Monoclonal antibody recognizing nmi protein and use thereof

Abstract

The application relates to the technical field of monoclonal antibodies, and discloses a monoclonal antibody for recognizing NMI protein and application thereof. The monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9. The monoclonal antibody for recognizing NMI protein and the chimeric antibody thereof have excellent specific recognition effects on hNMI protein and mNMI protein, and can be used for biological function research.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, and in particular to a monoclonal antibody that recognizes NMI protein and its applications. Background Technology

[0002] NMI (N-myc interactor) is a protein that interacts with N-MYC and C-MYC, as well as other transcription factors containing Zip, HLH, or HLH-Zip motifs. As an important regulator of signaling pathways, NMI participates in innate immune responses. For example, under the influence of IL-2 and IFN-γ, it can interact with STAT, activating the transduction of downstream genes involved in various developmental and homeostatic signals, and enhancing the recruitment of STAT1 and STAT5 by the CBP / p300 coactivator, leading to an increase in STAT1 and STAT5-dependent transcription. Under the influence of interferon IFN-α, it forms a complex with IFI35, preventing IFI35 degradation mediated by the proteasome pathway. Furthermore, when present in complex form with IFI35, it can inhibit virus-induced type I IFN-β production through ubiquitination mediated by the ubiquitin ligase TRIM21, or negatively regulate NF-κB signaling by inhibiting nuclear translocation, activation, and transcription of the NF-κB subunit p65 / RELA, leading to inhibition of endothelial cell proliferation, migration, and endothelialization of damaged arteries. Additionally, NMI negatively regulates virus-induced type I interferon production by inducing the degradation of the type I IFN transcription factor IRF7 via a protein-somatic-dependent pathway, thus affecting cellular antiviral responses.

[0003] Besides acting as a regulator of intracellular signaling pathways, NMIs, when actively released into the extracellular fluid during cell damage or pathogen invasion, can also promote inflammation as damage-associated molecular patterns (DAMPs). Macrophage-secreted NMIs bind to the Toll-like receptor TLR4, thereby activating NF-κB signaling in adjacent macrophages, inducing NF-κB translocation to the nucleus, and promoting the release of pro-inflammatory cytokines. NMI proteins have important physiological functions, and studies on their function often involve mouse animal experiments. Currently, there are limited detection methods for murine NMIs on the market, and most have not been validated with negative samples, leading to false-positive results in immunofluorescence methods for detecting murine or human NMI proteins, resulting in serious limitations in their application. Therefore, the development of detection methods for identifying NMI proteins has significant application value. Related fields also need detection reagents with both specificity and sensitivity for research on biological functions.

[0004] Therefore, there is an urgent need to develop a monoclonal antibody that specifically recognizes human and mouse NMI proteins. Summary of the Invention

[0005] The first aspect of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof that recognizes NMI.

[0006] A second aspect of the present invention aims to provide biological materials associated with the monoclonal antibody or its antigen-binding fragment that recognizes NMI.

[0007] A third aspect of the present invention is to provide a coupling.

[0008] A fourth aspect of the present invention is to provide a product.

[0009] A fifth aspect of the present invention aims to provide the use of monoclonal antibodies or antigen-binding fragments thereof that recognize NMI, biological materials or conjugates in the preparation of reagents for detecting human NMI protein and / or mouse NMI protein.

[0010] A sixth aspect of the present invention is to provide a method for preparing a monoclonal antibody or an antigen-binding fragment thereof that recognizes NMI.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof for recognizing NMI, comprising a heavy chain and a light chain;

[0013] The heavy chain includes a heavy chain variable region, and the heavy chain variable region contains complementarity determination regions CDR-H1, CDR-H2 and CDR-H3;

[0014] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.5;

[0015] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6;

[0016] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.7;

[0017] The light chain includes a light chain variable region, and the light chain variable region contains complementarity determination regions CDR-L1, CDR-L2 and CDR-L3;

[0018] The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.10;

[0019] The amino acid sequence of the CDR-L2 is shown in SEQ ID NO.11;

[0020] The amino acid sequence of the CDR-L3 is shown in SEQ ID NO.12.

[0021] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region comprises any one of A1) to A3):

[0022] A1) The amino acid sequence as shown in SEQ ID NO.4;

[0023] A2) An amino acid sequence of SEQ ID NO.4 with one or more amino acid substitutions and / or deletions and / or additions, which has the same function as the sequence shown in SEQ ID NO.3;

[0024] A3) An amino acid sequence that has 80%, 85%, or 90% or more homology with SEQ ID NO.4 and has the same function as the sequence shown in SEQ ID NO.4;

[0025] The amino acid sequence of the light chain variable region comprises any one of B1) to B3):

[0026] B1) The amino acid sequence as shown in SEQ ID NO.9;

[0027] B2) An amino acid sequence of SEQ ID NO.9 with one or more amino acid substitutions and / or deletions and / or additions, which has the same function as the sequence shown in SEQ ID NO.9;

[0028] B3) has 80%, 85% or 90% or more homology with SEQ ID NO.9 and has the same function as the sequence shown in SEQ ID NO.9.

[0029] In this document, the term "homology" refers to the similarity between amino acid sequences or nucleotide sequences, which can be evaluated using computer software. It is understood that an amino acid sequence having more than 80% homology with SEQ ID NO.4 and having the same function as the sequence shown in SEQ ID NO.4 refers to an amino acid sequence that is identical to the antibody heavy chain variable region in SEQ ID NO.4, obtained by adjusting the amino acids of the linker peptide.

[0030] In this document, the above 80% homology can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0031] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, and multispecific antibody.

[0032] In some embodiments of the present invention, the heavy chain further includes a heavy chain constant region; and / or

[0033] The light chain also includes a light chain constant region.

