Monoclonal antibody or antigen-binding fragment thereof against mouse liver sinusoidal endothelial cell oit3 protein and use thereof

Abstract

This invention discloses a monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or its antigen-binding fragment and its applications, belonging to the fields of biotechnology and medical immunology. The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO. 3~SEQ ID NO. 5; the light chain variable region contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO. 8~SEQ ID NO. 10. This monoclonal antibody and the recombinant fluorescent antibody exhibit high affinity and high specificity, and can be effectively applied to enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence detection. They can serve as important antibody tools for basic research on mouse hepatic sinusoidal endothelial cells and for exploring the mechanisms of liver diseases. Based on its variable region sequence, a recombinant fluorescent antibody, Oit3-scFv-GFP, was also constructed. This recombinant fluorescent antibody enables one-step direct immunofluorescence staining, providing a convenient tool for in situ visualization of mouse hepatic sinusoidal endothelial cells.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medical immunology, specifically relating to monoclonal antibodies against Oit3 protein in mouse hepatic sinusoidal endothelial cells or their antigen-binding fragments and their applications. Background Technology

[0002] Liver sinusoidal endothelial cells (LSECs) are a highly specialized type of endothelial cell in the liver, playing a crucial role in maintaining liver homeostasis, regulating substance exchange, mediating immune surveillance, and participating in pathophysiological processes such as liver injury and regeneration. Therefore, the specific identification, isolation, and study of LSECs are of great significance for a deeper understanding of liver physiology and disease mechanisms.

[0003] Currently, biomarkers used for identifying and studying LSECs mainly include Cdh5, LYVE-1, and CD31. However, these biomarkers suffer from significant specificity deficiencies in practical applications. For example, Cdh5 is widely expressed in all endothelial cells; while LYVE-1 is highly expressed in LSECs, it is also expressed in lymphoendothelial cells and certain macrophage subsets; CD31 is similarly widely expressed in vascular endothelial cells. This overlap in expression profiles makes it difficult to accurately distinguish LSECs from other cell types (such as vascular endothelial cells, lymphoendothelial cells, and macrophages) using these biomarkers, introducing uncertainties into the purification, functional studies, and targeted interventions of LSECs.

[0004] Oit3 (Uniprot ID: Q8R4V5) is a liver-associated protein that has attracted attention in recent years. Previous studies have shown that Oit3 can serve as one of the best marker genes for distinguishing mouse hepatic sinusoidal endothelial cells (LSECs). The Oit3-CreERT2-tdTomato mouse model established based on this provides an important tool for lineage tracing and functional studies of LSECs (see SignalTransduct Target Ther. 2023 Sep 12;8(1):344). These advances indicate that the potential of Oit3 as a specific marker for LSECs is gradually being recognized.

[0005] Despite the increasing value of Oit3 as a biomarker for liver secundum (LSECs), current antibody tools targeting this target remain significantly insufficient to meet diverse research needs. On one hand, most commercially available Oit3 antibodies are polyclonal, exhibiting significant batch-to-batch variability and inconsistent specificity. The affinity and sensitivity of a few monoclonal antibodies have not been systematically validated, and their applicability in different detection techniques such as ELISA, Western blotting, and immunofluorescence varies, limiting their application in demanding experimental settings. On the other hand, there are currently no fluorescently labeled antibodies against Oit3 protein, particularly recombinant fluorescent antibodies capable of one-step direct immunofluorescence detection. This restricts the dynamic observation and study of LSECs in their in situ microenvironment. Therefore, there is an urgent need to develop a high-performance, highly specific, and stable anti-Oit3 monoclonal antibody applicable to multiple detection techniques, especially a recombinant fluorescent antibody capable of direct immunofluorescence detection, to meet the needs of basic research on LSECs and the exploration of liver disease mechanisms. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or its antigen-binding fragment and its application, so as to solve the technical problems of insufficient specificity and inability to achieve stable detection in multiple scenarios of existing Oit3 antibody tools.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention discloses a monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 3 to SEQ ID NO. 5; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 8 to SEQ ID NO. 10.

[0008] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0009] Preferably, the heavy chain subtype of the monoclonal antibody is the IgG1 subtype, and the light chain is the kappa chain.

[0010] Preferably, the equilibrium dissociation constant KD of the monoclonal antibody or its antigen-binding fragment binding to the Oit3 protein is ≤2.63×10⁻⁶. -9 M.

[0011] Preferably, the rate constant ka for the binding of the monoclonal antibody or its antigen-binding fragment to the Oit3 protein, as determined by biomembrane interference technology, is 2.67 × 10⁻⁶. 5 M -1 s -1 The dissociation rate constant kd is 7.02 × 10⁻⁶. -4 s -1 .

[0012] In a second aspect, the present invention discloses a nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof that encodes the above-mentioned anti-mouse hepatic sinusoidal endothelial cell Oit3 protein.

[0013] A third aspect of the present invention discloses a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0014] A fourth aspect of the present invention discloses a host cell comprising the above-described recombinant expression vector.

[0015] A fifth aspect of the present invention discloses a recombinant fluorescent antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells, comprising the above-mentioned monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or its antigen-binding fragment, and a detectable marker fused to or conjugated thereto.

[0016] Preferably, the monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or its antigen-binding fragment is a single-chain antibody scFv, and the detectable marker is green fluorescent protein.

[0017] More preferably, the method for constructing the recombinant fluorescent antibody is as follows: by inserting a gene fragment encoding the monoclonal antibody or its antigen-binding fragment against the Oit3 protein of mouse hepatic sinusoidal endothelial cells into the pcDNA3.4-TOPO eukaryotic expression vector using molecular cloning technology, and then expressing and purifying the antibody to obtain the recombinant fluorescent antibody.

[0018] In a sixth aspect, the invention discloses the use of a monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or its antigen-binding fragment, or the aforementioned recombinant fluorescent antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells, in the preparation of a reagent for detecting Oit3 protein.

[0019] Preferably, the detection is an enzyme-linked immunosorbent assay (ELISA), immunoblotting, or immunofluorescence assay.

[0020] A seventh aspect of the present invention discloses an enzyme-linked immunosorbent assay (ELISA) method for detecting Oit3 protein in a sample, comprising the step of using a monoclonal antibody or its antigen-binding fragment that specifically binds to Oit3 protein in mouse hepatic sinusoidal endothelial cells as a detection antibody.

[0021] An eighth aspect of the present invention discloses an immunoblotting method for detecting Oit3 protein in a sample, comprising the step of using a monoclonal antibody or its antigen-binding fragment that specifically binds to Oit3 protein in mouse hepatic sinusoidal endothelial cells as a primary antibody.

