention provides a biomaterial, which includes the nucleic acid molecule of the present invention. The biomaterial includes 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 of the present invention, the hybridoma cell of the present invention, the isolated nucleic acid molecule of the present invention, or the biological material 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 China Microbiological Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is 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. 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, 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 It is a DNA marker, which consists 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: PlasmidDigested with XbaI-XhoI; M: DNA Marker;

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

[0024] Figure 5 This is a 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] Fig. 9 This is a diagram of the ScFv heavy chain sequence of strain 301;

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

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

[0031] Fig.12 Illustration of the three-dimensional simulated structure of Gephyrin;

[0032] Fig.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 the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope 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 has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" 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] The embodiment of the present invention provides a monoclonal antibody targeting Gephyrin protein, wherein 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 each subclone provides 0.1 ml of culture supernatant 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 was 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 in 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 a 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 times 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, and its deposit number is CGMCC No. 46029. Specifically, the hybridoma cell can secrete the monoclonal antibody targeting Gephyrin protein in the present invention.

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

[0051] Another embodiment of the present invention provides a biomaterial, which includes the isolated nucleic acid molecule of the present invention. The biomaterial includes 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, including 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, so it can 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 form an indirect ELISA detection system for detecting Gephyrin protein.

[0055] Another embodiment of the present invention provides a detection kit for detecting Gephyrin protein, which includes the monoclonal antibody targeting Gephyrin protein of the present invention 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, etc.

[0056] Another embodiment of the present invention provides a drug for treating cognitive dysfunction caused by abnormal Gephyrin expression, including 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 biological material 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 in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0060] 1. Obtaining the optimized gene sequence of Mouse Gephyrin

[0061] Gephyrin expression vector Figure 1 The amino acid sequence is shown below (SEQ ID NO.3):

[0062] MATEGMILTNHDHQIRVGVLTVSDSCFRNLAEDRSGINLKDLVQDPSLLGGTISAYKIVPDEIEEIKETLIDWCDEKELNLILTTGGTGFAPRDVTPEATKEVIEREAPGMALAMLMGSLNVTPLGMLSRPVCGIRGKTLIINLPGSKKGSQECFQFILPALPHAIDLLRDAIVKVKEVHDELEDLPSPPPPLSPPPTTSPHKQTEDKGVQCEEEEEEKKDSGVASTEDSSSSHITAAALAAKIPDSIISRGVQVLPRDTASLSTTPSESPRAQATSRLSTASCPTPKQIRRPDESKGVASRVGSLKARLPSCSSTYSVSEVQSRCSSKENILRASHSAVDITKVARRHRMSPFPLTSMDKAFITVLEMTPVLGTEIINYRDGMGRVLAQDVYAKDNLPPFPASVKDGYAVRAADGPGDRFIIGESQAGEQPTQTVMPGQVMRVTTGAPIPCGADAVVQVEDTELIRESDDGTEELEVRILVQARPGQDIRPIGHDIKRGECVLAKGTHMGPSEIGLLATVGVTEVEVNKFPVVAVMSTGNELLNPEDDLLPGKIRDSNRSTLLATIQEHGYPTINLGIVGDNPDDLLNALNEGISRADVIITSGGVSMGEKDYLKQVLDIDLHAQIHFGRVFMKPGLPTTFATLDIDGVRKIIFALPGNPVSAVVTCNLFVVPALRKMQGILDPRPTIIKARLSCDVKLDPRPEYHRCILTWHHQEPLPWAQSTGNQMSSRLMSMRSANGLLMLPPKTEQYVELHKGEVVDVMVIGRL(Carrier information: pET28a);

[0063] The optimized gene sequence (optimized by KMD Biotech (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] (I) Transformation of expression strains

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

[0072] (2) Take 20ul of the plasmid sent by the customer, add the plasmid 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 and immediately place on ice for 5 minutes. Take 30 μl and spread on LB plate (containing 50 μg / ml kan). Culture at 37°C for 14 hours (overnight culture) and then pick a single clone for activation culture.