[0034] In some embodiments of the present invention, the heavy chain constant region is the heavy chain constant region of rabbit immunoglobulin G1 (IgG1); the light chain constant region is the constant region of the rabbit kappa light chain.

[0035] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.24; and the light chain constant region is shown in SEQ ID NO.26.

[0036] A second aspect of the invention provides biological material associated with a monoclonal antibody or antigen-binding fragment thereof that recognizes NMI as described in any of the first aspects, said biological material comprising any one of C1) to C5);

[0037] C1) A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in any of the first aspects;

[0038] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0039] C3) A recombinant vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2);

[0040] C4) A recombinant microorganism containing the nucleic acid molecule described in C1), the expression cassette described in C2), or the recombinant vector described in C3);

[0041] C5) A recombinant cell containing the nucleic acid molecule described in C1), the expression cassette described in C2), or the recombinant vector described in C3), wherein the recombinant cell does not contain propagation material.

[0042] Those skilled in the art can use known methods (such as directed evolution and point mutation) to mutate nucleotide sequences. Those artificially modified nucleotide sequences that have a certain degree of identity with the monoclonal antibody or its antigen-binding fragment described in this invention, as long as they encode the monoclonal antibody or its antigen-binding fragment or variant and have the same function as the monoclonal antibody or its antigen-binding fragment, are all derived from and equivalent to the nucleotide sequences of this invention.

[0043] In some embodiments of the present invention, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

[0044] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO.3; and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO.8.

[0045] In some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the monoclonal antibody or its antigen-binding fragment in a host cell. This DNA may include not only a promoter to initiate transcription of the DNA molecule encoding the monoclonal antibody or its antigen-binding fragment, but also a terminator to terminate transcription of the DNA molecule encoding the monoclonal antibody or its antigen-binding fragment. Furthermore, the expression cassette may also include a replication start site, a transcription start sequence, an enhancer sequence, a selection element, or a reporter gene.

[0046] In some embodiments of the present invention, the recombinant vector may be a plasmid, bacteriophage, lentivirus, adenovirus, or adeno-associated virus. The recombinant vector is used to achieve the replication, integration, amplification, and / or expression of the exogenous target gene in recipient cells; it may be a cloning vector or an expression vector. For example, the recombinant vector in C3) may specifically be a recombinant plasmid obtained by inserting the nucleic acid molecule into a CMV vector.

[0047] In some embodiments of the present invention, the recombinant microorganism may be bacteria (such as Escherichia coli or Bacillus subtilis) or fungi (such as yeast or Aspergillus). For example, the recombinant microorganism in C4 may specifically be Escherichia coli DN5α.

[0048] In some embodiments of the present invention, the recombinant cells may be insect cells (such as S2 fruit fly cells or Sf9 cells), animal cells (such as B lymphocytes, 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells), or plant cells (such as Arabidopsis thaliana or tobacco).

[0049] In some embodiments of the present invention, the recombinant cells may specifically be hybridoma cells. The hybridoma cells are obtained by fusing spleen cells with SP2 / 0 cells.

[0050] A third aspect of the present invention provides a conjugate comprising: a monoclonal antibody or an antigen-binding fragment thereof that recognizes NMI as described in any one of the first aspects;

[0051] And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a radionuclide, and an enzyme.

[0052] In a fourth aspect, the present invention provides a product comprising at least one of D1) to D3);

[0053] D1) A monoclonal antibody or antigen-binding fragment thereof that recognizes NMI as described in any of the first aspects;

[0054] D2) Biomaterials as described in any of the second aspects;

[0055] D3) The coupling as described in the third aspect;

[0056] The product is selected from at least one of pharmaceuticals, test plates, chips, test strips, and reagent kits.

[0057] In some embodiments of the invention, the medicament further includes pharmaceutically acceptable excipients. The dosage used is non-toxic to the recipient.

[0058] In some embodiments of the present invention, the excipients include at least one selected from solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, stabilizers, buffers, isotonics, flavoring agents, and carriers. The excipients are used to promote the absorption of the monoclonal antibody or its antigen-binding fragment to exert preventive and / or therapeutic effects.

[0059] A fifth aspect of the invention provides the use of any one of E1) to E3) in the preparation of reagents for detecting human NMI proteins and / or mouse NMI proteins:

[0060] E1) A monoclonal antibody or antigen-binding fragment thereof that recognizes NMI as described in any of the first aspects;

[0061] E2) Biomaterials as described in any of the second aspects;

[0062] E3) Couplings as described in the third aspect.

[0063] A sixth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising: obtaining it by culturing the recombinant microorganism or the recombinant cell in the biological material described in the second aspect.

[0064] In some embodiments of the present invention, the antibody or its antigen-binding fragment may be obtained using the hybridoma technique first reported by Kohler et al. (Nature, 256:495, 1975), or may be obtained using recombinant DNA technology (see Journal of virological methods, 2009, 158(1-2):171-179).

[0065] The monoclonal antibody of the present invention that recognizes the NMI protein has at least the following beneficial effects:

[0066] The anti-NMI monoclonal antibody of the present invention exhibits high binding specificity and recognition sensitivity against both human and mouse NMI proteins. Based on its ability to recognize human and mouse NMI proteins simultaneously, it can be directly applied to cross-species comparative studies, reducing the need for consumables in experimental research.

[0067] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0068] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0069] Figure 1 This is an electrophoresis diagram of the recombinant hNMI protein and recombinant mNMI protein of the present invention.

[0070] Figure 2 This is a flowchart illustrating the subcloning and monocloning process in an embodiment of the present invention.

[0071] Figure 3 The binding curves of AB008 and AB018 with human recombinant NMI protein in the embodiments of the present invention are shown.

[0072] Figure 4 The binding curves of AB008 and AB018 with the mouse recombinant NMI protein in the embodiments of the present invention are shown.

[0073] Figure 5 The binding curves of rAB008 and rAB018 to human recombinant NMI protein in the embodiments of the present invention are shown.

