Neutralizing monoclonal antibody aiming at pig delta coronavirus S1 protein conserved linear epitope and application thereof

Abstract

The invention discloses a neutralizing monoclonal antibody aiming at a pig delta coronavirus S1 protein conserved linear epitope and application of the neutralizing monoclonal antibody, and belongs to the technical field of biology. The amino acid sequence of a light chain of the neutralizing monoclonal antibody is as shown in SEQ ID NO.9, and the amino acid sequence of a heavy chain of the neutralizing monoclonal antibody is as shown in SEQ ID NO.13. According to the invention, an insect baculovirus expression system is utilized to express the S1 protein of the porcine delta coronavirus and screen out a linear epitope with a neutralizing effect, and a neutralizing monoclonal antibody is harvested. The monoclonal antibody can be subjected to neutralization reaction with multiple strains of porcine delta coronaviruses, and is strong in neutralization activity and high in titer. The neutralizing monoclonal antibody provided by the invention can be used for efficiently preventing and treating the porcine delta coronavirus. The invention provides a new way for diagnosing, preventing and treating the porcine delta coronavirus, and provides a theoretical basis for development of related medicines and vaccines.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a neutralizing monoclonal antibody against a conserved linear epitope of porcine delta coronavirus S1 protein and its applications. Background Technology

[0002] Porcine deltacoronavirus (PDCoV), a type of porcine enteric coronavirus, can cause diarrhea in piglets, seriously threatening the healthy development of the pig industry. Infected 5-15 day old suckling piglets often exhibit symptoms such as diarrhea, vomiting, and rapid dehydration, with morbidity and mortality rates as high as 50%-100%. Growing pigs, adult pigs, and sows show relatively milder symptoms. Furthermore, studies have shown its potential for cross-species transmission; it has been detected in chickens, turkeys, cattle, mice, and even humans, posing significant public health risks.

[0003] The PDCoV S protein is a key structural protein for viral infection and invasion. As a type I membrane protein, it exists as a trimer on the viral envelope surface, determining not only the virus's host range and tissue tropism but also being the main protein inducing the production of neutralizing antibodies. However, there are currently few neutralizing epitopes targeting the S protein, primarily because the S protein is a glycoprotein with a complex tertiary structure, and prokaryotic expression systems cannot mimic its native conformation, resulting in low neutralizing antibody titers. Furthermore, existing research has largely focused on conformational epitopes, and neutralizing monoclonal antibodies targeting conserved linear epitopes have not yet been reported. Therefore, developing a neutralizing monoclonal antibody targeting a conserved linear epitope of the porcine delta coronavirus S1 protein is of great significance for the clinical prevention and treatment of PDCoV, neutralizing epitope research, and vaccine development. Summary of the Invention

[0004] The purpose of this invention is to provide a neutralizing monoclonal antibody targeting a conserved linear epitope of porcine delta coronavirus S1 protein and its application, thereby addressing the problems existing in the prior art. This invention utilizes an insect baculovirus expression system to express the porcine delta coronavirus S1 protein and screen for linear epitopes with neutralizing activity, resulting in a neutralizing monoclonal antibody. This monoclonal antibody can neutralize multiple strains of porcine delta coronavirus, exhibiting strong neutralizing activity and high titer. The neutralizing monoclonal antibody of this invention enables highly efficient prevention and control of porcine delta coronavirus. This invention provides a new approach for the diagnosis and prevention of porcine delta coronavirus and provides a theoretical basis for the development of related drugs and vaccines.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a neutralizing monoclonal antibody against a conserved linear epitope of porcine delta coronavirus S1 protein. The light chain of the neutralizing monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.10, CDR2 with an amino acid sequence as shown in SEQ ID NO.11, and CDR3 with an amino acid sequence as shown in SEQ ID NO.12.

[0007] The heavy chain of the neutralizing monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.14, CDR2 with an amino acid sequence as shown in SEQ ID NO.15, and CDR3 with an amino acid sequence as shown in SEQ ID NO.16.

[0008] Furthermore, the amino acid sequence of the light chain of the neutralizing monoclonal antibody is shown in SEQ ID NO.9, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.13.

