Monoclonal antibody of porcine CD163 protein and application thereof

Abstract

The invention provides a monoclonal antibody for detecting pig source CD163 protein and a corresponding antigen epitope peptide. Wherein the hybridoma cell strain used for preparing the monoclonal antibody is CCTCC (China Center For Type Culture Collection) NO: C2025258. The monoclonal antibody provided by the invention is good in specificity, has good affinity with pCD163, and is clear in epitope information. The antibody can significantly inhibit the infection efficiency of PRRSV pedigree strains in alveolar macrophages, has a good PRRSV in-vitro blocking effect, can be used for developing a high-specificity pCD163 detection reagent, and is expected to be used as a candidate drug for passive immune prevention and treatment of PRRS.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparation technology, specifically relating to an antigenic epitope peptide of porcine CD163 protein, a hybridoma cell line, and a monoclonal antibody. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) seriously threatens the healthy development of the global pig industry. Its pathogen, porcine reproductive and respiratory syndrome virus (PRRSV), exhibits high genetic variability, with significant sequence differences between different genotypes and subtypes, resulting in limited cross-protective efficacy of existing vaccines and antiviral control measures. Current research indicates that PRRSV possesses strict cell tropism, and porcine CD163 (pCD163) is an essential receptor for PRRSV infection of target cells. The SRCR5-9 domain of CD163 is a key functional region mediating viral entry into cells. Therefore, targeting the key pCD163 domain to block PRRSV invasion is considered a potentially broad-spectrum antiviral strategy.

[0003] CD163 belongs to the scavenger receptor cysteine-rich (SRCR) protein superfamily. It is a membrane receptor that binds to the globin-hemoglobin (Hp-Hb) complex, mediating the endocytosis of Hp-Hb by macrophages and clearing hemoglobin released during hemolysis, thereby preventing oxidative damage caused by heme iron. The extracellular region of pCD163 contains nine repeating SRCR domains (SRCR1-SRCR9) and two proline-serine-threonine (PST) rich motifs. PSTⅠ consists of 35 amino acids and is located between SRCR6 and SRCR7; PSTⅡ consists of 16 amino acids and connects SRCR9 to the transmembrane region and cytoplasmic tail.

[0004] Currently, PRRSV control measures rely on biosafety isolation and modified live virus (MLV) vaccines, which carry safety risks. While MLV can successfully induce a protective immune response against homologous viruses, it still suffers from insufficient cross-protection against heterologous viruses. Furthermore, MLV presents safety concerns regarding infection, transmission, immunosuppression, virulence reversion, and genetic recombination. Subunit vaccines, although showing better safety profiles, remain highly controversial regarding the protective antigens of PRRSV. Coupled with the diverse genetic variations of viral antigens, the immunoprotective efficacy of these vaccines is uncertain. Current products do not meet clinical needs. Therefore, there is an urgent need to screen epitope peptides from the key functional domains of pCD163 that possess good immunogenicity, high specificity, structural stability, and the ability to effectively block PRRSV infection. Based on these, specific monoclonal antibodies can be prepared, providing a new technical approach and theoretical basis for developing broad-spectrum antiviral control strategies against PRRSV. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody that can block porcine CD163 protein infection caused by PRRSV, and its corresponding epitope peptide. This monoclonal antibody was used for indirect immunofluorescence and Western blotting to analyze the expression and distribution of pCD163 in alveolar macrophages and overexpressing cells, providing a valuable tool for further research on the interaction mechanism between structural proteins and CD163 during PRRSV infection. Simultaneously, this invention provides a monoclonal antibody with broad-spectrum anti-PRRSV activity, offering a novel drug for the prevention and control of PRRS.

[0006] This invention first provides a hybridoma cell line, namely hybridoma cell line 1E5, with accession number CCTCC NO: C2025258, deposited on September 5, 2025; the depositary institution is the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0007] On the other hand, the present invention provides a monoclonal antibody that specifically binds to the pCD163 protein, said monoclonal antibody being produced by the hybridoma cell line described above.

[0008] The present invention provides that the amino acid sequence of the antigen epitope peptide recognized by the monoclonal antibody is DPSSSI (SEQ ID NO:1).

[0009] The present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of reagents for the detection of pCD163 protein.

[0010] The reagents described are preparations for Western blotting, immunohistochemistry, or immunofluorescence detection.

[0011] The present invention also provides a reagent for detecting pCD163 protein, the reagent comprising the above-mentioned monoclonal antibody.