[0074] Figure 6 The binding curves of rAB008 and rAB018 to the mouse recombinant NMI protein in the embodiments of the present invention are shown.

[0075] Figure 7 The fluorescence image of rAB018 identifying overexpression of mNMI and hNMI in 293t cells is shown in this embodiment of the invention. The scale bar is 20 μm.

[0076] Figure 8The fluorescence image shown is for the rAB008 recognition of overexpression of mNMI and hNMI in 293t cells in this embodiment of the invention. The scale bar is 20 μm. Detailed Implementation

[0077] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0078] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0079] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0080] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0081] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0082] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0083] Example 1: Preparation of hybridoma antibody recognizing recombinant human NMI (hNMI) protein

[0084] 1. Preparation of recombinant hNMI antigen

[0085] (1) Obtaining human NMI recombinant protein:

[0086] According to the UniProt database, the human NMI amino acid sequence is Q13287. The specific sequence information is as follows:

[0087] MEADKDDTQQILKEHSPDEFIKDEQNKGLIDEITKKNIQLKKEIQKLETELQEATKEFQIKEDIPETKMKFLSVETPENDSQLSNISCSFQVSSKVPYEIQKGQALITFEKEEVAQNVVSMSKHHVQIKDVNLEVTAKPVPLNSGVRFQVYVEVS KMKINVTEIPDTLHEDQMRDKLELSFSKSRNGGGEVDRVDYDRQSGSAVITFVEIGVADKILKKKEYPLYINQTCHRVTVSPYTEIHLKKYQIFSGTSKRTVLLTGMEGIQMDEEIVEDLINIHFQRAKNGGGEVDVVKCSLGQPHIAYFEE(SEQ ID NO.1), a total of 307 amino acids.

[0088] A recombinant expression vector was constructed based on the sequence shown in SEQ ID NO.1, and the human recombinant hNMI protein was expressed and purified using a prokaryotic expression system.

[0089] (2) Identification of recombinant hNMI protein:

[0090] The recombinant protein molecular size and aggregation were identified using FastPAGE gel (Qingke, TSP024). The specific method is as follows:

[0091] S1. Take 0.1 μg of recombinant hNMI protein to prepare a protein sample, add 5* loading buffer and mix thoroughly, then boil in a 95℃ metal bath for 10 minutes.

[0092] S2. Pour enough MOPS-SDS Running Buffer into the inner tank of the electrophoresis tank to cover the sample wells by 5-7 mm, and add the same MOPS-SDS electrophoresis buffer into the outer tank.

[0093] S3. Add 10 μl of protein sample to the FastPAGE gel wells, load with a protein marker (GenStar, ZM221), and perform electrophoresis. Electrophoresis is performed at 120V throughout the process.

[0094] S4. After electrophoresis, remove the gel plate from the gel plate and carefully insert a suitable pry bar into the gap between the gel plates; after prying open the clamp, remove the gel and place it in Coomassie Brilliant Blue staining solution, and gently shake and stain for 15 minutes at room temperature on a shaker.

[0095] S5. After staining, wash away the Coomassie Brilliant Blue staining solution adhering to the surface of the FastPAGE gel with water. Then place the gel in destaining solution (40% ethanol, 10% acetic acid, 50% dd H2O) and gently shake on a shaker at room temperature for 30 minutes. Change the destaining solution until the gel background is clean and the protein bands are clear.

[0096] The identification results of recombinant hNMI protein are as follows: Figure 1 As shown, the molecular size is consistent with expectations.

[0097] 2. Immunization of animals with recombinant hNMI antigen

[0098] In this experiment, the recombinant hNMI protein obtained above was used as an antigen for animal immunization. The specific method is as follows:

[0099] (1) Prepare recombinant hNMI protein as antigen, mix it with an equal volume of complete Freund's adjuvant, and emulsify it by repeatedly aspirating ≥20 times with a 1ml syringe until a uniform "water-in-oil" emulsion is formed. The standard is met if it does not spread when dropped into water.

[0100] (2) Female BALB / c mice about 6 weeks old were immunized subcutaneously at multiple sites using a dose of 100 μg / mouse (this strain is stable to protein antigens and female mice have low aggression, which can reduce mutual injury among cages). A total of 3 immunizations were performed, with an interval of 14 days between each immunization.

[0101] (3) On the 7th day after the completion of the 3 immunizations (the peak period of serum IgG antibodies), 150 μl of blood was collected from the tail vein to detect the production of anti-recombinant hNMI antibodies. The specific operation was as follows: the venous blood sample was placed at room temperature for 30 min, then centrifuged at 3000 rpm for 20 min at 4℃, and the upper yellow clear serum solution was taken for indirect ELISA to detect the serum antibody titer.

[0102] The specific procedure is as follows: Recombinant hNMI protein antigen (2 μg / ml) is coated onto a high-binding ELISA plate. Serially diluted serum (1:1000 to 1:512000) is added at 100 μl / well and incubated for 60 minutes. After washing the plate three times, horseradish peroxidase-labeled anti-mouse IgG secondary antibody is added and incubated. Finally, TMB substrate is used for color development. OD 450 The highest dilution with a value ≥ 2 times that of the negative control is the efficacy value. When the serum titer is ≥ 1:100000, the immunization efficiency can be considered to have met the standard.

[0103] (4) After confirming that the serum titer reached the target, a booster immunization was performed by intraperitoneal injection of 50 μg / mouse of adjuvant-free recombinant hNMI antigen (adjuvant removed to avoid excessive inflammatory response). Three days after immunization, the mice were sacrificed and their spleens were harvested.

