Specific monoclonal antibody against cap protein of porcine circovirus type 2 and application thereof

Abstract

This invention belongs to the field of biotechnology and relates to a specific monoclonal antibody against porcine circovirus type 2 (PCV2) Cap protein and its applications. The CDR of the heavy chain variable region of the monoclonal antibody includes CDR1, CDR2, and CDR3 as shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; the CDR of the light chain variable region of the monoclonal antibody includes CDR1, CDR2, and CDR3 as shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively. A sandwich ELISA method for protein content determination has been established, which is simple to operate, has good sensitivity and specificity, is widely applicable, and can perform high-throughput determination of multiple samples to detect the content of Cap protein, an effective antigen of various types of PCV2 vaccines, thus shortening the vaccine testing time.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and in vitro diagnostic technology, and specifically relates to a specific monoclonal antibody against the Cap protein of porcine circovirus type 2. Background Technology

[0002] Porcine Circovirus type 2 (PCV2) is one of the smallest single-stranded circular DNA genomes of any animal virus discovered to date, belonging to the genus *Circovirus* within the family Circoviridae. PCV2 is considered the causative agent of porcine circovirus disease (PCVD) or porcine circovirus-associated disease (PCVAD), including post-weaning multisystemic wasting syndrome (PMWS), dermatitis-nephritis syndrome (PDNS), proliferative necrotizing pneumonia (PNP), porcine respiratory disease syndrome (PRDC), and reproductive disorders. Besides infecting domestic and wild pigs, PCV2 is widely distributed in ruminants, rodents, canines, insects, and other species. In production, PCV2 is commonly co-infected with other porcine pathogens such as PRRSV, PRV, CSFV, and PEDV. PCV2 can modulate the innate immune response, making pigs more susceptible to secondary or concurrent viral or bacterial infections.

[0003] The PCV2 genome is predicted to have 11 ORFs. Of these, ORFs 1, 5, 7, and 10 are located on the sense strand of the PCV2 genome, while the remaining ORFs are encoded by the antisense strand. ORF2 encodes the Cap capsid protein, a key component of PCV2 subunit vaccines. Cap contains abundant antigenic epitopes and can serve as targets for neutralizing antibodies to prevent viral entry into host cells, making it an important target for PCV2 vaccine development.

[0004] Vaccination is currently a crucial means of controlling PCV2. At present, 18 porcine circovirus vaccines have obtained new veterinary drug certificates in China, along with 10 subunit vaccines expressing E. coli or baculoviruses, and 8 inactivated vaccines. Despite the development and investment in high-quality vaccines, PCV2 remains prevalent in major pig-producing countries and regions worldwide, causing significant economic losses to the pig industry. Vaccine efficacy is primarily assessed through animal challenge experiments, which requires a large number of animals and a considerable amount of time, and is prone to errors. Measuring the content of effective proteins is an excellent alternative method for determining vaccine efficacy. Efficient, accurate, and rapid determination of PCV2 Cap antigen content is of great significance for PCV2 vaccine development and formulation. Summary of the Invention

[0005] To address the aforementioned technical problems, the primary objective of this invention is to provide a specific monoclonal antibody against porcine circovirus type 2 (Cap) protein. The monoclonal antibody comprises a heavy chain constant region and a light chain constant region, a heavy chain variable region and a light chain variable region. The CDR of the heavy chain variable region of the monoclonal antibody includes CDR1 (amino acid sequence as shown in SEQ ID No. 1), CDR2 (amino acid sequence as shown in SEQ ID No. 2), and CDR3 (amino acid sequence as shown in SEQ ID No. 3). The CDR of the light chain variable region of the monoclonal antibody includes CDR1 (amino acid sequence as shown in SEQ ID No. 4), CDR2 (amino acid sequence as shown in SEQ ID No. 5), and CDR3 (amino acid sequence as shown in SEQ ID No. 6).

[0006] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8.

[0007] A second objective of the present invention is to provide a gene fragment encoding the monoclonal antibody, wherein the gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 9, and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 10.

[0008] A third objective of the present invention is to provide an expression vector containing the aforementioned gene fragment.

[0009] A fourth objective of this invention is to provide a host cell containing the aforementioned expression vector, or having the aforementioned gene fragment integrated into its genome.

