Anti-human foxp3 monoclonal antibodies, methods of making and uses thereof

Abstract

The present application relates to a kind of anti-human FOXP3 monoclonal antibody and its preparation method and application, belong to immunology technical field.The anti-human FOXP3 monoclonal antibody provided in the present application heavy chain variable region amino acid sequence as shown in SEQ ID NO.1, light chain variable region amino acid sequence as shown in SEQ ID NO.2.It also provides the nucleic acid molecule of the antibody coding, can be prepared using recombinant technology the monoclonal antibody.The anti-human FOXP3 monoclonal antibody prepared in the present application is excellent in specificity and stability, and has good specificity and reliability in the research of FOXP3 related diseases.

Description

Technical Field

[0001] This invention relates to an anti-human FOXP3 monoclonal antibody, its preparation method, and its application, belonging to the field of immunology technology. Background Technology

[0002] FOXP3 (Forkhead box protein P3), also known as scurfin protein, is encoded by the FOXP3 gene located on the X chromosome. It is a member of the forkhead box transcription factor family and is primarily expressed in regulatory T cells (Tregs). As a lineage-specific master transcription factor for Tregs, its expression directly determines the immunosuppressive capacity of Tregs. High FOXP3 expression promotes Treg cell maturation and stability, enhances their ability to secrete inhibitory cytokines such as IL-10 and TGF-β, and effectively suppresses excessive immune responses. Insufficient FOXP3 expression leads to Treg cell dysfunction, failing to effectively suppress effector T cell activation, thereby triggering autoimmune responses. Furthermore, FOXP3 protein participates in various physiological and pathological processes, such as cell differentiation, apoptosis, and inflammatory responses. Abnormal expression or loss of function of FOXP3 is associated with various diseases, including IPEX syndrome, autoimmune diseases, and tumors.

[0003] FOXP3 is primarily expressed in the cell nucleus and cannot be directly identified by surface markers. Therefore, detection using specific antibodies is a crucial method for studying its function and clinical applications. Detection of FOXP3 in tumor tissues... + Cellular proportions can serve as prognostic indicators. For example, high FOXP3 expression in esophageal cancer suggests a higher risk of tumor progression and can aid in clinical decision-making. In autoimmune diseases, assessing the number and functional status of Tregs helps determine disease activity. In autoimmune disease patients receiving low-dose IL-2 therapy, elevated FOXP3 expression levels can serve as a biomarker for Treg amplification and treatment efficacy. Monitoring FOXP3 in the tumor microenvironment is also important in cancer immunotherapy. + Changes in Tregs help assess whether CTLA-4 inhibitors have successfully interfered with Treg function. FOXP3 is an indispensable target in clinical pathology diagnosis and basic research.

[0004] The most widely used FOXP3 antibody on the market is Anti-FOXP3 antibody (clone number 236A / E7). This FOXP3 antibody is a recombinant mouse monoclonal antibody developed by Abcam. Its epitope is well-defined, recognizing the amino acid region from amino acid position 105 to 236 of the FOXP3 protein. However, its complete variable region sequence is not publicly available, and the complete amino acid or gene sequence of the light and heavy chains of the variable region of the FOXP3 antibody is not publicly available in public databases. This limits the direct application of the FOXP3 antibody in engineering, humanization, or recombinant expression.

[0005] A Chinese patent document with publication number CN107266568A also discloses a monoclonal antibody UMAB248 that specifically binds to the FOXP3 protein. This antibody uses the full-length FOXP3 sequence as the antigen sequence to prepare hybridoma cell lines and then produce antibodies. However, there is a risk of antibody gene loss, poor cell condition, or even cell death during the preservation of hybridoma cells, which limits its further industrial production and application. Summary of the Invention

[0006] The first objective of this invention is to provide an anti-human FOXP3 monoclonal antibody that is stable, has high experimental reproducibility, and exhibits good specificity and affinity.

[0007] A second objective of this invention is to provide the application of the above-mentioned anti-human FOXP3 monoclonal antibody in the preparation of FOXP3 in vitro detection reagents or kits.

[0008] A third objective of this invention is to provide a detection reagent or kit comprising the above-mentioned anti-human FOXP3 monoclonal antibody.

[0009] A fourth objective of this invention is to provide a nucleic acid molecule encoding the aforementioned anti-human FOXP3 monoclonal antibody.

[0010] A fifth objective of this invention is to provide an expression cassette, expression vector, recombinant cell, or recombinant bacterium containing the aforementioned nucleic acid molecules.

[0011] The sixth object of the present invention is to provide the use of the above-mentioned nucleic acid molecule, expression cassette containing the above-mentioned nucleic acid molecule, expression vector, recombinant cell or recombinant bacteria in the preparation of anti-human FOXP3 monoclonal antibody.

[0012] The seventh objective of this invention is to provide a method for preparing an anti-human FOXP3 monoclonal antibody.

