An anti-human smim45-107aa monoclonal antibody and uses thereof

Abstract

The application discloses an anti-human SMIM45-107aa monoclonal antibody and application thereof, the antibody can be specifically combined with SMIM45-107aa; in the monoclonal antibody, the sequence of CDR-H1 of the heavy chain variable region is as shown in SEQ ID No.3, the sequence of CDR-H2 of the heavy chain variable region is as shown in SEQ ID No.4, the sequence of CDR-H3 of the heavy chain variable region is as shown in SEQ ID No.5, the sequence of CDR-L1 of the light chain variable region is as shown in SEQ ID No.6, the sequence of CDR-L2 of the light chain variable region is as shown in SEQ ID No.7, and the sequence of CDR-L3 of the light chain variable region is as shown in SEQ ID No.8.The anti-human SMIM45-107aa monoclonal antibody prepared by the application has high specificity, can specifically recognize cells containing SMIM45-107aa protein, and is suitable for immunological detection, such as immunohistochemical detection, immunoblotting and immunofluorescence detection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an anti-human SMIM45-107aa monoclonal antibody and its uses. Background Technology

[0002] Small Integral Membrane Protein 45 (SMIM45-107aa) is a polypeptide encoded by LINC00634, belonging to the category of small molecular weight integrated membrane proteins, encoding 107 amino acids. These proteins typically possess one or more transmembrane domains and play important roles in cell signaling, cell-cell interactions, and maintaining cell structure. Compared to normal tissues, tumor tissues, such as liver cancer and colorectal cancer, show higher expression levels of LINC00634; however, the role and mechanism of its encoded polypeptide, SMIM45-107aa, in tumors have not yet been reported.

[0003] In recent years, lincRNA-encoded peptides have gradually become a research hotspot, playing a crucial role in the occurrence and development of diseases. These peptides can act as oncogenes or tumor suppressors, influencing disease progression. Therefore, these peptides can serve as therapeutic targets for tumors, and targeted antibody drugs can be developed for oncogenes. With the advancements in immunotherapy and targeted therapy in tumor treatment, targeted therapy using antibody drugs has broad application prospects.

[0004] Given the potential role of SMIM45-107aa in liver cancer, monoclonal antibodies targeting this protein have significant research and therapeutic value. However, currently, no human-mouse cross-reactivity, highly specific anti-SMIM45-107aa monoclonal antibodies have been found that can be used for immunoassay. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing an anti-human SMIM45-107aa monoclonal antibody and its uses.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect is to provide a monoclonal antibody against human SMIM45-107aa, comprising a heavy chain variable region and a light chain variable region, wherein the antibody can specifically bind to SMIM45-107aa; wherein the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID No. 3, the amino acid sequence of the heavy chain variable region CDR-H2 is shown in SEQ ID No. 4, the amino acid sequence of the heavy chain variable region CDR-H3 is shown in SEQ ID No. 5, the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID No. 6, the amino acid sequence of the light chain variable region CDR-L2 is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region CDR-L3 is shown in SEQ ID No. 8.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

[0009] Furthermore, the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID No. 9, and the amino acid sequence of the light chain is shown in SEQ ID No. 10.

[0010] Furthermore, both the heavy chain and the light chain include a constant region, which is a constant region of mouse or human IgG, preferably a constant region of IgG4.

[0011] The second aspect is to provide a nucleotide molecular composition comprising a nucleotide molecule as shown in sequence SEQ ID NO:11 and a nucleotide molecule as shown in sequence SEQ ID NO:12; wherein sequence SEQ ID NO:11 encodes the heavy chain variable region of the monoclonal antibody and sequence SEQ ID NO:12 encodes the light chain variable region of the monoclonal antibody.

[0012] The third aspect is to provide an expression vector containing the aforementioned nucleotide molecular composition.

[0013] The fourth aspect is to provide a host cell containing the above-mentioned expression vector, wherein the host cell is a eukaryotic cell, preferably a mammalian cell.