[0104] 3. Cell fusion

[0105] (1) Preparation of spleen single-cell suspension:

[0106] Spleens from immunized mice were collected and placed in a 10cm culture dish containing serum-free DMEM medium. The cells were gently ground using a disposable grinding stick, avoiding excessive force that could cause mechanical damage to the cells. The resulting liquid was filtered through a 200-mesh cell strainer to remove connective tissue fragments, retaining the lymphoid tissue pouch. The filtrate was then transferred to a 50ml centrifuge tube. The cells were washed three times with pre-cooled serum-free DMEM medium.

[0107] (2) Pretreatment of myeloma cells (SP2 / 0):

[0108] SP2 / 0 cells in the logarithmic growth phase were used. The SP2 / 0 cells were washed three times with serum-free DMEM to completely remove interference from fetal bovine serum-bound FBS. The SP2 / 0 cells were counted and the density adjusted to 1×10⁻⁶ cells / cells. 7 / ml.

[0109] (3) Cell mixing:

[0110] Myeloma SP2 / 0 cells were mixed with a suspension of lymphocytes filtered from the spleen at a ratio of spleen cells:SP2 / 0 = 1:10. After mixing, the culture supernatant was removed by centrifugation.

[0111] (4) PEG fusion:

[0112] a. Add 1 ml of 50% PEG-1500 solution at a constant rate and slowly stir the cell clusters to temporarily perforate the cell membrane and promote fusion.

[0113] b. After adding PEG, let stand at room temperature for 45 seconds.

[0114] c. Take 1 ml of serum-free DMEM medium and add it to the cell cluster at a constant rate over 1 min, then slowly and evenly dilute the PEG.

[0115] d. After dilution, slowly add serum-free culture medium to a total volume of 45 ml to terminate the PEG reaction.

[0116] (5)Planning:

[0117] After PEG fusion, centrifuge at 1200 rpm for 8 min and discard the supernatant. Prepare a complete culture medium containing 10% fetal bovine serum (FBS) and 10% hybridoma culture factor, resuspend the cell pellet, and seed it into 10 96-well plates.

[0118] (6) HAT screening and hybridoma amplification:

[0119] After 24 hours of cell growth, the medium was replaced with HAT selection medium to remove unfused SP2 / 0 cells and spleen cells. After approximately 7 days of selection in HAT medium, the medium was replaced with HT medium for another 7 days to allow for the transition and metabolic recovery of the hybridoma cells. Finally, the medium was replaced with standard complete medium to allow the fused cells to continue growing.

[0120] (7) Screening and validation of positive hybridomas:

[0121] After observing the cells until they reached approximately 1 / 10 of the size of the bottom of the covering plate (approximately 7-10 days after fusion to avoid insufficient antibody accumulation), the culture supernatant was aspirated and added to wells coated with recombinant hNMI protein. ELISA was used to screen hybridoma clones capable of secreting and binding the recombinant hNMI protein. Compared with the negative control (SP2 / 0 supernatant), OD... 450 A value ≥ 3 times that of the negative control can be judged as positive.

[0122] (8) Subcloning and Monocloning:

[0123] The processes of subcloning and monocloning are as follows: Figure 2 As shown, the positive maternal clones were expanded and cultured, followed by further subcloning using a 2-D dilution method to obtain single clones. Ultimately, multiple single-clonal hybridoma cell lines secreting anti-hNMI recombinant protein were obtained.

[0124] Example 2: Screening of hybridoma antibodies that simultaneously identify human and mouse NMI

[0125] 1. Preparation of recombinant mNMI antigen

[0126] Given that human natural NMI protein and mouse NMI protein share approximately 60% homology, in order to screen for recognition antibodies that can bind to both human and mouse NMI proteins simultaneously and facilitate subsequent animal experimental studies, it is necessary to obtain recombinant mouse NMI protein for dual-recognition antibody screening.

[0127] According to the UniProt database, the mouse NMI amino acid sequence with sequence number O35309 is as follows:

[0128] MDADKDNIKQACDERSAEMDDMRGEQSMGLVHEIMSENKELDEEIKKLEAELQSDAREFQIKENVPEKKLKLTSVESPKDGCHFSNSSCSFQVSSQILYELQEGQALITFEKEEVAQNVISMGNHVVQMEGTPVKVSAHPVPLNTGVRFQVHVDISKMK INVTGIPDELSEEQTRDKLELSFCKSRNGGGEVESVDYDRKSRSAVITFVETGVVDKILKKKTYPLYMNQKCHSVAVSPCIERCLEKYQVFSAVSKKTVLLTGLEGIPVDEETGEDLLNIHFQRKNNGGGEVEVVKCSLDQSFAAYFKEEARETI(SEQ ID NO.2), a total of 314 amino acids.

[0129] A recombinant expression vector was constructed based on the sequence shown in SEQ ID NO.2. The mouse-derived recombinant mNMI protein was expressed and purified using a prokaryotic purification system, and molecular identification was performed. The specific method referred to the identification method for recombinant mNMI protein in Example 1. The results are as follows: Figure 1 As shown, the molecular size is consistent with expectations.

[0130] 2. Screening of hybridoma antibodies combining human and mouse NMI proteins

[0131] To screen for monoclonal antibodies that can specifically recognize and bind to both human NMI (hNMI) and murine NMI (mNMI) proteins, this experiment used indirect enzyme-linked immunosorbent assay (indirect ELISA) to determine the binding ability of hybridoma antibodies to NMI proteins from two different species.

[0132] (1) Antigen coating:

[0133] Recombinant human NMI protein and mouse NMI protein, both expressed and purified, were used as coating antigens. These two proteins were coated at 2 μg / ml into highly binding 96-well microplates and incubated overnight at 4°C.

[0134] (2) Closed:

[0135] The following day, the coating solution inside the well plate was removed, and the plate was washed twice with PBST. 200 μl of blocking buffer containing 5% skim milk powder was added to each well, and the plate was incubated at room temperature for 2 hours to block non-specific binding sites.

