Porcine circovirus type 2 neutralizing antibody, hybridoma cell strain and application thereof

Abstract

The invention discloses a porcine circovirus type 2 neutralizing antibody and a hybridoma cell strain secreting the same. The invention also discloses sequence information of heavy chain and light chain variable regions of the neutralizing antibody and fully verifies a key site for binding the neutralizing antibody with Cap protein. The porcine circovirus type 2 neutralizing antibody can specifically recognize porcine circovirus type 2 and porcine circovirus type 2 Cap protein, has high neutralizing activity on PCV2b and PCV2d, and is of great significance in guiding PCV2 vaccine design and developing therapeutic antibodies.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a porcine circovirus type 2 neutralizing antibody, a hybridoma cell line, and applications of the antibody in research on mechanisms related to porcine circovirus type 2 neutralization. Background Art

[0002] Porcine circovirus type 2 (PCV2), a major member of the Circoviridae family and the genus Circovirus, was first discovered in 1998 and is a significant emerging pathogen of pigs. PCV2 was initially linked to postweaning multisystemic wasting syndrome (PMWS). Subsequent studies have further confirmed its widespread pathogenicity, causing not only reproductive dysfunction but also intestinal and respiratory diseases. These diseases are known as porcine circovirus–associated disease (PCVAD) in the United States and porcine circovirus disease (PCVD) in Europe. Importantly, PCV2 primarily attacks the pig immune system, causing lymphocyte apoptosis, cytokine disturbances, and a significant decrease in immune function. Ultimately, this leads to an immunosuppressive state and increases susceptibility to other pathogens. Pigs infected with PCV2 alone have mild clinical symptoms, but co-infection with other pathogens can enhance PCV2 infection and further aggravate porcine circovirus-related diseases.

[0003] PCV2 is a single-stranded circular DNA virus without an envelope. Each virion is composed of 60 nucleocapsid proteins. The viral genome is 1.7 kb in length, making it the smallest known mammalian virus. PCV2 primarily synthesizes its viral DNA using a "rolling-circle melting-pot" model, can integrate host genes, and has one of the fastest mutation rates among known single-stranded viruses. Nine PCV2 genotypes (PCV2a to PCV2i) have been identified, with PCV2b and PCV2d being the most prevalent worldwide. PCV2 contains 11 open reading frames (ORFs), with some overlap between them, enabling the virus to efficiently utilize limited genetic material to encode genes required for replication. The Cap protein, encoded by the ORF2 gene, is the only structural protein of PCV2, with a molecular weight of approximately 27.8 kDa. It is closely associated with PCV2 pathogenicity and is a key factor in triggering host immune responses and pathological damage. The conserved regions and common epitopes of the Cap protein can serve as important sites for monitoring viral evolution and mutation, and may also become potential targets for developing universal antiviral antibodies or drugs.

[0004] PCV2 is ubiquitous in pigs and has become a major pathogen threatening the global pig industry. The detection of porcine circovirus contamination in human rotavirus vaccines has raised concerns about the potential risk of cross-species transmission, suggesting that research on porcine circovirus has important public health implications. Vaccination is the most critical intervention in the current PCV2 prevention and control system, with neutralizing antibodies serving not only as core effector molecules in antiviral immunity but also as a key indicator for evaluating the efficacy of PCV2 vaccines. Due to its high mutation rate, PCV2 exhibits significant genetic diversity, and antigenic differences exist between different strains of porcine circovirus, resulting in a lack of localization of optimal PCV2 antigenic epitopes. Identifying the key sites where PCV2 neutralizing antibodies bind to antigens and studying the neutralization mechanisms mediated by neutralizing antibodies can provide important theoretical foundations for guiding PCV2 vaccine design and the development of therapeutic antibodies. Summary of the Invention

[0005] In this study, a monoclonal antibody 1G10 with neutralizing activity against the PCV2 Cap protein was screened and confirmed that the synergistic effect of amino acids R51 and D194 of the Cap protein determined the binding of the neutralizing antibody 1G10 to the Cap protein.

[0006] In order to obtain the above-mentioned neutralizing antibodies and determine their key binding sites, the present invention is achieved through the following technical solutions:

[0007] In one aspect of the present invention, a neutralizing antibody capable of neutralizing PCV2b and PCV2d is provided.

