3D protein monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to a 3D protein monoclonal antibody and application thereof. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the following three complementary determining regions CDR: CDR1 shown as SEQ ID No.5, CDR2 shown as SEQ ID No.6, and CDR3 shown as SEQ ID No.7; the heavy chain variable region comprises the following three complementary determining regions: CDR1 shown as SEQ ID No.5, CDR2 shown as SEQ ID No.6, and CDR3 shown as SEQ ID No.7; the light chain variable region comprises the following three complementary determining regions CDR: CDR1 as shown in SEQ ID No.8, CDR2 with the sequence of WAS, and CDR3 as shown in SEQ ID No.9. The light chain variable region has the advantages that the light chain variable region can be used for preparing the light chain variable region; the invention provides a specific monoclonal antibody of a foot and mouth disease virus non-structural protein 3D protein. A Western blotting test shows that the specific monoclonal antibody specifically reacts with the non-structural protein 3D; indirect immunofluorescence tests show that the antibody has good reactivity with an FMDV O-type strain.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to a 3D protein monoclonal antibody and its applications. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute and highly contagious disease caused by the foot-and-mouth disease virus (FMDV), primarily infecting cloven-hoofed animals such as pigs, cattle, and sheep. The disease spreads rapidly and widely, posing a serious threat to global livestock production, animal product trade, and biosecurity. It is listed as a notifiable animal disease by the World Organisation for Animal Health (OIE).

[0003] FMDV viral particles exhibit a non-enveloped spherical structure with typical icosahedral symmetry, ranging in diameter from approximately 25 nm to 30 nm, making them one of the smallest known animal RNA viruses. The viral genome is a single-stranded positive-sense RNA, approximately 8400 nucleotides in length, consisting of a complete open reading frame (ORF) and untranslated regions at both ends. The 5′ untranslated region (5′-UTR) is structurally complex, containing regulatory elements such as internal ribosome entry sites; while the 3′ untranslated region (3′-UTR) is relatively short. The viral ORF is first translated into a polyprotein precursor, which is then processed by the virus's own encoded protease (primarily L...). pro and 3C pro Under the influence of ) cascade cleavage, it ultimately generates 4 structural proteins (VP1, VP2, VP3, and VP4) and 8 non-structural proteins (L pro ,2A,2B,2C,3A,3B,3C pro and 3D pol During this process, several stable protein cleavage intermediates are also formed, which play important roles in viral genome replication, capsid assembly, and viral particle maturation.

[0004] The FMDV 3D protein (RNA-dependent RNA polymerase, RdRp) is a key catalytic subunit for viral genome replication, responsible for synthesizing both positive and negative sense RNA strands, and plays an indispensable role in the viral life cycle. This protein exhibits high sequence conservation across different serotypes and subtypes of FMDV. During replication, small RNA viruses utilize the precursor protein 3CD as a functional intermediate. Although 3CD contains an active 3C protease domain, the activity of the 3D polymerase remains silent until the protein precursor is fully processed.

[0005] Structural biology studies have shown that FMDV 3D polIn terms of overall folding and catalytic mechanisms, it shares similarities with RdRp of various RNA viruses. Its three-dimensional structure exhibits a typical "right-handed" conformation, with three domains—the "palm," "fingers," and "thumb"—forming the active center cavity, responsible for substrate localization and metal ion coordination. The catalytic center of all RdRp is located in the palm domain, which contains a core composed of five conserved motifs, featuring three antiparallel β-sheets and flanking α-helices, participating in key functions such as structural stability, nucleotide recognition, binding, and phosphate transfer. In contrast, the thumb domain, composed of the C-terminus of a polypeptide chain, exhibits higher sequence and structural diversity in RdRp from different viral origins.

[0006] RNA replication of small RNA viruses is initiated in a primer-dependent manner. The viral protein VPg serves as a protein primer, with its tyrosine hydroxyl group at 3D... pol Ureylation occurs with the assistance of 3CD, forming a VPg-pU-pU complex, which then initiates RNA synthesis. Biochemical and structural studies suggest that the binding site of VPg to polymerase differs among different small RNA viruses: in FMDV, VPg mainly binds to the cleft of the polymerase active site; in Coxsackievirus B3 (CVB3), it binds to the base of the thumb domain; and in Enterovirus 71 (EV71), it is located at the base of the palm domain. During replication and elongation, RdRp completes three steps—nucleotide selection, phosphodiester bond formation, and template translocation—through dynamic conformational changes.