[0136] (3) Antibody sample incubation:

[0137] After blocking, the blocking solution was removed, and the wells were washed twice with PBST. The antibodies obtained from the culture supernatants of the multiple hybridoma cell lines obtained in Example 1, after preliminary purification with Protein A, were serially diluted with dilution buffer. The diluted antibodies were added to wells coated with recombinant hNMI and recombinant mNMI in parallel, and incubated at room temperature or 37°C for 2 hours, with a negative control included. After antibody incubation, the plate was washed five times with PBST to thoroughly remove unbound antibodies.

[0138] (4) Detection:

[0139] Add 100 μl / well of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody diluted 1:1000 and incubate at room temperature for 1 hour. After incubation, wash the plate 5 times with PBST washing buffer to thoroughly remove unbound secondary antibody.

[0140] (5) Color development and reading:

[0141] Add TMB substrate to initiate the colorimetric reaction and incubate at room temperature in the dark for 15 minutes. After stopping the colorimetric reaction by adding stop solution (2M dilute H2SO4), immediately read the optical density (OD) value of each well at a specific wavelength (e.g., 450nm) on a microplate reader.

[0142] Two hybridoma cell lines were ultimately selected that could secrete both recombinant human NMI protein and mouse NMI antibody: AB008 and AB018. The monoclonal antibody secreted by hybridoma cell line AB008 was labeled AB008 antibody, and the monoclonal antibody secreted by hybridoma cell line AB018 was labeled AB018 antibody. The binding curves of both with recombinant human NMI protein are shown below. Figure 3 As shown, the binding curve with the murine recombinant NMI protein is as follows: Figure 4 As shown.

[0143] Example 3: Recombinant expression and modification of AB008 antibody recognizing human and mouse NMI protein

[0144] While hybridoma cells can continuously secrete antibodies, large-scale culture may face risks such as low yield and poor cell stability (e.g., genetic drift). Therefore, recombinant expression may be a better solution than using hybridoma cells to secrete antibodies.

[0145] 1. Hybridoma cell culture and RNA preparation

[0146] Hybridoma cells obtained from the above screening that secrete the target murine monoclonal antibodies AB008 and AB018 were cultured until the cells grew to approximately 1*102. 7 Subsequent collection. Total RNA was extracted from the collected cells using FreeZol RNA extraction reagent (Vazyme, R711-02) to ensure that the RNA purity met the requirements of subsequent experiments (A260 / A280 ratio between 1.8 and 2.1; A260 / A230 ratio between 2.0 and 2.5).

[0147] 2. cDNA synthesis

[0148] Using the extracted total RNA as a template, reverse transcription (RT) was performed using reverse transcriptase and Oligo(dT) primers to synthesize first-strand cDNA. At the same time, the mRNA information of AB008 antibody or AB018 antibody in hybridoma cells was converted into stable cDNA.

[0149] 3. Amplification of VH and VL genes

[0150] Specific primers were designed and synthesized to target the conserved framework region of the variable region of the murine antibody. Using the synthesized cDNA as a template, gene fragments encoding the antibody heavy chain variable region (VH) and light chain variable region (VL) were amplified by polymerase chain reaction (PCR). After verifying the correct size of the PCR products by agarose gel electrophoresis, the products were excised and purified. The purified products were ligated into a T-vector and then sequenced for analysis.

[0151] The sequencing results are as follows:

[0152] (1) Heavy chain variable region and light chain variable region of AB008 antibody:

[0153] The AB008 heavy chain variable region sequence is as follows:

[0154] GAGGTGCAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGA

[0155] AGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTTCAACATGTACTGGGTGAAG

[0156] CAGAGCCATGGAAAGAACCTTGAATTGATTGGATTTGTTAACCCTAATAATGGTGGTACT

[0157] ACTTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTCAACAAGTCCTCCAGCA

[0158] CAGCCTACATGGAGCTCCGCAGCCTGACATCGGAGGATTCTGCAGTCTATTACTGTGCAA

[0159] AAGATTTATTTCTACGGTCCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO. 3).

[0160] The corresponding amino acid sequence of the variable region of the AB008 heavy chain is as follows:

[0161] EVQLQESGPELVKPGASVKMSCKASGYTFTDFNMYWVKQSHGKNLELIGFVNPNNG GTTYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCAKDLFLRSWGTGTTVTVSS (SEQ ID NO. 4).

[0162] The AB008 heavy chain variable region includes the following complementary determinant region (CDR-H):

[0163] AB008 Heavy Chain Variable Region CDR-H1: DFNMY (SEQ ID NO.5);

[0164] AB008 Heavy Chain Variable Region CDR-H2: FVNPNNGGTTYNQKFKG (SEQ ID NO.6);

[0165] AB008 Heavy chain variable region CDR-H3:DLFLRS (SEQ ID NO.7).

[0166] The variable region sequence of the AB008 light chain is as follows:

[0167] GACATCCAGCTGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTC

[0168] ACCTTCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCTTGGTATCAGCAGCAAA

[0169] CAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCC

[0170] ATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCCTGAAGATCAACAGCCTGC

[0171] AGCCTGGCGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCTCTCACGTTCGG

[0172] TGCTGGGACCAAGCTGGAACTGAAA (SEQ ID NO. 8).

[0173] The corresponding amino acid sequence of the AB008 light chain variable region is as follows:

[0174] DIQLTQSPASSLSASVGETVTFTCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGV PSRFSGSGSGTQFSLKINSLQPGDFGSYYCQHHYGTPLTFGAGTKLELK (SEQ ID NO. 9).

[0175] The AB008 light chain variable region includes the following complementary determinant region (CDR-L):

[0176] AB008 Light chain variable region CDR-L1: RASENIYSYLA (SEQ ID NO.10);

[0177] AB008 Light chain variable region CDR-L2: NAKTLAE (SEQ ID NO.11);

[0178] AB008 Light chain variable region CDR-L3: QHHYGTPLT (SEQ ID NO.12).