[0014] The fifth aspect is to provide the application of the aforementioned monoclonal antibodies in the preparation of detection products for liver cancer, gastric cancer, breast cancer, and intrahepatic cholangiocarcinoma samples.

[0015] The sixth aspect is to provide a detection product comprising the aforementioned monoclonal antibody, wherein the detection product is selected from detection reagents, kits, immunohistochemical detection, and immunoblotting detection.

[0016] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0017] This invention selects the full-length amino acid sequence of human SMIM45-107aa protein as the antigen, and the protein expressed after conjugation with His is used as the immunogen. Mice are immunized using this immunogen, and mouse monoclonal antibodies against SMIM45-107aa protein, along with their heavy and light chain sequences, are obtained based on single B cell screening and culture monoclonal antibody development technology. The anti-human SMIM45-107aa monoclonal antibody prepared by this invention exhibits high specificity, specifically recognizing cells containing SMIM45-107aa protein, and is suitable for immunological detection, such as immunohistochemistry, Western blotting, and immunofluorescence. Furthermore, the anti-human SMIM45-107aa monoclonal antibody has a high affinity for human SMIM45-107aa, ensuring sensitivity and reliability in various immunological assays. Attached Figure Description

[0018] Figure 1 The results of serum titer analysis using ELISA were obtained for two immunized mice.

[0019] Figure 2 The results of sorting MBCs using flow cytometry.

[0020] Figure 3 Results of SDS-PAGE gel assay for the purity of SMIM45-107aa antibody.

[0021] Figure 4 Western blot analysis of the expression of anti-SMIM45-107aa monoclonal antibody in hepatocellular carcinoma tissue.

[0022] Figure 5 IHC was used to detect the expression of anti-SMIM45-107aa monoclonal antibody in liver cancer tissue.

[0023] Figure 6 IHC was used to detect the expression of anti-SMIM45-107aa monoclonal antibody in gastric cancer, breast cancer, and intrahepatic cholangiocarcinoma tissues.

[0024] Figure 7 Immunofluorescence detection of SMIM45-107aa in hepatocellular carcinoma cells. Detailed Implementation

[0025] The first aspect of this invention protects an anti-human SMIM45-107aa monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the antibody specifically binds to SMIM45-107aa; wherein the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID No. 3, the amino acid sequence of the heavy chain variable region CDR-H2 is shown in SEQ ID No. 4, the amino acid sequence of the heavy chain variable region CDR-H3 is shown in SEQ ID No. 5, the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID No. 6, the amino acid sequence of the light chain variable region CDR-L2 is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region CDR-L3 is shown in SEQ ID No. 8.

[0026] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

[0027] The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID No. 9, and the amino acid sequence of the light chain is shown in SEQ ID No. 10.

[0028] Preferably, both the heavy chain and the light chain further include a constant region, which is a constant region of mouse or human IgG, more preferably a constant region of IgG4.

[0029] The specific sequence of the anti-human SMIM45-107aa monoclonal antibody of this invention is as follows:

[0030] 1) Heavy chain variable region amino acid sequence (underlined regions are heavy chain CDR regions):

[0031] MDSRLNLVFLVLILKGVQCDVQLVES GGGLVQPGGSRK LSCAASGFTFSS FGMH WVRQAPEKGLE WVAYINFGSSTIYYADTVKGRFTISRDNPKNTLFLQM TS LRSEDTAMYYCVR CYGNYYAMDYWGQGTSVTVSS(SEQID NO:1)

[0032] CDR-H1 GGGLVQPGGSRK(SEQ ID NO:3)

[0033] CDR-H2 WVRQAPEKGLE(SEQ ID NO:4)

[0034] CDR-H3 LRSEDTAMYYCVR(SEQ ID NO:5)

[0035] 2) Amino acid sequence of the light chain variable region: (The underlined regions are the light chain CDR regions)

[0036] MDMRTPAQFLGILLLWFPGIKCDIKM TQSPSSMYASLG ERVTITCNASHDI NSYL SWFQQKPGKS PKTLIYRTNRLVDGVPSRFSGSGSGQDYSLTISSLEYED M GIYYCLQYDEFP LTFGAGTKLELK(SEQ ID NO:2)