[0007] Studies on the structure and function of RdRp have provided potential targets for the development of highly selective anti-FMDV drugs. Research indicates that some nucleoside analogs, such as 5-fluorouracil and ribavirin, can inhibit viral replication through mutagenesis. Specifically, 5-fluorouracil, as a pyrimidine analog, can induce mutagenesis against various RNA viruses, including FMDV; ribavirin also possesses RNA polymerase-guided mutagenesis activity and can be used to eliminate FMDV from persistently infected cells by enhancing mutagenesis pressure.

[0008] The lack of commercially available monoclonal antibodies against FMDV 3D protein has limited in-depth research into the protein's function and regulatory mechanisms. Summary of the Invention

[0009] In view of the above-mentioned shortcomings in the prior art, the purpose of this invention is to provide a 3D protein monoclonal antibody and its application.

[0010] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A 3D protein monoclonal antibody is provided, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes the following three complementary determinant regions (CDRs): CDR1 shown in SEQ ID No. 5, CDR2 shown in SEQ ID No. 6, and CDR3 shown in SEQ ID No. 7; The light chain variable region includes the following three complementary determinant regions (CDRs): CDR1 shown in SEQ ID No. 8, CDR2 with sequence WAS, and CDR3 shown in SEQ ID No. 9.

[0011] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1.

[0012] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

[0013] The present invention also provides a biomaterial comprising at least one of the following: 1) The nucleic acid encoding the monoclonal antibody; 2) An expression vector containing the nucleic acid; 3) Transform or transfect the host cells containing the expression vector; 4) Culturing the host cells to obtain a culture containing the monoclonal antibody.

[0014] The present invention also provides a labeled antibody, which includes a marker and the monoclonal antibody described above.

[0015] The present invention also provides a conjugate comprising a conjugation medium and the monoclonal antibody described above.

[0016] The present invention also provides the use of at least one of the following (i) to (iv) in the preparation of products for detecting foot-and-mouth disease virus: i. The monoclonal antibody mentioned above; ii. The aforementioned biomaterials; iii. The labeled antibody; iv. The aforementioned coupling.

[0017] This invention also provides a product for detecting foot-and-mouth disease virus, comprising at least one of the following ① to ④: ① The aforementioned monoclonal antibody; ② The aforementioned biomaterials; ③ The labeled antibody; ④ The aforementioned coupling material.

[0018] The present invention also provides the use of at least one of the following A-D in the preparation of products for the prevention and / or treatment of foot-and-mouth disease virus: A. The aforementioned monoclonal antibody; B. The aforementioned biomaterials; C. The labeled antibody mentioned above; D. The aforementioned coupling material.

[0019] The present invention also provides products for the prevention and / or treatment of foot-and-mouth disease virus, comprising at least one of the following a to d: a. The aforementioned monoclonal antibody; b. The aforementioned biomaterials; c. The labeled antibody; d. The aforementioned coupling.

[0020] The beneficial effects of this invention are as follows: This invention provides a specific monoclonal antibody against the foot-and-mouth disease virus (FMDV) non-structural protein 3D. Western blotting experiments showed that it specifically reacts with the non-structural protein 3D; indirect immunofluorescence assays showed that it has good reactivity with FMDV type O strain. The successful preparation of this antibody provides an important research tool for further exploring the interaction mechanism between the non-structural protein 3D and host proteins during FMDV infection. Attached Figure Description

