Monoclonal antibody of swine acute diarrhea syndrome coronavirus S1-CTD protein and application thereof

Abstract

The invention relates to a monoclonal antibody of porcine acute diarrhea syndrome coronavirus S1-CTD protein and application of the monoclonal antibody. According to the invention, S1-CTD protein is expressed in BL21 (DE3) cells by using a prokaryotic expression system, the purified S1-CTD protein is used as an immunogen, a BALB / c mouse is immunized by an immunological method, and the monoclonal antibody for resisting SADS-CoV S1-CTD protein is prepared through cell fusion. Two rounds of truncation are carried out by using an overlapping polypeptide method, the antigen epitope is identified by indirect ELISA (Enzyme-Linked Immunosorbent Assay) and Dot-ELISA, and the result shows that the linear B cell epitope region of the S1-CTD protein is PGFVLRVGRG. The monoclonal antibody and the B cell epitope lay a foundation for preparing a reagent for diagnosing or preventing SADS-COV.

Description

Technical Field

[0001] This invention relates to the field of bioimmunotechnology, specifically to monoclonal antibodies against SADS-CoV S1-CTD protein and their applications. Background Technology

[0002] Swine acute diarrhea syndrome (SADS-CoV) is an intestinal infectious disease of pigs caused by swine acute diarrhea syndrome coronavirus (SADS-CoV). The mortality rate in newborn piglets under 5 days old can reach over 90%, posing a significant threat to the pig industry. SADS-CoV has a wide host range and considerable potential for cross-species transmission. The main clinical symptoms of SADS-CoV infection are acute diarrhea and vomiting. Newborn piglets under 5 days old experience rapid weight loss after infection and generally die within 2-6 days of onset, with a mortality rate as high as 90%; the mortality rate in piglets aged 5-10 days is over 35%; infected sows generally experience mild diarrhea and recover within a few days. SADS-CoV is a newly emerging enteropathogenic coronavirus with an extremely high mortality rate in piglets and a strong ability to spread across species. Because it is a new threat to the pig industry, research and understanding of this virus are still in their early stages. Current knowledge of the structure and function of the proteins that make up SADS-CoV remains limited, leading to significant difficulties in the detection and vaccine development of SADS-CoV. Therefore, further research into the structure and function of porcine acute diarrhea syndrome coronavirus proteins is crucial for the development of SADS-COV detection reagents and vaccines, as well as for the prevention of African swine fever.

[0003] Viral recognition of corresponding receptors and successful entry into host cells are prerequisites for viral infection and pathogenesis. In porcine acute diarrhea syndrome coronavirus (SADS-CoV), both of these functions are performed by the S protein. The S protein is the largest structural protein of SADS-CoV, encoded by 1130 amino acids. The extracellular region of the SADS-CoV S protein is divided into S1 and S2 subunits. The S1 subunit contains the receptor-binding domain for viral binding to the cell receptor. Binding of the S1 subunit to the receptor causes a conformational change in the S protein, exposing cleavage sites. Subsequently, protease cleavage leads to the separation of the viral S1 and S2 subunits, prompting the S2 subunit fusion peptide to insert into the host cell membrane, resulting in membrane fusion. The viral genome is then released into the cytoplasm, ultimately utilizing the host cell's replication system for viral replication, transcription, and translation. The S1 subunit contains two main domains: the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD). The S1-NTD contains a sugar receptor-binding domain, and the S1-CTD contains a receptor-binding domain. The receptor-binding domain contains multiple non-overlapping epitopes, which are targets for the host to produce neutralizing antibodies. Therefore, the SADS-CoV S1-CTD protein is a promising target with good application prospects for the detection and vaccine development of SADS-CoV.

[0004] B-cell epitopes are amino acid clusters on the surface of antigen molecules that can be specifically recognized and bind to secreted antibodies and B-cell receptors, inducing cellular and humoral immune responses in the host. The identification of B-cell epitopes has significant biological implications, such as deepening our understanding of immune responses and autoimmune diseases, providing candidate epitopes for the development of peptide vaccines and the establishment of disease diagnostic methods, and helping to elucidate the mechanisms of action of therapeutic antibodies. Screening for immunodominant epitope regions of the S1-CTD protein is of great value for the diagnosis of this disease and the development of peptide vaccines. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against the SADS-CoV S1-CTD protein, which specifically recognizes the SADS-CoV S1-CTD protein. 345 PGFVLRVGRG 354 The sequence region.