[0037] CDR-L1 TQSPSSMYASLG(SEQ ID NO:6)

[0038] CDR-L2 SWFQQKPGKS(SEQ ID NO:7)

[0039] CDR-L3 MGIYYCLQYDEFP(SEQ ID NO:8)

[0040] 3) Heavy chain amino acid sequence:

[0041] MDSRLNLVFLVLILKGVQCDVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGM

[0042] HWVRQAPEKGLEWVAYINFGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLR

[0043] SEDTAMYYCVRCYGNYYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQT

[0044] NSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPS

[0045] STWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKD

[0046] VLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRS

[0047] VSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKE

[0048] QMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:9)

[0049] 4) Light chain amino acid sequence:

[0050] MDMRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTITCNASHDINSY

[0051] LSWFQQKPGKSPKTLIYRTNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIY

[0052] YCLQYDEFPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY

[0053] PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:10)

[0054] A second aspect of the present invention protects a nucleotide molecular composition comprising a nucleotide molecule as shown in sequence SEQ ID NO:11 and a nucleotide molecule as shown in sequence SEQ ID NO:12; wherein, sequence SEQ ID NO:11 encodes the heavy chain variable region of the above-mentioned monoclonal antibody; and sequence SEQ ID NO:12 encodes the light chain variable region of the above-mentioned monoclonal antibody.

[0055] Heavy chain variable region nucleic acid sequence:

[0056] ATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTATTTTAAAAGGTGTCCAGTGTGATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAATTTTGGCAGTAGTACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTACTACTGTGTAAGATGTTATGGTAACTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:11)

[0057] Light chain variable region nucleic acid sequence:

[0058] ATGGACATGAGGACCCCTGCTCAGTTTCTTGGAATCTTGTTGCTCTGGTTTCCAGGTATCAAATGTGACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAATGCGAGTCACGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTACAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:12)

[0059] A third aspect of this invention protects an expression vector comprising the aforementioned nucleotide molecular composition. The method for constructing the expression vector should be known to those skilled in the art; for example, the expression vector can be constructed using in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector in this invention generally refers to various commercially available expression vectors well-known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0060] A fourth aspect of this invention protects a host cell containing the above-described expression vector, wherein the host cell is a eukaryotic cell, preferably a mammalian cell. The method for constructing the host cell should be known to those skilled in the art, and may include, for example, one or more combinations of methods including, but not limited to, microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, and calcium phosphate precipitation.

[0061] The fifth aspect of this invention protects the use of the above-mentioned monoclonal antibody in the preparation of liver cancer detection products.

[0062] A sixth aspect of this invention protects a detection product comprising the above-described monoclonal antibody, wherein the detection product is selected from detection reagents, kits, chips, and membrane strips.

[0063] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0064] Example 1: Obtaining a mouse monoclonal antibody specific to SMIM45-107aa using the SingleB rapid monoclonal method.

[0065] 1.1 Animal Immunization

[0066] Step 1: Immunize mice according to the generally accepted method in the literature (E Harlow, D. Lane, Antibody: Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998). Use recombinant human SMIM45-107aa (where the human SMIM45-107aa protein sequence is selected as the amino acid sequence) as the immunogen.

[0067] Step two involves using a rapid immune adjuvant to enhance the immune response. Briefly, an antigen-protein solution is prepared with PBS, and the adjuvant and antigen are thoroughly mixed at a 1:1 volume ratio before injection into mice.

[0068] Step 3, serum titer detection: After final immunization, a small amount of tail blood is collected, and the serum titer is determined by indirect ELISA.

[0069] 1) Antigen coating: Dilute the antigen to 2 μg / ml with PBS buffer and add 100 μl to each well of a 96-well microplate. Incubate overnight at 4°C. Wash the coated microplate three times with a plate washer and dry it. Add 100 μl of blocking buffer to each well and incubate at 37°C for 2 hours. Then wash the blocked microplate three times with a plate washer and dry it.