[0021] Figure 1 For SDS-PAGE detection of FMDV 3D protein; M: protein molecular weight standard; 1: 3D protein after SUMO enzyme digestion; 2: 3D protein without SUMO tag; 3: 3D protein after E. coli induction; Figure 2 The results of serum titer testing in 6 immunized mice; Figure 3 3D antibody purification for SDS-PAGE detection; M: protein molecular weight standard; 1: purified 3D antibody; Figure 4 Purification of 3D antibody for Western blotting identification; M: protein molecular weight standard; 1: purified FMDV3D protein; Figure 5 Western blotting identification of eukaryotic expressed protein 3D; M: protein molecular weight standard; 1: 293T cell lysate transfected with pCAGGS-HA empty plasmid; 2: 293T cell lysate transfected with pCAGGS-HA-3D plasmid; Figure 6 Western blotting identification of 3D monoclonal antibodies for FMDV infection; M: protein molecular weight standard; 1: PK15 cell lysate uninfected with FMDV; 2-3: PK15 cell lysate infected with FMDV; Figure 7 To determine the expression and subcellular localization of FMDV 3D protein in transfected cells; Figure 8 To determine the expression and subcellular localization of FMDV 3D protein in virus-infected cells. Detailed Implementation

[0022] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0023] The genes, strains, plasmids, viruses, and experimental animals used in the following examples are as follows: The FMDV 3D gene sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd.; Escherichia coli (E. coli) DH5α and BL21(DE3) competent cells were purchased from TaKaRa; plasmids pCAGGS-HA, pCAGGS-Flag, pCAGGS-HA-3D, pCAGGS-flag-3D, 293T cells, PK15 cells, and FMDV type O strain were preserved in our laboratory. Eight-week-old female BALB / c mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0024] The main reagents used in the following examples are: restriction endonucleases. Bam HI and Χho I was purchased from TaKaRa; HA-tagged mouse monoclonal antibody, Flag-tagged mouse monoclonal antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody, Freund's incomplete adjuvant, Freund's complete adjuvant, fetal bovine serum, Flag beads, and Flag peptide were all purchased from Sigma; HisTrap FF nickel affinity chromatography column and protein G purification column were purchased from Cytiva; small ubiquitin-related modifier (SUMO) protease was purchased from Solarbio Science & Technology Co., Ltd. (Beijing); ECL colorimetric kit was purchased from Merck.

[0025] Example 1 Construction, expression and purification of FMDV 3D protein 1.3 Construction and Identification of Prokaryotic Expression Plasmids for the 3D Gene Based on the FMDV gene (Foot-and-mouth disease virus, accession number: JN998085.1) published by NCBI, codon optimization was performed on the 3D whole genome, and restriction enzyme sites were introduced at both ends of the expression sequence. Bam HI and Χho I. The gene sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and constructed into the prokaryotic expression vector pET-28a-His-SUMO. The recombinant plasmid was named pET-28a-His-SUMO-3D, and the plasmid was transformed into... E. coli DH5α competent cells, after plasmid extraction, were subjected to... Bam HI and Χho I double enzyme digestion identification, and sequencing verification by Beijing Qingke Biotechnology Co., Ltd. (Xi'an).

[0026] 2.3D protein expression in prokaryotes The recombinant plasmid pET-28a-His-SUMO-3D was transformed into... E. coli BL21(DE3) competent cells were selected, and positive single colonies were inoculated into 5 mL of LB liquid medium containing kanamycin (100 μg / mL). The cells were cultured overnight at 37 ℃ and 220 rpm to preserve glycerol-containing bacteria. The bacterial culture was then inoculated into 100 mL of LB liquid medium containing kanamycin at a ratio of 1:50. When the OD600 reached approximately 0.6, 100 μL of 1 M IPTG was added, and expression was induced at 16 ℃ for 14 h. The cells were collected by centrifugation at 8000 r / min for 10 min. The cell pellet was resuspended in a balanced buffer solution (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) and sonicated. The pellet was centrifuged at 8000 rpm for 15 min, and the supernatant and pellet were collected separately. The pellet was resuspended and dissolved in 8 M urea. After sample preparation, the expression of the 3D protein was observed by SDS-PAGE electrophoresis.

[0027] 3.3 Purification of the D protein The preserved glycerol bacteria were induced to express large quantities according to the above steps. The bacterial cells were collected by centrifugation, sonicated, and then centrifuged again to collect the supernatant. A nickel column was connected to an AKTA Pure 150 purifier. The collected supernatant was filtered through a 0.45 μm filter and purified using affinity chromatography. The protein was eluted sequentially with a linear gradient of imidazole buffer, and the eluent and elution buffers were collected separately. The purified fusion protein was digested with SUMO protease overnight at 4°C, and further purified using affinity chromatography to remove the His-SUMO protein tag. The purification status was analyzed by SDS-PAGE.