[0022] A ninth aspect of the present invention discloses an immunofluorescence method for detecting Oit3 protein in a sample, comprising the steps of using the above-described monoclonal antibody or antigen-binding fragment thereof that specifically binds to Oit3 protein in mouse hepatic sinusoidal endothelial cells, or the above-described recombinant fluorescent antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells.

[0023] Preferably, the sample is a slice of liver tissue.

[0024] Preferably, the method is a direct immunofluorescence staining method, using the above-mentioned recombinant fluorescent antibody targeting Oit3 protein in mouse hepatic sinusoidal endothelial cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or its antigen-binding fragment provided by this invention has the heavy chain subtype IgG1 and the light chain kappa chain, and possesses the following superior properties: 1) High specificity: ELISA verification shows that this monoclonal antibody can specifically recognize recombinant Oit3 protein, with no cross-reactivity with irrelevant protein BSA, and irrelevant isotype control antibodies also cannot recognize Oit3 antigen; Western blot verification shows that this monoclonal antibody only reacts with Oit3 protein in primary mouse hepatic sinusoidal endothelial cells, and has no effective cross-reactivity with hepatocytes, hepatic macrophages, and hepatic stellate cells; 2) High affinity: BLI technology determination shows that the equilibrium dissociation constant KD of this monoclonal antibody binding to Oit3 antigen is 2.63 × 10⁻⁶. -9 M, reaching nanomolar level of high affinity; ELISA method to determine EC 50 The value was 0.034 μg / mL. Therefore, this monoclonal antibody or its antigen-binding fragment can specifically recognize the Oit3 protein, solving the problems of insufficient specificity and inability to achieve stable detection in multiple scenarios in existing Oit3 antibody tools. It provides important tool support for basic research on LSECs and the exploration of the mechanisms of liver diseases, and has broad application prospects and significant industrial value.

[0026] The application of monoclonal antibodies or antigen-binding fragments of anti-Oit3 protein from mouse hepatic sinusoidal endothelial cells, or recombinant fluorescent antibodies against Oit3 protein from mouse hepatic sinusoidal endothelial cells, provided by this invention in the preparation of detection reagents for Oit3 protein, successfully applies monoclonal antibodies or antigen-binding fragments of Oit3 protein specifically bound to mouse hepatic sinusoidal endothelial cells to various immunoassay techniques, providing a systematic tool for the detection of Oit3 protein: 1) ELISA detection method: A highly specific and clear background indirect ELISA method was established. By setting PBS control, irrelevant protein coating control, and irrelevant antibody control, the high specificity of this monoclonal antibody in ELISA detection was confirmed, and it can be used for qualitative or semi-quantitative detection of Oit3 protein; 2) Western ELISA detection method... Blot detection method: An immunoblotting method was established for detecting Oit3 protein in Oit3-overexpressing cells and primary mouse LSECs. The target band was clear with a clean background, providing a reliable tool for molecular weight identification and expression analysis of Oit3 protein. 3) Immunofluorescence detection method: An immunofluorescence staining method was established for the localization of Oit3 protein at the cellular and tissue levels, clearly showing the distribution of LSECs in liver tissue. Therefore, this monoclonal antibody or its antigen-binding fragment can be used for various immunoassays, including enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunofluorescence detection, meeting the detection needs of Oit3 protein in different application scenarios.

[0027] The recombinant fluorescent antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells (LSECs) provided by this invention, based on the variable region sequence of an anti-Oit3 monoclonal antibody, successfully designed, expressed, and purified the Oit3-scFv-GFP recombinant fusion protein, which has the following advantages: 1) One-step direct detection: No secondary antibody incubation is required, simplifying the operation process, reducing non-specific background, and improving detection efficiency; 2) High specificity: In frozen liver tissue sections of the Oit3-CreERT2-tdTomato mouse primary liver cancer model, the green fluorescence signal of Oit3-scFv-GFP and the inherent tdTomato red fluorescence signal of Oit3-CreERT2-tdTomato mice are highly colocalized, confirming its specific recognition ability for LSECs; 3) In situ visualization: It can realize the direct observation of LSECs in the in situ microenvironment, providing a convenient tool for studying the biological functions of LSECs. Attached Figure Description

[0028] Figure 1 This is an SDS-PAGE image of the purified Oit3 recombinant protein of the present invention; wherein, lane M is the protein molecular weight standard, and lane 1 is the purified Oit3 recombinant protein; Figure 2This is a graph showing the serum antibody titer of mice on day 42 after immunization according to the present invention; where the vertical axis P / N represents the Positive / Negative ratio, and the horizontal axis represents the serum dilution ratio. Figure 3 This is a graph showing the indirect immunofluorescence detection results of the culture supernatant of 7 positive monoclonal cells of the present invention; wherein, the red fluorescence represents AF594-labeled Oit3 positive cells, scale bar: 50 μm; Figure 4 This is a graph showing the titer determination results of the seven anti-Oit3 monoclonal antibodies of this invention; Figure 5 This is an SDS-PAGE image of the purified anti-Oit3 monoclonal antibody (5E30) of the present invention; wherein lane M is the protein molecular weight standard and lane 1 is the purified monoclonal antibody. Figure 6 This is a graph showing the binding activity analysis of the anti-Oit3 monoclonal antibody (5E30) of the present invention with the Oit3 antigen; Figure 7 This is a graph showing the results of the affinity constant determination between the anti-Oit3 monoclonal antibody (5E30) of this invention and the Oit3 antigen; Figure 8 This is a structural simulation diagram of the Oit3-5E30 antigen-antibody complex of the present invention; Figure 9 This is a graph showing the results of Western blotting detection of the specificity of the anti-Oit3 monoclonal antibody (5E30) with overexpressing cell lysates, using the anti-Oit3 monoclonal antibody (5E30) as the primary antibody. Figure 10 This is a diagram showing the results of Western blotting, which uses anti-Oit3 monoclonal antibody (5E30) as the primary antibody to detect its specificity in recognizing mouse LSECs at the primary cell level. Figure 11 This is a diagram showing the results of the direct immunofluorescence (IF) assay of the Oit3-scFv-GFP antibody as a detection probe to detect its specificity in recognizing mouse LSECs at the tissue level. In the diagram, green fluorescence represents Oit3-scFv-GFP, red fluorescence represents tdTomato-labeled LSECs, and blue represents DAPI nuclear staining. Scale bar: 100 μm. Detailed Implementation

[0029] To enable those skilled in the art to understand the features and effects of the present invention, the following description and definitions are only general descriptions of the terms and expressions mentioned in the specification. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0030] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0031] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0032] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0033] In this article, the term "antibody" includes whole antibodies and any antigen-binding fragment thereof. Antibodies include murine antibodies, humanized antibodies, bispecific antibodies, or chimeric antibodies. Antibodies can also be Fab, F(ab)2, Fv, or ScFv (single-chain antibodies). Antibodies can be naturally occurring antibodies or antibodies that have been altered (e.g., by mutation, deletion, substitution, etc.).