[0070] 2) Incubation of primary antibody: Dilute the serum of immunized mice and the serum of non-immunized mice (the serum of non-immunized mice is a negative control) with PBS buffer at dilution ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000. Add 100 μl of the diluted serum to each well and incubate at 37°C for 1 h. After incubation, wash the ELISA plate three times in a plate washer.

[0071] 3) Incubation with secondary antibody: Add 100 μl of HRP-labeled goat anti-mouse secondary antibody to each well and incubate at 37°C for 30 min. Then, wash the plate three times in a plate washer.

[0072] 4) Color development: Add 100 μl of the mixed color development solution to each well of the 96-well plate and incubate at 37°C for 15 min.

[0073] 5) Termination and Reading: Add 100 μl of stop solution to each well of a 96-well plate, then place it in a microplate reader for reading. Set the detection wavelength to 450 nm. A positive result is considered achieved when the OD value is greater than 2.1 times that of the negative control. See below for the test results. Figure 1 .

[0074] Step four: Based on the serum titer test results, 2 to 3 booster immunizations are usually required after the initial immunization to achieve a good immune effect. Select immunized mice with high serum titers and administer an intraperitoneal injection as the final pulse immunization before sorting.

[0075] 1.2 In vitro expansion of single B cells to obtain monoclonal antibodies

[0076] Step 1, Single-cell suspension preparation: Before sorting B cells, euthanize immunized mice and obtain the spleen in a sterile environment, removing the surrounding connective tissue. Place the spleen on a cell filter and gently crush it with a syringe needle core to obtain a cell suspension. Add Ficoll-Paque PLuS cell separation solution and the cell suspension to a test tube at a volume ratio of 1:2. Centrifuge at 1500 rpm for 15 minutes. The middle layer of the test tube after centrifugation is generally a mononuclear cell layer. Aspirate the mononuclear cell layer, add SOL024 solution, centrifuge at 1500 rpm for 10 minutes, remove the supernatant, and resuspend the cells in 500 μL of SOL024 solution.

[0077] Step 2, Flow cytometry sorting of MBCs: Utilizing the property that the BCRs on the surface of MBCs can specifically bind to antigen proteins, the antigen proteins are fluorescently labeled. MBCs specifically bound to the antigen proteins can then be sorted from the cell suspension one by one using flow cytometry. The sorting results are shown below. Figure 2 .

[0078] Step 3: DNA cloning and sequencing, and sequence analysis of anti-SMIM45-107aa antibody.

[0079] Step four: Extract total RNA from individual B cells using TRIZOL reagent (Invitrogen, CAT#15596-018). Simply collect 5 x 10⁻⁶ cells. 6 Transfer the cells to 1.5 ml centrifuge tubes and aspirate the supernatant. Add 600 μl of TRIZOL reagent and pipette, incubate at room temperature for 10 min to lyse the cells. Add 120 μl of chloroform to each tube, vortex vigorously for 30 seconds, incubate at room temperature for 10 min, and centrifuge at 12000g for 15 min at 4°C. Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix well, and incubate on ice for 20 min to precipitate RNA. Then centrifuge at 12000g for 15 min at 4°C, discard the supernatant, leaving the precipitate at the bottom of the tube, and wash twice with pre-chilled 75% ethanol. Finally, dry at room temperature for 5 min to evaporate residual ethanol, and add 30 μl of DEPC-treated water to dissolve the RNA.