[0009] Furthermore, the neutralizing monoclonal antibody can recognize the antigenic epitope in the porcine delta coronavirus S1 protein, as shown in SEQ ID NO. 17.

[0010] The present invention also provides the use of the above-mentioned neutralizing monoclonal antibody in the preparation of medicaments for the prevention and / or treatment of porcine delta coronavirus infection.

[0011] The present invention also provides a medicament for the prevention and / or treatment of porcine delta coronavirus infection, the medicament comprising the above-described neutralizing monoclonal antibody and pharmaceutically acceptable excipients.

[0012] The present invention also provides the application of the above-mentioned neutralizing monoclonal antibody in the preparation of reagents or kits for detecting porcine delta coronavirus.

[0013] The present invention also provides a porcine delta coronavirus S1 protein antigenic epitope peptide, the amino acid sequence of which is shown in SEQ ID NO.17.

[0014] The present invention also provides the use of the above-mentioned antigenic epitope peptide in the preparation of a vaccine to prevent porcine delta coronavirus infection.

[0015] The present invention also provides a vaccine for preventing porcine delta coronavirus infection, the vaccine comprising the above-mentioned antigenic epitope peptide and a pharmaceutically acceptable carrier.

[0016] The present invention discloses the following technical effects:

[0017] This invention utilizes an insect baculovirus expression system to express the porcine delta coronavirus S1 protein and screen for linear epitopes with neutralizing activity, yielding a neutralizing monoclonal antibody. This monoclonal antibody has a Kappa-type light chain and an IgG1-type heavy chain. Using a truncated PDCoV S1 protein, the antibody epitope recognized by this monoclonal antibody was identified as FTSPTGELYAF, which is highly conserved in PDCoV strains. This indicates that the monoclonal antibody of this invention can neutralize multiple porcine delta coronavirus strains, and experiments demonstrate that the antibody exhibits strong neutralizing activity and high titer. The neutralizing monoclonal antibody of this invention can achieve highly efficient prevention and control of porcine delta coronavirus. This invention provides a new approach for the diagnosis and prevention of porcine delta coronavirus and provides a theoretical basis for the development of related drugs and vaccines. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Electrophoresis diagram of the PDCoV S1 target gene amplification product; M is the marker; lane 1 is the PDCoV S1 target gene amplification product; lane 2 is the negative control (water);

[0020] Figure 2 Electrophoresis diagram of pFASTbac HTA-S1 recombinant plasmid; M is the marker; lane 1 is the pFASTbac HTA-S1 recombinant plasmid; lane 2 is the empty pFASTbac HTA vector; lane 3 is the negative control (water);

[0021] Figure 3 Electrophoresis diagram of the Bacmid-PDCoV S1 genome; M is the marker; lane 1 is the Bacmid-PDCoV S1 genome; lane 2 is the empty pFASTbac HTA vector; lane 3 is the negative control (water);

[0022] Figure 4 Cell morphology diagram during the rescue process of recombinant baculovirus; scale bar is 50 μm;

[0023] Figure 5 The image shows the IFA identification results of recombinant proteins in insect cells.

[0024] Figure 6Figure 1 shows the SDS-PAGE (A) and Western blot (B) results of recombinant protein identification in insect cells; M is the marker; lane 1 is the supernatant of broken cells; lane 2 is the pellet of broken cells; lane 3 is the supernatant of culture medium.

[0025] Figure 7 The images show the SDS-PAGE (A) and Western blot (B) identification results during the purification process of recombinant proteins in insect cells; M is the marker; lane 1 is the cell culture supernatant; lanes 2 and 4 are the cell lysate supernatant; lanes 3 and 5 are the purified recombinant proteins;

[0026] Figure 8 Animal immunization flowchart;

[0027] Figure 9 The image shows the results of IFA identification in mouse immune serum.

[0028] Figure 10 A: Western blot identification results of mouse immune serum; B: Western blot identification results of PDCoV S1 immunized mouse serum; C: Western blot identification results of porcine PDCoV positive serum; M: Marker; Lane 1: PDCoV virus (HNZK-02, GenBank: MH708123.1); Lane 2: The porcine kidney proximal tubular epithelial cells (LLC-PK1);

[0029] Figure 11 The image shows the results of IFA identification specific to monoclonal antibodies.