[0028] The FMDV 3D protein was successfully purified by SDS-PAGE analysis using affinity chromatography. The purified protein size was consistent with the expected molecular weight. (See attached figure.) Figure 1 It has 473 amino acid residues, a protein size of 53 kDa, and an isoelectric point of 5.84. Its amino acid sequence is shown in SEQ ID No. 10.

[0029] SEQ ID No. 10: GLIVDTRDVEERVHVMRKTKLAPTVAHGVFNPEYGPAALSNKDPRLNEGVVLDEVIFSKHKGDTKMSPEDKALFRRCAADYASRLHSVLGTANAPLSIYEAIKGVDGLDAMEPDTAP GLPWALQGKRRGALIDFENGTVGPEVQAALELMEKREYKFACQTFLKDEIRPMEKVRAGKTRIVDVLPVEHILYTRMMIGRFCAQMHSNNGPQIGSAVGCNPDVDWQRFGTHFAQYRN VWDVDYSAFDANHCSDAMNIMFEEVFRTDFGFHPNAEWILKTLVNTEHAYENKRITVEGGMPSGCSATSIINTILNNIYVLYALRRHYEGVELDTYTMISYGDDIVVASDYDLDFEAL KPHFKSLGQTITPADKSDKGFVLGHSITDVTFLKRHFHMDYGTGFYKPVMASKTLEAILSFARRGTIQEKLISVAGLAVHSGPDEYRRLFEPFQGLFEIPSYRSLYLRWVNAVCGDA.

[0030] Example 2 Preparation of monoclonal antibodies 1. Immunization of mice with purified protein 3D To prepare a monoclonal antibody against FMDV 3D protein, the purified 3D protein was emulsified with adjuvant in a 1:1 ratio to prepare a vaccine, which was then used to immunize 6 mice. Eight-week-old female BALB / c mice were immunized once with Freund's complete adjuvant emulsion, and the second to fourth immunizations were emulsions with Freund's incomplete adjuvant emulsion. The immunizations were performed on days 0, 14, 28, and 36, with each immunization using 50 μg of protein per mouse. Fourteen days after the fourth immunization, blood was collected by tail amputation, and serum was obtained to determine the antibody titer.

[0031] 2. Evaluation of mouse serum titers using indirect ELISA Using FMDV 3D protein as the coating antigen, the antigen was diluted with coating buffer and added to ELISA microplates at a concentration of 100 ng / well, 100 μL / well, and incubated overnight at 4°C. The ELISA plates coated with the antigen were removed, the buffer was discarded, and the plates were washed 5 times with 1×PBST and blotted dry. 2% bovine serum albumin (BSA) was added at 200 μL / well, and the plates were blocked at 37°C for 2 h. The plates were washed 5 times with 1×PBST and blotted dry. Mouse ascites fluid prepared with SP2 / 0 was used as a negative control. Mouse serum (serially diluted 2-fold starting at 1:1000) was used as the primary antibody and serially diluted 2-fold with 1×PBST (starting at 1:1000). The buffer was added to ELISA plates at a concentration of 100 μL / well. The plates were incubated at 37°C for 1 h, the buffer was discarded, and the plates were washed 5 times with 1×PBST and blotted dry. HRP-labeled goat anti-mouse IgG was diluted with 1×PBST (1:10,000) and added to each well at 100 μL / well. The plate was incubated at 37°C for 1 h, the solution was discarded, and the plate was washed 5 times and patted dry. TMB substrate chromogenic solution was added at 50 μL / well, and the plate was incubated at 37°C in the dark for 15 min. Stop solution was added at 50 μL / well, and the OD450 value was measured on a microplate reader within 5 min. (OD values ​​of experimental sample wells) (OD value of blank control) / (OD value of negative control) When the blank control OD value is ≥2.1, that is, when S / N ≥2.1, it is judged as positive.

[0032] The indirect ELISA detection results of this embodiment are as follows: Figure 2 As shown, the serum titers of all six immunized mice were positive, indicating that they could be used for cell fusion.