[0080] Step 5: Reverse transcription of RNA into cDNA was performed using Takara's reverse transcription kit (cat#6110A). The experimental system was as follows: 1 μg total RNA (approximately 3 μl), 1 μl Oligo(dT) primer, and water (RNase-free water) to a final volume of 10 μl. The mixture was incubated at 65°C for 5 min, and then immediately cooled on ice. Subsequently, 4 μl of 5*PrimeScript Buffer, 0.5 μl of RNase Inhibitor, and 1 μl of PrimeScript RTase were added, and water (RNase-free water) to a final volume of 20 μl was added. The mixture was slowly mixed, and reverse transcription was performed at 42°C for 30-60 min, followed by incubation at 95°C for 5 min to complete cDNA synthesis. Further, poly-G was added to the 3' end of the cDNA. The reaction system was prepared as follows: 5 μl cDNA, 33.5 μl ddH2O, 5 μl 10*TdT buffer, 5 μl CoCl2, 1 μl dGTP, and 0.5 μl terminal deoxynucleotidyl transferase (total volume 50 μl). The mixture was incubated at 37°C for 30 minutes, followed by incubation at 70°C for 10 minutes to complete the poly-G tailing.

[0081] Step 6: Amplify the antibody variable region using the tailed cDNA as a template. For amplifying the antibody heavy chain variable region sequence, prepare the PCR reaction system as follows: 5 μl 10*Taq enzyme buffer, 0.5 μl universal poly C primer (forward primer), 0.5 μl mouse IgG reverse primer, 1 μl dNTP, 1 μl Taq polymerase, 1 μl cDNA, and 41 μl ddH2O. The PCR amplification conditions for the antibody heavy and light chain variable regions are as follows: 1) Pre-denaturation: 95℃, 5 min; 2) Denaturation: 95℃, 20 sec; 3) Annealing: 58℃, 15 sec; 4) Extension: 72℃, 30 sec; 5) Storage: 20℃, 60 min.

[0082] The PCR products were analyzed by 1.5% agarose gel electrophoresis. The corresponding bands (VH 414bp, VK 387bp) were cut out and the Fab region fragments of the heavy chain and light chain were obtained by Tiangen Biotech Gel Extraction Kit.

[0083] Step 7, Construction of expression plasmids and transfection: The amplified paired antibody light and heavy chain Fab base sequences were ligated into the pcDNA3.4 vector containing the mouse IgG1 Fc fragment to construct the light and heavy chain recombinant expression plasmids. Plasmid extraction was performed using a commercially available plasmid extraction kit; refer to the plasmid extraction instructions for each kit for specific steps.

[0084] Step 8, Instantaneous Expression

[0085] 1) Seed cells the day before transfection to ensure that cells grow to 3.0-5.0 × 10⁻⁶ on the day of transfection. 6 Cells / mL, viability 98%, cell suspension diluted with culture medium, 400 μl per well in 96-well plates.

[0086] 2) Spread 100 μl of transfection buffer into each well of a 96-well plate, then add the plasmid to the transfection buffer and mix by pipetting. Add PEI max to the buffer and mix by pipetting. Incubate at 37°C for 10 min and then add to the 96-well plate for further cell culture.

[0087] 3) 20 h after transfection, add 25 μl of feed to each well. After 4-5 days of expression, centrifuge at 4000 rpm for 10 min and collect the supernatant.

[0088] Step 9: ELISA detection of cell supernatant and selection of candidate clones; indirect ELISA detection to determine if the cell supernatant is positive.

[0089] 1) Antigen coating: Dilute the antigen to 2 μg / ml with PBS buffer and add 100 μl to each well of a 96-well microplate. Incubate overnight at 4°C. Wash the coated microplate three times with a plate washer and dry it. Add 100 μl of blocking buffer to each well and incubate at 37°C for 2 hours. Then wash the blocked microplate three times with a plate washer and dry it.

[0090] 2) Incubation with primary antibody: Add 50 μl of cell supernatant to each well, use an equal amount of immune serum as a positive control, and PBS buffer as a negative control. Incubate at 37°C for 1 hour. After incubation, wash the ELISA plate three times in a plate washer and then dry it.

[0091] 3) Incubation with secondary antibody: Add 100 μl of HRP-labeled goat anti-mouse secondary antibody to each well, incubate at 37°C for 30 min, wash the plate three times in a plate washer and then dry it.

[0092] 4) Color development: Add 100 μl of the mixed color development solution to each well of the 96-well plate and incubate at 37°C for 15 min.