[0034] In this article, the terms "variable region" and "constant region" refer to the sequence regions near the N-terminus of the antibody heavy and light chains, which are called the variable regions (V regions), and the remaining amino acid sequences near the C-terminus, which are relatively stable and are called the constant regions (C regions). The variable regions include 3 complementarity-determining regions (CDRs) and 4 framework regions (FRs). Each light chain variable region and heavy chain variable region consists of 3 CDR regions and 4 FR regions. The 3 CDR regions of the heavy chain are represented by HCDR1, HCDR2, and HCDR3, respectively, and the 3 CDR regions of the light chain are represented by LCDR1, LCDR2, and LCDR3, respectively.

[0035] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0036] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0037] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0038] Example 1: Preparation of Oit3 recombinant protein This embodiment aims to prepare high-purity Oit3 recombinant protein to provide an immunogen and detection antigen for subsequent screening and identification of monoclonal antibodies.

[0039] I. Construction of the eukaryotic expression vector pcDNA3.1-Oit3 Based on the reference sequence of the Oit3 protein in the UniProt database (UniProt ID: Q8R4V5), the entire gene encoding the Oit3 protein was synthesized, and codon bias optimization was performed for mammalian cell expression systems. To facilitate subsequent protein detection and purification, nucleotide sequences encoding the (GGS)3 flexible linker peptide and the 6×His tag were sequentially added to the 3' end of the optimized gene sequence. The designed and synthesized target gene fragment (the gene encoding the recombinant Oit3 protein, nucleotide sequence shown as SEQ ID NO.11 in Table 1) was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into the pcDNA3.1(+) eukaryotic expression vector using restriction endonucleases NheⅠ and NotⅠ to construct the recombinant expression plasmid, named pcDNA3.1-Oit3.

[0040] II. Expression and purification of Oit3 recombinant protein The pcDNA3.1-Oit3 recombinant plasmid, whose sequencing accuracy was verified, was transfected into HEK293F suspension cells for transient expression. 72 h post-transfection, the cell culture supernatant was collected. A small amount of the supernatant was used for anti-His Western blot analysis to verify successful expression of the target protein. After confirming expression, the expanded cell culture was centrifuged at 10,000 rpm for 20 min at 4°C to remove cell debris. The collected supernatant was filtered through a 0.22 μm sterile filter and purified using a nickel-NTA affinity chromatography column. The elution fraction containing the target protein was collected using an imidazole gradient elution to obtain the purified Oit3 recombinant protein.

[0041] The purified products were identified by SDS-PAGE gel electrophoresis and stained with Coomassie Brilliant Blue. Results are as follows: Figure 1 As shown, under reducing conditions, the Oit3 recombinant protein appears as a single, clear band with a molecular weight between 60 and 70 kDa, consistent with theoretical expectations. Gel image analysis software confirmed that the purified Oit3 recombinant protein had a purity greater than 95%, indicating that high-purity Oit3 recombinant protein was successfully obtained and can be used for subsequent antibody preparation experiments.

[0042] Example 2: Preparation of anti-Oit3 monoclonal antibody This embodiment aims to use the prepared Oit3 recombinant protein as an immunogen to prepare a monoclonal antibody that specifically recognizes Oit3 through hybridoma technology.

[0043] I. Animal Immunization The Oit3 recombinant protein purified in Example 1 was used as the immunogen. For the first immunization, the Oit3 recombinant protein was thoroughly emulsified with Freund's complete adjuvant at a volume ratio of 1:1. Six 6-8 week old female BALB / c mice were selected and immunized via subcutaneous injection at multiple sites on the back, with an immunization dose of 10 μg / mouse (total volume 200 μL / mouse). Booster immunizations were then performed at 14-day intervals. For the second and third immunizations, Freund's incomplete adjuvant was used to emulsify the antigen, while maintaining the same immunization dose and route.

[0044] Blood was collected from the tail tip of mice on day 28 (14 days after the second immunization) and day 42 (14 days after the third immunization), and serum was separated. Serum antibody titers were detected by indirect ELISA. Based on the test results, mice with the highest serum antibody titers were selected for pulse immunization. Pulse immunization was performed via intraperitoneal injection of recombinant Oit3 protein without any adjuvant at a dose of 50 μg / mouse.

[0045] II. Determination of Antibody Titer in Immunized Mice Serum The titer of anti-Oit3 antibody in mouse serum collected on days 28 and 42 was determined using an indirect ELISA method. The specific steps are as follows: (1) Antigen coating: Dilute the Oit3 recombinant protein to 1 μg / mL with ELISA coating buffer, add 100 μL / well to a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer, wash three times with PBST, and pat dry. (2) Blocking: Add 200 μL of blocking buffer (PBST containing 5% skim milk powder) to each well and block at 37°C for 2 h. Washing is the same as in step (1); (3) Primary antibody incubation: The serum to be tested was serially diluted 2-fold with PBST, and 100 μL / well was added to the microplate and incubated at 37°C for 1 h. The serum diluent was discarded and the plate was washed 6 times with PBST. (4) Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgG antibody diluted 1:2000 with PBST to each well, and incubate at 37°C for 45 min. Discard the secondary antibody dilution buffer and wash 6 times with PBST; (5) Color development and termination: Add 100 μL of single-component TMB color development solution to each well and react at room temperature in the dark for 20 min. Then add 50 μL of ELISA termination solution to each well to terminate the reaction. (6) Data reading: Immediately measure the absorbance (OD) of each well at a wavelength of 450 nm using a microplate reader. 450 The data was organized and analyzed using Excel software, and the serum antibody titers of each mouse were calculated.

[0046] The serum antibody titer of each mouse on day 42 (14 days after the third immunization) is as follows: Figure 2 As shown, the serum titers of two BALB / c mice reached 1:102400. Mouse number 6 was randomly selected for a shock immunization for subsequent cell fusion experiments.