[0179] (2) Heavy chain variable region and light chain variable region of AB018 antibody:

[0180] The AB018 heavy chain variable region sequence is as follows:

[0181] GAGGTGAAGCTGCAGGAGTCAGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGA

[0182] AACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATGGAATACACTGGGTTCGTC

[0183] AGGCTCCAGAGAAGGGGCTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAGTACCATC

[0184] TACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACAC

[0185] CCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTACAA

[0186] GGCCTACTATAGTAACTACAATGGCCCACTGGGGTCAAGGAACCTCAGTCACCGTCTCC

[0187] TCA (SEQ ID NO.13).

[0188] The corresponding amino acid sequence of the AB018 heavy chain variable region is as follows:

[0189] EVKLQESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEKGLEWVAYISSGSSTIY YADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCTRPTIVTTMAHWGQGTSVTVSS (SEQ ID NO. 14).

[0190] The AB018 heavy chain variable region includes the following complementary determinant region (CDR-H):

[0191] AB018 Heavy Chain Variable Region CDR-H1: DYGIH (SEQ ID NO.15);

[0192] AB018 Heavy chain variable region CDR-H2: YISSGSSTIYYADTVKG (SEQ ID NO.16);

[0193] AB018 Heavy chain variable region CDR-H3:PTIVTTMAH (SEQ ID NO.17).

[0194] The variable region sequence of the AB018 light chain is as follows:

[0195] GACATCCAGCTGACTCAGTCTCCTCAGGCTGTCTCCTCAGGTTGCCTCCTCAAAAT

[0196] GAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGT

[0197] TGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTC

[0198] TTGCAGCTCTAGTCAGAGCCTTGTACACAGTAGTGGAGACACCTTTTTTCATTGGTACCT

[0199] ACAGAAGCCAGGCCAGTTCCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTG

[0200] GGGTCCCAGACAGGTTCAGTGGCATGGATCAGGGACAGATTTCACACTCAAGATCAGT

[0201] AGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGCT

[0202] CACGTTCGGTACTGGGACCAAGCTGGAGCTGAAG (SEQ ID NO. 18).

[0203] The corresponding amino acid sequence of the variable region of the AB018 light chain is as follows:

[0204] DIQLTQSPQAVSSGCLLKMKLPVRLLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASIS CSSSQSLVHSSGDTFFHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDTLKISRVEAEDLGVYFCSQSTHVPLTFGTGTKLELK (SEQ ID NO. 19).

[0205] The AB018 light chain variable region includes the following complementary determinant region (CDR-L):

[0206] AB018 Light chain variable region CDR-L1: SSSQSLVHSSGDTFFH (SEQ ID NO.20);

[0207] AB018 Light chain variable region CDR-L2: KVSNRFS (SEQ ID NO.21);

[0208] AB018 Light chain variable region CDR-L3: SQSTHVPLT (SEQ ID NO.22).

[0209] 4. Construction of chimeric expression vectors

[0210] The mammalian expression vector pcDNA3.1 was selected as the basic backbone. The vector was pre-loaded with the constant regions of the heavy chain (CH1, CH2, CH3) of rabbit immunoglobulin G1 (IgG1) and the constant region (CL) of the light chain of rabbit kappa.

[0211] Rabbit heavy chain constant region nucleic acid sequence Rabbit IgG1 (CH1-3):

[0212] GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAA ACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAA(SEQ IDNO.23)。

[0213] Amino acid sequence of rabbit heavy chain constant region Rabbit IgG1 (CH1-3):

[0214] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO.24).

[0215] Amino acid sequence of rabbit light chain constant region Rabbit kappa CL:

[0216] GGGGATCCCGTGGCCCCCACCGTGCTGATCTTTCCTCCCGCCGCTGACCAAGTGGCCACCGGCACCGTGACCATCGTGTGCGTGGCCAACAAGTACTTCCCCGACGTGACCGTGACCTGGGAGGTGGACGGCACCACACAGACCACCGGCATCGAGAACAGCAAGACCCCTCAGAACAGCGCCGACTGCACCTACAACCTGAGCAGCACCCTGACCCTGACAAGCACACAGTACAACAGCCACAAGGAGTACACCTGCAAGGTGACCCAAGGCACCACAAGCGTGGTGCAGAGCTTCAACAGAGGCGACTGC (SEQ ID NO.25).

[0217] Amino acid sequence of rabbit light chain constant region Rabbit kappa CL:

[0218] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQN SADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO. 26).

[0219] The mouse VH and VL gene fragments were optimized and synthesized, and then constructed into the aforementioned expression vectors using a seamless cloning method. Ultimately, two independent expression plasmids for rAB008 were obtained: one containing the mouse VH-rabbit CH1-3 chimeric heavy chain gene, and the other containing the mouse VL-rabbit CL chimeric light chain gene; and two independent expression plasmids for rAB018 were obtained: one containing the mouse VH-rabbit CH1-3 chimeric heavy chain gene, and the other containing the mouse VL-rabbit CL chimeric light chain gene.

[0220] 5. Expression of chimeric antibodies in eukaryotic HEK293F cells

[0221] The constructed chimeric heavy and light chain expression plasmids were co-transfected into human embryonic kidney suspension cells (293F) at a heavy chain to light chain ratio of 1:1. Transfection was performed via PEI-mediated transfection using the highly efficient eukaryotic protein expression system of 293F cells. Transfected cells were cultured under suitable conditions for 7 days to express and secrete chimeric antibody molecules into the cell culture supernatant.