[0013] To achieve the above objectives, the technical solution adopted by the anti-human FOXP3 monoclonal antibody in this invention is as follows: An anti-human FOXP3 monoclonal antibody, wherein the heavy chain variable region of the anti-human FOXP3 monoclonal antibody has an amino acid sequence as shown in SEQ ID NO.1, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO.2.

[0014] The beneficial effects of the above technical solution are as follows: This invention provides an anti-human FOXP3 monoclonal antibody and discloses the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the antibody. The antibody can be prepared by recombination technology, avoiding the risk factors that occur in the traditional monoclonal antibody production and storage process.

[0015] To achieve the above objectives, the technical solution adopted in this invention for the application of an anti-human FOXP3 monoclonal antibody in the preparation of FOXP3 in vitro detection reagents or kits is as follows: Application of an anti-human FOXP3 monoclonal antibody in the preparation of FOXP3 in vitro detection reagents or kits.

[0016] The beneficial effects of the above technical solution are as follows: Experimental verification shows that the anti-human FOXP3 monoclonal antibody has good specificity and affinity, and can be used to prepare FOXP3 detection reagents or kits to detect the expression of FOXP3 in tissues and cells, and can further study the function of FOXP3.

[0017] To achieve the above objectives, the technical solution adopted in the detection reagent or kit containing the above-mentioned anti-human FOXP3 monoclonal antibody in this invention is as follows: A detection reagent or kit containing the above-mentioned anti-human FOXP3 monoclonal antibody.

[0018] The beneficial effects of the above technical solution are as follows: by utilizing the anti-human FOXP3 monoclonal antibody with good specificity and affinity of the present invention, it can be directionally prepared into detection reagents or kits for detecting FOXP3 in tissues or cells, such as immunohistochemical staining, immunocellular staining, Western blot or ELISA, according to the needs of users, which helps to meet diverse market demands.

[0019] Preferably, the detection reagent or kit includes an immunohistochemical detection reagent or kit.

[0020] To achieve the above objectives, the technical solution adopted in this invention for a nucleic acid molecule encoding the aforementioned anti-human FOXP3 monoclonal antibody is as follows: A nucleic acid molecule encoding the aforementioned anti-human FOXP3 monoclonal antibody.

[0021] The beneficial effects of the above technical solution are as follows: This invention provides a nucleic acid molecule encoding an anti-human FOXP3 monoclonal antibody, which preserves the anti-human FOXP3 monoclonal antibody in the form of DNA, resulting in higher stability and facilitating subsequent exogenous expression and large-scale industrial production.

[0022] Specifically, the nucleotide sequence of the heavy chain variable region gene of the anti-human FOXP3 monoclonal antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region gene of the anti-human FOXP3 monoclonal antibody is shown in SEQ ID NO.4.

[0023] This nucleic acid molecule can be obtained through genetic engineering or chemical synthesis. The variant sequences of the heavy chain variable region nucleotide sequence and / or light chain variable region nucleotide sequence obtained by mutating the above-mentioned nucleic acid molecule provided in this invention through one or more nucleotide additions, deletions, substitutions, modifications, etc., and whose encoded amino acid sequences form single-chain antibodies, chimeric monoclonal antibodies, modified monoclonal antibodies, or other forms of monoclonal antibodies or antibody fragments, still retain the ability to specifically bind to FOXP3.

[0024] To achieve the above objectives, the technical solution adopted in this invention for an expression cassette, expression vector, recombinant cell, or recombinant bacterium containing the aforementioned nucleic acid molecules is as follows: An expression cassette, expression vector, recombinant cell or recombinant bacteria containing the above-mentioned nucleic acid molecules.

[0025] The beneficial effects of the above technical solution are as follows: The present invention provides an expression cassette, expression vector, recombinant cell or recombinant bacteria containing a nucleic acid molecule encoding an anti-human FOXP3 monoclonal antibody, which can rapidly, stably and on a large scale produce anti-human FOXP3 monoclonal antibodies through genetic engineering.

[0026] To achieve the above objectives, the technical solution adopted in this invention for the application of the above-mentioned nucleic acid molecule, expression cassette containing the above-mentioned nucleic acid molecule, expression vector, recombinant cell or recombinant bacteria in the preparation of anti-human FOXP3 monoclonal antibody is as follows: The use of the above-mentioned nucleic acid molecule, expression cassette containing the above-mentioned nucleic acid molecule, expression vector, recombinant cell or recombinant bacteria in the preparation of anti-human FOXP3 monoclonal antibody.

[0027] The beneficial effects of the above technical solution are as follows: This invention provides a nucleic acid molecule encoding an anti-human FOXP3 monoclonal antibody, an expression cassette containing the above nucleic acid molecule, an expression vector, and recombinant cells or recombinant bacteria. Based on this, the anti-human FOXP3 monoclonal antibody of this invention can be obtained by conventional genetic engineering methods, avoiding the risks that may exist in the hybridoma technology production process, such as gene mutation leading to decreased antibody stability, small fluctuations in culture conditions leading to cell population heterogeneity, and lack of antigen selection pressure leading to decreased antibody specificity.