[0047] III. Cell Fusion Three days after the initial immunization, mice (size 6) were euthanized by cervical insemination (blood was collected from the orbital cavity before euthanasia to serve as a positive control for subsequent testing), and their bodies were disinfected in 75% alcohol. Cell fusion was then performed aseptically in a laminar flow hood, following these steps: (1) Preparation of myeloma cells: Collect mouse Sp2 / 0 myeloma cells in the logarithmic growth phase, and count them to approximately (2~5) × 10⁻⁶. 7 Each was placed in a centrifuge tube; (2) Preparation of spleen cell suspension: The spleen of the mouse was aseptically removed and placed on a 200-mesh sterile filter. It was then thoroughly minced with ophthalmic scissors, and simultaneously rinsed and ground with 1640 basal culture medium to filter the spleen cells into a sterile beaker below. The collected spleen cell suspension was transferred to a centrifuge tube and 1640 culture medium was added to bring the total volume to 40 mL. (3) Washing: Centrifuge the spleen cell suspension and Sp2 / 0 myeloma cells together at 1200 rpm for 10 min and discard the supernatant; (4) Mixing: Resuspend spleen cells and Sp2 / 0 myeloma cells in 10 mL of 1640 medium, respectively. Transfer the spleen cell suspension to Sp2 / 0 cell tubes and mix. Add 1640 medium to 40 mL. Centrifuge again at 1200 rpm for 10 min and discard the supernatant. (5) Fusion: Gently break up the cell clusters, and slowly add 1 mL of fusion agent (50% PEG1500) preheated to 37°C over 1 min, and let stand for 90 s. Then, slowly add 15 mL of 1640 medium over 2 min to terminate the fusion (add 1 mL in the first 30 s, 3 mL in the second 30 s, and then gradually increase the rate of addition of the remaining 11 mL). Stabilize the cell suspension in a 37°C water bath for 5 min, add 1640 medium to 40 mL, and centrifuge at 1000 rpm for 10 min; (6) Resuspension: Discard the supernatant, gently break up the cell clusters, add HAT selection medium, and mix gently (avoid vigorous blowing to avoid damaging the fused cells). (7) Plating: The cell suspension was seeded into 96-well cell culture plates at 250 μL / well and cultured in a 37°C, 5% CO2 incubator; (8) Culture and observation: After 3-4 days of culture, small cell clusters can be observed under a microscope. After about 9-12 days of culture, the supernatant of hybridoma cells can be collected for testing.

[0048] IV. Screening and Identification of Hybridoma Cells On day 11 after cell fusion, the supernatant of hybridoma cells was screened using indirect ELISA and indirect immunofluorescence assay.

[0049] 1. Indirect ELISA screening (1) Coating: Dilute the Oit3 recombinant protein to an appropriate concentration with ELISA coating buffer, add 50 μL / well (containing 50 ng antigen) to a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer, wash three times with PBST, and pat dry. (2) Blocking: Add 200 μL of blocking solution to each well, block at 37°C for 2 h, and wash as before; (3) Primary antibody incubation: Add 50 μL of hybridoma cell culture supernatant to each well, and set up a positive control (pre-fusion mouse serum) and a negative control (pre-immunization mouse serum). Incubate at 37°C for 30 min. Discard the supernatant and wash 6 times with PBST; (4) Secondary antibody incubation: Add 50 μL of HRP-labeled goat anti-mouse IgG antibody diluted with PBST at a ratio of 1:5000 to each well, incubate at 37°C for 30 min, and wash 6 times; (5) Color development: Add 50 μL of single-component TMB color development solution to each well, react at room temperature in the dark for 20 min, and observe the color development results.

[0050] 2. Verification by indirect immunofluorescence method (1) Cell seeding: 293T cell line overexpressing Oit3 (positive control) and empty 293T cell line (negative control) were seeded into 96-well cell culture plates, 100 μL of cell suspension per well, and cultured overnight at 37°C in a 5% CO2 incubator. (2) Fixation and blocking: Discard the culture supernatant and wash each well three times with PBS. Add PBS solution containing 4% paraformaldehyde and fix at room temperature for 15 min. Discard the fixative and wash three times with PBS. Add QuickBlock. TM Western blot solution, block at room temperature for 15 min; (3) Primary antibody incubation: Discard the blocking solution, add 100 μL of hybridoma cell culture supernatant to each well as the primary antibody, and incubate overnight at 4°C. Discard the supernatant, wash three times with PBS for 5 min each time; (4) Secondary antibody incubation: Add 100 μL of AF594-labeled goat anti-mouse IgG (H+L) antibody diluted 1:1000 with blocking buffer to each well, and incubate at room temperature in the dark for 1 h. Discard the secondary antibody, and wash 3 times with PBS for 5 min each time; (5) Microscopic observation: Add DAPI staining solution to each well and stain at room temperature in the dark for 5 min. After rinsing 3 times with PBS, add 100 μL of PBS to each well and observe under an inverted fluorescence microscope. If red fluorescence appears in Oit3 overexpressing cells incubated with hybridoma supernatant, while no red fluorescence appears in blank 293T cells, it is considered a positive clone and can proceed to the next step of cloning.

[0051] Based on the above test results, highly reactive positive hybridoma cells were selected and transferred to 24-well cell culture plates, where HT selection medium was added for expansion culture. After 2-3 days of culture, cell growth was observed, and secondary screening was performed using indirect ELISA and indirect immunofluorescence. Wells with positive results and cell density exceeding 80% were transferred to 6-well cell culture plates, at which point the medium was replaced with standard 1640 medium containing 10% FBS for further culture. Secondary screening was performed after each transfer to ensure the stability of positive hybridoma cells.

[0052] V. Subcloning of hybridoma cells (limiting dilution method) Cells from 6-well plates that were finally identified as positive in step four, showed good growth, and had a cell density of over 50% were used for subcloning using the limiting dilution method. The specific procedures are as follows: (1) Preparation of feeder cells: One day before subcloning, normal mouse peritoneal macrophages were prepared as feeder cells and seeded into multiple 96-well cell culture plates at 100 μL / well for later use. (2) Cell dilution: Gently blow up and mix the cells to be subcloned, add 200 μL of cell suspension to the first column of a 96-well plate, and then perform serial dilutions of 2-fold with 1640 medium containing 10% FBS. Observe the number of cells in each well under an inverted microscope, and select the dilution wells with 10-15 cells in the field of view (i.e., the total number of cells in the whole well is less than 100). (3) Plating: Add all the cells from the dilution wells to 10 mL of 1640 medium containing 10% FBS, mix thoroughly, and then seed 100 μL / well into a 96-well plate pre-coated with feeder cells (theoretically ensuring no more than one cell per well). Subcloning of each hybridoma cell line is performed using the same method; (4) Culture and identification: Culture for 7-10 days. When the cell cluster grows to 1 / 5 of the bottom area of ​​the well, use the method described in step four for detection and identification. (5) Repeated subcloning: Subcloning is performed on the positive wells again, and the above steps are repeated until a hybridoma cell line that stably secretes monoclonal antibodies is obtained (i.e., the positive well rate in the 96-well plate reaches 100%).

[0053] After three limiting dilutions and subcloning, seven hybridoma cell lines stably secreting anti-Oit3 monoclonal antibodies were obtained in this experiment, named 2B27, 3C35, 4D36, 5E30, 6F13, 7G17, and 8H22. The results of indirect immunofluorescence experiments on the cell culture supernatants of each cell line are shown below. Figure 3 As shown, all results exhibit good specificity.