[0093] 5) Termination and reading: Add 50 μl of stop solution to each well of the 96-well plate, then place it in a microplate reader for reading. Set the detection wavelength to 450 nm and read the results. An OD value greater than 2.1 times that of the negative control well is considered positive.

[0094] The serum titers of 20 candidate positive clones (1M1E1, 1M1C3, 1M1E3, 1M1G3, 1M1G4, 1M1G7, 1M1B8, 1M1C10, 1M1D10, 1M1B12, 1M1D12, 1M1F12, 1M3A2, 1M3A3, 1M3D4, 1M3C8, 1M3C9, 1M3D10, 1M3A11, 1M3C12) were measured, and the results are shown in Table 1. The clone with the highest serum titer, 1M3C8, was selected for expression and purification, and the antibody was used for subsequent functional validation.

[0095] Table 1

[0096]

[0097]

[0098] Step 10: Antibody Expression and Purification

[0099] 1) Expression plasmid transient transduction expression: The selected clone 1M3C8 was expressed using the same steps as above.

[0100] 2) Antibody purification: Centrifuge the cell culture medium after 6 days of transfection and culture, filter the secreted supernatant through a filter membrane, dialyze to buffer at 4°C, and purify with Protein A column after dialysis to a purity of >90%.

[0101] 3) SDS-PAGE gel assay for SMIM45-107aa antibody purity: SDS-PAGE gel preparation: Install vertical electrophoresis equipment; prepare a 15% lower separating gel and incubate at room temperature for 30 minutes to allow gel polymerization; prepare a 5% upper stacking gel, insert a 10-well comb, and allow it to solidify; Sample loading: Add the prepared sample with SDS-loading buffer to the gel wells; Electrophoresis: 80V constant voltage for 10 minutes, then switch to 120V constant voltage for 60 minutes. Coomassie brilliant blue staining: After electrophoresis, stain with Coomassie brilliant blue solution for 30 minutes; Destaining: After destaining with destaining solution, take a picture and save it. Figure 3 The results showed that the purified SMIM45-107aa antibody had two clear bands, one for the heavy chain and one for the light chain, with almost no impurities, indicating that the purified SMIM45-107aa antibody had high purity.

[0102] Example 2: Detection of SMIM45-107aa monoclonal antibody specificity using Western blot, IHC, and immunofluorescence.

[0103] 2.1 Western blot analysis of SMIM45-107aa expression in hepatocellular carcinoma tissue and cell samples. The procedure was simple: prepare an SDS-PAGE gel; load the sample (with SDS loading buffer) into the gel wells; electrophoresis: 80V constant voltage for 10 min, then 120V constant voltage for 60 min; transfer: wet transfer was performed using the sponge-filter paper-gel-membrane-filter paper-sponge method, at a constant current of 280mA for 60 min; antibody incubation: 5% milk blocking for 1 hour; incubation with diluted primary antibody (1:1000) for 2 hours; after rinsing the membrane 5 times, goat anti-mouse secondary antibody was added at a ratio of 1:5000, and incubation for 1 hour; development: positive energy imaging was used for development and storage. Figure 4 The results showed that the SMIM45-107aa monoclonal antibody had clear bands in purified SMIM45-107aa protein samples, SK-Hep1 liver cancer cells overexpressing SMIM45-107aa, and liver cancer tissue samples, indicating that the SMIM45-107aa antibody can recognize the SMIM45-107aa natural antigen.

[0104] 2.2 Functional Qualification and Application of SMIIM45 Using IHC:

[0105] Dewaxing: Set the oven to 70 degrees Celsius and bake the liver tissue sections for 30 minutes. Continue dewaxing with xylene for a second time. Then pass the sections through alcohol (95%, 95%, 75%) for 5 minutes.

[0106] Antigen retrieval: Boil the sections in EDTA retrieval solution on an induction cooker for 20 minutes, cool to room temperature, and rinse with tap water; then soak the tissue in 3% hydrogen peroxide for 30 minutes.