[0008] Another aspect of the present invention is to analyze the mechanism of action of the neutralizing antibody and determine the key site of its binding to the antigen.

[0009] Preferably, the antibody is the monoclonal antibody 1G10, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is:

[0010] QVQLKQSGPGLVQPSQSLSITCTVS GFSLPRYS LHWVRQSPGKGLEWLGV IWSGGST DYNAAFISRLTISKDNSRSQVFFKMNSLQANDTAIYYC AKSRPPDG YYGAMDY WGQGTSVTVSS (SEQ ID NO. 1);

[0011] The amino acid sequence of the light chain variable region is:

[0012] DIVMTQSQKFMSTSIGDRVSVTYKAS QNVGTN VAWYQQRPGQSPKTLIY SAS YRSSGVPDRFTGGGSGTDFTLTINNVQSEDLAEYFC QQNNTYPYT FGGGT KLEIK (SEQ ID NO. 2), wherein the underlined portion is CDR1-3.

[0013] The monoclonal antibody against the porcine circovirus type 2 Cap protein of the present invention is secreted and produced by the monoclonal antibody hybridoma cell line 1G10, can efficiently and specifically recognize the porcine circovirus type 2 Cap protein, and can neutralize PCV2 infection on PK-15 cells and in mice; monoclonal antibody 1G10 can inhibit the adsorption of the virus on the surface of PK-15 cells and prevent the spread of the virus. The amino acids R51 and D194 of the Cap protein determine the binding of the neutralizing antibody to the antigen. This study provides new ideas and new materials for guiding the design of a broad-spectrum PCV2 vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0015] Figure 1 The mAb-1G10 ( Figure 1 A); ELISA detection of purified mAb-1G10 ( Figure 1 B); Western blot detection of purified mAb-1G10 ( Figure 1 C).

[0016] Figure 2The IFA identification of purified mAb-1G10 in Example 1 of the present invention shows the ability to recognize PK-15 cells infected with PCV2b and PCV2d, respectively.

[0017] Figure 3 The neutralizing activity of mAb-1G10 against PCV2b and PCV2d infected PK-15 cells identified by IFA in Example 1 of the present invention ( Figure 3 A and Figure 3 C); calculate the half-maximal inhibitory concentration of mAb-1G10 against PCV2b and PCV2d infection ( Figure 3 B and Figure 3 D).

[0018] Figure 4 The neutralization effect of mAb-1G10 on PCV2b and PCV2d infection was detected by Western blot in Example 1 of the present invention ( Figure 4 A); Determination of progeny virus titers after mAb-1G10 neutralized PCV2b and PCV2d (as shown in Figure 4 B).

[0019] Figure 5 The qPCR detection of mouse lymph nodes (such as Figure 5 A) and mouse spleen (as Figure 5 B) of PCV2 content; immunohistochemical detection of PCV2 antigen in mouse spleen (as Figure 5 C).

[0020] Figure 6 The IFA in Example 2 of the present invention identified that mAb-1G10 blocked the adsorption of PCV2b and PCV2d to PK-15 cells.

[0021] Figure 7 The flow cytometry analysis of Example 2 of the present invention identified that mAb-1G10 inhibited the spread of PCV2b and PCV2d to uninfected cells.

[0022] Figure 8 This is the sequencing result of the heavy chain and light chain variable regions of mAb-1G10 in Example 2 of the present invention.

[0023] Figure 9 This is the molecular docking analysis of Example 2 of the present invention, showing the interactions between mAb-1G10 and Cap (2b) and Cap (2d).

[0024] Figure 10 These are the key binding sites of mAb-1G10 identified by IFA in Example 2 of the present invention with Cap (2b) and Cap (2d), respectively.

[0025] Figure 11 The expression of Cap mutant protein was identified by SDS-PAGE in Example 2 of the present invention (eg Figure 11 A shown in the figure below), Western blot (as Figure 11 A) and indirect ELISA (as shown above) Figure 11 (B) Identification of the binding ability of mAb-1G10 to various Cap mutant proteins. DETAILED DESCRIPTION

[0026] In the following examples, experimental methods without specific conditions were generally performed under conventional conditions, such as those described in the "Compendium of Molecular Biology Experiments" (edited by FM Osborne, RE Kingston, JG Seidman, etc., translated by Ma Xuejun and Shu Yuelong. Beijing: Science Press, 2004).