[0033] 3. Screening of hybridoma cells Spleen cells from mice with the highest antibody levels were used for fusion experiments with SP2 / 0 cells. After fusion, an indirect ELISA method was used, with hybridoma cell supernatant as the primary antibody and HRP-labeled goat anti-mouse IgG antibody as the secondary antibody, to screen for positive hybridoma cell lines reacting with FMDV 3D. The positive hybridoma cell lines were cloned, purified, and passaged three times for expansion culture. Cells were collected and cultured at 2×10⁻⁶ cells / year. 6 Each cell / branch is preserved in liquid nitrogen.

[0034] 4. Preparation and purification of monoclonal antibodies One week prior to injection of the monoclonal cell line, sterile liquid paraffin was injected intraperitoneally into each BALB / c mouse (500 μL). Immunoreactive hybridoma monoclonal cells were collected, counted, and then adjusted to a cell density of 1 × 10⁻⁶. 6Cells / ml. Subsequently, the cell suspension was injected into the peritoneal cavity of BALB / c mice, and ascites fluid was collected multiple times over 10–14 days. The mouse ascites fluid was centrifuged at 12000 rpm for 10 min, filtered to remove lipids, tissue debris, and other impurities, and clear ascites fluid was obtained. Antibodies were purified by affinity chromatography using the following purification method: (1) Pretreatment of the chromatography column: Wash the Protein G affinity chromatography column with 10 column volumes of deionized water 3-5 times at a flow rate of 1 ml / min (2) Equilibration: Wash the column 3-5 times with 10 column volumes of equilibration buffer (0.02M PB + 0.3M NaCl, pH 7.0) at a flow rate of 1 ml / min until the pH of the effluent remains constant.

[0035] (3) Sample loading: Dilute the ascites with 0.02M PB, filter with a 0.22 μm filter membrane, and load the sample at a flow rate of 0.6mL / min.

[0036] (4) Washing: After loading the sample, wash the chromatography column with 5-10 times the column volume of equilibration buffer and collect the eluent.

[0037] (5) Elution: Elute with 5-10 times the amount of elution buffer (0.1M glycine, pH 3.0) and collect the eluent.

[0038] (6) pH adjustment: After elution, immediately adjust the pH of the elution product to neutral with saturated sodium carbonate.

[0039] (7) Clean the chromatography column: Rinse the chromatography column with 5-10 column volumes of pure water, and then immediately rinse with 5 times the equilibration buffer to equilibrate the chromatography column to neutral.

[0040] (8) Chromatography column storage: After washing the affinity chromatography column with 5 column volumes of 20% ethanol, store at 4°C.

[0041] (9) Sample concentration: Use a 10 kDa ultrafiltration tube to concentrate the eluted product to about 1-5 mL.

[0042] (10) Dialysis: Add all the concentrated sample into a dialysis bag and place it in 5 L of buffer (0.01M PBS, pH=7.4) for dialysis overnight. Change the buffer once on the second day.

[0043] (12) SDS-PAGE analysis was performed to determine antibody purity and protein content.

[0044] The SDS-PAGE analysis results are shown below. Figure 3 The antibody heavy chain and light chain bands were observed at 50 kDa and 25 kDa, respectively, and the purified 3D monoclonal antibody concentration was 7.5 mg / mL.

[0045] (13) The results of Western blotting identification are shown in [the table below]. Figure 4 This indicates that the screened 3D monoclonal antibody has good reactivity with the purified 3D protein.

[0046] 5. Monoclonal antibody subtype identification and sequencing (1) The monoclonal antibody Ig subtype was identified using a mouse monoclonal antibody subtype identification kit (proteintech), and the IgG1 subtype of the 3D monoclonal antibody was determined.

[0047] (2) Resuscitation of hybridoma cells: Take out a cryopreservation tube of hybridoma cells containing a specific monoclonal antibody against foot-and-mouth disease virus 3D protein from the liquid nitrogen tank, hold it with tweezers and quickly put it into a 37°C water bath. Shake it continuously to make the cells thaw quickly. Aseptically open the cryopreservation tube and transfer the cell solution into a 10mL DMEM medium. Place it in a 37°C incubator containing 5% CO2 until the hybridoma cells are completely attached to the wall. Then change the medium for the cells.