[0054] VI. Preparation and purification of ascites fluid containing anti-Oit3 monoclonal antibody The seven stable antibody-secreting hybridoma cell lines obtained from the final screening were expanded and cultured, and then cultured at a rate of (1~2)×10⁻⁶. 6 Cell / tube density was used for cryopreservation. Female BALB / c mice aged 12-14 weeks were selected, and each mouse was injected intraperitoneally with 500 μL of sterile paraffin oil. After 7-10 days, the expanded hybridoma cell lines were collected by centrifugation, resuspended in PBS, and divided into doses of approximately 1 × 10⁻⁶ cells per mouse. 7 A specific number of cells were injected intraperitoneally. Mice were closely monitored for condition and abdominal distension. Ascites fluid was collected approximately 10 days later. The ascites fluid was centrifuged at 4000 rpm for 20 min to remove the upper oil layer and the lower cell precipitate, collecting the clear yellow liquid in the middle. Antibody purification was performed using a HiTrap Protein G pre-packed column and an ÄKTA pure chromatography system.

[0055] The titers of the seven purified antibodies were compared using an indirect ELISA method, and the results are as follows: Figure 4As shown in the figure, the hybridoma cell line 5E30 secreted the highest monoclonal antibody titer, reaching 1:256000. The purity of the anti-Oit3 monoclonal antibody (5E30) was determined by SDS-PAGE gel electrophoresis, and the results are as follows. Figure 5 As shown, under reducing conditions, a clear band of approximately 50 kDa immunoglobulin heavy chain and approximately 25 kDa light chain are visible, with a purity greater than 95%, indicating that a high-purity anti-Oit3 monoclonal antibody has been successfully obtained.

[0056] Example 3: Identification of anti-Oit3 monoclonal antibody subtypes This embodiment aims to determine the immunoglobulin isotype of the anti-Oit3 monoclonal antibody (5E30) obtained in Example 2, providing a reference for its subsequent purification process optimization and functional application. A commercial mouse monoclonal antibody isotype identification kit (Proteintech) was used for detection, and the specific operation steps are as follows: (1) Sample dilution: Dilute the anti-Oit3 monoclonal antibody (5E30) with 1×PBST buffer to 200 ng / mL; (2) Sample addition and secondary antibody incubation: Add 50 μL of diluted antibody sample to the sample wells of the ELISA strip. Then add 50 μL of horseradish peroxidase (HRP)-labeled antibodies specific to various mouse immunoglobulin subtypes to each well. Gently mix the ELISA plate on a micro-shaker and incubate at room temperature for 1 h; (3) Washing: After incubation, discard the liquid in the well, add 1×PBST buffer to each well and wash 3 times, and pat dry the remaining liquid on absorbent paper; (4) Color development and termination: Add freshly prepared TMB color development solution to each well and develop the color at room temperature in the dark for 10-20 min. After the color has fully developed, add 50 μL of termination solution to each well to terminate the reaction. (5) Reading and interpretation: Immediately measure the absorbance (OD) of each well at a wavelength of 450 nm using an ELISA reader. 450 Antibody subtypes are determined based on the colorimetric results of the corresponding wells for each subtype.

[0057] The test results showed that the heavy chain subtype of the anti-Oit3 monoclonal antibody (5E30) was IgG1, and the light chain type was kappa chain.

[0058] Example 4: Affinity determination of anti-Oit3 monoclonal antibody This embodiment aims to determine the binding affinity between the anti-Oit3 monoclonal antibody (5E30) prepared in Example 2 and the Oit3 antigen protein using enzyme-linked immunosorbent assay (ELISA) and bio-layer interference (BLI) technology.

[0059] I. Antibody Binding Activity Analysis Based on ELISA The binding activity of the anti-Oit3 monoclonal antibody (5E30) to the recombinant Oit3 protein was determined by indirect ELISA, and the half-maximal effective concentration (EC50) was calculated. 50 Assess its relative affinity. The specific steps are as follows: (1) Antigen coating: The Oit3 recombinant protein prepared in Example 1 was serially diluted with ELISA coating buffer and added to a 96-well microplate at 100 μL / well. The plate was incubated overnight at 4°C. The coating buffer was discarded, and the plate was washed three times with PBST and patted dry. (2) Blocking: Add 200 μL of blocking buffer (PBST containing 5% skim milk powder) to each well and block at 37°C for 2 h. Washing is the same as in step (1); (3) Primary antibody incubation: Dilute the anti-Oit3 monoclonal antibody (5E30) with PBST to 1 μg / mL, add 100 μL / well to the microplate, and set up negative control wells (add an equal volume of PBS instead of primary antibody). Incubate at 37°C for 1 h. Discard the antibody dilution solution, wash 6 times with PBST, and pat dry. (4) Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgG antibody diluted 1:5000 to each well and incubate at 37°C for 45 min. Discard the secondary antibody dilution buffer, wash 6 times with PBST, and pat dry; (5) Color development and termination: Add 100 μL of single-component TMB color development solution to each well and react at room temperature in the dark for 15 min. Then add 50 μL of ELISA stop solution to each well to terminate the reaction; (6) Data reading and analysis: Immediately measure the absorbance (OD) of each well at a wavelength of 450 nm using a microplate reader. 450 The binding curve was plotted using a four-parameter logistic fitting model, and the half-maximal effective concentration (EC50) was calculated. 50 )value.

[0060] Test results as follows Figure 6 As shown. Through fitting calculations, the EC50 of the anti-Oit3 monoclonal antibody (5E30) binding to the Oit3 antigen was calculated. 50 The value was 0.034 μg / mL, indicating that the antibody has good binding activity with the Oit3 antigen.

[0061] II. Determination of Affinity Constant Based on Biomembrane Interferometry Biomembrane interferometry (BLI) was used to further determine the kinetic binding constant and equilibrium dissociation constant between the anti-Oit3 monoclonal antibody (5E30) and the Oit3 antigen. This technique accurately calculates the kinetic parameters of molecular interactions by real-time monitoring the optical interference spectral shift caused by the binding and dissociation of molecules immobilized on the surface of the biosensor with the analyte in solution. The specific experiments were performed on the Fortebio Octet® molecular interaction analysis system, and the operating procedures are as follows: (1) Ligand immobilization: The anti-Oit3 monoclonal antibody (5E30) was diluted to an appropriate concentration with kinetic buffer and immobilized on the surface of the biosensor by amino coupling or capture. (2) Baseline equilibration: Immerse the antibody-immobilized sensor in kinetic buffer to establish a stable baseline signal; (3) Binding kinetics determination: The sensor was immersed in a solution containing a series of gradient concentrations of Oit3 antigen protein, and the binding process was monitored in real time and the binding rate was recorded; (4) Dissociation kinetics determination: The sensor was transferred to a kinetic buffer solution without antigen, the dissociation process was monitored in real time, and the dissociation rate was recorded; (5) Data analysis: Use the instrument software to perform global fitting of the binding and dissociation curves, and calculate the binding rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (KD = kd / ka).