[0074] (II) Expression identification

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

[0076] (2) SDS-PAGE analysis of bacterial samples under various conditions before and after induction of expression;

[0077] (III) Amplification and affinity purification

[0078] (1) Amplification of expression: A BL21 strain capable of expressing the target protein was selected from the above expression identification, inoculated into 1LLB medium (containing 50ug / ml kan), cultured at 220rpm and 37°C until OD600 reached 0.6-0.8, then IPTG with a final concentration of 1mM was added for induction culture at 30°C for 12h, and the cells were collected by centrifugation at 4000rpm for later use;

[0079] (2) Collect the obtained bacterial sludge, resuspend every 2 g of bacterial sludge in 40 ml of 20 mM PB + 150 mM Nacl, pH 7.2; disrupt by ultrasonication at 420 W in an ice bath, ultrasonication for 3 seconds, interval of 5 seconds, for 15 minutes; centrifuge at 12000 rpm, 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; wherein, the immunization dose was 100ug / mouse, the immunization method was 3-4 subcutaneous multi-point injections, and 1 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 Dilution 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 Dilution 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 preparation of hybridoma is as follows:

[0110] (1) Preparation of splenocytes

[0111] Balb / c mice were killed by cervical dislocation and soaked in 75% alcohol for 3-5 minutes; the spleen was removed by aseptic operation and placed in a plate containing 5mL of incomplete culture medium, and washed 3 times to remove fat and connective tissue on the surface of the spleen; the washed spleen was cut into 3-5 small pieces with scissors, and then the spleen was crushed and passed through a cell sieve to collect cells; the spleen cell suspension was centrifuged at 1000r / min for 5 minutes and the supernatant was discarded. Wash and centrifuge again in the same way; the precipitated cells were resuspended in 10mL of incomplete culture medium, the number of living cells was counted, and 10^8 spleen lymphocyte suspensions were taken for use.

[0112] (2) Preparation of myeloma cells

[0113] Select a myeloma cell line, take cells in the logarithmic growth phase of in vitro culture or isolate myeloma cells from tumors growing in vivo; centrifuge the logarithmic growth myeloma cells and wash them twice with serum-free culture medium; prepare a cell suspension and count the number of live cells; adjust the cell concentration and take 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 RPMI-1640 culture medium was added; centrifuged at 1000 r / min for 8 minutes, the supernatant was discarded, and the residual liquid was gently sucked out with a dropper; the bottom of the centrifuge tube was gently hit with a finger to disperse the precipitated cells; the centrifuge tube was placed in a 37°C water bath, 1 mL of preheated 50% PEG was drawn, and it was slowly dripped into the centrifuge tube, and the addition was completed within 30 seconds, and it was gently stirred while adding, and the effect was 1-5 minutes; preheated incomplete culture medium was dripped along the wall of the tube to terminate the PEG effect, and 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 10 mL were added every 2 minutes, and the centrifuge tube was gently rotated while dripping; centrifuged at 800 r / min for 5-10 minutes, and the supernatant was discarded; the precipitated cells were gently suspended in HAT selection culture medium containing 20% ​​calf serum to resuspend the cells. Be sure not to blow vigorously to avoid dispersing the fused cells; 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 (1.0-1.5mL per well) or 96-well culture plates (0.10-0.15mL 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 culture medium, unfused myeloma cells cannot synthesize DNA by salvage pathways due to lack of HGPRT and TK and die. Unfused B lymphocytes have HGPRT and TK, but they cannot survive in vitro for a long time and gradually die. Only hybridomas fused with myeloma cells and immune B cells have the ability of unlimited proliferation of myeloma cells and obtain the gene products of HGPRT and TK from immune B cells, so they can survive and reproduce in HAT selection culture medium; within 1 to 2 days of HAT selection culture, a large number of tumor cells will die, and after 3 to 4 days, the tumor cells will disappear, and small colonies of round and translucent hybridoma cells similar to myeloma cells can be seen; observe the growth of hybridoma cells, and when they cover 1 / 10 of the bottom of the well, the culture supernatant can be taken to detect specific antibodies.