[0048] (3) PCR amplification of the variable region of monoclonal antibody: Cells were collected, centrifuged at 1500 rpm for 10 min, and RNA was extracted using the Trizol method. RNA concentration and integrity were assessed by NanoDrop spectrophotometer and agarose gel electrophoresis. Subsequently, RNA was reverse transcribed into cDNA using SMART Scribe reverse transcriptase (Vazyme), combined with oligo-dT primers and template switching primers (TSO), following the manufacturer's recommended procedure. The prepared cDNA was used as a template to amplify its gene fragments using high-fidelity polymerase. The forward primers used for amplification were anchored to the TSO sequence, while the reverse primers targeted the constant regions of the immunoglobulin heavy or light chains. Partial P5 and P7 adapter sequences were introduced into the 5′ ends of the forward and reverse primers, respectively. In the first round of PCR amplification, the heavy and light chain fragments were amplified separately in independent reaction systems; in the second round of PCR, the products from the first round were tailed using index primers to construct a TruSeq library with a double-indexed structure. After purification with magnetic beads, the concentration of the library was determined using the Qubit quantitative PCR system, and paired-end sequencing was performed on the Illumina MiSeq PE300 or BGI G99 sequencing platform according to the instrument operation manual.

[0049] (4) The sequencing results were compared with the antibody gene library (IMGT / v-quest). The complementarity determining region (CDR) sequences of the heavy chain variable region and light chain variable region of the anti-FMDV 3D protein monoclonal antibody prepared in this embodiment are shown in Table 1.

[0050] Table 1 Antibody variable region sequence The amino acid sequence of the heavy chain variable region is (SEQ ID No. 1): EVQLVESGGGLVKPGGSLKVSCAASGFTFSSYAMSWVRQTPEKRLEWVATISSGATYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAIYYCGRHDSSGYVPYAYWGQGTLVTVSA.

[0051] The gene sequence encoding the variable region of the heavy chain is (SEQ ID No. 3): GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAAGTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGCTACTTACACCTACTAT CCAGACAGTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATATATTACTGTGGAAGACATGACAGCTCGGGCTACGTCCCTTATGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0052] The amino acid sequence of the light chain variable region is (SEQ ID No. 2): DIVMTQSHKFMSTSVGDRISITCKASQDVSTAVEWYQQKQGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCLQHYNTPYTFGGGTKLEIK.

[0053] The gene sequence encoding the variable region of the light chain is (SEQ ID No. 4): GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGATCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTTGAGTGGTATCAACAAAAACAAGGGCAATCTCCTAAACTGCTGATTTACTGGGCATCCACCCG GCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACTGATTATACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCACTGTATTACTGTCTACAACACTATAACACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA.

[0054] Example 3 Monoclonal antibodies specifically react with non-structural proteins in 3D 1. Western blotting to detect the expression of 3D proteins in overexpressing cells The eukaryotic expression plasmid pCAGGS-HA-3D and the empty vector plasmid pCAGGS-HA were transfected into 293T cells, respectively. Cell samples were collected and processed, and Western blotting was used to detect their reactivity with the 3D monoclonal antibody. The specific experimental procedure is as follows: (1) The eukaryotic expression plasmid pCAGGS-HA-3D and the empty vector plasmid pCAGGS-HA were transfected into 293T cells, and the cells were collected after being cultured at 37℃ for 24 h.

[0055] (2) Sample preparation: Cells were lysed using lysis buffer, and after obtaining protein samples by centrifugation at 12,000 rpm for 15 min, 5× protein loading buffer was added, mixed thoroughly, and then in a 100℃ metal bath for 10 min. After cooling, the mixture was centrifuged at 12,000 rpm for 2 min.

[0056] (3) Electrophoresis: Fix the SDS-PAGE gel (12% separating gel, 5% stacking gel) on the electrophoresis apparatus, add 1×Tris-glycine electrophoresis buffer, and load 10µL of sample into each lane. The initial electrophoresis voltage is 80 V. After the sample enters the separating gel, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue reaches the bottom of the separating gel, then terminate the electrophoresis.