[0006] The second objective is to provide a method for preparing monoclonal antibodies against the SADS-CoV S1-CTD protein.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A monoclonal antibody against the S1-CTD protein of porcine acute diarrhea syndrome coronavirus, wherein the heavy chain variable region includes CDR1 with the amino acid sequence shown in SEQ ID NO.1, CDR2 with the amino acid sequence shown in SEQ ID NO.2, and CDR3 with the amino acid sequence shown in SEQ ID NO.3;

[0009] The light chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.6.

[0010] The monoclonal antibody has a heavy chain constant region of IgG2a and a light chain constant region of Kappa.

[0011] The immunogen for preparing anti-SADS-CoV S1-CTD protein monoclonal antibody was obtained by expressing S1-CTD protein in the BL21(DE3) cell line using a prokaryotic expression system to obtain the active protein.

[0012] The monoclonal antibody prokaryotic expression system BL21(DE3) cell line expresses S1-CTD protein, which is used as an immunogen to immunize mice and screened by indirect ELISA using cell fusion technology.

[0013] The aforementioned monoclonal antibody against SADS-CoV S1-CTD protein can specifically recognize the B-cell epitope on the SADS-CoV S1-CTD protein. 345 PGFVLRVGRG 354 Sequence region.

[0014] The regional sequence of the B-cell epitope of the S1-CTD protein of a porcine acute diarrhea syndrome coronavirus is as follows: 345 PGFVLRVGRG 354 .

[0015] The application of the monoclonal antibody in the preparation of reagents for the diagnosis, detection or prevention of SADS-CoV.

[0016] The application of the B-cell epitopes in the preparation of reagents for the diagnosis or prevention of SADS-COV.

[0017] The beneficial effects of this invention are:

[0018] The SADS-CoV S1-CTD protein monoclonal antibody of the present invention is used to immunize mice with S1-CTD protein as an immunogen. Using cell fusion technology and with S1-CTD protein as a detection antigen, monoclonal cell lines anti-SADS-CoV S1-CTD protein are obtained through indirect ELISA screening. The monoclonal antibody produced by this cell line can specifically recognize and bind to the SADS-CoV S1-CTD protein. 345 PGFVLRVGRG 354 Sequence region.

[0019] The monoclonal antibody of this invention has a titer of 1:1024000 and good specificity, and this monoclonal antibody has good application prospects in the detection of SADS-CoV.

[0020] This invention utilizes the overlapping peptide method for two rounds of truncation, and identifies the antigenic epitopes using indirect ELISA and Dot-ELISA, demonstrating that the linear B-cell epitope region of the S1-CTD protein is... 345 PGFVLRVGRG 354 Experiments show that this B-cell epitope is conserved, laying the foundation for the detection of SADS-CoV pathogen.

[0021] The monoclonal antibody and B-cell epitope of the present invention have good application value in the preparation of reagents for the prevention, detection and diagnosis of SADS-CoV. Attached Figure Description

[0022] Figure 1 Results of SDS-PAGE (A) and Western Blot (B) identification of S1-CTD protein;

[0023] Figure 2 Identification results of purified S1-CTD protein by SDS-PAGE;

[0024] Figure 3 Western blot identification results of S1-CTD protein expression;

[0025] Figure 4 S1-CTD protein immunization mouse serum titer before fusion;

[0026] Figure 5 Western Blot analysis of the reactivity of the monoclonal antibody with prokaryotic expression of S1-CTD protein;

[0027] Figure 6 IFA-assayed reactivity of monoclonal antibodies with eukaryotic expression of S1-CTD protein;

[0028] Figure 7 Ascites purification results of monoclonal antibody;

[0029] Figure 8 Ascites titer determination of monoclonal antibody 10B3;

[0030] Figure 9 Affinity determination of monoclonal antibody 10B3;

[0031] Figure 10 Schematic diagram of truncated expression of S1-CTD protein;

[0032] Figure 11 Results of indirect ELISA and Dot-ELISA experiments with monoclonal antibody 10B3;

[0033] Figure 12 Indirect ELISA and Dot-ELISA identification of key amino acids. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments.

[0035] Unless otherwise specified, the instruments and equipment involved in the embodiments are all conventional instruments and equipment, the reagents involved are all commercially available conventional reagents, and the experimental methods involved are all conventional methods.

[0036] Example 1: Immunogen Preparation

[0037] The SADS-CoV S1-CTD protein plays a crucial role in the adsorption and invasion of the SADS-CoV virus. Since the S1-CTD protein lacks a transmembrane region, we designed and constructed a prokaryotic expression vector for S1-CTD to prepare the antigen protein using a prokaryotic expression system. Specifically, the immunogen preparation includes the following steps:

[0038] 1.1 Primer Design

[0039] The SADS-CoV S gene sequence was obtained from the SADS-CoV / CN / GDGL / 2017 strain (GenBan: MG775250.1) published on NCBI. The S gene sequence was truncated according to relevant literature to obtain the original S1-CTD gene sequence. Based on the SADS-CoV S1-CTD gene sequence, upstream and downstream primers were designed to amplify the target gene. The restriction enzyme sites for the upstream and downstream primers were EcoRI and XhoI (underlined portion), respectively, and the primers were constructed into the pET-32a vector. The primer sequences are shown in Table 1.