[0027] The present invention uses the Shanghai isolate (SH) of porcine circovirus type 2 as the material. After large-scale amplification in PK-15 cells, the virus particles are concentrated by sucrose density gradient centrifugation. The purified PCV2 virus particles are used to immunize 6-8 week-old BABL / c mice, and monoclonal antibodies are prepared using traditional hybridoma technology. After screening, a neutralizing antibody 1G10 (mAb-1G10) that can neutralize PCV2b and PCV2d is obtained. The 1G10 monoclonal antibody hybridoma cell line is injected into the mouse peritoneal cavity to prepare monoclonal antibody ascites. After purification by Protein G affinity chromatography, SDS-PAGE is performed for identification. At the same time, the binding ability of mAb-1G10 to PCV2 Cap protein is detected by indirect ELISA, Western blot, and IFA experiments. The neutralizing activity of mAb-1G10 against porcine circovirus type 2 is explored in vitro (PK-15 cells) and in vivo (BABL / c mice). Based on the variable region sequence of mAb-1G10, molecular docking and alanine scanning techniques were used to identify the key binding sites of mAb-1G10 to the Cap protein as R51 and D194. The purified mouse ascites prepared from the monoclonal hybridoma cell line 1G10 in the present invention is capable of specifically recognizing PCV2 and the PCV2 Cap protein, and neutralizing PCV2 in vitro and in vivo. Ultimately, it was determined that the synergistic action of amino acids R51 and D194 determines the binding of mAb-1G10 to the Cap protein. This neutralizing antibody exhibits strong affinity for the porcine circovirus type 2 Cap protein, strong specificity, and neutralizing activity against porcine circovirus type 2.

[0028] Example 1 Purification and specificity identification of mAb-1G10

[0029] 1. Preparation of mAb-1G10 Mouse Ascites

[0030] (1) Prepare five 8-week-old BALB / c female mice and inject each mouse intraperitoneally with 500 μL of liquid paraffin oil.

[0031] (2) After 7 days, the purified 1G10 monoclonal antibody hybridoma cells (5×10 5 cells / cells);

[0032] (3) Observe the condition of the mice daily. When the abdomen of the mice is swollen and the condition of the mice is not good, extract the mouse ascites at one time (or extract the mouse ascites in several times). The collected mouse ascites is centrifuged at 4°C and 6000 rpm for 10 minutes. Take the middle layer (the upper layer is the lipid layer, the middle layer is the ascites, and the lower layer is cells and other impurities) of the ascites, package it, and store it in a -80°C refrigerator for later use.

[0033] 2. Purification of mAb-1G10

[0034] (1) Remove the prepared mouse ascites from the -80°C freezer, thaw, and dilute with binding / washing buffer at a 1:1 ratio to ensure that the sample solution has the appropriate ionic strength and pH value;

[0035] (2) Filter the diluted sample through a 0.22 μm filter membrane and collect the filtrate;

[0036] (3) Fix the prepacked column on the bracket, remove the plugs at the upper and lower ends in turn, and drain the protective solution in the prepacked column;

[0037] (4) Column equilibration: Add 5 mL of binding buffer to the pre-packed column. After the binding buffer has drained, repeat twice.

[0038] (5) Add the treated ascites sample to the prepacked column and collect the effluent for SDS-PAGE analysis of protein binding;

[0039] (6) Add 5 mL of wash buffer to the prepacked column to wash the column tube to remove non-specifically adsorbed impurities. After the wash buffer is drained, repeat this step 5 times and collect the wash buffer each time for SDS-PAGE analysis of protein washing.