[0010] A fifth objective of this invention is to provide the application of the monoclonal antibody in the preparation of reagents, test strips, or kits for detecting porcine circovirus type 2.

[0011] The sixth objective of this invention is to provide a type 2 porcine circovirus double antibody sandwich ELISA detection kit, the kit comprising the aforementioned monoclonal antibody.

[0012] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

[0013] The seventh objective of this invention is to provide a double-antibody sandwich ELISA method for detecting porcine circovirus type 2 antigen for non-diagnostic purposes. The method includes: coating an enzyme-labeled plate with a rabbit polyclonal antibody against porcine circovirus type 2 Cap protein, blocking the plate, adding the sample to be tested and incubating, washing the plate, adding the enzyme-labeled monoclonal antibody, washing again, adding a chromogenic reagent for color development, adding a stop solution to terminate the reaction, reading the absorbance at 450 nm, and determining the detection result based on the P / N ratio.

[0014] Preferably, the method includes the following steps:

[0015] (1) Coating: The rabbit polyclonal antibody against porcine circovirus type 2 Cap protein was diluted with phosphate buffer at pH 9.6 at a ratio of 1:10000. 100 μl of the solution was added to each well of a 96-well plate and coated at 4°C for 12 h. The plate was then washed with 300 μl of PBST.

[0016] (2) Blocking: Add 100 μl of 3% BSA to each well, block at 37℃ for 1 h, and wash with 300 μl of PBST;

[0017] (3) Add the test sample: Add the test sample and standard to the 96-well plate at 100 μl / well and incubate at 37°C for 60 min;

[0018] (4) Washing: The monoclonal antibody was diluted 1:7500 with phosphate buffer at pH 7.2 and added to the wells respectively. The mixture was reacted at 37°C for 60 min and then washed with 300 μl PBST.

[0019] (5) Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse enzyme-labeled secondary antibody 1:5000 with phosphate buffer at pH 7.2, add it to each well, react at 37℃ for 30 min, and add 300 μl PBST to each well;

[0020] (6) Color development: Add 50 μl TMB and develop color for 15 min at room temperature in the dark;

[0021] (7) Termination: The reaction was terminated by adding 50 μl of 1.5 M H2SO4, OD 450nm reading;

[0022] (8) Result calculation and judgment: Subtract the OD value of the blank control from the value of the standard, input the result into Excel for curve fitting, and obtain the corresponding concentration-OD value conversion formula. Substitute the OD value of the sample to be tested minus the blank control into the formula to calculate the Cap protein concentration of the sample to be tested.

[0023] The beneficial effects of this invention are: (1) This invention obtains a specific monoclonal antibody against type 2 porcine circovirus Cap protein; (2) This invention uses rabbit polyclonal antibody against type 2 porcine circovirus Cap protein as the capture antibody and monoclonal antibody against type 2 porcine circovirus Cap protein as the detection antibody to establish a double-antibody sandwich ELISA method for detecting type 2 porcine circovirus based on polyclonal antibody and specific monoclonal antibody; (3) This detection method is simple to operate, has good specificity, high sensitivity, short time consumption, and large application space; (4) This invention can accurately and quickly determine the PCV2 Cap antigen content in multiple samples simultaneously, and complete the Cap protein antigen content determination experiment, data processing, and result judgment within 4 hours, thereby effectively ensuring the quality of the vaccine, reducing the testing cost and labor intensity, improving the detection efficiency, and realizing high-throughput vaccine efficacy detection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0025] Figure 1 SDS-PAGE detection of PCV2 Cap protein.

[0026] Note: M: Protein molecular weight standard; 1: Supernatant of sonicated fragments from insect cells not infected with recombinant baculovirus; 2: Supernatant of sonicated fragments from High Five cells infected with recombinant baculovirus; 3: Purified PCV2 Cap protein

[0027] Figure 2 Transmission electron microscopy observation of PCV2 Cap forming VLPs

[0028] Figure 3 ELISA assessment of serum antibody levels in mice after five immunizations

[0029] Figure 4 Western blot analysis confirmed that the PCV2 Cap monoclonal antibody 5C4 specifically recognizes the PCV2 Cap protein.