[0057] (4) Transfer: Pre-cool the membrane transfer buffer at 4°C. After removing the stacking gel, wash the surface with deionized water. Cut a PVDF membrane to the same size as the gel and soak it in 100% methanol for 1-2 minutes. Stack the membranes neatly in the order of "black side - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - white side", making sure to remove air bubbles between the layers. Place the membrane in a wet transfer tank, add sufficient transfer buffer, and transfer at a constant voltage of 100V for 2 hours.

[0058] (5) Sealing: After the transfer is completed, take out the PVDF membrane and seal it with 5% skim milk powder at room temperature for 1-2 hours.

[0059] (6) Incubation of primary antibody: Add 5 mL of anti-3D mouse monoclonal antibody diluted with 5% skim milk powder (1:4000 dilution) and incubate overnight at 4°C.

[0060] (7) Incubation of secondary antibody: Wash 5 times with 1×PBST, shake for 5 min each time under room temperature conditions, add 5 mL of horseradish peroxidase-labeled goat anti-mouse IgG antibody diluted with 5% skim milk powder (1:5000), and incubate at room temperature for 1 h.

[0061] (8) Development: Wash with 1×PBST 5 times, shaking for 5 minutes each time under room temperature conditions. Mix ECL substrate colorimetric reagent A and reagent B in a 1:1 ratio. Add an appropriate amount of colorimetric solution to the PVDF membrane, scan and expose, and save the image.

[0062] Test results are shown Figure 5 The 3D protein monoclonal antibody provided by this invention specifically reacts with the FMDV non-structural protein 3D, and no band is observed at the same location in 293T cells transfected with the empty vector, indicating that the monoclonal antibody can specifically bind to the 3D protein. This demonstrates that the monoclonal antibody 3D provided by this invention can specifically react with the FMDV non-structural protein 3D.

[0063] 2. Western blotting detection of 3D protein expression in FMDV-infected cells PK15 cells were infected with FMDV at a MOI of 0.1 and cultured at 37°C for 12 h. PK15 cells were then collected, with uninfected cells used as a negative control. Cells were lysed using NP-40 lysis buffer, centrifuged at 12000 rpm for 15 min to obtain protein samples, and then loaded with protein loading buffer. The mixture was incubated at 100°C for 10 min and then subjected to SDS-PAGE gel electrophoresis using a 12% protein gel. The membrane was transferred at a constant voltage of 100 V for 1 h. After transfer, the PVDF membrane was removed and blocked with 5% skim milk powder at room temperature for 1 h. 3D monoclonal antibody diluted with blocking buffer was added, and the membrane was incubated overnight at 4°C. The membrane was washed 5 times with 1×PBST, and diluted HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) was added. The membrane was incubated at room temperature for 1 h. After washing 5 times with 1×PBST, an appropriate amount of ECL chromogenic solution was added to the PVDF membrane, and the image was scanned, exposed, and saved.

[0064] The results of Western blotting are shown below. Figure 6 The 3D protein monoclonal antibody detected a clear and specific reaction band in protein samples derived from infected PK15 cells, while uninfected PK15 cells did not show this band at the same location.

[0065] Example 4 Assay of monoclonal antibody activity against FMDV nonstructural protein 3D 1. Immunofluorescence detection of 3D protein expression and subcellular localization analysis in overexpressed cells PK15 cells were loaded at a rate of 1×10 5 Cells were seeded at a density of / wells in confocal dishes and transfected with eukaryotic expression plasmid pCAGGS-HA-3D and empty vector plasmid pCAGGS-HA for 24 h. Cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized with 0.2% Triton X-100 at room temperature for 10 min, and blocked with blocking buffer containing 2% BSA at room temperature for 1 h. Cells were then incubated overnight at 4 °C with FMDV 3D monoclonal antibody (1:500 dilution); followed by incubation with goat anti-mouse 555 red fluorescent secondary antibody (1:500 dilution) at 37 °C for 1 h; washed four times with PBS; and finally stained nuclei with DAPI at room temperature for 5 min, washed four times with PBS, and observed and photographed under a confocal microscope. Confocal results ( Figure 7 The results showed that the monoclonal antibody bound well to the FMDV 3D overexpression product in PK15 cells and had high specificity, while the control group transfected with the empty vector pCAGGS-HA showed no fluorescence. In cells transfected with pCAGGS-HA-3D, the 3D protein was mainly distributed in the cytoplasm.