[0062] Test results as follows Figure 7 As shown, the binding rate constant ka between the anti-Oit3 monoclonal antibody (5E30) and the Oit3 antigen is 2.67 × 10⁻⁶. 5 M -1 s -1 The dissociation rate constant kd is 7.02 × 10⁻⁶. -4 s -1 The equilibrium dissociation constant KD is 2.63 × 10⁻⁶. -9 The M.KD value is at the nanomolar level, indicating that the antibody has a high affinity for the Oit3 antigen.

[0063] In summary, through dual verification by ELISA and BLI methods, the anti-Oit3 monoclonal antibody (5E30) prepared in this invention exhibits high affinity binding to the Oit3 antigen, laying the foundation for subsequent immunoassay and functional research applications.

[0064] Example 5: Cloning and Bioinformatics Analysis of the Variable Region Gene of Anti-Oit3 Monoclonal Antibody In order to resolve the structural features of the anti-Oit3 monoclonal antibody (5E30) at the molecular level and to provide a sequence basis for subsequent genetic engineering (such as constructing Oit3-scFv-GFP), the variable region gene of the anti-Oit3 monoclonal antibody (5E30) was cloned, sequenced and analyzed by bioinformatics in this embodiment.

[0065] I. Total RNA extraction and variable region gene amplification from hybridoma cells Total RNA was extracted from the hybridoma cell line 5E30, which secretes anti-Oit3 monoclonal antibody, and cDNA was synthesized by reverse transcription. Using this cDNA as a template, polymerase chain reaction (PCR) was performed using universal primers for the variable region (VH) and variable region (VL) of mouse IgG heavy chain to amplify the antibody variable region gene. The PCR reaction program was as follows: 98°C pre-denaturation for 5 min; followed by 35 cycles (98°C denaturation for 10 s, 60°C annealing for 15 s, 72°C extension for 1 min); and a final extension at 72°C for 10 min. The amplified products were separated by agarose gel electrophoresis, purified by gel extraction, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0066] II. Gene Sequencing and Bioinformatics Analysis The sequenced nucleotide sequences were analyzed for homology and gene structure using the International Immunogenetic Information System (IMGT / V-QUEST, http: / / www.imgt.org / IMGT_vquest / vquest) and the IGBLAST database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / igblast / igblast.cgi). Simultaneously, the tertiary structure of the antibody was predicted using the SWISS-MODEL (https: / / swissmodel.expasy.org) homology modeling server, and molecular docking simulations were performed with the target antigen Oit3 protein. Finally, the simulated antigen-antibody complex structure was visualized and analyzed using the PyMOL molecular visualization system.

[0067] III. Sequencing Results and Structural Analysis Sequencing and analysis results showed that the variable region sequence of the anti-Oit3 monoclonal antibody (5E30) had high homology (>95%) with the reported variable region sequences of mouse antibodies, confirming it as a mouse antibody. Specific sequence information is shown in Table 1. Table 1 Sequence List

[0068] Heavy chain variable region (VH): Its nucleotide sequence is shown in SEQ ID NO.1 in Table 1, and the corresponding amino acid sequence is shown in SEQ ID NO.2 in Table 1. This variable region contains three complementarity-determining regions (CDRs) and four frame regions (FRs); among them, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 in Table 1, respectively.

[0069] Light chain variable region (VL): Its nucleotide sequence is shown in SEQ ID NO.6 of Table 1, and the corresponding amino acid sequence is shown in SEQ ID NO.7 of Table 1. This variable region also contains three complementarity-determining regions (CDRs) and four frame regions (FRs); among which, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 of Table 1, respectively.

[0070] Structural analysis revealed that the VH and VL genes are structurally complete, each containing three complementarity-determining regions (CDRs) and four framework regions (FRs). Homology modeling and molecular docking simulations showed that the CDR regions of the heavy and light chains are spatially close to each other, forming a stable antigen-binding pocket. This is the molecular basis for the antibody's ability to specifically recognize the Oit3 protein. Figure 8 The simulated structure of the Oit3-5E30 antigen-antibody complex is clearly shown, and the interaction mode between the antigen epitope and the antibody complementarity-determining region is intuitively presented.

[0071] This embodiment successfully resolved the molecular structural features of the anti-Oit3 monoclonal antibody (5E30), identified the key sequence of its variable region, provided a structural basis for understanding its antigen-binding specificity, and also laid the foundation for subsequent genetic engineering modification and application development based on this antibody.

[0072] Example 6: Application of anti-Oit3 monoclonal antibody in enzyme-linked immunosorbent assay (ELISA) detection This embodiment aims to verify the specificity and effectiveness of the prepared anti-Oit3 monoclonal antibody (5E30) as a detection antibody in enzyme-linked immunosorbent assay (ELISA). Its ability to recognize the target protein is evaluated by detecting the reactivity of this antibody with recombinant Oit3 protein. Specific experimental steps are as follows: (1) Antigen coating: The Oit3 recombinant protein prepared in Example 1 was diluted to 1 μg / mL with ELISA coating buffer and added to a 96-well microplate at 100 μL / well. The plate was incubated overnight at 4°C. An irrelevant protein coating control was also set up. Bovine serum albumin (BSA) was diluted to 1 μg / mL with ELISA coating buffer and coated in parallel at 100 μL / well. The coating buffer was discarded, and the plate was washed three times with PBST for 3 min each time, and then patted dry. (2) Blocking: Add 200 μL of blocking buffer (PBST containing 5% skim milk powder) to each well and block at 37°C for 2 h. Discard the blocking buffer, wash 3 times with PBST, and pat dry. (3) Primary antibody incubation: Dilute the anti-Oit3 monoclonal antibody (5E30) and another unrelated isotype control antibody to 1 μg / mL with PBST, add 100 μL / well to the microplate, and incubate at 37°C for 1 h. Discard the antibody dilution solution, wash 6 times with PBST, and pat dry; (4) Secondary antibody incubation: Add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody diluted 1:2000 with PBST to each well, and incubate at 37°C for 45 min. Discard the secondary antibody dilution buffer, wash 6 times with PBST, and pat dry; (5) Color development and termination: Add 100 μL of single-component TMB color development solution to each well and react at room temperature in the dark for 15 min. Then add 50 μL of ELISA stop solution to each well to terminate the reaction; (6) Result reading: Immediately measure the absorbance (OD) of each well at a wavelength of 450 nm using a microplate reader. 450 ).