[0012] The present invention also provides a method for detecting pCD163 protein in a sample, the method comprising the step of detecting pCD163 protein using the above-mentioned monoclonal antibody.

[0013] In one embodiment, the sample is selected from lysates of 293T cells.

[0014] The present invention also provides the use of the pCD163 monoclonal antibody as described in any of the preceding embodiments in the preparation of a medicament for blocking porcine reproductive and respiratory syndrome virus.

[0015] The present invention also provides a method for detecting pCD163 protein in a sample, the method comprising the step of detecting pCD163 protein using the above-mentioned antibody.

[0016] The monoclonal antibody provided by this invention exhibits good specificity, strong affinity for pCD163, and clearly defined epitope information. This antibody significantly inhibits the infection efficiency of various PRRSV lineages in alveolar macrophages, demonstrating excellent in vitro PRRSV blocking efficacy. It can be used to develop highly specific pCD163 detection reagents and holds promise as a candidate drug for passive immunization and treatment of PRRS. Attached Figure Description

[0017] Figure 1 This is a PAGE image of prokaryotic expression of pCD163 SRCR5-9 protein in an embodiment of the present invention. The target band is between 55-70 kDa.

[0018] Figure 2 This invention relates to the purity identification of SRCR5-9 after prokaryotic expression and gel purification in an embodiment of the invention; M: Marker; 1: Purified SRCR5-9; 2: Inclusion bodies before purification; Figure 3 This is the IFA result of detecting exogenously overexpressed pCD163 protein using 1E5 mAb in an embodiment of the present invention;

[0019] Figure 4 This is the Western blotting result of detecting pCD163 protein using 1E5 mAb in an embodiment of the present invention; M: Marker; 1: pCD163 protein sample; 2: Negative control;

[0020] Figure 5This is the electrophoresis result of SDS-PAGE detection of purified 1E5 mAb in an embodiment of the present invention; M: Marker; 1: Ascites fluid before purification; 2: Antibody after purification;

[0021] Figure 6 This refers to the IFA results of the antigen epitope identified by the monoclonal antibody in the embodiments of the present invention;

[0022] Figure 7 This is the localization result of the antigen epitope recognized by the monoclonal antibody in the embodiments of the present invention;

[0023] Figure 8 The images show the IFA results of monoclonal antibodies used in this invention to detect overexpressed and endogenous pCD163 protein. The left image shows the detection of pCD163 in BHK-21 cells after a 1:1000 dilution of 1E5-AF647; the right image shows the detection of pCD163 in PAMs after a 1:500 dilution of 1E5-AF647.

[0024] Figure 9 This is an IFA diagram of monoclonal antibody blocking infection of alveolar macrophages by various lineages of PRRSV strains in an embodiment of the present invention.

[0025] Figure 10 This is a graph showing the titer determination of the monoclonal antibody blocking PRRSV-2 JXwn06 strain infection of alveolar macrophages according to an embodiment of the present invention;

[0026] Figure 11 The IC50 of the monoclonal antibody against PRRSV-2 JXwn06 in this embodiment of the invention is shown. 50 Measurement chart;

[0027] Figure 12 This invention relates to the EC5 of monoclonal antibody 1E5 against an antigenic epitope peptide. 50 Measurement;

[0028] Figure 13 This is a model diagram for predicting the spatial structure of antigen epitopes in an embodiment of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will be further explained and illustrated below through specific embodiments. The following description is merely illustrative and not intended to limit the scope of the invention. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0030] Example 1: Preparation of antigen protein

[0031] 1. Plasmid construction

[0032] pCD163 SRCR5-9 (Gene ID: 397031; mRNA: NM_213976.1) was constructed into the pET-28a(+) prokaryotic expression vector via homologous recombination, and then transformed into DH5α Escherichia coli competent cells for positive clone identification and plasmid extraction.

[0033] 2. Protein Expression and Purification

[0034] ① Induction of expression: The prepared pET-28a-pCD163-SRCR5-9 plasmid was transformed into BL21 competent cells. Single colonies were cultured and induced to express when the OD value reached approximately 0.6. The induction conditions were 0.1 mM and 0.5 mM IPTG at 16℃ for 18 h. After induction, the bacterial cells were collected and sonicated. The supernatant and inclusion bodies were sampled separately for PAGE analysis. The identification results are as follows: Figure 1 The pCD163 SRCR5-9 protein is mainly found in inclusion bodies.