[0040] Table 1 Primer sequence list

[0041]

[0042] 1.2 PCR amplification of the S1-CTD gene

[0043] Using the synthesized S1-CTD gene sequence as a template, PCR amplification was performed. The amplification system is as follows:

[0044] Table 2 PCR amplification system

[0045]

[0046] 1.3 Purification and recovery of PCR products

[0047] After amplification of the target gene, the PCR products were identified by nucleic acid electrophoresis using a 1% nucleic acid gel. Finally, the PCR products at the target band location were selected using a DNA gel extraction kit and purified by gel cutting. The concentration of the recovered DNA solution was determined using Nano Drop and stored at -20°C for later use.

[0048] 1.4 Double digestion of target gene and vector

[0049] (1) The S1-CTD target gene and pET-32a vector were digested with EcoRI and XhoI to prepare for subsequent ligation. The enzyme digestion reaction system is shown in Table 3 below.

[0050] Two reaction systems were set up: one for target gene digestion and one for vector digestion. The digestion reactions were carried out separately.

[0051] Table 3. Double enzyme digestion reaction system

[0052]

[0053] (2) After mixing all components, place them on a 37℃ thermostat for 4 hours for enzyme digestion, and then inactivate them in a 65℃ water bath for 20 minutes.

[0054] 1.5 Recovery of enzyme digestion products

[0055] The enzyme digestion products were recovered using a DNA gel recovery and purification kit. 1.5 μL of the recovered product was aspirated, the DNA concentration was determined, and the product was stored at -20°C for later use.

[0056] 1.6 Construction of Recombinant Plasmids

[0057] (1) S1-CTD gene ligated to pET-32a vector

[0058] The connection system is shown in Table 4. Add the following components to the centrifuge tubes, mix well, and then place them in a connection apparatus at 16°C overnight for connection.

[0059] Table 4 Connection System

[0060]

[0061] (2) Transformation

[0062] Add 10 μL of the ligation product to 100 μL of DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, incubate on ice for 2-5 min, add 500 μL of antibiotic-free LB liquid medium, and incubate at 37℃ and 220 rpm for 45 min with shaking. After recovery, take 150 μL of bacterial culture, spread it evenly on LB solid plates containing ampicillin resistance, and incubate upside down in a 37℃ incubator for 12 h.

[0063] (3) Screening positive clones by bacterial culture PCR

[0064] Select a single round colony and inoculate it into LB liquid medium containing ampicillin resistance. Incubate at 37℃ and 220r / min for 4-6h. Use the turbid bacterial solution as a PCR template. The PCR reaction system is shown in Table 5.

[0065] Table 5 PCR reaction system

[0066]

[0067] Mix the above components thoroughly and place them in a PCR instrument for PCR amplification. After amplification, identify the products using a 1% nucleic acid gel, and select monoclonal strains whose band size matches the target gene of SADS-CoV S1-CTD for sequencing at Anhui General Biotechnology Co., Ltd.

[0068] (4) Extraction of recombinant plasmids

[0069] Selected positive strains that successfully underwent sequencing were inoculated into 500 ml of LB liquid medium containing ampicillin and cultured at 37°C and 220 rpm for 12 h. The pET-32a-S1-CTD recombinant plasmid was extracted using the Kangwei Endotoxin-Free Plasmid Extraction Kit, following the kit's instructions.

[0070] 1.7 Expression of the target protein

[0071] The protein induction and extraction of pET-32a-S1-CTD-BL21(DE3) expression bacteria, using a 20ml culture system (100ml culture flask) as an example, are detailed below:

[0072] a. Material preparation: pET-32a-S1-CTD-BL21 expression bacteria, LB liquid medium containing ampicillin, bacterial resuspension buffer (1×PBS);

[0073] b. Primary activation: The pET-32a-S1-CTD-BL21 expression bacteria stored at -80℃ were inoculated into 5 mL of LB medium containing ampicillin at a ratio of 1:1000 and cultured overnight at 37℃ and 220 rpm in a constant temperature shaker.