[0073] Table 2 Results of enzyme-linked immunosorbent assay (ELISA) for the reactivity of primary antibody with recombinant Oit3 protein.

[0074] The experimental results are shown in Table 2. The experimental group using anti-Oit3 monoclonal antibody (5E30) as the primary antibody and recombinant Oit3 protein as the coating antigen had the highest OD value. 450 The OD values ​​were significantly higher than those of the negative control groups. Specifically, the OD values ​​of the PBS-substituted primary antibody control group, the BSA-coated control group, and the BSA-coated control group were significantly higher. 450 The values ​​were all at background levels, in stark contrast to the experimental group.

[0075] The results of this embodiment demonstrate that the anti-Oit3 monoclonal antibody (5E30) prepared in this invention can specifically recognize and bind to the recombinant Oit3 protein coated on a solid-phase support, exhibits no cross-reactivity with the irrelevant protein BSA, and the irrelevant isotype control antibody also fails to recognize the Oit3 antigen. Therefore, this antibody can be effectively applied to immunoassays based on the ELISA principle, providing a reliable tool with high specificity and clear background for the detection of Oit3 protein.

[0076] Example 7: Application of anti-Oit3 monoclonal antibody in Western blot detection I. Application of anti-Oit3 monoclonal antibody in Western blot detection of Oit3 overexpressing cells This embodiment aims to verify the specificity and efficacy of the prepared anti-Oit3 monoclonal antibody (5E30) in Western blotting. Its ability to recognize the target protein is evaluated by detecting the reactivity of this antibody with cell lysates overexpressing Oit3 protein. Specific experimental steps are as follows: (1) Cell sample preparation: The eukaryotic expression plasmid pcDNA3.1-Oit3 carrying the Oit3 gene and the empty control plasmid pcDNA3.1 were transfected into 293T cells. After culturing at 37°C for 48 h, the culture medium was discarded, and the cells were washed three times with PBS. Cell lysis buffer was added to lyse the cells, and the cells were centrifuged at 12000 rpm for 15 min at 4°C. After determining the protein concentration of the supernatant, an appropriate amount of protein loading buffer was added and mixed thoroughly. The cells were heated in a metal bath at 100°C for 10 min to denature the protein. After cooling, the cells were centrifuged at 12000 rpm for 2 min and set aside. (2) SDS-PAGE electrophoresis and transfer: Equal amounts of prepared cell protein samples were subjected to SDS-PAGE gel electrophoresis. After electrophoresis, the proteins were transferred to a PVDF membrane using a wet transfer method. The transfer jacket was assembled in the order of "cathode plate-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-anode plate", and the membrane was transferred for 2 h at a constant voltage of 100 V. (3) Blocking: After the transfer is completed, the PVDF membrane is taken out and placed in a blocking solution containing 5% skim milk powder. It is blocked at room temperature for 2 hours to block non-specific binding sites. (4) Primary antibody incubation: The blocked PVDF membrane was incubated overnight at 4°C with anti-Oit3 monoclonal antibody (5E30) diluted with 5% skim milk powder (dilution ratio of 1:5000); (5) Secondary antibody incubation: Remove the PVDF membrane and wash it 5 times with 1×PBST buffer at room temperature for 5 min each time. Then, incubate the membrane with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody diluted with 5% skim milk powder (1:2000 dilution) at room temperature for 1 h; (6) Imaging detection: After incubation with the secondary antibody, wash five times with 1×PBST buffer at room temperature, 5 min each time. Mix equal volumes of ECL chemiluminescent substrate solution A and solution B, and add them evenly to the PVDF membrane. Use a chemiluminescence imaging system to detect the antigen signal.

[0077] Experimental results are as follows Figure 9 As shown, the anti-Oit3 monoclonal antibody (5E30) described in this invention only detected a specific target band in 293T cell samples transfected with pcDNA3.1-Oit3 plasmid. The band was clear, had high signal intensity, and a clean background. In contrast, no non-specific bands were observed at the corresponding positions in control cell samples transfected with empty vector plasmids. This result fully demonstrates that the anti-Oit3 monoclonal antibody (5E30) prepared in this invention can highly specifically recognize and bind to the Oit3 protein, exhibiting excellent immunoblotting detection performance.

[0078] II. Application of anti-Oit3 monoclonal antibody in the detection of primary mouse hepatic sinusoidal endothelial cells by Western blotting This embodiment further validates the specificity of the anti-Oit3 monoclonal antibody (5E30) in recognizing mouse hepatic sinusoidal endothelial cells (LSECs) at the primary cell level. Primary hepatocytes, kupffer cells (KCs), LSECs, and hepatic stellate cells (HSCs) were isolated from wild-type mouse livers using an optimized two-step collagenase perfusion method. Proteins were extracted from each of these four cell types to prepare samples. Subsequent SDS-PAGE electrophoresis, transfer, blocking, antibody incubation, and development were performed in accordance with Example 6.

[0079] Experimental results are as follows Figure 10As shown, the results indicate that the anti-Oit3 monoclonal antibody (5E30) of this invention detected a clear Oit3-specific band only in protein samples from primary mouse LSECs; while no obvious bands were observed at the corresponding positions in protein samples from hepatocytes, KCs, and HSCs tested under the same conditions. This result confirms that Oit3 protein is mainly expressed in mouse hepatocytes in LSECs, and that the anti-Oit3 monoclonal antibody (5E30) of this invention can specifically recognize endogenously expressed Oit3 protein, and can serve as a reliable molecular tool for accurately identifying and distinguishing mouse LSECs at the primary cell level.

[0080] Example 8: Application of anti-Oit3 monoclonal antibody in immunofluorescence (IF) detection I. Construction, Expression and Purification of Oit3-scFv-GFP Fusion Antibody To expand the application of anti-Oit3 monoclonal antibody (5E30) in immunofluorescence detection, especially to achieve direct immunofluorescence detection without secondary antibody incubation, this embodiment designs and prepares the fusion expression product Oit3-scFv-GFP of recombinant single-chain antibody (single-chain fragment variable, scFv) and green fluorescent protein (GFP) based on the variable region gene sequence obtained by sequencing in Example 5.