[0040] (7) Elute the target antibody with 20 mL of elution buffer, collect 1 tube for every 5 mL, and detect in sections to ensure the complete elution of the bound antibody and obtain high-purity and high-concentration antibodies. Note that the eluent is acidic and the pH value of the elution component can be adjusted according to the needs of the purified sample. If there is no special need for the purified sample, it is recommended to use 1 / 10 of the volume of the elution component (1MTris-HCL pH=8.5) for neutralization to adjust the pH of the eluent to neutral to maintain the activity of the antibody;

[0041] (8) Washing and regeneration of pre-loaded strains: Alternately equilibrate the medium with 5 mL of combined buffer and 5 mL of deionized water, repeat twice, and then equilibrate once with 5 mL of 20% ethanol. Then, store the pre-loaded strain in an equal volume of 20% ethanol, reinstall the upper and lower stoppers, and store at 4°C to prevent bacterial contamination of the medium.

[0042] 3. SDS-PAGE Identification of mAb-1G10 Purification Results

[0043] Take 80 μL of purified antibody and add 20 μL of 5×SDS-PAGE protein loading buffer, mix well and boil the sample in boiling water for 10 minutes; then use 12.5% ​​protein gel for gel electrophoresis at 80V constant voltage. After the end, place the protein gel in a solution containing an appropriate amount of Coomassie Brilliant Blue and stain at room temperature for 1 hour; after staining, change to destaining solution and destain at room temperature for 1 hour, changing the destaining solution twice during the period until the protein bands are clear and the background is free of color. Figure 1 As shown in A, specific bands of the heavy chain and light chain were observed at 50 kDa and 25 kDa, and the antibody concentration was 500 μg / mL as determined by the BCA assay.

[0044] 4. Indirect ELISA assay to identify mAb-1G10 purification results

[0045] PCV2 Cap protein and control protein (sPRA) were diluted to 4 μg / mL with antigen coating solution and added to a 96-well ELISA plate (100 μL / well). The plates were coated at 4°C for 12 h, washed 3 times with 1×PBST, and 100 μL of blocking solution containing 5% skim milk was added to each well. The plates were blocked at 4°C for 12 h, washed 3 times with 1×PBST, and the water in the wells was patted dry for later use. The purified 1G10 monoclonal antibody was diluted to 20 μg / mL, 10 μg / mL, 1 μg / mL, and 10 μg / mL, respectively. -1 μg / mL, 10 -2 μg / mL, 10 -3 μg / mL, 10 -4 μg / mL and 10 -5 μg / mL, diluted monoclonal antibody (100 μL / well) was added, and the cells were incubated at 37°C for 1 hour; washed 3 times with 1×PBST, each time for 5 minutes, and 100 μL HRP-labeled goat anti-mouse IgG (H+L) (1:5000 dilution) was added, and the cells were incubated at 37°C for 45 minutes; washed 3 times with 1×PBST, 100 μL TMB color development solution was added in the dark, and the color was developed at room temperature for 15 minutes, and then 2 mol / L H2SO4 was directly added to terminate the color reaction (50 μL / well); the cells were immediately placed in a microplate reader to read the OD 450nm Numeric value. Figure 1As shown in Figure B, when mAb-1G10 was diluted to 10-3 μg / mL, the OD450nm value of the indirect ELISA was around 1.3, and there was no reaction with the isotype control group, confirming that mAb-1G10 can specifically bind to PCV2 Cap protein.

[0046] 5. Western blot analysis of mAb-1G10 purification results

[0047] PK-15 cells were evenly plated in 12-well plates. When the cell density reached approximately 60%, PCV2b and PCV2d viruses were inoculated (MOI = 1) and allowed to adsorb for 2 hours in a 37°C incubator. After washing three times with PBS, the cells were replaced with fresh MEM medium containing 2% NBS and 3 mmol / L D-glucosamine. 48 hours after infection, the supernatant was discarded, and the cells were washed three times with PBS. An appropriate amount of RIPA lysis buffer was added to thoroughly lyse the cells on ice. 5× SDS-PAGE protein loading buffer was added according to the proportion, and the samples were thoroughly mixed and boiled in boiling water for 10 minutes. The samples were then separated on a 12.5% ​​SDS-PAGE gel. After electrophoresis, the proteins were transferred to a NC membrane using semi-dry transfer, and then blocked with 5% skim milk for 2 hours at room temperature. The diluted 1G10 monoclonal antibody (1:5000 dilution) was used as the primary antibody and incubated with the NC membrane at 4°C overnight, washed three times with 1×TBST, each time for 10 minutes, and then HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 hour. After washing three times with 1×TBST, the binding of the antibody was detected using enhanced ECL luminescence solution, and the membrane was observed and photographed in a gel imaging system. Figure 1 In Figure C, the purified mAb-1G10 as a primary antibody can specifically recognize the Cap protein expressed after PCV2b and PCV2d infected PK-15 cells.