[0074] c. Secondary activation: The above live bacterial solution was inoculated into 20 mL of LB liquid medium containing ampicillin at a ratio of 1:100;

[0075] d. Place the bacterial culture on a 37°C constant temperature shaker and incubate at 220 rpm for 1.5–2 hours. Measure the OD of the bacterial culture using a spectrophotometer. 600 Value. When OD 600 When the value reading is between 0.6 and 0.8, add 0.2 mmol / L of the inducer IPTG and incubate at 16℃ and 220 rpm for 16 h;

[0076] e. After centrifugation at 8000 rpm for 10 min, collect the bacterial cells. Resuspend the collected bacterial cells in 1×PBS at a ratio of 1 mL of 1×PBS per 0.1 g of bacterial cell weight.

[0077] f. Use an ultrasonic disruptor to disrupt the bacterial cells, then clean the ultrasonic probe. First, sterilize with 75% alcohol, then rinse with deionized water to remove residual alcohol, wash with 1×PBS resuspending bacterial buffer, and finally sonicate the resuspended bacterial cells.

[0078] g. The ultrasonically disrupted sample was centrifuged at 12000 rpm for 10 min, the supernatant was collected, and the precipitate after centrifugation was resuspended with an equal volume of 1×PBS.

[0079] 1.8 Identification and purification of the target protein

[0080] Take 80 μL of the supernatant and precipitate after disruption, add 20 μL of 5×SDS loading buffer, boil for 10 min, and then perform SDS-PAGE and Western Blot analysis, respectively. The results are as follows: Figure 1 .

[0081] Figure 1 A represents the SDS-PAGE result. Figure 1 B represents the results of Western blotting.

[0082] In the figure, M is the protein marker, 1 is the whole bacteria after lysis, 2 is the S1-CTD supernatant, and 3 is the S1-CTD precipitate.

[0083] In Western blotting, HRP-labeled Goat-Mouse 6×His monoclonal antibody was diluted 1:5000 and incubated at room temperature for 1 h. After incubation, the PVDF membrane was washed 5 times with PBST and then exposed to ECL chemiluminescence solution (Synthetium hydroxychloroquine).

[0084] The supernatant after disruption was filtered through a 0.45 μm filter and purified by nickel-ion affinity chromatography. The purification conditions were as follows: equilibration buffer was 20 mM Tris + 150 mM NaCl, pH 8.0; wash buffer was 20 mM Tris + 150 mM NaCl + 20 mM imidazole, pH 7.5; and elution was performed using 20 mM Tris + 150 mM NaCl + 200 mM imidazole at pH 7.5 and 20 mM Tris + 150 mM NaCl + 500 mM imidazole at pH 7.5, respectively. 80 μL of each of the lysed supernatant, column run-through buffer, wash buffer, and target protein eluent were collected sequentially, and 20 μL of 5×SDS loading buffer was added. After boiling for 10 min, SDS-PAGE was performed. Collect the eluent and dialyze overnight using a dialysis bag with 20 mM Tris + 150 mM NaCl as the dialysate. After dialysis, centrifuge at 5000 rpm for 10 min, collect the supernatant, and take 80 μL. Add 20 μL of 5×SDS loading buffer, boil for 10 min, and then perform SDS-PAGE and Western Blot analysis. Figure 2 , Figure 3 As shown.

[0085] Figure 2 The results of SDS-PAGE identification of purified S1-CTD protein; Figure 2 In the table, M represents protein marker, 1 represents the area before S1-CTD purification, 2 represents flow bleed, 3 represents washing with 20 mmol / L imidazole, 4 represents elution with 200 mmol / L imidazole, and 5 represents elution with 500 mmol / L imidazole.

[0086] Figure 3 The results of Western Blot identification of S1-CTD protein expression are shown in the figure. M is the protein marker, 1 is S1-CTD incubated with SADS-COV positive serum, and 2 is pET32a empty vector expression supernatant.

[0087] Western blot identification used HRP-labeled Goat-Mouse 6×His monoclonal antibody 1:5000 and SADS-CoV positive serum as primary antibodies, and incubated at room temperature for 1 h. After incubation, the PVDF membrane was washed 5 times with PBST and exposed to ECL chemiluminescence solution. SDS-PAGE and Western blot results showed that the purity of the dialyzed SADS-CoV S1-CTD recombinant protein reached over 90%.

[0088] Example 2: Analysis of mouse immunization and immunogenicity of SADS-CoV S1-CTD recombinant protein

[0089] 2.1 Preliminary evaluation of mouse immunization and its effects

[0090] Two 6-8 week old BALB / c mice were used, each immunized with 20 μg of S1-CTD protein, for a total of three immunizations, with each immunization occurring every two weeks. For the first immunization, S1-CTD protein / PBS and Freund's complete adjuvant were mixed at a 1:1 (v / v) and thoroughly emulsified. For the second and third immunizations, S1-CTD protein / PBS and Freund's incomplete adjuvant were mixed at a 1:1 (v / v) and thoroughly emulsified. Blood samples were collected from the tail vein of the mice at 14, 21, and 35 days after the first immunization, and the immunization efficacy was evaluated by indirect ELISA.