[0035] ② The inclusion bodies after ultrasonic disruption were loaded onto a single-well comb and subjected to SDS-PAGE according to standard methods. After electrophoresis, the protein gel was stained with 0.3M KCl solution for 5 min. The target band, which had turned silvery-white, was cut off and washed three times with PBS. The gel strip was crushed and subjected to three freeze-thaw cycles at -80℃. It was then centrifuged at 12000 rpm at 4℃ for 10 min. The supernatant was collected and concentrated using an ultrafiltration tube. The protein content was measured using a spectrophotometer. After further testing for protein purity by SDS-PAGE, the protein was stored at -80℃ for later use. The PAGE results are as follows. Figure 2 As shown.

[0036] Example 2: Preparation and Identification of Monoclonal Antibodies

[0037] Monoclonal antibodies were prepared using conventional hybridoma techniques.

[0038] 1. Immunize BALB / c mice

[0039] The antigen protein prepared in Example 1 was used to immunize 6-8 week old BALB / c mice according to the immunization schedule shown in Table 1. One week after the third immunization, blood was collected from the orbital sinus and serum was separated. The serum was diluted 1:40 with PBS buffer. IFA was performed using BHK-21 cells transfected with pCAGGS-HA-pCD163 plasmid as the antigen. The negative control was negative serum from unimmunized mice. Mice that tested positive for IFA were given a booster immunization.

[0040] Table 1: Immunization Schedule

[0041] Number of immunizations interval Immune pathway Immunization dose adjuvant First-time exemption Multiple subcutaneous injections on the neck and back 50μg / each Equal volume Freund's complete adjuvant Secondary immunization 2 weeks Multiple subcutaneous injections on the neck and back 100μg / each Equal volume Freund's complete adjuvant Three immunizations 2 weeks Multiple subcutaneous injections on the neck and back 100μg / each Equal volume Freund's complete adjuvant Strengthen immunity 2-3 weeks Intraperitoneal injection 100μg / each No adjuvants

[0042] 2. Establishment of hybridoma cell lines

[0043] ① Preparation of myeloma cells: 7 days before fusion, resuscitate SP2 / 0 cells and resuspend them in DMEM medium containing 20% ​​serum, then add them to a 25cm² cell culture flask for culture. 2 days before fusion, place the SP2 / 0 cells to be passaged in a 75cm² cell culture flask and expand their culture in a 37℃, 5% CO2 cell culture incubator. Change the medium 6 hours before fusion. At fusion, wash the cells and resuspend them in a small amount of serum-free DMEM, then count them for later use.

[0044] ② Preparation of feeder cells: Feeder cells were prepared from 4-6 week old female BALB / c mice 1 day before fusion; mouse peritoneal macrophages were perfused with HAT selective medium to adjust the cell density to 2×10⁻⁶. 5 Cells / mL. Spread the cell suspension into 10 96-well plates, 100 μL per well, and incubate at 37°C in a 5% CO2 cell culture incubator.

[0045] ③ Preparation of spleen cells from immunized mice: On the day of fusion, mice that have undergone booster immunization for 3-4 days are taken. The spleens of the mice are separated and repeatedly blown, aspirated, and irrigated until the spleen is empty and no longer has any blood color. Single-cell suspensions are prepared and counted for later use.

[0046] ④ Cell fusion and selective culture of hybridoma cells: Mix SP2 / 0 cells with spleen cells at a ratio of 1:10 to 1:5, and slowly and evenly add 1 mL of preheated 50% PEG solution (37℃) along the tube wall; after incubating in a 37℃ water bath for 60 seconds, slowly add 10 mL of serum-free DMEM medium; after incubating in a 37℃ water bath for 5 minutes, centrifuge at 800 rpm for 6 minutes and discard the supernatant; resuspend thoroughly in HAT medium containing 20% ​​FBS. Seed 100 μL per well in a 96-well plate with feeder cells; after 5 days, replace half of the original medium with HAT selective medium; after approximately 7-10 days, replace with HT medium containing 20% ​​FBS. During the above selective culture period, when hybridoma cells cover 1 / 10 of the bottom area of ​​the well, specific antibody detection can begin to screen for the desired hybridoma cell line. During the selective culture period, half of the culture medium is generally replaced every 2-3 days.