[0028] To achieve the above objectives, the technical solution adopted in the preparation method of the anti-human FOXP3 monoclonal antibody in this invention is as follows: A method for preparing an anti-human FOXP3 monoclonal antibody involves introducing the above-mentioned nucleic acid molecules into host cells, collecting the cell supernatant, and purifying and ultrafiltration the supernatant.

[0029] The beneficial effects of the above technical solution are as follows: Compared with traditional monoclonal antibody preparation methods, the method for preparing anti-human FOXP3 monoclonal antibodies provided by this invention has advantages such as known antibody sequences, long-term preservation of antibody genes, stable antibody properties, and good experimental reproducibility. It is a standardized antibody production process that avoids the risk factors that occur in the traditional monoclonal antibody production and preservation process. Utilizing recombinant technology to prepare FOXP3 antibodies ensures the controllability and traceability of the antibody sequence, solves the batch-to-batch differences and preservation problems that may occur in traditional antibody production, and provides a more stable and reliable antibody tool suitable for long-term and standardized clinical diagnostic reagent development and basic research applications.

[0030] Preferably, the host cell includes mammalian cells, including HEK293 cells and CHO cells. Attached Figure Description

[0031] Figure 1 The results of Western blot detection of anti-human FOXP3 monoclonal antibody in Example 3 of this invention are as follows (Cho indicates that the sample added to this lane is CHO cell lysate, Foxp3 is the sample electrophoresis result, GAPDH is the internal standard electrophoresis result, and the numbers on the left indicate the protein size in kDa). Figure 2 The immunohistochemical detection result of human tonsil tissue using anti-human FOXP3 monoclonal antibody in Example 3 of this invention (1000×). Figure 3 The results of immunohistochemical detection of human lung cancer tissue using anti-human FOXP3 monoclonal antibody in Example 3 of this invention (1000×). Figure 4 The images show the detection results of anti-human FOXP3 monoclonal antibody and 236A / E7 antibody in human tonsil tissue in Example 4 of this invention. The left image shows the detection result of 236A / E7 antibody (1000×), and the right image shows the detection result of anti-human FOXP3 monoclonal antibody (1000×). Figure 5 The images show the detection results of anti-human FOXP3 monoclonal antibody and 236A / E7 antibody in human lung tissue in Example 4 of this invention. The left image shows the detection result of 236A / E7 antibody (1000×), and the right image shows the detection result of anti-human FOXP3 monoclonal antibody (1000×). Detailed Implementation

[0032] This invention provides an anti-human FOXP3 monoclonal antibody, and the technical solution adopted is as follows: 1. Selection of FOXP3 protein sequence: The amino acid sequence was obtained according to the FOXP3 protein accession number Q9BZS1 published by Uniprot. The full-length FOXP3 was selected for expression using bioinformatics analysis.

[0033] 2. Fusion expression: The full-length FOXP3 gene was synthesized, inserted into the prokaryotic expression vector pET-28a, expressed and purified, and the recombinant protein was collected.

[0034] 3. Preparation and identification of anti-human FOXP3 monoclonal antibodies: 6-8 week old female BalB / c mice were immunized with 100 μg of the recombinant FOXP3 protein expressed above. Spleen cells from these mice were fused with sp2 / 0 cells, and monoclonal cells were obtained using limiting dilution. Positive hybridoma cells were screened using indirect ELISA to obtain hybridoma cell lines that secrete anti-FOXP3 specific antibodies. Anti-human FOXP3 monoclonal antibodies were obtained by purifying ascites fluid using a Protein A column. The specificity and sensitivity of the antibody were verified using Western blotting (WB) and immunohistochemistry (IHC), respectively. CHO cell lysate samples were selected for Western blotting identification. Human tonsil tissue and lung cancer tissue were used for immunohistochemical staining to identify the effectiveness of the anti-human FOXP3 monoclonal antibody in recognizing FOXP3 in tissues.

[0035] 4. Preparation and identification of recombinant antibodies: RNA was extracted from hybridoma cell lines, and cDNA was obtained by reverse transcription, followed by plasmid preparation; anti-FOXP3 recombinant antibody was expressed using CHO cells, and the antibody was purified, including pre-transfection cell culture, preparation of transfected cells, transient transfection, and product expression and detection; finally, antibody verification was performed by Western blotting and IHC.

[0036] The anti-human FOXP3 monoclonal antibody provided by this invention has an amino acid sequence as shown in SEQ ID NO.1 for its heavy chain variable region and an amino acid sequence as shown in SEQ ID NO.2 for its light chain variable region. Based on the amino acid sequence of the antibody provided by this invention, modifications such as the addition, deletion, or substitution of one or more amino acids can be used to obtain its active fragment or conserved variant, laying the foundation for further improving the specificity and affinity of the antibody.