[0030] Figure 12 The image shows the results of Western blot identification of monoclonal antibody specificity; M is the marker; lane 1 is PDCoV; lane 2 is PEDV; lane 3 is TGEV; lane 4 is PSV.

[0031] Figure 13 The image shows the Western blot results for the reactivity of monoclonal antibodies; M is the marker; lane 1 represents eukaryotic expression of S1 protein; lane 2 represents PDCoV virus; lane 3 represents prokaryotic expression of S1 protein.

[0032] Figure 14 The image shows the IFA identification results for the neutralizing activity of monoclonal antibodies.

[0033] Figure 15 The graph shows the results of the monoclonal antibody neutralization titer assay.

[0034] Figure 16 This is a graph showing the results of monoclonal antibody subtype identification;

[0035] Figure 17 Electrophoresis diagrams of the light and heavy chain amplification products of monoclonal antibodies; M is the marker; lanes 1-3 are heavy chain amplification products; lanes 4-6 are light chain amplification products;

[0036] Figure 18 A is a diagram of the identification of antigenic epitopes by monoclonal antibodies; A is a diagram of the process of identifying antigenic epitopes by monoclonal antibodies; BG is a diagram of the SDS-PAGE and Western blot identification results of each truncated antigenic epitope.

[0037] Figure 19 Figure 1 shows the results of monoclonal antibody conservation analysis; A shows the comparison results of monoclonal antibody recognition epitopes with different PDCoV strains; B shows the comparison results of monoclonal antibody recognition epitopes with different generations of PDCoV; C shows the comparison results of monoclonal antibody recognition epitopes with different PEDV strains; D shows the comparison results of monoclonal antibody recognition epitopes with different TGEV strains. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0044] Example 1

[0045] 1. PDCoV S1 gene amplification

[0046] Using the reverse-transcribed PDCoV (HNZK-02, GenBank: MH708123.1) genome as a template, the PDCoV S1 gene was amplified according to the PCR amplification system described in Table 1, using primers F / R. The amplification cycle conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles, followed by a final extension at 72℃ for 5 min, and then termination at 4℃. The primer sequences are as follows:

[0047] F: 5'-gcggccgctttcgaatctagaGTTTGATGTTGGCGTTCTTCCTG-3' (SEQ ID NO. 1);

[0048] R: 5'-cttggtaccgcatgcctcgagTGTTCCCACAATTGCACCATC-3' (SEQ ID NO. 2).

[0049] Table 1 PCR amplification system

[0050]

[0051]

[0052] The amplified target gene was subjected to agarose gel electrophoresis, such as... Figure 1As shown. Cut off the agarose gel containing the target gene, add an equal volume of sol solution, and incubate at 55°C for approximately 10 minutes. Add the dissolved liquid to the adsorption column, centrifuge at 13000g for 1 minute, discard the filtrate, add 300μL of sol solution, centrifuge at 13000g for 1 minute, discard the filtrate, add 700mL of SPW Buffer, centrifuge at 13000g for 1 minute, repeat twice, discard the filtrate, centrifuge at 13000g for 2 minutes to remove residual liquid from the adsorption column, mount the adsorption column to a clean 1.5mL centrifuge tube, add 20mL of Elution Buffer to the binding column matrix, incubate at room temperature for 2 minutes, and centrifuge at 13,000×g for 1 minute to elute the target gene.

[0053] 2. Construction of pFASTbac HTA-S1 recombinant plasmid and Bacmid-PDCoV S1 genome

[0054] The pFASTbac HTA vector was digested with enzymes in the following manner: 1 μL Xhol and Xbal restriction endonucleases, 5 μL Cusmart buffer, 1 μg pFASTbac HTA vector, and water was added to bring the volume to 50 μL. After transient centrifugation, the vector was digested at 37°C for 90 min.