[0091] 2.2 Indirect ELISA determination of serum titer of S1-CTD protein in mice immunized with mice

[0092] The S1-CTD recombinant protein CBS was diluted to 2 μg / mL and coated onto a 96-well plate at 100 μL per well. The plate was incubated overnight at 4°C. The coating solution was discarded, the plate was washed three times with PBST, blocked with 5% skim milk, and incubated at 37°C for 1 h. The blocking solution was discarded, and the plate was washed three times with PBST.

[0093] The experiment was divided into three groups: mouse No. 1, mouse No. 2, and a negative control. The first well was filled with serum from mice immunized for the first time at a concentration of 1:800, which was initially diluted 35 days after the first immunization. Simultaneously, PBS-immunized mouse serum was used as a negative control. The serum was serially diluted from left to right and incubated at 37°C for 1 hour. After washing three times with PBST, HRP-labeled goat anti-mouse antibody diluted 1:1000 was added as a secondary antibody and incubated at 37°C for 1 hour. After washing three times with PBST, TMB chromogenic solution was added and the mixture was incubated in the dark for 5 minutes. Then, 2 mol / L H2SO4 was added to stop the chromogenic process. The OD450nm value was measured to evaluate the immunization effect.

[0094] like Figure 4 As shown, the titer of mouse 1 (1:204800) was slightly higher than that of mouse 2 (1:102400), indicating that the immunized mice produced a higher titer of specific antibody against the S1-CTD protein.

[0095] Example 3: Preparation and Identification of S1-CTD Recombinant Protein Monoclonal Antibody

[0096] 3.1 Cell Fusion

[0097] Mice were hyperimmunized 3-4 days before cell fusion. During hyperimmunization, the mice were directly injected intraperitoneally. The serum titer of the S1-CTD-immunized mice was determined by indirect ELISA. The mice with the highest titer, S1-CTD-1, were selected, and 40 μg of recombinant S1-CTD protein was injected intraperitoneally without adjuvant. Cell fusion was performed three days after hyperimmunization.

[0098] Collect well-growing mouse myeloma SP2 / 0 cells into 50 mL sterile centrifuge tubes for later use; the cell concentration is 2 × 10⁻⁶. 7 ~5×10 7 Spleen cells / mL. Mice were euthanized by cervical dislocation after hyperimmunization and disinfected by immersion in 75% alcohol. In a laminar flow hood, the epidermis was cut open with sterile scissors and forceps. A second set of scissors was used to cut open the peritoneum, and the spleen was removed and placed on a 200-mesh sterile nylon mesh. The spleen was ground with scissors, rinsed with GNK washing solution, and allowed to filter the spleen cells into a sterile beaker. The spleen cell suspension was transferred to a centrifuge tube, and GNK was added to a final volume of 40 mL. The mixture was centrifuged together with tumor cells at 1000 rpm for 10 min. The supernatant was discarded, and the cell clumps were deflated. 10 mL of GNK was added to each cell clump. The spleen cell suspension was transferred to a tumor cell flask, and GNK was added to a final volume of 40 mL. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded.

[0099] Gently disperse the cell clusters, add 1 mL of 50% PEG1500 dropwise over 1 minute, let stand for 90 seconds, then slowly add 15 mL of GNK to terminate the confluence. Stabilize in a 37°C water bath for 5 minutes, add GNK to bring the total volume to 40 mL, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, gently disperse the cell clusters, add 500 mL of RPMI-1640 medium containing HAT and 10% fetal bovine serum, gently resuspend the cells, and spread them evenly in a 96-well cell culture plate. Add 250 μL of cell suspension to each well and incubate for 7 days.

[0100] 3.2 Screening of hybridoma cell positive wells

[0101] Seven days after cell fusion, when the cell clusters reached a relatively large size, their cell supernatant was measured using an indirect ELISA. Pre-fusion mouse ocular serum served as a positive control, and PBS-immunized mouse serum served as a negative control. 2 μg / mL S1-CTD protein was used as the detection antigen; 100 μL of CBS diluted in this solution was coated per well and incubated overnight at 4°C. 200 μL of 5% skim milk was added to each well, and the plate was blocked at 37°C for 2 h. 50 μL of cell supernatant was collected as the primary antibody and incubated at 37°C for 30 min. 50 μL of HRP-labeled goat anti-mouse antibody was added to each well after a 1:1000 dilution and incubated at 37°C for 30 min. After washing the plate three times with PBST, 100 μL of TMB chromogenic buffer was added to each well, and the reaction was carried out in the dark for 5 min. The reaction was terminated with 2 mol / L H2SO4. The wells with the strongest chromogenic reaction were transferred to 48-well plates for further culture, and the same method was used for measurement. Measurements were performed three times, and the wells with stable positive hybridoma cell reactions were subcloned. Monoclonalization was performed using the limiting dilution method to ensure the acquisition of hybridoma cell lines that stably secrete monoclonal antibodies.