[0037] This invention also provides a nucleic acid molecule encoding the aforementioned anti-human FOXP3 monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region gene of the anti-human FOXP3 monoclonal antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region gene is shown in SEQ ID NO.4. This nucleic acid molecule can be obtained through genetic engineering or chemical synthesis. The variant sequences of the heavy chain variable region nucleotide sequence and / or light chain variable region nucleotide sequence obtained by mutation of the above-mentioned nucleic acid molecule provided by this invention through one or more nucleotide additions, deletions, substitutions, modifications, etc., whose encoded amino acid sequences form single-chain antibodies, chimeric monoclonal antibodies, modified monoclonal antibodies, or other forms of monoclonal antibodies or antibody fragments, still retain the ability to specifically bind to the FOXP3 protein.

[0038] This invention provides the amino acid and nucleotide sequences of the variable regions of the heavy and light chains of an anti-human FOXP3 monoclonal antibody. Based on these sequences, the monoclonal antibody of this invention can be obtained using conventional genetic engineering methods. Accordingly, this invention provides an expression cassette, expression vector, recombinant cells, and recombinant bacteria comprising the above-mentioned nucleic acid molecule encoding the anti-human FOXP3 monoclonal antibody. The expression cassette, expression vector, recombinant cells, and recombinant bacteria are all commonly used tools in genetic engineering. The expression vector can be selected from prokaryotic or eukaryotic expression vectors, specifically including bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors; the recombinant cells are selected from mammalian cells, insect cells, plant cells, etc.; and the recombinant bacteria are selected from bacteria, yeast, filamentous fungi, etc.

[0039] Using the above-mentioned nucleic acid molecules, expression cassettes, expression vectors, recombinant cells, and recombinant bacteria, the anti-human FOXP3 monoclonal antibody of the present invention can be prepared by recombination technology.

[0040] This invention also provides the application of anti-human FOXP3 monoclonal antibodies in the preparation of FOXP3 in vitro detection reagents or kits. The anti-human FOXP3 monoclonal antibody provided by this invention has good specificity and affinity, and can be specifically prepared into detection reagents or kits for detecting FOXP3 in tissues or cells according to user needs. Specifically, the anti-human FOXP3 monoclonal antibody of this invention can be made into a working solution, and combined with appropriate reagents to prepare detection reagents or kits for immunohistochemical staining, immune cell staining, Western blot, or ELISA.

[0041] The anti-human FOXP3 monoclonal antibody of this invention has undergone detailed validation, including Western blotting and IHC assays. Compared to commonly used polyclonal antibodies prepared from recombinant proteins or synthetic peptides, the anti-human FOXP3 monoclonal antibody prepared in this invention exhibits superior specificity and stability, and also demonstrates good specificity and reliability in studies of FOXP3-related diseases.

[0042] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art, and the equipment and raw materials used are commercially available or commonly used in the art.

[0043] Example 1: Anti-human FOXP3 monoclonal antibody This embodiment provides an anti-human FOXP3 monoclonal antibody, which is obtained through antigen preparation, animal immunization, cell fusion, screening, and cloning, as detailed below: 1. Antigen sequence synthesis and vector construction Based on the FOXP3 sequence and structure published by Uniprot, the full-length sequence was selected for the preparation of the FOXP3 immunogen using bioinformatics analysis. The Uniprot accession number for human FOXP3 is Q9BZS1, and the full-length amino acid sequence of FOXP3 is shown in SEQ ID NO. 5. The full-length FOXP3 nucleic acid sequence was artificially synthesized, and restriction enzyme sites NdeI and XhoI were introduced at both ends of the sequence. The synthesized tandem nucleic acid sequence was cloned into the pET-28a expression vector to construct the recombinant plasmid pET-28a-FOXP3.

[0044] 2. Transformation and Expression The pET-28a-FOXP3 recombinant plasmid constructed above was transformed into BL21(DE3) *E. coli* competent cells. Single colonies were picked and inoculated into 4 mL of LB broth containing kanamycin (50 μg / mL) for culture. Subsequently, IPTG was added to a final concentration of 1 mM to induce expression analysis, and strains expressing the FOXP3 target protein were preserved. Strains expressing the FOXP3 target protein were inoculated into 350 mL of LB broth containing kanamycin (50 μg / mL) and cultured at 37°C until OD500. 600 When the concentration reached 0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 37°C for 4 hours.

[0045] 3. Bacterial cell collection and ultrasonic disruption The induced bacterial cells were collected by centrifugation and resuspended in 10 mM PBS at ice temperature. The cells were then sonicated (350 W, 20 min, 3 sec on, 3 sec rest). After sonication, the cells were centrifuged at 12000 g for 10 min, and the supernatant was collected.

