Monoclonal antibody 2F10-36A5 resisting HpFT protein, product based on monoclonal antibody 2F10-36A5 and application of monoclonal antibody 2F10-36A5

Abstract

The invention discloses a monoclonal antibody 2F10-36A5 for resisting HpFT protein, a product based on the monoclonal antibody 2F10-36A5 and application of the monoclonal antibody 2F10-36A5, and belongs to the technical field of monoclonal antibodies. The invention discloses amino acid sequences of variable regions of a light chain and a heavy chain of an anti-HpFT protein monoclonal antibody, and experiments prove that the anti-HpFT protein monoclonal antibody 2F10-36A5 has high affinity, can specifically recognize target antigen HpFT protein, can be used for preparing a product for detecting the HpFT protein level or preparing a cell marking system, and can be used for detecting the HpFT protein level. The product comprises a detection reagent, a kit or detection test paper.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies, and in particular relates to a monoclonal antibody against HpFT protein and products and applications based thereon. Background Art

[0002] Fucosyltransferase (FT) is a class of enzymes that catalyze fucosylation modification and is widely present in eukaryotic and prokaryotic organisms. It can catalyze the transfer of fucose from the sugar nucleotide donor GDP-Fuc to the acceptor molecule and participate in a variety of physiological and pathological processes. In Helicobacter pylori (Hp), FT can be divided into different subtypes according to the type of acceptor glycosyl group it catalyzes and the connection position of the formed fucosidic bond, such as α1,3-FT and α1,4-FT (Ma et al., 2006, 281(10):6385-6394). Among them, α1,3-FucT plays a key role in the biosynthesis pathway of bacterial lipopolysaccharide (LPS), mainly responsible for catalyzing the Lewis X (Lewis X) on the terminal glycan chain of the O-antigen domain of the LPS molecule. x ) and LewisY(Le y ) Fucosylation modification of antigenic epitopes (Sun et al., 2007; 282(13): 9973-9982). Lewis antigens formed by these fucosylation modifications are crucial in pathogen-host interactions. They give Helicobacter pylori the ability of molecular mimicry, that is, the Lewis antigen structure on the bacterial surface is highly similar to the corresponding antigen expressed by the host gastrointestinal epithelial cells. This similarity interferes with the host immune system's effective recognition of the pathogen, is an important mechanism for Helicobacter pylori to achieve immune evasion, and is involved in the process of establishing and maintaining chronic infection (Wang et al., 2000, 36(6): 1187-1196).

[0003] Recombinant Helicobacter pylori 26695α1,3-FT (HpFT) has been widely used in research fields such as fucose oligosaccharide synthesis, antibody-cell conjugates (ADCs) (Li et al., 2018, 4(12):1633-1641) development, and cell surface labeling systems (Zhou et al., 2025, 296:139701) due to its key role in catalyzing the synthesis of Lewis antigens and specific fucosylated structures (Kadirvelraj R et al., 2023, 19(8):1022-1030). However, specific monoclonal antibodies (mAbs) against HpFT have not yet been reported. mAbs have been widely used in the fields of high-sensitivity detection of target proteins, cell sorting and identification, disease diagnosis, etc. due to their high specificity and precise targeting ability, and are an indispensable core tool in modern biomedical research. Therefore, the development of specific monoclonal antibodies against HpFT is of urgent need and great value. Summary of the Invention

[0004] The purpose of the present invention is to provide a monoclonal antibody with high affinity and capable of specifically recognizing the target antigen HpFT protein, as well as products based on the monoclonal antibody and applications of the monoclonal antibody.

[0005] The present invention provides an anti-HpFT protein monoclonal antibody (designated 2F10-36A5), comprising a light chain and a heavy chain, wherein the light chain is of kappa type and the heavy chain is of IgG1 type; wherein: the amino acid sequences of the three complementary determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; and the amino acid sequences of the three complementary determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11, respectively.

[0006] Furthermore, the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region respectively have homologous sequences with at least 90% sequence identity to the amino acid sequences shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7; the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region respectively have homologous sequences with at least 90% sequence identity to the amino acid sequences shown in SEQ ID No.12, SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15.

[0007] Furthermore, the light chain variable region VL of the monoclonal antibody 2F10-36A5 has a homologous sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID No. 8; the heavy chain variable region VH has a homologous sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID No. 16.