[0107] Blocking: Block with 5% BSA prepared in PBS for 1 hour;

[0108] Antibody incubation: Remove the blocking solution, dilute the primary antibody with PBS at a ratio of 1:200, add it to the tissue section, and incubate overnight at 4°C; remove the primary antibody, wash 3 times with PBS, add an appropriate amount of secondary antibody, incubate at room temperature for 30 min, and wash 3 times with PBS.

[0109] Color development: Prepare DAB color development solution for color development, rinse 3 times with tap water; stain with hematoxylin for 1 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 10 s, rinse with tap water; pass through 95% and 100% alcohol for 5 min in sequence.

[0110] Covering: Add 3 drops of neutral resin, cover with a coverslip and blot off any excess resin.

[0111] Immunohistochemical results are shown in Figure 5 , Figure 6 .Depend on Figure 5The presence of SMIM45-107aa in the cytoplasm of liver cancer cells indicates that the purified SMIM45-107aa antibody has high titer and good specificity, making it suitable for IHC experiments. Meanwhile, Figure 6 This indicates that SMIM45 can also be used in IHC experiments for gastric cancer, breast cancer, and intrahepatic cholangiocarcinoma.

[0112] 2.3 Validation and application of antibody function using immunofluorescence

[0113] Cell slides: Slide the cells onto a slide and incubate overnight;

[0114] Fixation and permeabilization: Wash twice with PBS, permeabilize with 0.01% Triton for 5 min, wash twice with PBS, and fix with pentylene glycol for 15 min;

[0115] Blocking: Wash 3 times with PBS for 2 minutes each time, then block with 5% fetal bovine serum for 30 minutes;

[0116] Antibody incubation: Add primary antibody at a ratio of 1:200, incubate at room temperature for 2 hours, wash 5 times with PBS for 2 minutes each time; add fluorescent secondary antibody at a ratio of 1:500, incubate at room temperature for 1 hour, wash 5 times with PBS;

[0117] Mounting: Place a DAPI-containing mounting medium on the slide, cover with a coverslip, and take a photograph for record-keeping.

[0118] Immunofluorescence results are shown in Figure 7 ,Depend on Figure 7 It can be seen that SMIM45-107aa exhibits green fluorescence in the cytoplasm of the QGY-7701 hepatocellular carcinoma cell line, indicating that it is localized within the cytoplasm. It can be used for immunofluorescence experiments.

[0119] In summary, the obtained monoclonal antibody SMIM45-107aa specifically recognizes the SMIM45-107aa protein antigen and can be used for immunoblotting, immunohistochemistry, and immunofluorescence experiments.

[0120] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against human SMIM45-107aa, comprising a heavy chain variable region and a light chain variable region, characterized in that, The antibody can specifically bind to the SMIM45-107aa antigen; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The anti-human SMIM45-107aa monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID No. 9, and the amino acid sequence of the light chain is shown in SEQ ID No.

10.

3. The anti-human SMIM45-107aa monoclonal antibody according to claim 2, characterized in that, Both the heavy chain and the light chain include a constant region, which is a constant region of mouse or human IgG.

4. The anti-human SMIM45-107aa monoclonal antibody according to claim 3, characterized in that, The constant region is the constant region of IgG4.

5. A nucleic acid molecular composition, characterized in that, It includes nucleic acid molecules as shown in SEQ ID NO:11 and nucleic acid molecules as shown in SEQ ID NO:12; wherein the nucleic acid molecules shown in SEQ ID NO:11 and SEQ ID NO:12 respectively encode the heavy chain variable region and the light chain variable region of the monoclonal antibody of claim 2.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecular composition as described in claim 5.

7. A host cell comprising the expression vector as described in claim 6, characterized in that, The host cell is a eukaryotic cell.

8. The use of the monoclonal antibody as described in any one of claims 1-4 in the preparation of a kit for analyzing the localization and expression of SMIM45-107aa protein in liver cancer, gastric cancer, breast cancer, and intrahepatic cholangiocarcinoma samples by immunohistochemistry.

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