[0081] 1. Construction of recombinant eukaryotic expression vectors Based on the heavy chain variable region (VH, amino acid sequence as shown in SEQ ID NO.2) and light chain variable region (VL, amino acid sequence as shown in SEQ ID NO.7) sequences of the anti-Oit3 monoclonal antibody (5E30) obtained by sequencing in Example 5, a molecular structure was designed according to the sequence: "secretory signal peptide - heavy chain variable region (VH) - linker peptide (G4S)3 - light chain variable region (VL) - linker peptide (G4S)3 - enhanced green fluorescent protein (EGFP) - linker peptide (GGS)2 - 6×His tag". The Oit3-scFv-GFP fusion gene fragment encoding the above structure (nucleotide sequence as shown in SEQ ID NO.12 in Table 1) was synthesized using whole-genome synthesis technology and cloned into the pcDNA3.4-TOPO eukaryotic expression vector using restriction endonucleases BamHⅠ and XhoⅠ to construct the recombinant expression plasmid pcDNA3.4-Oit3-scFv-GFP.

[0082] 2. Expression and purification of Oit3-scFv-GFP The recombinant plasmid, verified by sequencing, was introduced into 293T cells for expression via transient transfection. After transfection, cells were cultured for another 96 h, and the cell culture supernatant was collected. To remove cell debris and other impurities, the collected culture supernatant was centrifuged at 10,000 rpm for 20 min at 4°C and filtered through a 0.22 μm filter membrane.

[0083] Purification was performed using a nickel-NTA affinity chromatography column with a 6×His tag at the C-terminus of the recombinant protein. The treated cell culture supernatant was loaded onto a pre-equilibrated affinity chromatography column. After binding, the sample was washed with a buffer containing an appropriate amount of imidazole to remove non-specifically bound proteins. Finally, the sample was eluted with a high concentration of imidazole elution buffer, and the target protein fraction was collected to obtain the purified Oit3-scFv-GFP fusion antibody.

[0084] II. Application of Oit3-scFv-GFP in the detection of mouse hepatic sinusoidal endothelial cells by direct immunofluorescence method This embodiment aims to verify the application effect of the prepared Oit3-scFv-GFP fluorescent antibody in direct immunofluorescence, and to evaluate its specific labeling ability for Oit3-positive cells in mouse liver tissue. Using the Oit3-scFv-GFP antibody as a detection probe, frozen liver tissue sections from an Oit3-CreERT2-tdTomato mouse primary liver cancer model were stained using direct immunofluorescence staining. The specific recognition ability of this antibody for hepatic sinusoidal endothelial cells (LSECs) was analyzed by observing the colocalization of the green fluorescence signal of Oit3-scFv-GFP and the inherent red fluorescence signal of tdTomato in mice. The specific experimental steps are as follows: (1) Sample preparation and fixation: Frozen liver tissue sections of the prepared Oit3-CreERT2-tdTomato mouse primary liver cancer model were placed at room temperature for 30 min to rewarm, and then fixed with 4% paraformaldehyde for 30 min. (2) Antigen retrieval: Immerse the slides in a retrieval box containing citrate retrieval solution (pH 6.0) and microwave on medium heat for 5 min. After natural cooling, place the slides in PBS buffer and wash three times on a decolorizing shaker for 5 min each time; (3) Endogenous peroxidase blocking: The slices were transferred into 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min to inactivate endogenous peroxidase. After incubation, the slices were washed three times with PBS buffer on a decolorizing shaker for 5 min each time. (4) Blocking: Gently shake the section dry and draw a circle around the tissue with a histochemical pen. Add QuickBlock™ immunostaining blocking solution to the circle and incubate at room temperature for 15 min; (5) Primary antibody incubation: Discard the blocking solution and add Oit3-scFv-GFP antibody diluted at a ratio of 1:200 to the tissue section. Place the section stably in a humidified chamber and incubate overnight at 4°C; (6) Nuclear counterstaining: Discard the antibody working solution, place the slide in PBS buffer, and wash 3 times on a decolorizing shaker for 5 min each time. After slightly drying, add DAPI staining solution to the tissue circle and incubate at room temperature in the dark for 5 min; (7) Mounting: Place the slides in PBS buffer and wash them three times on a decolorizing shaker for 5 min each time. After slightly drying, mount them with anti-fluorescence quenching mounting medium; (8) Image acquisition and analysis: Observe the staining results and acquire images under a fluorescence microscope.

[0085] Experimental results are as follows Figure 11 As shown, in frozen liver tissue sections of an Oit3-CreERT2-tdTomato mouse primary liver cancer model, the green fluorescent signal generated by the Oit3-scFv-GFP antibody exhibits a typical hepatic sinusoidal reticular distribution and is highly co-localized with the Cre recombinase-mediated tdTomato red fluorescent signal in the mouse liver tissue. This result demonstrates that the prepared Oit3-scFv-GFP fluorescent antibody can specifically recognize and bind to Oit3-positive cells, namely hepatic sinusoidal endothelial cells (LSECs), in mouse liver tissue.

[0086] In summary, this embodiment demonstrates that the Oit3-scFv-GFP antibody, as a novel fluorescently labeled probe, can be applied with high specificity to direct immunofluorescence staining of mouse liver tissue sections. This method is simple to operate, produces a clear background, and effectively enables in situ visualization of LSECs, providing an important detection tool for subsequent studies on the tissue localization, quantity statistics, and biological functions of mouse LSECs under physiological and pathological conditions.

[0087] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against mouse liver sinusoidal endothelial cell Oit3 protein, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO.3 to SEQ ID NO.5; and the light chain variable region contains LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO.8 to SEQ ID NO.

10.

2. The monoclonal antibody or antigen-binding fragment thereof against mouse liver sinusoidal endothelial cell Oit3 protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

3. The monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells according to claim 1 or 2, or its antigen-binding fragment, is characterized in that... The heavy chain subtype of the monoclonal antibody is IgG1, and the light chain is kappa chain.

4. The monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells according to claim 1 or 2, or its antigen-binding fragment, is characterized in that... The equilibrium dissociation constant KD of the monoclonal antibody or antigen-binding fragment thereof binding to the Oit3 protein is ≤ 2.63 x 10 -9 M.

5. A nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof against the Oit3 protein of mouse hepatic sinusoidal endothelial cells as described in any one of claims 1 to 4.

6. A recombinant expression vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the recombinant expression vector of claim 6.

8. A recombinant fluorescent antibody targeting Oit3 protein in mouse hepatic sinusoidal endothelial cells, characterized in that, The monoclonal antibody or its antigen-binding fragment against the Oit3 protein of mouse hepatic sinusoidal endothelial cells as described in any one of claims 1 to 4, and a detectable marker fused to or conjugated thereto.

9. The use of the monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells as described in any one of claims 1 to 4, or the antigen-binding fragment thereof, or the recombinant fluorescent antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells as described in claim 8, in the preparation of a reagent for detecting Oit3 protein.

10. The application according to claim 9, characterized in that, The detection methods are enzyme-linked immunosorbent assay (ELISA), immunoblotting, or immunofluorescence assay.

Images