[0222] 6. Verification of the affinity between chimeric antibody and NMI protein

[0223] Activity assay: Cell culture supernatant containing expressed antibodies was collected. To assess whether chimerism (i.e., replacing the original mouse constant region with a rabbit constant region) affected the antigen-binding ability of the antibodies, enzyme-linked immunosorbent assay (ELISA) was used to detect the binding activity of chimeric antibodies rAB008 (recombinant AB008) and rAB018 (recombinant AB018) to their target antigens (recombinant proteins).

[0224] Test results as follows Figure 5 , Figure 6 As shown in Table 1.

[0225] Table 1: EC5 of recombinant proteins 50 value

[0226] AB008 rAB008 AB018 rAB018 Recombinant murine NMI 0.2048 1.612 0.1339 2.591 Recombinant human NMI 0.3023 0.9662 1.591 30.53

[0227] The test results showed that compared with the parental mouse monoclonal antibody AB008, the chimeric antibody rAB008 exhibited a slight decrease in binding ability to the recombinant target protein, but the overall effect was not significant (as shown in Table 1). This indicates that the chimeric antibody constructed by combining the mouse variable region (VH / VL) and rabbit constant region (CH / CL) of the AB008 antibody successfully retained the key antigen recognition characteristics of the parental antibody. Compared with the parental mouse monoclonal antibody AB018, the recombinant chimeric rAB018 showed a slight decrease in affinity for mouse NMI protein, but a significant decrease in affinity for human NMI protein.

[0228] Example 4: Anti-NMI recombinant expression of rAB008 and rAB018 antibodies used in immunofluorescence experiments

[0229] Immunofluorescence assay is a common method for locating and visualizing intracellular target proteins using antigen-antibody specific binding and fluorescent labeling techniques. In this study, recombinant human and mouse NMI proteins were transfected into 293T cells and then recognized by rAB008 / rAB018 antibodies to verify their specificity. The specific contents of this experiment are as follows.

[0230] 1. Cell preparation and inoculation

[0231] (1) Experimental Groups:

[0232] Negetive group: Overexpression of red fluorescent protein (mCherry) plasmid.

[0233] 293tmNMI-OE: Overexpression of mouse recombinant NMI protein (mNMI), which has a red fluorescent protein (mCherry) tag fused to its C-terminus.

[0234] 293thNMI-OE: Overexpression of human recombinant NMI protein (hNMI), which has an mCherry red fluorescent protein (mCherry) tag fused to its C-terminus.

[0235] (2) Confocal dish plating:

[0236] The two groups of cells were seeded at an appropriate density (ensuring monolayer growth and avoiding overcrowding) into dedicated confocal culture dishes. The dishes were then incubated at 37°C in a 5% CO2 incubator. Cell status was closely monitored, and culture was terminated when cells adhered well and reached approximately 60-80% confluence (i.e., cells covering about 60-80% of the bottom of the culture dish).

[0237] (3) Cell fixation:

[0238] Carefully aspirate the complete culture medium from the culture dish. Gently rinse the cells three times with pre-chilled phosphate-buffered saline (PBS, pH 7.4), approximately one minute each time, to remove residual culture medium and serum proteins to avoid affecting fixation. Add sufficient freshly prepared, pre-chilled 4% paraformaldehyde (PFA) solution (dissolved in PBS) to the culture dish, ensuring complete coverage of the cell layer. Incubate at room temperature for 30 minutes. This step fixes intracellular proteins and structures through cross-linking, maintaining cell morphology. After fixation, thoroughly aspirate the PFA solution.

[0239] (4) Cell permeability:

[0240] Wash cells thoroughly three times with PBS for 5 minutes each time to completely remove residual fixative. After discarding the PBS, add 0.5% Triton X-100 solution, ensuring complete coverage of the cell layer. Incubate at room temperature for 10 minutes. Triton X-100 is a non-ionic detergent that dissolves lipids on cell membranes and some organelle membranes, increasing cell membrane permeability and allowing subsequently added macromolecular antibodies to enter the cell and bind to the target antigen (NMI protein). After permeabilization, discard the Triton X-100 solution.

[0241] (5) Blocking of nonspecific binding sites:

[0242] Wash cells three times with PBS for 5 minutes each time to remove permeabilizer. Add sufficient blocking buffer containing 5% BSA to the culture dish. Incubate at room temperature for 1 hour to block non-specific protein binding sites, thereby reducing subsequent non-specific antibody adsorption and lowering background fluorescence signal. After incubation, carefully aspirate the blocking buffer.

[0243] 2. Incubation of rAB008 and rAB018 antibodies

[0244] Dilute the target antibody to a final concentration of 5.0 μg / mL using the blocking buffer containing 5% BSA. Add the diluted target antibody working solution and negative control solution to the corresponding cell culture dishes, ensuring the liquid covers the cell layer. Incubate at room temperature for 2 hours. After incubation, carefully aspirate the primary antibody solution.

[0245] Wash cells thoroughly three times with PBS for 5 minutes each time to remove any free primary antibody molecules that are not bound to the target antigen and prevent false positive signals.

[0246] 3. Incubation with fluorescent secondary antibody

[0247] Select a specific fluorescently labeled secondary antibody that matches the species origin of the target antibodies rAB008 and rAB018 (Alexa Fluor 488 labeled Goat anti-Rabbit IgG secondary antibody against rabbit IgG).

[0248] After discarding the final PBS wash, add a drop of the 1:500 diluted fluorescent secondary antibody working solution to the culture dish, ensuring complete coverage of the cell layer. Incubate at room temperature for 1 hour in the dark. After incubation, carefully discard the secondary antibody solution. Wash the cells thoroughly three times with PBS for 5 minutes each time in the dark to completely remove unbound fluorescent secondary antibody and minimize background.

[0249] 4. Nuclear staining

[0250] After aspirating the PBS, add a drop of DAPI (4',6-diamidinyl-2-phenylindole) solution (working concentration is usually 1 μg / mL, dissolved in PBS) to cover the cell layer. Incubate at room temperature in the dark for 5 minutes. DAPI is a fluorescent dye that binds to the minor groove of the DNA double helix, efficiently and specifically labeling the cell nucleus and exhibiting blue fluorescence under a fluorescence microscope.