[0047] 3. Hybridoma cell screening

[0048] ① 24-36 h after transfection with pCAGGS-HA-pCD163 plasmid, BHK-21 cells were fixed with anhydrous ethanol at room temperature for 15 min; washed 3 times with PBS buffer and then blocked with 2% BSA solution at room temperature for 30 min.

[0049] ② Incubate the hybridoma cell supernatant culture medium at 37°C for 1 hour, wash 3 times with PBS buffer, add FITC-labeled goat anti-mouse fluorescent secondary antibody in the dark, incubate in the dark for 1 hour, and then determine the positive or negative result.

[0050] ③ Select cells with strongly positive IFA results, change all the medium, and perform IFA testing again the next day. Cells with positive results are used for subcloning. Select the positive hybridoma cell wells, dilute the remaining cells with HT selective medium according to the cell count results, 1-2 cells / 100μl, and culture in 96-well cell culture plates. When the cells grow to 1 / 10-1 / 5 in about a week, aspirate the supernatant for IFA testing, and select cells from positive wells for secondary subcloning.

[0051] ④ The entire subcloning process is repeated approximately three times or more until all cells test positive, in order to screen for hybridoma cell lines that stably secrete specific monoclonal antibodies.

[0052] The final selected hybridoma cell line was named Hybridoma cell line 1E5 and was deposited on September 5, 2025, at the China Center for Type Culture Collection, Wuhan University, China, with accession number CCTCC NO: C2025258.

[0053] 4. Preparation and identification of monoclonal antibodies

[0054] ① Monoclonal antibody preparation: Three days before injection of hybridoma cells, 7-9 month old multiparous BALB / c mice were intraperitoneally injected with 500 μl of Freund's incomplete adjuvant per mouse; each mouse was injected with approximately 5 × 10⁶ positive hybridoma cells. 5 -10 6 One week after injection, when the abdominal circumference of the mice increased significantly, the ascites fluid was collected, centrifuged at 8000 r / min for 5 min, and the ascites fluid layering device was stored at -80℃; the collected hybridoma cell supernatant and its ascites fluid were serially diluted, and the titers were detected by indirect immunofluorescence.

[0055] ② IFA identification of monoclonal antibodies: IFA was performed on BHK-21 cells transfected with pCAGGS-HA-pCD163 plasmid after a 1:10000 dilution of ascites fluid. The results are as follows: Figure 3 As shown, the antibody reacts with pCD163 with good specificity.

[0056] ③ Western blotting identification of monoclonal antibodies: pCAGGS-HA-pCD163 plasmid was transfected into 293T cells. Cell lysates were collected after 24 hours as the antigen. Ascites fluid was diluted 1:10000 and used as the primary antibody. After incubation at room temperature for 1 hour, the cells were incubated with goat anti-mouse HRP secondary antibody. The monoclonal antibody was able to undergo Western blotting, indicating that it recognizes the linear epitope of pCD163. The results are as follows: Figure 4 As shown.

[0057] Example 3: Purification of monoclonal antibody 1E5

[0058] The monoclonal antibody purification in this embodiment includes the following specific steps:

[0059] 1. Prepare the Buffer

[0060] Binding / washing buffer: 0.15M NaCl, 20mM Na2HPO4, pH 6.0, pH = 7.4;

[0061] Elution Buffer: 0.1M glycine, pH=3.0;

[0062] Neutralization solution: 1M Tris-HCl, pH=8.5;

[0063] Stop solution: 50mM Tris, pH=7.5.

[0064] 2. Purification of Ascites Fluid: Add an appropriate amount of equilibrated rProtein A / G Beads to a 2ml centrifuge tube, add diluted ascites fluid, invert at room temperature for 30 minutes, centrifuge at 800 rpm for 1 minute, collect the supernatant for later analysis. Add 0.5ml of washing buffer to wash and remove non-specifically adsorbed proteins, centrifuge at 800 rpm for 1 minute, and discard the supernatant. Repeat twice. Add 5 column volumes of elution buffer to the centrifuge tube, pipette five times to mix, then invert at room temperature for 10 minutes, centrifuge at 800 rpm for 1 minute, and collect the elution fraction from the supernatant. Immediately add 1 / 10 volume of neutralization solution to the supernatant to adjust the pH of the elution fraction to 7-8.0.

[0065] 3. Monoclonal antibody purity identification:

[0066] SDS-PAGE electrophoresis analysis showed a purity of over 95%, and the results were as follows: Figure 5 As shown.