[0008] The present invention further provides use of the above-mentioned anti-HpFT protein monoclonal antibody 2F10-36A5 in preparing a product for detecting HpFT protein levels or preparing a cell labeling system.

[0009] The present invention provides a nucleic acid molecule encoding the above-mentioned anti-HpFT protein monoclonal antibody 2F10-36A5 or a functional fragment thereof.

[0010] The present invention provides a recombinant expression vector containing the above nucleic acid molecule.

[0011] The present invention provides a host cell, which contains the above-mentioned recombinant expression vector.

[0012] The present invention further provides a product for detecting the level of HpFT protein, which comprises the above-mentioned nucleic acid molecule, recombinant expression vector and host cell.

[0013] Furthermore, in the product for detecting the level of HPFT protein, the recombinant expression vector has a signal peptide connected to the antibody and contains a transcriptional regulatory element; the host cell is selected from mammalian cells, specifically 293T cells, CHO cells, Expi293F cells, TM cell.

[0014] The above-mentioned product for detecting HpFT protein levels is a detection reagent, a detection kit or a test strip, wherein the detection reagent is a reagent for performing an antigen-antibody reaction or a reagent for a detection reaction; the reagent for performing an antigen-antibody reaction includes a buffer (PBS, etc.), a salt (NaCl, etc.), and a diluent (Binding buffer, Elution buffer, etc.); the kit is any one of a colloidal gold immunoassay kit, a chemiluminescence detection kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, a fluorescence immunoassay kit, and a microfluidic chip.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The anti-HpFT protein monoclonal antibody 2F10-36A5 provided by the present invention has high affinity and can specifically recognize the target antigen HpFT protein. It can be used to prepare products for detecting HpFT protein levels. In studying the biological functions of HpFT protein and strengthening other application fields related to HpFT protein, such as in the fields of immune cell interaction, intercellular signal transduction and tumor immunotherapy, the HpFT monoclonal antibody is expected to become an important tool for studying intercellular interactions and developing new immunotherapy strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the result of titer determination in tail tip blood of mice immunized with HpFT protein.

[0018] Figure 2 This is the subtype identification result of monoclonal antibody 2F10-36A5.

[0019] Figure 3 This is a representative electrophoresis result diagram of the purified monoclonal antibody 2F10-36A5; where M is the marker; 1 is before purification; 2 is the purification flow-through; 3 is the Binding buffer wash; 4 is the Elution buffer elution; 5 is the beads; 6 is the ultrafiltration flow-through; and 7 is the concentrated antibody after purification.

[0020] Figure 4 This is the result of ELISA to detect the affinity of monoclonal antibody 2F10-36A5.

[0021] Figure 5 This is the affinity constant Kaff result chart calculated based on the affinity of monoclonal antibody 2F10-36A5 detected by ELISA.

[0022] Figure 6 This is the result of ELISA to verify the specificity of monoclonal antibody 2F10-36A5.

[0023] Figure 7 The figure shows the representative results of the western blot method for detecting HpFT expression in CHO transfected cells using the monoclonal antibody 2F10-36A5; M is a marker; 1 is an empty vector; and 2 is the expression vector pcDNA3.1-HpFT.

[0024] Figure 8 The figure shows a representative result of detecting HpFT expression in 293T transfected cells using the monoclonal antibody 2F10-36A5 based on the western blot method; M is a marker; 3 is an empty vector; and 4 is a eukaryotic expression vector pcDNA3.1-HpFT.

[0025] Figure 9Representative results of immunofluorescence detection of HpFT expression using monoclonal antibody 2F10-36A5. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0027] Example 1: Preparation of HpFT protein

[0028] The HpFT gene sequence was synthesized into the PUC-GW vector, and the target gene was cloned into the PET-24b vector. The plasmid was extracted, identified by double enzyme digestion, and sequenced for alignment. The constructed prokaryotic expression plasmid was transformed into Escherichia coli BL21(DE3) competent cells. A single colony was selected and inoculated into LB medium. The cells were shaken at 37°C to an OD value of 0.5-1. IPTG was added and expression was induced overnight at 16°C. The cells were harvested, ultrasonically disrupted, and centrifuged, followed by collection of the supernatant. The protein was purified by Ni affinity chromatography, dialyzed, and concentrated to obtain a high-concentration HpFT protein.