[0251] 5. Sealing and Preservation

[0252] Discard the DAPI solution. Quickly rinse the cells twice with PBS to remove excess dye. Discard most of the PBS, leaving only a very small amount of liquid to cover the cells.

[0253] Add an appropriate amount of anti-fluorescence quenching mounting medium to the cell layer. Carefully cover with a coverslip, tilting the coverslip and slowly lowering it to avoid air bubbles. Carefully seal the edges of the coverslip with clear nail polish to prevent the mounting medium from drying out and air from entering, which could lead to sample oxidation and fluorescence quenching. Place the sealed sample in the dark and observe it after the mounting medium has completely solidified.

[0254] 6. Image Acquisition and Analysis

[0255] After the mounting medium has solidified, place the sample on the stage of a fluorescence microscope or a laser scanning confocal microscope.

[0256] Select the excitation and emission filter groups:

[0257] For detection of mCherry (negetive control group with simple red fluorescent protein and NMI overexpression labeling): excitation light 587nm, emission light approximately 610nm (red fluorescence).

[0258] For detecting the fluorescent secondary antibody (Alexa Fluor 488, labeled rAB008 and rAB018 binding): excitation light 495nm, emission light approximately 519nm (green fluorescence).

[0259] For detecting DAPI (nuclear marker): excitation light 358nm, emission light 461nm (blue fluorescence).

[0260] Adjust microscope parameters to obtain images with the optimal signal-to-noise ratio. Images were acquired for the experimental group (cells expressing hNMI or mNMI) and the negative control group, respectively.

[0261] 7. Experimental Results

[0262] Figure 7 This study demonstrates that rAB018 recognizes overexpressed mNMI and hNMI in 293t cells. However, in untreated wild-type 293t cells and 293t cells expressing red fluorescent (mCherry) labeled hNMI or mNMI, rAB018 exhibits non-specific binding, disrupting observation. While overlapping green fluorescent signals from rAB018 binding to the fluorescent secondary antibody (Alexa Fluor 488) were successfully observed in 293t cells overexpressing red fluorescent (mCherry) labeled mNMI, no enhanced overlapping green fluorescent signals were observed in 293t cells successfully overexpressing red fluorescent (mCherry) labeled hNMI, indicating that rAB018 cannot recognize the hNMI protein.

[0263] Figure 8 This study demonstrates that rAB008 recognizes overexpressed mNMI and hNMI in 293t cells. A green fluorescent signal emitted by rAB008 bound to the fluorescent secondary antibody (Alexa Fluor 488) was successfully observed in 293t cells expressing red fluorescent (mCherry) labeled hNMI or mNMI. More importantly, a significant spatial overlap (co-localization) was observed between the green fluorescent signal (the location where rAB008 recognizes NMI) and the red fluorescent signal (the location of the NMI protein itself) within the cell. This co-localization phenomenon provides direct evidence that the rAB008 antibody specifically recognizes hNMI and mNMI proteins, indicating that the rAB008 antibody has excellent specific recognition efficacy for both hNMI and mNMI proteins.

[0264] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A monoclonal antibody or its antigen-binding fragment for recognizing NMI protein, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain; The heavy chain includes a heavy chain variable region, and the heavy chain variable region contains complementarity determination regions CDR-H1, CDR-H2 and CDR-H3; The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.5; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.7; The light chain includes a light chain variable region, and the light chain variable region contains complementarity determination regions CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.10; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO.11; The amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region comprises any one of A1) to A3): A1) The amino acid sequence as shown in SEQ ID NO.4; A2) An amino acid sequence of SEQ ID NO.4 with one or more amino acid substitutions and / or deletions and / or additions, which has the same function as the sequence shown in SEQ ID NO.3; A3) An amino acid sequence that has 80%, 85% or 90% or more homology with SEQ ID NO.4 and has the same function as the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain variable region includes any one of B1) to B3): B1) The amino acid sequence as shown in SEQ ID NO.9; B2) An amino acid sequence of SEQ ID NO.9 with one or more amino acid substitutions and / or deletions and / or additions, which has the same function as the sequence shown in SEQ ID NO.9; B3) is an amino acid sequence that has 80%, 85% or 90% or more homology with SEQ ID NO.9 and has the same function as the sequence shown in SEQ ID NO.

9.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises at least one of full-length antibody, Fab, Fab', F(ab')2, Fv, and scFv.

4. The monoclonal antibody or its antigen-binding fragment according to claim 3, characterized in that, The heavy chain further includes a heavy chain constant region; and / or The light chain also includes a light chain constant region.

5. A biological material relating to the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, characterized in that, The biomaterial comprises any one of C1) to C5); C1) A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2); C4) A recombinant microorganism containing the nucleic acid molecule described in C1), the expression cassette described in C2), or the recombinant vector described in C3); C5) A recombinant cell containing the nucleic acid molecule described in C1), the expression cassette described in C2), or the recombinant vector described in C3), wherein the recombinant cell does not contain propagation material.

6. The biomaterial according to claim 5, characterized in that, The nucleic acid molecule includes a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

7. A product characterized in that, The product contains at least one of D1) to D2): D1) The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4; D2) The biomaterial as described in claim 5 or 6; The product is selected from at least one of pharmaceuticals, test plates, chips, test strips, and reagent kits.

8. Application of E1) or E2) in the preparation of reagents for detecting human NMI protein and / or mouse NMI protein: E1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4; E2) Biomaterials as described in claim 5 or 6.

9. A method for preparing a monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, characterized in that, Obtained by culturing the recombinant microorganisms or recombinant cells as described in claim 5 or 6.

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