[0102] 3.3 Western Blot Identification

[0103] Western blotting was used to identify the S1-CTD recombinant protein in the cell supernatant of selected positive hybridoma cell lines. The primary antibody was the cell supernatant from 10B3 positive hybridoma cells; the secondary antibody was Goat Anti-Mouse IgG / HRP (1:5000). AEC chromogenic assay was then performed, and images were taken for recording. Results are as follows: Figure 5 .

[0104] Figure 5 Figure 1 shows the supernatant from S1-CTD incubation with monoclonal antibody, indicating that the selected positive hybridoma cell lines can react with S1-CTD recombinant protein.

[0105] 3.4 Identification by indirect immunofluorescence (IFA) assay

[0106] HEK293T cells were seeded at 70-80% confluence in 6-well plates, 2 mL per well. After 48 h, immunofluorescence staining was performed as follows: 1 mL of pre-chilled methanol was added to each well, and the cells were fixed for 20 min. The methanol was carefully removed and the cells were air-dried. 1 mL of PBS was used to wash the 6-well plate, and 2 mL of 5% skim milk was added to each well. The plates were blocked at 37°C for 1 h. The supernatant of positive hybridoma cells was added to each well, with a 1:500 dilution of fused mouse ocular serum as a positive control. The plates were incubated at 37°C for 1 h. After washing three times with PBS, FITC-labeled goat anti-mouse IgG (1:500) was added, and the plates were incubated at 37°C in the dark for 1 h. After washing three times with PBS, 80 μL of DAPI was added to each well, and the plates were incubated for 15 min. After washing three more times with PBS, 1 mL of ddH2O was added to each well. The results were observed under a fluorescence microscope. Figure 6 As shown.

[0107] Figure 6 In the diagram, PC represents a positive control for positive serum detection, and NC represents the supernatant of hybridoma cells containing irrelevant proteins, indicating that the monoclonal antibody 10B3 of the present invention can specifically react with the S1-CTD recombinant protein.

[0108] 3.5 Large-scale preparation of monoclonal antibodies

[0109] After subcloning, hybridoma cells from the selected positive wells were screened again using the same indirect ELISA method described above. The cells from the selected positive wells were then expanded and cultured in 12-8F wells. Sterile liquid paraffin was injected intraperitoneally into multiparous BALB / c mice. One week after paraffin injection, the positive well hybridoma cells were diluted with RPMI-1640 basal medium and counted. The number of cells injected per mouse was approximately 1.0 × 10⁻⁶. 6 Ten days after injection, the mice's abdomens were significantly enlarged, indicating the production of ascites. The ascites was collected.

[0110] 3.6 Monoclonal antibody ascites purification

[0111] Ascites fluid was purified using a Protein A agarose gel purification column. The ascites fluid was removed from a -80°C freezer, slowly thawed on ice, and centrifuged at 3000 rpm for 10 min. The supernatant was collected. The fluid was washed three times with equilibration buffer (1×PBS) to equilibrate the column (1×PBS was filtered through a 0.22 μm filter). The purified ascites fluid was transferred to a 10 mL centrifuge tube, diluted to 10 mL with 1×PBS, filtered through a 0.22 μm filter, and then added to the column at a flow rate of 1 mL / min. The column was washed three times with equilibration buffer (1×PBS) to remove unbound proteins, and an indicator was used to check for complete elution of proteins. The antibody was eluted with 0.1 mol / L glycine (pH 2.9) elution buffer. Before elution, ten 1.5 mL centrifuge tubes were prepared, and 70 μL of 0.1 mol / L Tris-HCl (pH 9.0) neutralization solution was added to each tube. The elution buffer was then brought to a final volume of 1 mL, and the pH was adjusted to between 7.0 and 7.5. SDS-PAGE analysis was used to identify the purified SADS-CoV S1-CTD monoclonal antibody.

[0112] Figure 7 The image shows the ascites fluid purification results for the monoclonal antibody. In the figure, M: Marker; 1 represents the ascites fluid before purification; 2 represents the ascites fluid after purification.