[0055] The target gene recovered from the gel was ligated to the enzyme-digested vector at a molar ratio of 2:1. 10 μL of 2×CEMix was added, and water was added to bring the volume to 20 μL. Ligation was carried out at 50°C for 5 min. The ligation product was then added to DH-5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, incubated on ice for 10 min, and then 600 μL of antibiotic-free LB was added. The cells were incubated at 37°C with shaking for 1 h. A control group with an empty vector was also included. The bacterial culture was plated onto a solid medium containing ampicillin, and plasmids were extracted from single colonies. After PCR identification, electrophoresis was performed to detect plasmids. Figure 2 As shown.

[0056] Further PCR and sequencing confirmed the successful construction of the pFASTbac HTA-S1 recombinant plasmid. Centrifuge 5 mL of bacterial culture containing pFASTbac HTA-S1 recombinant plasmid at 8000 rpm for 2 min to collect the bacterial cells. Add 250 μL of Solution / Rnase A mixture to resuspend the bacterial cells. Add 250 μL of Solution II and lyse for 3 min. Add 350 μL of Solution III, gently invert, and centrifuge at 12000 rpm for 10 min at room temperature. Transfer the supernatant to an adsorption column, centrifuge at 10000 g for 1 min, discard the filtrate, add 500 μL of HB Buffer, centrifuge at 10000 g for 1 min, discard the filtrate, add 700 μL of DNA Wash Buffer, centrifuge at 10000 g for 1 min, repeat once, centrifuge at 13000 g for 2 min, dry the column, transfer the column to a new 1.5 mL centrifuge tube, add 30 μL of Solution Buffer, let stand for 2 min, centrifuge at 13000 g for 1 min, and elute the pFASTbac HTA-S1 plasmid.

[0057] The pFASTbac HTA-S1 plasmid and empty vector plasmid were transformed into DH-10bac competent cells using the transformation method described above. The cells were shaken at 37°C for 5 hours. The bacterial culture was then plated onto solid LB medium containing gentamicin, tetracycline, and kanamycin. PCR identification and electrophoresis were performed using universal primers for the pFASTbac HTA vector (F: TATTCCGGATTATTCATACC, SEQ ID NO.3; R: ACAAATGTGGTATGGCTGA, SEQ ID NO.4). Figure 3 As shown, the Bacmid-PDCoV S1 genome was successfully constructed according to OMEGA EZNA. TM The Bacmid-PDCoV S1 genome and empty vector plasmid were extracted using the BAC / PAC DNA Kit.

[0058] 3. Recombinant baculovirus rescue

[0059] Take clean, sterile centrifuge tubes and add 100 μL of serum- and antibiotic-free insect cell culture medium to dilute the Bacmid-PDCoV S1 genome / empty vector plasmid and transfection reagent, respectively. Add 16 μg of Bacmid-PDCoV S1 genome / empty vector plasmid to one tube and gently mix with a pipette; add 8 μL of LipoInsect gel to the other tube. TMMix the transfection reagent by gently pipetting. Incubate the diluted Bacmid-PDCoV S1 / empty vector plasmid and transfection reagent at room temperature for 30 min. Then, gently add the diluted Bacmid-PDCoV S1 to the diluted LipoInsect solution using a pipette. TM In the transfection reagent, gently invert the centrifuge tube and let it stand at room temperature for 30 minutes. Add the transfection mixture to the insect cells. Replace the cells with fresh culture medium after 5 hours. After 3 days, the cells will become larger and rounder. Figure 4 As shown, cells transfected with Bacmid-PDCoV S1 developed cytopathic effects, while cells transfected with the empty vector (Control) did not. The virus was passaged three times consecutively, followed by protein expression and purification.

[0060] 4. Protein expression identification and purification

[0061] 4.1 Indirect immunofluorescence assay (IFA) for protein expression identification

[0062] Insect cells infected with the Bacmid-PDCoV S1 genome were fixed with pre-cooled anhydrous ethanol when 70% lesions were observed. After blocking with 5% BSA at 37°C for 2 hours and washing three times with PBS, the cells were incubated with his monoclonal antibody (1:500) and porcine PDCoV-positive serum (1:500) as primary antibodies, and FITC-labeled goat anti-mouse IgG (1:200) and FITC-labeled goat anti-pig IgG (1:200) diluted with 5% BSA as secondary antibodies. The cells were incubated at 37°C for 1 hour, washed three times with PBST, and incubated with DAPI at room temperature for 10 minutes. After three washes with PBST, an anti-fluorescence quencher was added, and the cells were observed and photographed using a fluorescence microscope. Figure 5 ).