[0119] (5) Hybridoma cell cloning

[0120] Among the hybridoma cells grown in HAT medium, only a few are cells that secrete the predetermined specific monoclonal antibody, so they must be screened and cloned. Cloning - that is, the cells in the culture well are cultured and propagated from single cells to make them monoclonal, and the antibodies they secrete are uniform; the hybridoma cells to be cloned are gently blown dry from the culture wells and counted; the number of cells is adjusted to 3-10×10^3 / mL; take the 96-well cell culture plate with feeder cells prepared in advance, divide the 96-well plate into 4 groups, each with 24 wells, dilute the cell suspension into 4 groups of cells by multiple dilution method, that is, 12.5, 25, 50, 100 / mL per group, add 100μL of diluted cells to each well, and incubate at 37℃, 5% CO2; after 8-9 days, cell clones can be seen with the naked eye, and antibody activity can be detected in time.

[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 used to carry out cell fusion with spleen cells of the preferred mouse, and after fusion, a hybridoma cell that met the experimental requirements was obtained through culture, observation, detection and positive and negative control tests. According to the cell line screening, the present invention summarizes the fusion of Gephyrin-3 mice.

[0124] 1. Integration

[0125] After the mice were fused, multiple positive wells were obtained, and this stage met the requirements of the present invention, and the next stage of screening was entered (Gephyrin is Gphn). The fusion plate detection data is 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] (II) Cloning

[0140] In the cloning stage, a number of 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] (III) Plant determination

[0154] After fusion, primary cloning by limiting dilution method and supernatant detection, multiple cell lines were obtained, and 5 cell lines were selected for subsequent ascites preparation. The data of cell expansion supernatant detection are shown in the table (K: thousand, W: ten thousand, X: times), and the original data of cell supernatant detection 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 detection 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] (I) Preparation of 96-well plates

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

[0166] (2) On the second 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] (II) EC50 titration

[0168] (1) Dilute the antibody protein into 16 gradients using the 5-fold gradient dilution method. Add 100 μl of sample to 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 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 450 nm.

[0174] The EC50 test results of the 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 showed that the EC50 value 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 showed that the EC50 value 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 showed that the EC50 value was 55.62nM when the antibody concentration was 0.05g / l and diluted 5.8 times.

[0175] 6. Sequencing

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

[0177] (I) Primers

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

[0179] Table 20 Amplification primers

[0180] Primer Name Sequence (5'to3') 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] (ii) RNA extraction (QIAGEN, RNeasy Mini Kit)

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

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

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

[0185] (4) Add 350 μl of 70% ethanol and mix well 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 min.

[0190] (9) Leave the tube empty for 1 min 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 Company was used; the amplification system and reaction procedure are shown in Table 21.

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

[0195]

[0196] (IV) PCR amplification of antibody genes

[0197] (1) Using cDNA formed by reverse transcription of 2ug-20ug total RNA as a template, a PCR reaction system was configured according to Table 22 below to amplify antibody light chain and heavy chain genes (30 total systems, 27 reaction system 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 about 330 bp. After recovery and quantification, the next round of PCR was performed or frozen at -20°C for later use.

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

[0202] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. 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 solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should 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 method comprises the nucleic acid molecule of claim 3, and the biological material comprises 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 as claimed in claim 1.

6. The detection reagent for detecting Gephyrin protein according to claim 3, 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 3 or 4.

8. A drug for treating cognitive dysfunction caused by abnormal Gephyrin expression, characterized in that: The invention comprises the monoclonal antibody targeting Gephyrin protein as claimed in claim 1.

9. 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 biological material according to claim 4 in preparing a detection reagent for detecting Gephyrin protein.

10. 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 drug for treating cognitive dysfunction caused by abnormal Gephyrin expression.

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