[0067] Example 4: Identification of 1E5 mAb epitopes

[0068] Using pET-28a-pCD163-SRCR5-9 plasmid as a template, PCR primers were designed to truncate pCD163 SRCR5-9, and pCAGGS-HA-SRCR5-9 and the truncated expression plasmid were constructed. Each truncated region contained different domains of SRCR5-9, named SRCR5, SRCR5-6-PSTⅠ, SRCR5-7, and SRCR5-8, respectively. Plasmids with different domains were transfected into BHK-21 cells and identified by IFA. Figure 6As shown in Figure A, 1E5 mAb reacts with the truncated SRCR5-6-PSTⅠ protein but not with SRCR5. Therefore, this invention further divides SRCR6-PSTⅠ into three regions: SRCR6-1, SRCR6-2, and SRCR6-3. Simultaneously, a truncated expression plasmid pCAGGS-HA-PSTⅠ-SRCR7 is constructed and transfected into BHK-21 cells for IFA identification. Figure 6 As shown in Figure B, 1E5 mAb reacts with truncated SRCR6-3 and PSTⅠ-SRCR7 proteins, indicating that 1E5 recognizes the overlapping region PSTⅠ. This invention further divides PSTⅠ into 14 regions and fuses them with SRCR6 and SRCR7 for expression, as shown in Figure B. Figure 6 As shown in Figure C, 1E5 mAb reacts with SRCR6-PSTⅠ(21N) and PSTⅠ(20C)-SRCR7 truncated proteins, confirming the amino acid sequence in the CD163 PSTⅠ region. 16 DPSSSI 21 The epitope recognized by the 1E5 monoclonal antibody is located at 698-703aa in the full length of pCD163.

[0069] To illustrate the antigen epitope identification process more intuitively and clearly, a schematic diagram of monoclonal antibody identification is drawn based on the IFA identification results (see...). Figure 7 The red area represents the PSTⅠ domain, the blue area represents the truncated version recognized by monoclonal antibody 1E5, and the yellow highlighted area represents the key amino acid residues recognized by monoclonal antibody 1E5.

[0070] Example 5: Preparation of 1E5 mAb-AF647 direct-labeled antibody

[0071] After adjusting the pH of purified pCD163 (1 mg / mL) to 8.3, it was added to Alexa Fluor 647 NHS ester at a dye:antibody molar ratio of 10:1. The mixture was incubated at room temperature in the dark for 30 min–2 h. 1.5 M hydroxylamine (pH=8.5) was added to a final concentration of 100 mM, and the reaction was terminated by incubation at room temperature for 10 min. The mixture was then centrifuged at 14,000 × g for 10 min using an ultrafiltration tube, repeated three times to remove free dye. The absorbance of A280 (protein) and A650 (dye) was measured using a spectrophotometer, and the dye / protein molar ratio (DOL) was calculated. The direct-labeled antibody was stored in PBS containing 0.03% Proclin-300, 50% glycerol, and 1% BSA for later use. The pCD163 overexpressed in BHK-21 cells and the endogenous pCD163 in PAMs were detected at dilutions of 1:1000 and 1:500, respectively. The results are as follows: Figure 8 As shown, 1E5 mAb exhibits good specificity, and this direct-labeling antibody can be used for the detection of endogenous pCD163 and overexpressed pCD163.

[0072] Example 6: Detection of antiviral activity of 1E5 mAb

[0073] 1. Micro-neutralization experiment

[0074] The blocking ability of 1E5 mAb against various PRRSV lineages was evaluated on PAMs using a micro-neutralization assay. Purified 1E5 mAb was serially diluted 4-fold from 100 μg / ml to 1.56 μg / ml in 2% RPMI 1640 medium and pre-incubated in PAMs for 30 min. PRRSV lineage dilutions (MOI=0.1) were then added to different concentrations of antibody and co-incubated with the cells for 1 h. After washing three times with PBS and replacing with maintenance medium, the cells were incubated at 37℃ for 24–36 h in a 5% CO2 incubator, after which the maintenance medium was discarded for fixation.

[0075] 2. Toxicity determination: The purified 1E5 mAb was serially diluted 25-fold from 100 μg / ml to 0.16 μg / ml in 2% RPMI 1640 medium and added to PAMs for pre-incubation for 30 min. PRRSV JXwn06 strain dilution with MOI=0.1 was added to different concentrations of antibody and co-incubated with cells for 1 h. After washing three times with PBS and changing to maintenance medium, the cells were incubated at 37℃ in a 5% CO2 incubator for 24-36 h. After freeze-thaw, the PAMs supernatant was subjected to TCID assay on MARC-145 cells. 50 Measurement.