[0029] Example 2: Preparation of monoclonal antibodies

[0030] 1. Immunization of mice with HpFT protein

[0031] Step 1: 1 female Balb / c mouse aged 5 to 8 weeks; after one week of acclimatization, 50 μg of HpFT protein was mixed with Freund's complete adjuvant in a 1:1 ratio, completely emulsified to an oil-in-water state, and then injected subcutaneously at 4 points to complete the first immunization; three weeks later, 50 μg of HpFT protein was mixed with Freund's incomplete adjuvant in a 1:1 ratio, completely emulsified to an oil-in-water state, and then injected subcutaneously at 4 points to complete the second immunization; three weeks later, 50 μg of HpFT protein was diluted with PBS and injected intraperitoneally to complete the third immunization.

[0032] Step 2: ELISA to detect the titer of tail tip blood of immunized mice

[0033] Three weeks after the third immunization, tail blood was collected to collect serum, and its titer was tested by ELISA. The specific steps were as follows: HpFT protein was diluted to 1 μg / mL and added to the ELISA plate, 100 μL / well, and coated at 4°C overnight; washed 3 times with PBST, 2 minutes each time, and patted dry; 3% BSA-PBST Block at 37°C for 2h at 200μL / well; wash 3 times with PBST, 2min / time, and pat dry; add diluted mouse serum (dilution multiples are: 1000, 2000, 4000, 8000, 16000, 32000, 64000), 100μL / well, incubate at 37°C for 1h; wash 3 times with PBST, 2min / time, and pat dry; add goat anti-mouse IgG-HRP, 100μL / well, incubate at 37°C for 40min; wash 3 times with PBST, 2min / time, and pat dry; add color development solution, 100μL / well, incubate at room temperature for 10min, then stop with stop solution, and measure the absorbance on a microplate reader, where the measurement wavelength is 450nm and the reference wavelength is 630nm. Figure 1 The results of antibody titer testing of immunized mice used in subsequent experiments are presented. These results show that compared to unimmunized normal mice, the antibody titer of the immunized mice was higher than 1:128,000. This result indicates that the immunized mice have produced an immune response to the immunogen HpFT protein and can be used for subsequent experiments.

[0034] 2. Cell fusion and screening of positive hybridoma cell lines

[0035] Step 1. Cell fusion

[0036] Myeloma SP2 / 0 cells were acclimated with 20% fetal bovine serum for one week. Three days before fusion, 50 μg of HpFT protein was mixed with PBS and injected intraperitoneally from the immunized mice described above for pulse immunization. On the day of fusion, the mice were sacrificed and the spleens were sterilely removed, triturated, and washed twice with serum-free medium. Splenocytes were mixed with SP2 / 0 cells at a ratio of 10:1 and centrifuged at 1200 rpm for 6 minutes. The supernatant was discarded and the bottom of the centrifuge tube was gently tapped to loosen the cell pellet. Over 30 seconds, 1 mL of 45% PEG solution preheated to 37°C was slowly added. After standing at room temperature for 90 seconds, incomplete medium preheated to 37°C was slowly added. The mixture was mixed thoroughly and centrifuged at 800 rpm for 6 minutes. The supernatant was discarded and 40 mL of complete medium preheated to 37°C was added. The mixture was then added to a 96-well cell culture plate seeded with peritoneal macrophages and cultured at 37°C in a 5% CO2 incubator. From day 2 to 7 after fusion, HAT and HT were added every other day to screen for fused cells.

[0037] Step 2: ELISA detection of hybridoma cells and cloning of positive hybridoma cells

[0038] Observe hybridoma cell growth 7-10 days after fusion. When cells in the well exceed 1 / 3 of the well bottom, test hybridoma antibody secretion. The test steps are as follows: HpFT protein is diluted to 1 μg / mL and added to the ELISA plate at 100 μL / well. Coat overnight at 4°C; wash three times with PBST for 2 minutes each time and pat dry; block with 3% BSA-PBST at 37°C for 2 hours; wash three times with PBST for 2 minutes each time and pat dry; add hybridoma cell supernatant at 100 μL / well and incubate at 37°C for 1 hour; wash three times with PBST for 2 minutes each time and pat dry; add goat anti-mouse IgG-HRP at 100 μL / well and incubate at 37°C for 40 minutes; wash three times with PBST for 2 minutes each time and pat dry; add colorimetric solution at 100 μL / well and incubate at room temperature for 10 minutes. Stop the reaction with the stop solution and measure absorbance on a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. The clones with the highest OD values ​​were subcloned and screened again by ELISA 7-10 days later. After three rounds of cloning, one positive clone was finally identified and named 2F10-36A5.