[0113] 3.7 Subclass Identification and Variable Region Sequencing of Monoclonal Antibodies

[0114] The 10B3 monoclonal antibody was identified as an IgG2a type using the Mouse Monoclonal Antibody Isotype Identification Kit (Proteintech) according to the kit instructions. The results showed that the 10B3 monoclonal antibody of this invention has a heavy chain of Kappa. Sequencing revealed that the 10B3 heavy chain variable region CDR1 is DSYIN (SEQ ID NO.1), the 2nd CDR2 is WISHGGANSNYNEKFKG (SEQ ID NO.2), the 3rd CDR3 is LDDDYAMDY (SEQ ID NO.3), the 4th CDR1 is RASKSVSTSGYSYMH (SEQ ID NO.4), the 5th CDR2 is LVSNLES (SEQ ID NO.5), and the 6th CDR3 is QHIR (SEQ ID NO.6).

[0115] 3.8 Monoclonal Antibody Titer Determination

[0116] The titer of monoclonal antibodies was determined by indirect ELISA. 100 μL of 2 μg / mL S1-CTD protein was diluted with CBS and coated per well, incubated overnight at 4°C. 200 μL of 5% skim milk was added to each well, and the plates were blocked at 37°C for 2 h. The purified monoclonal antibody was diluted 1:1000 and added to the first well, followed by serial dilutions from left to right. Mouse serum immunized with PBS was used as a negative control, and the plates were blocked at 37°C for 1 h. HRP-labeled goat anti-mouse antibody was used as a secondary antibody, diluted 1:1000, and 100 μL was added to each well, and the plates were blocked at 37°C for 1 h. After washing the plates three times with PBST, 100 μL of TMB chromogenic buffer was added to each well, and the reaction was carried out in the dark for 5 min. The reaction was terminated by adding 2 mol / L H2SO4, and the OD value at 450 nm was recorded.

[0117] The results are as follows Figure 8 As shown, the monoclonal antibody titer of the present invention reaches 1:1024000.

[0118] 3.9 Determination of monoclonal antibody affinity

[0119] S1-CTD recombinant protein was used as the coating agent, and 100 μL of the protein was applied to each well at concentrations of 2 μg / mL (200 ng) and 1 μg / mL (100 ng) respectively, and incubated overnight at 4°C. The plates were then blocked with 5% skim milk at 37°C for 2 h. The primary antibody was purified monoclonal antibody, serially diluted from 1:1000 to 2048000 (100 μL / well), and the antibody titers at different protein concentrations were measured. Based on data read from the ELISA reader, the OD values ​​were plotted with monoclonal antibody concentration on the x-axis. 450 Plot a scatter plot on the ordinate and record the OD. 450 The value at which the values ​​gradually stabilized was taken as 1. The concentration of the purified monoclonal antibody was determined using a NANODROP 2000c full-wavelength spectrophotometer. The x-axis was plotted as the reciprocal of the monoclonal antibody concentration, and the OD value was plotted as follows: 450 A double reciprocal curve was constructed with the reciprocals of the values ​​as the ordinate. The regression equation and correlation coefficient of the double reciprocal curve were analyzed using GraphPad Prism. The results are shown below. Figure 9 The regression equations for the resulting double reciprocal curves are y = 0.0047x + 0.3414 (R²). 2 =0.9926) and y =0.0042x + 0.2914(R) 2 =0.9927), and its affinity was calculated to be 1.97 × 10⁻⁶. 9 mol / L.

[0120] Example 4: Localization of the linear B-cell epitope of SADS-CoV S1-CTD protein

[0121] 4.1 Truncation and Identification of the S1-CTD Protein Amino Acid Sequence of SADS-CoV

[0122] Truncation method: The full-length S1-CTD protein (402 aa) was truncated into seven segments, named C1, C2, C3, C4, C5, 4-1, and 4-2. C1–C5: cover the 275–402 aa region (25–57 aa / segment, partially overlapping); 4-1 / 4-2: fine-mapping epitopes (4-1: 345–354 aa; 4-2: 353–359 aa);

[0123] Construction of prokaryotic expression vector: Using the S1-CTD protein gene as a template, a seven-segment truncated protein gene sequence was amplified by PCR, followed by double enzyme digestion, ligation, and transformation experiments. Finally, the target gene sequence was inserted into the pET-32a vector to construct a prokaryotic expression vector for epitope identification of the seven-segment truncated protein. A schematic diagram of the S1-CTD protein truncated expression is shown below. Figure 10 The amino acid sequences have been labeled.