[0046] 4. Purification by nickel agarose gel The collected supernatant was filtered through a 0.45 μm filter, and the filtrate was purified by nickel-agarose gel electrophoresis. The filtrate was loaded into an equilibrated nickel-agarose gel column at a flow rate of 1 mL / min and washed with 10 mM PBS to remove unbound impurity proteins. Then, linear elution was performed with PBS containing 0.5 M imidazole, and different elution peaks were collected for SDS-PAGE to determine protein purity. Samples with a purity greater than 90% were ultrafiltered, and the buffer was replaced with 10 mM PBS for subsequent experiments.

[0047] 5. Immunization and Antibody Screening The previously purified FOXP3 protein was mixed with an equal volume of Freund's complete adjuvant and administered subcutaneously to immunize 6-8 week old BalB / c mice. Each mouse received 100 μg of this immunization. Two weeks later, a second immunization was administered via intraperitoneal injection, with the antigen emulsified with Freund's incomplete adjuvant at the same dose. After the second immunization, tail blood was collected and serum titers were determined using a serially diluted indirect ELISA method. Mice with the highest antibody titers were selected for a tail vein pulse immunization at a dose of 50 μg per mouse.

[0048] 6. Cell fusion Sp2 / 0 myeloma cells derived from BalB / c mice were used, and the cells should be in the logarithmic growth phase. Spleens were aseptically harvested from mice after a shock immunization, and a single-cell suspension of spleen cells was prepared. Mouse spleen cells and myeloma cells were mixed at a ratio of 1:5, and 1 mL of 50% PEG (pH 8.0) at 37°C was added. After mixing, incomplete culture medium was added. After centrifugation, the supernatant was discarded, HAT medium was added, and the volume was adjusted to 50 mL. The mixture was then added to 96-well cell culture plates. The culture plates were incubated at 37°C in a 5% CO2 incubator.

[0049] 7. Screening and Cloning Within 7-10 days post-fusion, cell clones are selected and screened using methods including ELISA, IHC, and WB. Positive well cells are subjected to limiting dilutions, with ELISA values ​​measured 5-6 days after each dilution to select OD cells. 450 The wells with high positive values ​​were further limited diluted until the entire 96-well plate was positive by ELISA. Finally, the monoclonal cell lines with the highest positive values ​​were selected for confirmation.

[0050] 8. Preparation and purification of ascites monoclonal antibodies Eight- to ten-week-old BalB / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin. One week later, each mouse was intraperitoneally injected with a 1 mL syringe containing a suspension of monoclonal cells washed and resuspended in PBS. Each cell line was injected with 5 × 10⁻⁶ cells. 6Cells / mouse, a total of 3 mice were injected. After ascites fluid accumulated in the mice, it was collected, centrifuged, and the supernatant was obtained. The ascites fluid was purified using Protein A column chromatography, and the concentration of the purified monoclonal antibody was determined. The purified anti-human FOXP3 monoclonal antibody with a concentration of 1 mg / mL was aliquoted and stored at -20°C.

[0051] Example 2: Variable region sequence of anti-human FOXP3 monoclonal antibody This embodiment provides a method for obtaining the amino acid and nucleotide sequences of the light and heavy chain variable regions of anti-human FOXP3 monoclonal antibodies. Total RNA was extracted from hybridoma cells screened in step 7 of Example 1, and the sequence information of anti-human FOXP3 monoclonal antibodies was obtained by 5' RACE. The specific operation is as follows: 1. Total RNA extraction The hybridoma cell lines screened in step 7 of Example 1 were revived, and the cells were collected after the cell titer was tested. 1 mL of Trizol was added to the cell suspension, and the mixture was thoroughly mixed by pipetting. The cells were allowed to stand at room temperature for 5-10 minutes until complete lysis. Then, the cells were centrifuged at 12000 rpm for 5 minutes, and the precipitate was discarded. 200 μL of chloroform was added to the supernatant, and the mixture was vigorously inverted and mixed. The mixture was allowed to stand for 15 minutes, and then centrifuged at 12000 rpm at 4°C for 15 minutes. The upper aqueous phase was collected. An equal volume of isopropanol was added, and the mixture was gently mixed. After standing for 10 minutes, the mixture was centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was discarded. 1 mL of pre-chilled 75% ethanol was added to the precipitate, and the mixture was gently tapped to mix. The mixture was centrifuged again at 12000 rpm at 4°C for 5 minutes, and the supernatant was discarded. After air-drying at room temperature for several minutes, 30-50 μL of pre-chilled DEPC water was added until dissolved, yielding the RNA product. The RNA product can be stored at -80°C for an extended period. Masks and gloves must be worn throughout the RNA extraction process to prevent RNase contamination.