[0048] 6. Identification of mAb-1G10 Purification Results by Indirect Immunofluorescence Assay

[0049] The cells were evenly spread in a 12-well plate. When the cell density reached about 50%, the original culture medium was discarded and PCV2d and PCV2b viruses were inoculated (MOI = 1), respectively. At the same time, an uninoculated group was set up and placed at 37°C for adsorption for 2 hours. Subsequently, the medium was replaced with MEM maintenance medium containing 2% NBS + 3 mmol / L D-glucosamine and cultured for another 48 hours. After washing the cells three times with PBS, 300 μL of 4% paraformaldehyde fixative was added to each well, fixed at room temperature for 10 minutes, and washed three times with 1×PBST; 300 μL of 0.5% TritonX-100 was added to each well for permeabilization for 10 minutes at room temperature, and washed three times with 1×PBST; 500 μL of 3% BSA blocking solution was added to each well, blocked at 37°C for 30 minutes, and washed three times with 1×PBST; the purified 1G10 monoclonal antibody was diluted at a ratio of 1:100, 1:500, 1:1000, and 1:2000, and 300 μL of the diluted antibody was added to each well, incubated at 37°C for 1 hour, and washed three times with 1×PBST; Alexa Fluor 500 was added to each well under dark conditions. 488-AffiniPureGoat Anti-Mouse (H+L) fluorescent secondary antibody (1:1000 dilution) was incubated at 37°C for 1 hour, washed 3 times with 1×PBST, and the liquid in the wells was discarded. The wells were observed and photographed under a Nikon inverted fluorescence microscope. Figure 2 As shown, no green fluorescence was observed in the blank control group, while mAb-1G10 at all dilutions could specifically recognize the Cap protein of the virus in PK-15 cells infected with PCV2b and PCV2d.

[0050] Example 2 Neutralization Activity Identification Experiment of mAb-1G10

[0051] 1. Identification of the Virus Neutralizing Activity of mAb-1G10 by Indirect Immunofluorescence Assay

[0052] To test the neutralizing activity of purified mAb-1G10, a virus neutralization assay was performed in PK-15 cells. PK-15 cells were evenly plated in a 96-well cell culture plate, and virus neutralization assays were performed when the cell density reached approximately 50%.

[0053] Virus dilution: Dilute PCV2b and PCV2d viruses to 200 TCID in EP tubes 50 ;

[0054] Antibody dilution: In a new 96-well cell culture plate, mAb-1G10 was serially diluted with blank MEM medium to 9.6 μg / mL, 4.8 μg / mL, 2.4 μg / mL, 1.2 μg / mL, 0.6 μg / mL, 0.3 μg / mL, and 0 μg / mL, adding 100 μL to each well. At the same time, an isotype control monoclonal antibody group was set up and diluted to a concentration of 9.6 μg / mL. 100 μL of 200 TCID 50 The virus was mixed thoroughly; the final antibody concentrations in each well were 4.8 μg / mL, 2.4 μg / mL, 1.2 μg / mL, 0.6 μg / mL, 0.3 μg / mL, 0.15 μg / mL, and 0 μg / mL, respectively. The concentration of the isotype control monoclonal antibody was 4.8 μg / mL. The amount of virus in all wells was 100 TCID 50 3 replicates were set up for each group; after mixing, the virus and antibody were incubated in a 37°C incubator for 1 hour, and then added to PK-15 cells at a density of 50%, 200 μL per well; after adsorption for 2 hours, the liquid in the well was discarded, and the cells were washed 3 times with PBS and replaced with MEM containing 2% NBS + 3mmol / L D-glucosamine for maintenance, and cultured for another 48 hours. The progeny virus was analyzed by IFA, Western blot, and TCID 50 The neutralizing activity of 1G10 monoclonal antibody was determined. The cells in the above 96-well plate were fixed, permeabilized, and blocked before IFA experiment. After the secondary antibody incubation, they were washed 3 times with 1×PBST. 100 μL DAPI (1:5000 dilution) was added to each well for cell nucleus staining. The cells were stained at room temperature for 10 minutes, washed 3 times with 1×PBST, and observed under a fluorescence microscope and photographed. The fluorescence images were counted using Image J software, and the number of positive cells in each field of view was counted to calculate the neutralization rate of mAb-1G10. The half-maximal inhibitory concentration (IC) of mAb-1G10 was calculated using Graphpad Prime 9 software. 50 )The result is as follows Figure 3 shown.