[0063] 4.2 Western blot identification of protein expression

[0064] Insect cells with 90% disease severity were centrifuged at 500×g for 10 min to collect cell pellet and cell culture supernatant. Cells were resuspended in 1 / 6 volume of 0.25Mm Tris HCl (pH=8.25) culture medium, sonicated and lysed, and centrifuged at 12000×g for 20 min. The lysed cell supernatant and pellet were collected, filtered through a 0.45μm filter membrane, and detected by SDS-PAGE electrophoresis.

[0065] After SDS-PAGE electrophoresis, Western blot was performed. The protein gel was placed on the wet transfer electrode, with the transfer sponge, NC membrane, protein gel, and semi-dry transfer sponge placed in that order near the positive electrode. The transfer was performed at a constant current of 250 A for 80 min. After transfer, the NC membrane was blocked in 5% skim milk at room temperature for 2 h. It was then incubated with mouse His monoclonal antibody (1:2000) as the primary antibody for 1 h at room temperature, washed three times with TBST, and incubated with HRP-labeled goat anti-mouse IgG antibody as the secondary antibody for 1 h at room temperature, washed three times with TBST, and then developed. Figure 6 ).

[0066] 4.3 Protein purification

[0067] Insect cells with 90% disease severity were centrifuged at 500×g for 10 min to collect the cell pellet and cell culture supernatant. The cells were resuspended in 1 / 6 volume of 0.25M Tris HCl (pH=8.25) culture medium, sonicated, and centrifuged at 12000×g for 20 min. The supernatant of the fragmented cells was collected, filtered through a 0.45 μm filter, and resuspended in Q Sepharose Fast Flow anion exchange medium. The column was then loaded with the resuspending medium, and the gravity column was opened to allow ethanol to flow out. Ten column volumes of filtered deionized water were added to the column to wash away residual anhydrous ethanol. The column was then equilibrated with ten column volumes of Tris HCl pH=7.25 buffer. After binding the sample to the packing material, the column was incubated in a 3D mixer at 4℃ for 2 h. After incubation, the packing material was added back to the gravity column, and the gravity column was opened. The flow-through was collected and treated with 0.01M, 0.06M, 0.15M, 0.2M, 0.3M, and 0.4M Tris HCl buffer, respectively. After elution with HCl-NaCl eluent for five column volumes, the column was thoroughly eluted with 10 column volumes of Tris-HCl buffer (pH 7.25) containing 1 M NaCl. The column was then washed with 10 column volumes of filtered deionized water, and stored at 4°C with 20% anhydrous ethanol. The purified recombinant protein was then concentrated and replaced using a 10 kDa ultrafiltration tube. SDS-PAGE and Western blot were used to analyze the cell culture supernatant, lysed cell supernatant, and purified recombinant protein. Figure 7 As shown. Protein concentration was measured using the BCA protein assay kit and stored at 80°C.

[0068] 5. Immunization of mice and detection of immune serum activity

[0069] according to Figure 8The immunization procedure shown involved 8-week-old female Balb / c mice immunized with the recombinant protein purified in step 4. Serum was collected before immunization, with each mouse receiving 50 μg per immunization. A second immunization was performed two weeks after the first, followed by a third two weeks after the second. Two weeks after the third immunization, blood was collected from the tail vein, and serum was collected. Mice with high antibody levels were selected for intraperitoneal pulse immunization. Three days later, spleen lymphocytes were harvested for cell fusion. The collected serum (immunized with PDCoV S1 protein) was analyzed using an indirect ELISA method to detect S1 protein antibody levels. Protein expression was identified using indirect immunofluorescence (IFA) and Western blot methods as described in "4.1 Indirect Immunofluorescence Assay (IFA)" and "4.2 Western blot Identification of Protein Expression." Results are as follows. Figure 9 and Figure 10 As shown.