[0039] 3. Identification of monoclonal antibody 2F10-36A5 subtypes

[0040] Add 100 ng / well of HpFT protein to the ELISA plate and coat overnight at 4°C; wash 3 times with PBST, 2 min / time, and pat dry; block with 3% BSA-PBST at 37°C for 2 h; wash 3 times with PBST, 2 min / time, and pat dry; add 100 μL / well of hybridoma cell supernatant and incubate at 37°C for 1 h; wash 3 times with PBST, 2 min / time, and pat dry; add 100 μL / well of HRP-labeled rabbit anti-mouse secondary antibody (IgG1, IgG2a, IgG2b, IgG3, IgM, kappa) and incubate at 37°C for 40 min; wash 3 times with PBST, 2 min / time, and pat dry; add 100 μL / well of colorimetric solution and incubate at room temperature for 10 min, then stop with stop solution and measure absorbance on a microplate reader. The measurement wavelength is 450 nm and the reference wavelength is 630 nm. The experimental results are shown in Figure 2. Figure 2 As shown in the figure, the OD values ​​of IgG1 and kappa subtypes of 2F10-36A5 are much higher than those of other subtypes and the negative control. Therefore, the heavy chain of monoclonal antibody 2F10-36A5 is IgG1 subtype, and the light chain is kappa subtype.

[0041] 4. Ascites Preparation and Antibody Purification

[0042] Step 1: Ascites preparation

[0043] 500 μL of ascites adjuvant was injected into the peritoneal cavity of 10-week-old Balb / c mice. After 7 days, the hybridoma cells were cultured to the logarithmic growth phase and the cell density was adjusted to 2×10 6500 μL was injected intraperitoneally and ascites was collected 7 to 10 days later.

[0044] Step 2: Purification of monoclonal antibody 2F10-36A5

[0045] Binding: The ascites was diluted with binding / wash buffer (pH 7.0), centrifuged to obtain the supernatant, added with protein A beads, mixed well, and bound on a shaker at 4°C overnight. Equilibration: Equilibrate the chromatography column twice with binding / wash buffer. Loading: Add the binding solution that has been bound overnight to the chromatography column. Washing: After the protein A beads in the binding solution have sunk to the bottom of the chromatography column, wash the chromatography column twice with binding / wash buffer. Elution: Elute the antibodies bound to protein A with Elution buffer (pH 3.0), and immediately add 1 / 10 volume of neutralization buffer (pH 8.5) to the eluate and mix well. Ultrafiltration replacement: Use an ultrafiltration tube to concentrate the obtained elution neutralization solution, and gradually replace the liquid with an antibody storage solution in PBS (pH 7.2). After determining the concentration using the BCA method, take 10-fold and 20-fold diluted antibodies and the original ascites for SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. The results are as follows: Figure 3 As shown in the figure, the heavy chain and light chain bands of the antibody can be clearly seen, and the impurity proteins are significantly reduced after purification, and the purity of the antibody is significantly improved.

[0046] 5. Affinity detection of monoclonal antibody 2F10-36A5

[0047] Dilute the coating antigen to 1 μg / mL and 2 μg / mL with coating buffer, add 100 μL to each well, and coat overnight at 4°C. Use indirect ELISA to determine the titer of monoclonal antibodies at different coating concentrations. Draw two ELISA reaction curves with the antibody concentration on the x-axis and the OD on the y-axis. 450nm Value. Select Figure 4 OD corresponding to the flat area of ​​the middle curve 450nm The value was used as the reference point of 100%. Then two reciprocal curves were drawn, where the x-axis represents the reciprocal of the antibody concentration (1 / concentration) and the y-axis represents the OD 450nm The reciprocal of the value (1 / OD450 value). Figure 5 The equations derived from these curves were used to calculate the 50% OD 450nmThe antibody concentration corresponding to the value was converted into molar concentration. Finally, the affinity constant (Kaff) was calculated using the formula Kaff = (n-1) / 2(n[Ab']t-[Ab]t), where n = [Ag]t / [Ag']t, [Ag]t and [Ag']t represent two different antibody concentrations, and [Ab]t and [Ab']t represent 50% OD at each antibody concentration. 450nm The results show that the affinity constant of monoclonal antibody 2F10-36A5 can reach 10 -10 M.