[0052] 2. Acquiring antibody genes via 5'RACE ① The 5' sequence of the FOXP3 antibody was isolated using the SMARTer RACE 5' / 3' Kit (Clontech, Cat. No. 634859). Using 1 μg of total RNA as a template, first-strand cDNA synthesis was performed according to the instructions using the 5'-CDS primer A, SMART II A oligo, and 3'-CDS primer A provided in the kit to prepare 5'-RACE-Ready cDNA. Then, PCR amplification was performed using these cDNAs as templates, i.e., rapid amplification of the cDNA ends (RACE). The 5'-RACE PCR reaction used UPM (Universal Primer) and GSP (gene-specific primer) primers designed based on the FOXP3 gene-specific sequence.

[0053] ② The first round of PCR reaction system was 50 μL, with the following composition: 2.5 μL 5'-RACE-Ready cDNA, 5 μL 10×UPM, 1 μL 10 μM GSP, 25 μL 2×SeqAmp buffer, 1 μL SeqAmp DNA polymerase, and 15.5 μL ddH2O. The PCR reaction program was as follows: Cycles 1-5: 94℃ for 30 seconds, 72℃ for 3 minutes; Cycles 6-10: 94℃ for 30 seconds, 70℃ for 30 seconds, 72℃ for 3 minutes; Cycles 11-35: 94℃ for 30 seconds, 68℃ for 30 seconds, 72℃ for 3 minutes.

[0054] ③ Store the PCR product at 4℃. Take 5 μL of the PCR product and perform electrophoresis on a 1.0% agarose gel. If the electrophoresis result shows a single band, cut the gel to recover the remaining product and clone it into the pRACE vector. Then select positive clones for sequencing. If the electrophoresis result shows multiple bands, nested PCR is required for further amplification. After that, clone the product into the pRACE vector, select positive clones for sequencing, and finally obtain the FOXP3 antibody gene.

[0055] 3. Identification of antibody heavy chain and light chain gene information and analysis of the IMGT database. Sequencing analysis revealed that the nucleotide sequence of the heavy chain variable region gene of the anti-human FOXP3 monoclonal antibody of the present invention is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.1; the nucleotide sequence of the light chain variable region gene of the anti-human FOXP3 monoclonal antibody is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.2.

[0056] Example 3: Preparation method of anti-human FOXP3 monoclonal antibody This embodiment provides a method for preparing anti-human FOXP3 monoclonal antibodies using recombinant technology. Based on the light and heavy chain sequence information of the anti-human FOXP3 monoclonal antibody obtained in Example 2, the variable region genes of the heavy and light chains are amplified by PCR, constructed into an expression vector, transformed into host cells, and expressed and purified to obtain the anti-human FOXP3 monoclonal antibody. The specific operation is as follows: 1. Construction of recombinant carriers Based on the light and heavy chain sequence information of the anti-human FOXP3 monoclonal antibody obtained in Example 2, the fragment was amplified by PCR; the pTT5 vector was digested with EcoRI / HindIII for 3 hours, and then recovered by agarose gel electrophoresis. The antibody expression vector was then constructed by recombination, and the positive clones with correct sequencing were extracted for plasmid extraction for transfection.

[0057] 2. Expression and purification of anti-human FOXP3 monoclonal antibody 1) Cell culture before transfection and preparation of transfected cells CHO-K1 cells were placed in a 5% CO2 constant-temperature shaker and cultured at 37°C and 120 rpm. Before passage, cell counts were performed to confirm the density; after confirmation, centrifugation was not required, and the cell suspension could be directly added to the culture medium in the correct proportion. If an excessive number of dead cells were observed, the entire batch of cells should be discarded, and new cells should be reused.

[0058] Before transient transfection, determine cell density and viability; without centrifugation, directly dilute the cell density to 3 × 10⁻⁶. 6 Transfection was performed after incubating the cells at 37°C and 120 rpm for 10 minutes with shaking at 37°C and 120 rpm.

[0059] KPM (Catalog No.: K03125), TA-CHO (Catalog No.: K20002), KE-CHO (Catalog No.: K30002), and KT-Feed (Catalog No.: K40001) used for transient transfection and expression were all purchased from Zhuhai Kerry Biotechnology Co., Ltd.

[0060] 2) Instantaneous transfection Taking 100 mL of cell suspension as an example: Use a 15 mL sterile centrifuge tube to mix 5 mL of KPM and 100 μg of sterile plasmid DNA (heavy chain:light chain plasmid ratio 1:1), and gently pipette to mix. In another 15 mL centrifuge tube, add 5 mL of KPM and 500 μL of TA-CHO transfection reagent, and gently pipette to mix. Combine the contents of the two centrifuge tubes and let stand for 10 minutes to prepare the plasmid-vector complex. Then, add the complex to the cell suspension and return to a 37°C CO2 incubator for shaking culture. After 3 hours, an appropriate amount of antibiotic can be added as needed.