[0070] 6. Monoclonal antibody screening and specificity identification

[0071] Two flasks of 80% SP2 / 0 cells and mouse spleen cells were added to centrifuge tubes and centrifuged at 1000 rpm for 6 min. The supernatant was discarded, and the cell pellet was gently tapped at the bottom of the tube in the palm of the hand to loosen it and mix the two cell types evenly. The centrifuge tubes were placed in a 37°C water bath, and 1 mL of fusion agent PEG 1420 (preheated to 38°C) was slowly and evenly added. The tubes were then incubated in a 37°C water bath for 90 s. Then, preheated ordinary culture medium was added to terminate the fusion, following the principle of adding slowly first and then quickly, adding 30 mL of 37°C preheated ordinary culture medium. After mixing, the mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. 5 mL of HAT culture medium containing feeder cells was added first, and the cell pellet was gently aspirated to suspend it. The cell culture was then added to 400 mL of HAT culture medium containing feeder cells and mixed well. The cells were resuspended in HAT culture medium, and 100 μL was added to each well of a 96-well cell culture plate containing feeder cells. The plates were then incubated at 37°C in a 5% CO2 cell culture incubator. The culture medium containing HT was replaced on day 7 and day 10, and the antibody titer of the cell culture supernatant was detected by indirect ELISA. Positive hybridoma cells were then cloned.

[0072] After four subcloning processes, a monoclonal antibody was selected. Using PDCoV (HNZK-02, GenBank: MH708123.1), PEDV (HN2021, GenBank: OR707084.1), TGEV (HN-2012, GenBank: OP434397.1), and PSV (HNHB-01 / CHN / 2016, GenBank: MN939541.1) viruses, the monoclonal antibody was identified by indirect immunofluorescence and Western blot. The results are shown below. Figure 11 and Figure 12The antibody showed good reactivity in both indirect immunofluorescence and Western blot, and exhibited no cross-reactivity with PEDV, TGEV, or PSV. Western blot results showed that the monoclonal antibody reacted well not only with the recombinant S1 protein prepared in this invention, but also with the prokaryotically expressed S1 protein (obtained by ligating the PDCoV S1 gene of this invention to the pet-32a vector, transforming it into BL-21 bacteria, and purifying it after induction expression) and PDCoV. Figure 13 ).

[0073] 7. Determination of neutralizing activity and neutralizing titer of monoclonal antibodies

[0074] The selected monoclonal antibody ascites fluid was first diluted 1:16, then serially diluted 2-fold. The serially diluted ascites fluid was mixed with 200 TCID50 / 0.1 mL of PDCoV virus at a 1:1 ratio and incubated at 37°C for 1 h. Cells in 96-well plates were washed twice with Dhanks solution, 100 μL per well, and incubated at 37°C for 1 h. After discarding the solution, the cells were washed twice with Dhanks solution, and MEM maintenance medium containing 2.5% T was added. Cells were fixed with anhydrous ethanol 24 h after inoculation and detected using indirect immunofluorescence. Figure 14 As shown, the fluorescence in the monoclonal antibody incubation wells was significantly reduced, indicating that the monoclonal antibody has a neutralizing effect on PDCoV virus and belongs to the category of neutralizing antibodies.

[0075] The selected monoclonal antibody was serially diluted 2-fold in ascites fluid and mixed with 200 TCID50 / 0.1 mL of PDCoV virus at a 1:1 ratio. The mixture was incubated at 37°C for 1 h. Cells in 96-well plates were washed twice with Dhanks solution, 100 μL per well, and incubated at 37°C for 1 h. After discarding the solution, the cells were washed twice with Dhanks solution, and MEM maintenance medium containing 2.5% T was added. After 72 h of inoculation, the neutralizing titer of the neutralizing antibody was calculated to be 10. -3.225 ( Figure 15 ).

[0076] 8. Monoclonal antibody subtype identification, sequencing, and antigenic epitope identification

[0077] Using a monoclonal antibody subtype detection kit, 50 μL of hybridoma cell supernatant was added to each well, followed by 50 μL of 1* goat anti-mouse IgM+IgG-HRP without incubation. The mixture was incubated at room temperature for 1 h, washed three times with PBST, and then 100 μL of TMB chromogenic solution was added per well. The mixture was incubated in the dark for 10 min, and 100 μL of stop solution was added to each well. The OD450 values ​​were read using a microplate reader. Results are as follows: Figure 16 As shown, the light chain of this monoclonal antibody is of the Kappa type, and the heavy chain is of the IgG1 type.