[0055] 2. Western blot identification of the virus neutralization activity of mAb-1G10

[0056] After the neutralization test was performed as described above, the cell supernatant in the 96-well plate was discarded, and an appropriate amount of RIPA lysis buffer was added to completely lyse the cells on ice. Then, an appropriate amount of 5× SDS-PAGE protein loading buffer was added proportionally, mixed, and boiled in boiling water for 10 min. Western blot experiments were then performed. The primary antibody was the PCV2 monoclonal antibody 4B6 stored in the laboratory, such as Figure 4 As shown in A.

[0057] 3. TCID of progeny virus50 Determination

[0058] After the virus neutralization test, the 96-well cell culture plate was placed at -80°C and repeatedly frozen and thawed three times. The liquid in the wells was removed and centrifuged at 6000rpm for 10 minutes. The supernatant after centrifugation was carefully aspirated as the virus liquid. The collected virus liquid was diluted 10 times in a gradient, with a total of 12 gradients, and 4 replicates were performed for each dilution. At the same time, a non-infected group was set up; the diluted virus was inoculated into PK-15 cells with a density of about 50%. After adsorption for 2 hours, it was replaced with MEM maintenance solution containing 2% NBS + 3mmol / L D-glucosamine. The culture was continued for 48 hours, and the IFA experiment was performed. The cell wells stained with green fluorescent substance were judged to be PCV2 infection positive, and the TCID of the progeny virus was calculated using the Reed-Muench method. 50 ,like Figure 4 As shown in B.

[0059] 4. Evaluation of the protective effect of mAb-1G10 against PCV2 infection in mice

[0060] Fifteen mice aged 6 to 8 weeks were randomly divided into three groups, with 5 mice in each group, and the experiment was performed according to the protocol in Table 1. Pretreatment of PBS+PCV2 group and 1G10+PCV2 group: 100 μL PBS and 150 μg mAb-1G10 were mixed with 100 μL PCV2 (TCID 50 / mL=10 6.5 ) were mixed and incubated in a 37°C incubator for 1 hour. The mice were then injected intramuscularly according to the order in which they were grouped. The PBS+MEM group received the same treatment. 28 days after challenge, the mice's eyeballs were removed for blood collection, and their lymph nodes and spleens were removed by dissection.

[0061] Table 1 Mouse experimental groups and challenge scheme

[0062]

[0063] 5. Determination of viral load in lymph nodes and spleen of mice after challenge

[0064] Weigh the collected mouse lymph nodes and spleen, place them in magnetic beads and add an appropriate amount of PBS, place them in a freezing grinder for thorough grinding, centrifuge them twice at 12000 rpm, 10 min each time, collect the supernatant, extract DNA from the tissues, and detect the viral load in the mouse lymph nodes and spleen by fluorescent quantitative PCR. Figure 5 As shown in A and 5B.

[0065] 6. Immunohistochemical Analysis

[0066] Mouse spleens were removed and trimmed to an appropriate size. They were fixed with 4% paraformaldehyde at room temperature for at least 24 hours, followed by immunohistochemical analysis. The primary antibody was the PCV2 monoclonal antibody 4B6, prepared and maintained in our laboratory. Scanned immunohistochemical images were analyzed, with spleens from mice in the PBS + MEM group serving as negative controls and spleens from mice in the PBS + PCV2 group serving as positive controls. Positive results were determined if significant brownish-yellow staining was observed; negative results were determined if no brownish-yellow staining was observed. Figure 5 As shown in C.