[0124] C1: 275–299 aa; C2: 296–320 aa; C3: 317–341 aa; C4: 338–362 aa; C5: 345–402 aa; 4-1: 345–354 aa and 4-2: 353–362 aa. The identification results of the S1-CTD truncated expression of C1–C5 proteins are as follows: Figure 11 As shown in Figure A, Western blotting results indicated that all five truncated protein segments were successfully expressed. The amino acid sequence from positions 1 to 402 of the S1-CTD protein is SEQ ID NO. 9: MKLFTVFTLLASIRVLYGCESVDFNLFNTIFSTHRGLSNTTSVITGAYPSTNKSDWSCNTRTGHLSGSGFGIGLYVQTPREQYQYDGSGAGGYTIAVSPIHVTNLTWE LWIHRKWGVNSVVTVRLCRWWQFMSFNSTSHAADAGPTNAFECLINGSYPTHRNTGYMFGVTWYNDLVRIVFPPTVLEMQLDGLQWERVQFNSPVNAGHATRFNVVKDISTVLVETNSGGSVFRYSYCADGFVNGLQCKLRLFDIPP GVYSNSEVEYPTALYTVVHNMSACPERPDSYCGSNSCPFKRAVFSNCIVNYTTWVNPDQRDFQHLILPNGKFNPFTECNGLNRIVDGCVPGFVLRVGRGKAVNRTIVTPYLKPYECFGWSWNDNQDSIYDWWIADFVSTGAFVCESN

[0125] 4.2 Identification of Monoclonal Antibody 10B3 by Indirect ELISA and Dot-ELISA

[0126] Using the truncated S1-CTD proteins as coating agents, indirect ELISA was performed to quantitatively detect the binding titer of monoclonal antibody 10B3 to each truncated protein. The results showed that monoclonal antibody 10B3 reacted with both C4 and C5 segments of the C1–C5 truncated proteins. Figure 11 B). Subsequently, 2 μg / well of the truncated protein was spotted onto the NC membrane, and binding activity was qualitatively verified by Dot-ELISA. The Dot-ELISA results are shown in [Figure 1]. Figure 11 C10B3 reacted with both C4 and C5 segments, consistent with indirect ELISA results. Further analysis revealed that the truncated protein C4 was divided into two segments, 4-1 and 4-2, which were successfully expressed by Western blotting. Figure 11 D).

[0127] Further via indirect ELISA ( Figure 11 E) and Dot-ELISA Figure 11 F) Identification showed that monoclonal antibody 10B3 specifically recognized 4-1 (345-354aa) and did not react with 4-2. Figure 11 The results (E–F) were consistent with those of Peptide-ELISA. The results indicate that the linear epitope of monoclonal antibody 10B3 is located in region 345–354aa (4-1 region) of the S1-CTD.

[0128] Figure 11 In Figure A, M is the protein marker, and wells 1-5 are for truncated CTD1-5 proteins, respectively. Figure B shows the reaction of 10B3 monoclonal antibody with truncated CTD1-5 proteins detected by indirect ELISA. Figure C shows the reaction of 10B3 monoclonal antibody with truncated CTD1-5 proteins detected by Dot-ELISA. In Figure D, M is the protein marker, and wells 1-2 are for truncated 4-1 and 4-2 proteins, respectively. Figure E shows the reaction of 10B3 monoclonal antibody with truncated 4-1 and 4-2 proteins detected by indirect ELISA. Figure F shows the reaction of 10B3 monoclonal antibody with truncated 4-1 and 4-2 proteins detected by Dot-ELISA.

[0129] Further identification of key amino acids was performed by scanning for mutations. The alanine mutation status is shown in Table 6 below.

[0130] Table 6. Amino acid sequences of alanine mutations

[0131]

[0132]

[0133] Using 1A to 10A as coating antigens, indirect ELISA and Dot-ELISA were performed for identification. The results showed that R350, R353, and G354 were the key amino acids. Figure 12 ).

Claims

1. A monoclonal antibody against the S1-CTD protein of porcine acute diarrhea syndrome coronavirus, characterized in that, The heavy chain variable region of the monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; The light chain variable region of the monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

6.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes and binds to the B-cell epitope on the SADS-CoV S1-CTD protein. 345 PGFVLRVGRG 354 Sequence region.

3. A B-cell epitope of the S1-CTD protein of porcine acute diarrhea syndrome coronavirus, characterized in that, The region sequence of the B cell epitope is as follows: 345 PGFVLRVGRG 354 .

4. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody has a heavy chain constant region of IgG2a type and a light chain constant region of Kappa type.

5. A method for preparing the monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody was obtained by immunizing mice with S1-CTD protein expressed in the BL21(DE3) prokaryotic expression system cell line, and then screening them using an indirect ELISA method via cell fusion technology.

6. The use of the monoclonal antibody of claim 1 in the preparation of reagents for the diagnosis, detection or prevention of SADS-CoV.

7. The use of the B-cell epitope according to claim 3 in the preparation of reagents for the diagnosis or prevention of SADS-COV.

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