[0078] Heavy chain and light chain primers were designed based on antibody subtypes. RNA was extracted from monoclonal antibody cell lines, and cDNA was reverse transcribed using these primers to amplify light and heavy chain gene fragments. The experiment was repeated three times, and electrophoresis was performed for detection. Figure 17 As shown. The amplification product was recovered and sequenced to determine the light chain variable region and heavy chain variable region sequences of the monoclonal antibody. The specific sequences are as follows:

[0079] Heavy chain primer F: GGCTAGCCACCATGGCGGAGGTGAAGCTG (SEQ ID NO.5);

[0080] Heavy chain primer R: CTTCAAGCTTTGGGGGTGTCGTTTTGGCCG (SEQ ID NO.6);

[0081] Light chain primer F: AAGCAGCGTAGACATTGTGCTGACCCAATCTC (SEQ ID NO.7);

[0082] Light chain primer R: CCAGCGGCCGCGGATACAGTTGGTGCAG CATC (SEQ ID NO.8).

[0083] Light chain: ARVDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQP PRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK (SEQ ID NO.9); among them, CDR1 is RASKSVSTSGYSYMH (SEQ ID NO.10), and CDR2 is LVSNLES (SEQ ID NO.10) NO.11), CDR3 is QHIRELTR (SEQID NO.12).

[0084] Heavy chain: MAEVKLSCKASGYTFTDYEMHWVKQTPVHGLEWIGTVDPETGGSAYN QKFKDKATLTADKSSSTAYMELRSLTSEDSAVYYCTRLLRYWGQGTTLTVSS (SEQ ID NO.13); where CDR1 is DYEMH (SEQ ID NO.14), CDR2 is TVDPETGGSAYNQKFKD (SEQ ID NO.15), and CDR3 is LLRY (SEQ ID NO.16).

[0085] according to Figure 18The PDCoV S1 protein was truncated to determine the antigenic epitope recognized by the monoclonal antibody. The results showed that the antigenic epitope was FTSPTGELYAF (SEQ ID NO. 17). Comparison with different PDCoV strains and different generations of PDCoV revealed that this antigenic epitope was completely conserved among PDCoV strains. The epitope showed 50% and 42% conservation with different PEDV strains and different TGEV strains, respectively, indicating high conservation. Figure 19 ).

[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A neutralizing monoclonal antibody targeting a conserved linear epitope of porcine delta coronavirus S1 protein, characterized in that, The light chain of the neutralizing monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.10, CDR2 with an amino acid sequence as shown in SEQ ID NO.11, and CDR3 with an amino acid sequence as shown in SEQ ID NO.12; The heavy chain of the neutralizing monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.14, CDR2 with an amino acid sequence as shown in SEQ ID NO.15, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

16.

2. The neutralizing monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain of the neutralizing monoclonal antibody is shown in SEQ ID NO.9, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

13.

3. The neutralizing monoclonal antibody according to claim 1, characterized in that, The neutralizing monoclonal antibody can recognize the antigenic epitope in the porcine delta coronavirus S1 protein, as shown in SEQ ID NO.

17.

4. The use of the neutralizing monoclonal antibody according to any one of claims 1-3 in the preparation of a medicament for the prevention and / or treatment of porcine delta coronavirus infection.

5. A drug for the prevention and / or treatment of porcine delta coronavirus infection, characterized in that, The drug comprises the neutralizing monoclonal antibody as described in any one of claims 1-3 and pharmaceutically acceptable excipients.

6. The use of the neutralizing monoclonal antibody according to any one of claims 1-3 in the preparation of reagents or kits for detecting porcine delta coronavirus.

7. A porcine delta coronavirus S1 protein antigenic epitope peptide, characterized in that, The amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO.

17.

8. The use of the antigenic epitope peptide according to claim 7 in the preparation of a vaccine for the prevention of porcine delta coronavirus infection.

9. A vaccine for preventing porcine delta coronavirus infection, characterized in that, The vaccine comprises the antigenic epitope peptide of claim 7 and a pharmaceutically acceptable carrier.

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