[0076] PRRSV infection status was detected using IFA with N protein-specific antibody. Results are as follows: Figure 9 and Figure 10 As shown, 1E5 mAb significantly reduces PRRSV-specific fluorescence in PAMs and has an inhibitory effect on all PRRSV lineages. Taking the JXwn06 strain as an example, 1E5 mAb reduces the PRRSV titer by about 100 times.

[0077] 3. Determination of half-maximal inhibitory concentration

[0078] The recombinant PRRSV JXwn06 strain (RvJX-Nsp2) carrying a complementary Nanoluc luciferase tag was used. 325 A micro-neutralization assay was performed using 1E5 mAb against PRRSV JXwn06 strain using PAMs. The purified 1E5 mAb was serially diluted 10-fold from 100 μg / ml to 0.1 ng / ml in 2% RPMI 1640 medium and pre-incubated in PAMs for 30 min. RvJX-Nsp2 with an MOI of 0.1 was then added. 325HiBiT strain dilutions were added to different concentrations of antibody and co-incubated with cells for 1 hour. After washing three times with PBS and replacing with maintenance medium, the cells were incubated at 37°C with 5% CO2 for 24-36 hours. Cells were then frozen and thawed, and 50 μL of cell supernatant was added to each well of a 96-well plate. An equal volume of Nano-Glo® HiBiT LyticReagent was then added, and the mixture was thoroughly shaken and mixed for 10 minutes at room temperature. The fluorescence intensity was then measured using a luminescence detector. The fluorescence intensity of each sample was measured at least three times. Results are as follows: Figure 11 1E5 mAb for PRRSV IC 50 It is 4.48 nM.

[0079] Although the present invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes and modifications can be made without departing from the spirit and scope of the invention. All such changes and modifications fall within the protection scope of the present invention.

[0080] Example 7: SSSS based on CD163 antigenic epitope peptide 16 DPSSSI 21 IS assay of EC5 of monoclonal antibody 1E5 50

[0081] ELISA detection of 1E5 against the antigenic epitope peptide SSSS 16 DPSSSI 21 IS's EC 50 The antigenic epitope peptide was coated onto an ELISA plate at a concentration of 1 μg / ml at 37°C for 1 h. After washing three times with PBST, the plate was blocked with 5% skim milk. The purified 1E5 antibody was serially diluted 5-fold from 250 μg / ml to 3.2 ng / ml, with two replicates for each dilution. The plate was incubated at 37°C for 1 h. After washing three times with PBST, 100 μl of a 1:10000 dilution of goat anti-mouse HRP secondary antibody was added to each well, and the plate was incubated at 37°C for 1 h. After washing three times with PBST, 100 μl of TMB chromogenic buffer was added to each well, and the plate was incubated for 5 min. The reaction was terminated with 2M H₂SO₄ solution, and the OD value was read at 450 nm. The results are as follows: Figure 12 As shown, monoclonal antibody 1E5 and antigenic epitope peptide SSSS 16 DPSSSI 21 IS's EC 50 It is 1.59 μg / mL.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line described has the accession number CCTCC NO: C2025258.

2. The use of the hybridoma cell line according to claim 1 in the preparation of monoclonal antibodies.

3. A monoclonal antibody, characterized in that, The monoclonal antibody is prepared using the hybridoma cell line described in claim 1.

4. The use of the monoclonal antibody according to claim 3 in the preparation of reagents for detecting pCD163 protein.

5. The application as described in claim 4, characterized in that, The reagents described are preparations for Western blotting, immunohistochemistry, or immunofluorescence detection.

6. A reagent for detecting pCD163 protein, characterized in that, The reagents include the monoclonal antibody as described in claim 3.

7. A method for detecting pCD163 protein in a sample, characterized in that, The method utilizes the monoclonal antibody described in claim 3 for detection.

8. The use of the monoclonal antibody according to claim 3 in the preparation of a medicament for blocking porcine reproductive and respiratory syndrome virus.

9. An antigenic epitope peptide, characterized in that, The amino acid sequence of the antigenic epitope peptide is DPSSSI.

10. The use of the antigenic epitope peptide according to claim 9 in the preparation of a formulation for detecting porcine CD163.

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