[0067] Example 3 Study on the mechanism of mAb-1G10 neutralizing PCV2 virus

[0068] 1. Effect of mAb-1G10 on the adsorption of PCV2b and PCV2d on the surface of PK-15 cells

[0069] To investigate whether 1G10 works by blocking virus adsorption, the effect of mAb-1G10 on PCV2b and PCV2d cell adsorption was evaluated by indirect immunofluorescence assay: 50 The virus was incubated with mAb-1G10 (or isotype control monoclonal antibody IC) at 37°C for 1 hour, and then infected into PK-15 cells at 4°C for 1 hour. After washing three times, the virus particles on the cell surface were labeled with anti-PCV2 monoclonal antibody 4B6. Figure 6 As shown, no green fluorescence signal was detected on the surface of PK-15 cells in the mAb-1G10-treated group, while a clear green fluorescence signal was observed in the isotype control group and no fluorescence signal was observed in the unvaccinated group. These results indicate that mAb-1G10 exerts its neutralizing effect by blocking the adsorption of PCV2b and PCV2d virions to the cell surface.

[0070] 2. Effect of mAb-1G10 on the spread of PCV2 to uninfected cells

[0071] After 293T cells were transfected with pCDNA3.1-EGFP-Cap (2b) and pCDNA3.1-EGFP-Cap (2d) plasmids, they were incubated with different concentrations of mAb-1G10 (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL and 80 μg / mL) at 37°C for 1 hour. Then, 293T cells were co-cultured with PK-15 cells for 24 hours, and the fluorescence intensity was detected by flow cytometry. The results are shown in Figure 2. Figure 7 As shown, mAb-1G10 inhibited the spread of PCV2b and PCV2d to uninfected cells in a dose-dependent manner, and the fluorescence intensity could be reduced to 50% when the concentration of mAb-1G10 reached 80 μg / mL.

[0072] 3. mAb-1G10 variable region sequence determination

[0073] After the 1G10 monoclonal cell line was fully lysed with an appropriate amount of Trizol Reagent, total cell RNA was extracted, reverse transcribed into cDNA, and transferred to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The antibody heavy chain (VH) and light chain (VL) sequencing results were compared with the antibody database (IMGT, etc.) to obtain relevant information such as CDR1 / 2 / 3 and determine the gene sequence of the variable region. The variable region sequences of mAb-1G10 heavy chain (VH) and light chain (VL) are as follows Figure 8 shown.

[0074] 4. Molecular docking simulation of antigen-antibody binding sites

[0075] Based on the sequencing results of the light chain and heavy chain variable regions of mAb-1G10, Cap (2b) and Cap (2d) were homologously modeled with mAb-1G10, and the conformation of Top1 was energy minimized using AMBER18 under the ff14SB force field. Finally, the binding energy of the protein complex conformation after energy minimization was evaluated using the online tool prodigy1 and visualized using PyMOL2.5.3. The protein-protein binding affinity was obtained based on prodigy. The smaller the binding affinity value, the stronger the binding. The binding affinity values ​​of Cap (2b) / 1G10-VH / 1G10-VL and Cap (2d) / 1G10-VH / 1G10-VL were both negative, indicating a strong binding interaction. The binding modes of mAb-1G10 with the PCV2b and PCV2d Cap protein complexes were obtained using AlphaFold3 prediction. Further analysis revealed hydrogen bonds occurring during the formation of the Cap(2b) / 1G10-VH / 1G10-VL complex. E210, G54, K58 on the Cap(2b) protein formed hydrogen bonds with N28, S50, D1, and Y94 on the 1G10-VL. In addition, R51 and D194 on the Cap(2b) protein formed hydrogen bonds with Y105 and S53 on the 1G10-VH protein. T56, D78, R73, D168, D194 on the Cap(2d) protein formed hydrogen bonds with Y105, R99, Y32, S53, and R31 on the 1G10-VH. In addition, T56, G54, T52, R51, N77 on the Cap(2d) protein formed hydrogen bonds with T31, N32, S50, and Y49 on the 1G10-VL protein. Figure 9As shown. Hydrogen bonding is an important non-covalent binding interaction and is very important for protein-protein electrostatic complementarity. Numerous hydrogen bonding interactions indicate that the docked Cap(2b) / 1G10-VH / 1G10-VL and Cap(2d) / 1G10-VH / 1G10-VL binding is very strong. Table 2 summarizes the sites where mAb-1G10 binds to Cap(2b) and Cap(2d), respectively. Combined with the analysis of the PK-15 cell experiments in which mAb-1G10 can neutralize PCV2b and PCV2d infection, three common sites for mAb-1G10 to bind to Cap protein were finally obtained, namely R51, G54 and D194.