[0048] Example 3: Application experiment of monoclonal antibodies

[0049] 1. Detection of target protein HpFT by ELISA based on monoclonal antibody 2F10-36A5

[0050] The expressed and purified HpFT protein and the laboratory-available GFAP, Tau-His, and BSA proteins were diluted to 1 μg / mL in coating buffer and added to the ELISA plate. The diluent was also added to the ELISA plate at 100 μL / well and incubated at 4°C overnight. The plate was washed with PBST three times, 2 min each time, and patted dry. The plate was then incubated with 3% BSA-PBST. 200ul / well blocked at 37℃ for 2h; washed 3 times with PBST, 2min / time, pat dry; added hybridoma cell 2F10-36A5 supernatant, 100μL / well, incubated at 37℃ for 1h; washed 3 times with PBST, 2min / time, pat dry; added goat anti-mouse IgG-HRP, 100μL / well, incubated at 37℃ for 40min; washed 3 times with PBST, 2min / time, pat dry; added color development solution, 100μL / well, incubated at room temperature for 10min, then stopped with stop solution, and measured absorbance on a microplate reader, where the measurement wavelength is 450nm and the reference wavelength is 630nm. Table 1 and Figure 6 The experimental results show that the monoclonal antibody 2F10-36A5 can specifically recognize the target antigen HpFT protein.

[0051] Table 1 ELISA verification of the specificity of monoclonal antibody 2F10-36A5

[0052] Coating antigen HpFT GFAP Tau-His BSA OD1 1.3153 0.0157 0.0184 0.0158 OD2 1.2969 0.0130 0.0174 0.0189

[0053] 2. Detection of target protein HpFT by western blot based on monoclonal antibody 2F10-36A5

[0054] Total protein from 293T and CHO cells was collected and quantified by BCA assay. 20 μg of protein was subjected to SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% skim milk powder, the membrane was incubated with the purified monoclonal antibody 2F10-36A5. After washing with TBST three times, the membrane was incubated with goat anti-mouse IgG-HRP. After washing with TBST three times, the membrane was developed with ECL. Figure 7 、 8 The results showed that HpFT-specific bands appeared in all cellular protein samples in the 50-80 kDa band range, and the monoclonal antibody 2F10-36A5 produced bands consistent with those of commercial anti-HpFT antibodies. This result demonstrates that the monoclonal antibody 2F10-36A5 can specifically recognize the HpFT protein and can be used for detection of HpFT protein in samples by western blot.

[0055] 3. Immunofluorescence detection of target protein HpFT based on monoclonal antibody 2F10-36A5

[0056] CHO-HpFT and CHO-HpFT-Vec cells were seeded on slides, fixed with paraformaldehyde, washed three times with PBST, and incubated with 3% BSA-TBST (containing 0.1% Triton100) at room temperature for 1 hour. After washing once with PBST, the cells were incubated with 2F10-36A5HpFT ascites at room temperature for 2 hours. After washing three times with PBST, the cells were incubated with Alexa Fluor 488-labeled goat anti-mouse IgG antibody at room temperature for 60 minutes. After washing with PBST, the slides were mounted with a mounting medium containing DAPI, and the staining results were observed under a fluorescence microscope. Figure 9 The experimental results showed that after the two types of cells were incubated with antibodies, significant green ( Figure 9 HpFT) and DAPI-stained blue nuclei ( Figure 9 DAPI), and the two can overlap well ( Figure 7 This result indicates that the monoclonal antibody 2F10-36A5 can specifically stain HpFT protein and can be used for immunofluorescence staining to detect HpFT protein in samples.