[0061] 3) Expression and purification of the product 600 μL of KE-CHO and KT-Feed were added 24 hours after transfection; cell supernatant was collected 6-8 days after transfection. The cell supernatant was purified by Protein A column, the antibody was collected, and ultrafiltered to PBS (pH 7.4) for concentration to obtain a 1 mg / mL anti-human FOXP3 monoclonal antibody prepared using recombinant technology.

[0062] 3. Antibody verification 3.1 Western blotting to detect the specificity of anti-human FOXP3 monoclonal antibody 1) Sample preparation: Prepare CHO cells and lyse them using RAPI lysis buffer containing 1 mM PMSF. After lysis, mix the sample with 5×SDS-PAGE Loading buffer at a 1:1 volume ratio for sample preparation.

[0063] 2) Electrophoresis and Transfer: Load 30 μg of each sample for SDS-PAGE electrophoresis at an initial voltage of 80 V for 20 minutes. Then adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue dye reaches the bottom of the gel. After electrophoresis, perform the transfer procedure: soak the NC membrane and filter paper in transfer buffer beforehand. Place the sample and membrane in a sandwich configuration in the wet transfer apparatus in the following order: electrode (-) - sponge - filter paper - gel - NC membrane - filter paper - sponge - electrode (+). Transfer at a constant voltage of 90 V for 90 minutes.

[0064] 3) Blocking: Immerse the NC membrane in blocking solution (TBST containing 5% skim milk) and incubate at 37°C for 2 hours to block the membrane and reduce non-specific binding.

[0065] 4) Primary antibody incubation: Place the NC membrane in blocking buffer containing anti-human FOXP3 monoclonal antibody (diluted 1:2000) and incubate at 37°C for 1 hour. After incubation, wash the membrane three times with TBST for 5 minutes each time to remove unbound primary antibody.

[0066] 5) Secondary antibody incubation: Place the NC membrane in HRP-labeled goat anti-mouse IgG secondary antibody (diluted 1:5000) and incubate at 37°C in a shaker for 30 minutes. After incubation, wash the membrane three times with TBST for 5 minutes each time to remove unbound secondary antibody.

[0067] 6) Color development: Mix component A and component B of the ECL luminescent solution in a 1:1 ratio, add the mixture to the NC membrane, and perform color development. Expose the NC membrane using an exposure device, observe and record the FOXP3 protein bands.

[0068] The results are as follows Figure 1 As shown, GAPDH was used as an internal standard, and the band was clear, indicating that the electrophoresis results were reliable. In the lanes of CHO cell lysate samples, there was only a single target band at around 50 kDa, with no other extraneous bands. This indicates that the anti-human FOXP3 monoclonal antibody of the present invention specifically binds to FOXP3 in human tissue samples, and the antibody has good specificity.

[0069] 3.2 Immunohistochemical (IHC) detection of the specificity of anti-human FOXP3 monoclonal antibody 1) Tissue section preparation: Paraffin-embedded blocks of human tonsils and lung cancer tissue were obtained and sectioned using a Leica microtome. The section thickness was set to 3 μm. After sectioning, the sections were dry-baked at 65°C for 2 hours to remove excess paraffin and perform preliminary fixation.

[0070] 2) Immunohistochemical staining: Immunohistochemical staining with anti-human FOXP3 monoclonal antibody was performed using manual immunohistochemistry. The specific steps are as follows: ① Use DAKO HIGH pH repair solution for high-temperature antigen repair, with a repair time of 20 minutes.

[0071] ② After the sections have cooled, add 100 μL of endogenous peroxidase blocking solution and incubate at room temperature for 5 minutes to block endogenous peroxidase activity. Then, soak the sections twice with washing solution (PBS or TBS), 5 minutes each time.

[0072] ③ Primary antibody incubation: Use anti-human FOXP3 monoclonal antibody, diluted at a ratio of 1:1000. Take 100 μL of the diluted antibody and add it to the slide, then incubate at 37°C for 30 minutes. After incubation, soak the slide twice with washing buffer for 5 minutes each time to remove unbound primary antibody.

[0073] ④ Secondary antibody incubation: Add 100 μL of enzyme-labeled polymer and incubate at room temperature for 20 minutes. After incubation, soak the sections twice more with washing buffer, 5 minutes each time.

[0074] ⑤ Perform color development using DAB color development solution: Add 100 μL of DAB color development solution and incubate at room temperature for 5 minutes. After color development, soak the sections in purified water twice, 5 minutes each time.

[0075] ⑥ Counterstaining: Add 100 μL of hematoxylin to counterstain the sections and incubate at room temperature for 5 minutes. After counterstaining, rinse the sections with tap water for bluing treatment.

[0076] 3) Dehydration, clearing and sealing ① Rinse the slides with deionized water for 3 minutes.

[0077] ② Perform dehydration treatment sequentially: 85% ethanol for 1 minute; 95% ethanol for 1 minute; 100% ethanol for 1 minute, repeat 2 times.