[0076] Table 2 Analysis of binding sites between mAb-1G10 and Cap protein

[0077]

[0078] 5. Identification of the key binding sites between Cap protein and mAb-1G10 using alanine scanning technology

[0079] Alanine scanning technology was used to mutate the three amino acid sites R51, G54 and D194 on the Cap protein to alanine (alanine is the simplest non-polar amino acid with only one methyl group on the side chain, which has little effect on the overall conformation of the protein). Single mutant plasmids (R51A, G54A, D194A), double mutant plasmids (RG, RD, GD) and triple mutant plasmids (RGD) were constructed and connected to the pCDNA3.1-EGFP vector and transfected into 293T cells. After 24 hours, IFA experiments were performed to identify the recognition ability of 1G10 for the mutant Cap protein. The primary antibody was purified mAb-1G10. The results are shown in Figure 3. Figure 10 As shown in the figure, mAb-1G10 can recognize the Cap mutant protein when a single amino acid among R51, G54 and D194 is mutated to alanine. However, when both R51 and D194 are mutated to alanine at the same time, mAb-1G10 cannot recognize the Cap mutant protein, which proves that the synergistic effect of the two amino acid sites R51 and D194 is crucial for the binding of Cap protein to mAb-1G10.

[0080] 6. Western blot and ELISA identification of mAb-1G10 binding to mutant protein

[0081] The amino acids R51, G54, and D194 were mutated to alanine, and prokaryotic expression plasmids with single, double, and triple mutations were constructed. After induction and purification, the binding ability of 1G10 monoclonal antibody to the mutant proteins was identified by Western blot. The purified Cap protein and each mutant protein were diluted to different concentrations and coated on ELISA plates. ELISA experiments were performed using mAb-1G10 as the primary antibody (1:1000 dilution). Figure 11 As shown in the results, mAb-1G10 can only recognize Cap protein with amino acid mutation at position G54 and Cap protein without mutation, which proves that the amino acid mutation at position G54 does not affect the binding of mAb-1G10 to Cap protein, but the changes of amino acids at positions R51 and D194 affect the binding of mAb-1G10 to the mutant protein, which once again proves that the two amino acid sites R5 and D194 are critical for mAb-1G10 to recognize Cap protein.

[0082] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A monoclonal antibody 1G10 with neutralizing activity against PCV2 Cap protein, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1G10 is: QVQLKQSGPGLVQPSQSLSITCTVS GFSLPRYS LHWVRQSPGKGLEWLGV IWSGGST DYNAAFISRLTISKDNSRSQVFFKMNSLQANDTAIYYC AKSRPPDGYYGAMDY WGQGTSVTVSS The amino acid sequence of the light chain variable region is: DIVMTQSQKFMSTSIGDRVSVTYKAS QNVGTN VAWYQQRPGQSPKTLIY SAS YRSSGVPDRFTGGGSGTDFTLTINNVQSEDLAEYFC QQNNTYPYT FGGGT KLEIK, wherein the underlined portion is CDR1-3.

2. Use of the monoclonal antibody according to claim 1 in the preparation of a medicament for treating and / or preventing porcine circovirus.

3. A pharmaceutical composition for treating and / or preventing porcine circovirus, comprising an effective amount of the monoclonal antibody according to claim 1, wherein the pharmaceutical composition can neutralize PCV2 infection, inhibit the adsorption of the virus on the surface of PK-15 cells, and prevent the spread of the virus.

4. Use of the antibody according to claim 1 in studying key binding sites of PCV2 Cap protein, wherein the key binding sites are R51 and D194.

5. Use of the monoclonal antibody 1G10 according to claim 1 in preparing a reagent for detecting and identifying porcine circovirus.

6. A reagent for detecting porcine circovirus, characterized in that The reagent is a test kit and / or a test strip.

7. A test strip for detecting porcine circovirus, characterized in that: The test strip comprises the monoclonal antibody according to claim 1.

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