[0057] Example 4: Antibody variable region sequencing and gene cloning

[0058] 1. Sequencing of the variable region of monoclonal antibody 2F10-36A5

[0059] Total RNA was extracted from hybridoma cells and reverse transcribed to obtain cDNA. The variable region fragments of the antibody heavy and light chains were amplified using the 5' RACE method. The amplified fragments were subcloned into the pBM16A vector, and the plasmids were extracted and sequenced to obtain the sequenced light and heavy chain sequences of the monoclonal antibody 2F10-36A5. The complementary determining regions of the antibody amino acid sequence were marked using the Kabat method. The results are shown in Table 2.

[0060] The amino acid sequence of the light chain variable region VL of the monoclonal antibody 2F10-36A5 is shown in SEQ ID No. 8, wherein the amino acid sequences of the three complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the amino acid sequences of the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region are shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, respectively.

[0061] The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody 2F10-36A5 is shown in SEQ ID No. 16, wherein the amino acid sequences of the three complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively, and the amino acid sequences of the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region are shown in SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15, respectively.

[0062] Table 2

[0063]

[0064]

[0065] 2. Construction of recombinant plasmid of monoclonal antibody 2F10-36A5

[0066] The amplified fragment containing the 36A5-VH sequence was ligated with the linearized pCDH-hG1 vector by homologous recombination. The ligation product was transformed into E. coli DH5α competent cells, and the plasmid was extracted. Successful recombination was confirmed by sequencing and named pCDH-36A5-VH-hG1. The pCDH-36A5-VL-hk vector was constructed using the same steps as above.

[0067] 3. Recombinant expression of monoclonal antibody 2F10-36A5

[0068] The constructed pCDH-36A5-VH-hG1 and pCDH-36A5-VL-hk expression vectors were co-transfected into HEK 293 cells for expression. Antibodies recognizing the HpFT protein were obtained in the culture supernatant 72 hours after transfection. The target antibodies were purified from the culture supernatant using Protein A resin gel, and the activity of the recombinant antibodies was further tested by ELISA.

[0069] In summary, the monoclonal antibody 2F10-36A5 provided in the present invention has good binding activity with the HpFT protein and strong specificity, and can be used for the detection of the HpFT protein. In studying the biological functions of the HpFT protein and strengthening other application fields related to the HpFT protein, such as in the fields of immune cell interactions, intercellular signaling and tumor immunotherapy, the HpFT monoclonal antibody is expected to become an important tool for studying intercellular interactions and developing new immunotherapy strategies.

[0070] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A monoclonal antibody 2F10-36A5 against HpFT protein, characterized in that: The monoclonal antibody 2F10-36A5 comprises a light chain and a heavy chain, wherein the light chain is of kappa type and the heavy chain is of IgG1 type; wherein: The amino acid sequences of the three complementarity determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; The amino acid sequences of the three complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively.

2. The anti-HpFT protein monoclonal antibody 2F10-36A5 according to claim 1, characterized in that: The framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region have homologous sequences with at least 90% sequence identity to the amino acid sequences shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, respectively; The framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region have homologous sequences with at least 90% sequence identity to the amino acid sequences shown in SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15, respectively.

3. The anti-HpFT protein monoclonal antibody 2F10-36A5 according to claim 1 or 2, characterized in that: The light chain variable region has a homologous sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID No. 8; the heavy chain variable region has a homologous sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID No.

16.

4. Use of the anti-HpFT protein monoclonal antibody 2F10-36A5 according to any one of claims 1 to 3 in the preparation of a product for detecting HpFT protein levels or a cell labeling system.

5. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the anti-HpFT protein monoclonal antibody 2F10-36A5 according to any one of claims 1 to 3 or a functional fragment thereof.

6. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that: The host cell contains the recombinant expression vector according to claim 6.

8. A product for detecting HpFT protein levels, characterized by: Comprising the nucleic acid molecule according to claim 5, the recombinant expression vector according to claim 6, and the host cell according to claim 7.

9. The product for detecting HpFT protein levels according to claim 8, characterized in that: The recombinant expression vector has a signal peptide connected to the antibody and contains a transcriptional regulatory element; the host cell is selected from 293T cells, CHO cells, Expi293F TM Any type of cell.

10. The product for detecting HpFT protein levels according to claim 8 or 9, characterized in that: The product includes any one of a detection reagent, a detection kit, and a detection test paper.

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