[0066] 2. Immunofluorescence detection of 3D protein expression and subcellular localization in FMDV-infected cells PK15 cells were adsorbed with FMDV at an MOI of 0.1 for 1 hour, with uninfected cells used as a negative control. The confocal dish was gently agitated every 20 minutes. After 1 hour of adsorption, the cells were cultured in complete medium containing 10% fetal bovine serum for 12 hours. Cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed four times with PBS, and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 minutes. The solution was discarded, and cells were blocked with blocking buffer containing 2% BSA at room temperature for 1 hour. FMDV 3D monoclonal antibody was diluted with the above blocking buffer and incubated overnight at 4°C. Cells were washed four times with PBS, and goat anti-mouse 555 red fluorescent secondary antibody was diluted with PBS and incubated at 37°C in the dark for 1 hour. Cells were washed four times with PBS, and nuclei were stained with DAPI at room temperature in the dark for 5 minutes. Cells were washed four times with PBS and observed and photographed under a confocal microscope. Results ( Figure 8 The results showed that the 3D monoclonal antibody exhibited good specificity in reacting with protein samples derived from FMDV-infected PK15 cells, and showed no non-specific binding in uninfected PK15 cells. The study also found that the 3D protein was mainly distributed in the cytoplasm of FMDV-infected cells.

[0067] In summary, this invention successfully prepared a specific monoclonal antibody against the FMDV 3D protein and systematically evaluated its effectiveness in indirect immunofluorescence and Western blotting detection. Furthermore, the expression and subcellular localization of the FMDV 3D protein in overexpressing cells and virus-infected cells were analyzed. The successful preparation of this antibody provides an important research tool for further exploring the interaction mechanism between the non-structural protein 3D and host proteins during FMDV infection.

[0068] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0069] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A 3D protein monoclonal antibody, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises the following three complementarity determining regions (CDRs): CDR1 shown in SEQ ID No. 5, CDR2 shown in SEQ ID No. 6, and CDR3 shown in SEQ ID No. 7; The light chain variable region comprises the following three complementarity determining regions (CDRs): CDR1 shown in SEQ ID No. 8, CDR2 with sequence WAS, and CDR3 shown in SEQ ID No.

9.

2. The 3D protein monoclonal antibody according to claim 1, characterized by, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

1.

3. The 3D protein monoclonal antibody according to claim 1, characterized by, The amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

4. Biomaterials characterized in that, At least one of the following: 1) nucleic acid encoding the monoclonal antibody of any one of claims 1-3; 2) an expression vector comprising the nucleic acid; 3) a host cell transformed or transfected with the expression vector; 4) culturing the host cell to obtain a culture containing the monoclonal antibody.

5. A labeled antibody characterized in that, The monoclonal antibody of any one of claims 1-3.

6. Conjugate characterized in that, The monoclonal antibody of any one of claims 1-3.

7. Use of at least one of the following i-iv in the preparation of a product for detecting foot-and-mouth disease virus: i) the monoclonal antibody of any one of claims 1-3; ii) the biological material of claim 4; iii) the labeled antibody of claim 5; iv) the conjugate of claim 7.

8. A product for the detection of foot-and-mouth disease virus, characterized in that, At least one of the following: i) the monoclonal antibody of any one of claims 1-3; ii) the biological material of claim 4; iii) the labeled antibody of claim 5; iv) the conjugate of claim 7.

9. Use of at least one of the following A-D in the preparation of a product for preventing and / or treating foot-and-mouth disease virus: A) the monoclonal antibody of any one of claims 1-3; B) the biological material of claim 4; C) the labeled antibody of claim 5; D) the conjugate of claim 7.

10. A product for the prevention and / or treatment of foot-and-mouth disease virus, characterized in that, At least one of the following: a) the monoclonal antibody of any one of claims 1-3; b) the biological material of claim 4; c) the labeled antibody of claim 5; d) the conjugate of claim 7.

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