[0078] ③ Treat the slices with xylene for 1 minute, repeat twice, to achieve a transparent effect.

[0079] ④ Finally, seal the slide with neutral resin and cover it with a coverslip.

[0080] 4) Microscopic observation: Observe the staining results through a microscope.

[0081] Tonsil tissue test results as follows Figure 2 As shown, the lung cancer tissue examination results are as follows: Figure 3 As shown, FOXP3 protein is localized in the cell nucleus and exhibits specific expression in tonsil and lung cancer tissues, with staining results showing specific positive signals within the nucleus. The number of dark brown dotted areas in the tonsil tissue was significantly greater than that in the lung cancer tissue, with a stronger positive signal, showing differential expression, which is consistent with the expected results.

[0082] Example 4: Application of anti-human FOXP3 monoclonal antibody in the preparation of FOXP3 in vitro detection reagents or kits This embodiment provides the application of anti-human FOXP3 monoclonal antibody in the preparation of FOXP3 in vitro detection reagents or kits, as detailed below: This embodiment provides an in vitro detection reagent for FOXP3, including a working solution of anti-human FOXP3 monoclonal antibody. The working solution of anti-human FOXP3 monoclonal antibody uses TBS buffer, the concentration of anti-human FOXP3 monoclonal antibody in the working solution is 1 μg / mL, the concentration of BSA is 10 mg / mL, and the preservatives Proclin 300 and Proclin 950 are added at a ratio of 1:1000 (v / v).

[0083] Based on the above-mentioned anti-human FOXP3 monoclonal antibody working solution, a FOXP3 detection kit is constructed by combining DAKO HIGH pH restoration solution, endogenous peroxidase blocking solution, enzyme-labeled polymer, DAB chromogenic solution, hematoxylin, etc.

[0084] This invention also provides specific application examples of the above-mentioned FOXP3 in vitro detection reagents or kits. Currently, the most widely used FOXP3 antibody on the market is the FOXP3 antibody with clone number 236A / E7. The anti-human FOXP3 monoclonal antibody prepared in this invention is compared with the 236A / E7 antibody (abcam, ab20034). Specifically, the above-mentioned FOXP3 in vitro detection reagents, kits, and the 236A / E7 antibody are used to perform immunohistochemical staining on human tonsil and lung tissues. Detailed steps are described in section 3.2 of Example 3 regarding the specificity of the anti-human FOXP3 monoclonal antibody detected by immunohistochemistry (IHC). In the IHC experiment, the concentration of the anti-human FOXP3 monoclonal antibody of this invention is 1 μg / mL, and the concentration of the 236A / E7 antibody is 2 μg / mL.

[0085] Human tonsil tissue test results as follows Figure 4 As shown, the results of human lung tissue examination are as follows: Figure 5 As shown, the anti-human FOXP3 monoclonal antibody of the present invention, at a working concentration of half that of the 236A / E7 antibody, exhibits a positive signal intensity comparable to that of the 236A / E7 antibody. The anti-human FOXP3 monoclonal antibody provided by the present invention has higher sensitivity and specificity compared to the 236A / E7 antibody, and can be used as a domestically produced alternative to the 236A / E7 antibody.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-human FOXP3 monoclonal antibody, characterized in that, The heavy chain variable region of the anti-human FOXP3 monoclonal antibody has the amino acid sequence shown in SEQ ID NO.1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.

2.

2. The use of the anti-human FOXP3 monoclonal antibody as described in claim 1 in the preparation of FOXP3 in vitro detection reagents or kits.

3. A detection reagent or kit comprising the anti-human FOXP3 monoclonal antibody as described in claim 1.

4. The detection reagent or kit according to claim 3, characterized in that, The detection reagents or kits include immunohistochemical detection reagents or kits.

5. A nucleic acid molecule encoding the anti-human FOXP3 monoclonal antibody as described in claim 1.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleotide sequence of the heavy chain variable region gene of the anti-human FOXP3 monoclonal antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region gene of the anti-human FOXP3 monoclonal antibody is shown in SEQ ID NO.

4.

7. An expression cassette, expression vector, recombinant cell, or recombinant bacterium comprising the nucleic acid molecule as described in claim 5 or 6.

8. The use of a nucleic acid molecule as described in claim 5 or 6, or an expression cassette, expression vector, recombinant cell, or recombinant bacterium as described in claim 7, in the preparation of an anti-human FOXP3 monoclonal antibody.

9. A method for preparing an anti-human FOXP3 monoclonal antibody, characterized in that, The nucleic acid molecule as described in claim 5 or 6 is introduced into a host cell, the cell supernatant is collected, and purified by ultrafiltration.

10. The method for preparing an anti-human FOXP3 monoclonal antibody according to claim 9, characterized in that, The host cells include mammalian cells, including HEK293 cells and CHO cells.

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