[0030] M: Protein molecular weight standard; 1: Supernatant of sonicated fragments from insect cells not infected with recombinant baculovirus; 2: Supernatant of sonicated fragments from SF9 cells infected with recombinant baculovirus; 3: Supernatant of sonicated fragments from High Five cells infected with recombinant baculovirus.

[0031] Figure 5IFA assay showed that the PCV2 Cap monoclonal antibody 5C4 bound well to PCV2.

[0032] Figure 6 SDS-PAGE assay for antibody purification.

[0033] M: Protein molecular weight standard; 1: Clarified ascites fluid; 2: Equilibration effluent; 3: Washing effluent; 4: Elution effluent

[0034] Figure 7 ELISA assay for sensitivity of monoclonal antibody 5C4

[0035] Figure 8 Monoclonal antibody specific ELISA test

[0036] Figure 9 Establishment of marker curves for double-antibody sandwich ELISA Detailed Implementation

[0037] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0038] The experimental animals and cells used in the following examples were purchased from:

[0039] The New Zealand white rabbits and BALB / c mice used in the experiments were purchased from the Laboratory Animal Center of Lanzhou Veterinary Research Institute. The PK15 and sf9 cells used were purchased from the China Center for Type Culture Collection, Wuhan University. High Five cells were purchased from Thermo Fisher Scientific.

[0040] The main reagents used in the following examples were purchased from Thermo Fisher Scientific: BamHI, Hind III endonuclease, T4 DNA ligase, and Prime STAR Max Premix high-fidelity polymerase; DH5α and DH10bac competent cells were purchased from Biomed Biotechnology Co., Ltd.; LipoInsect transfection reagent and baculovirus plasmid extraction kit were purchased from Beyotime Biotechnology Co., Ltd.; BCA protein quantification kit was purchased from Yaxin Biotechnology Co., Ltd.; gel extraction kit, plasmid mini-preparation kit, Protein A / G Resin, and Ni-NTA packing material were purchased from TransGen Biotech Co., Ltd.; and mouse monoclonal antibody subtype identification kit was purchased from Proteintech Co., Ltd.

[0041] Example 1: Preparation, Expression, and Purification of PCV2 Cap Protein

[0042] 1. Preparation

[0043] The pfastbac-dual-Cap recombinant plasmid was constructed using conventional methods. The PCV2 Cap target fragment was amplified by PCR, with BamHI and HindIII restriction sites added at 5' and 3', respectively. The amplified target fragment, matching the expected size, was double-digested with the pfastbac-dual vector using BamHI and HindIII restriction enzymes. The digested products were ligated using T4 DNA ligase at 16°C for 30 min. The ligation products were transformed into DH5α competent cells and cultured at 37°C with shaking at 225 rpm for 1 h. The cells were then plated on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12 h. Five to eight white positive monoclonal colonies were randomly selected and identified by PCR. Plasmids identified as positive were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0044] 2. Expression

[0045] (1) The constructed pfastbac-dual-Cap recombinant shuttle plasmid was transformed into DH10bac (Beijing Bomeide Company) competent cells and cultured at 37℃ with shaking at 225 rpm / min for 4 h. -1 10 -2 10 -3 After dilution, the solution was spread onto LB agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL x-gal, and 40 μg / mL IPTG. The plates were incubated upside down at 37°C for 24–36 h for blue-white screening.

[0046] (2) Randomly select 3-5 white positive monoclonal colonies and perform secondary plating on LB agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL x-gal, and 40 μg / mL IPTG. Incubate at 37°C upside down for 24-36 h and perform blue spot screening. Select another 3-5 white positive monoclonal colonies for PCR identification. Primers M13 Forward (5'-3'): GTTTTCCCAGTCACGAC, M13 Reverse (5'-3'): CAGGAAACAGCTATGCA. Select PCR-positive monoclonal colonies and extract recombinant baculovirus plasmids using a baculovirus plasmid extraction kit (Beyotime Biotechnology Co., Ltd.).

[0047] (3) Culture sf9 insect cells at a density of approximately 8 × 10⁸ cells per well. 5 Pre-seed the cells into six-well plates to achieve a cell density of approximately 70-90%. Take two sterile 1.5ml centrifuge tubes and add 100μl of serum- and antibiotic-free insect cell culture medium to each. Add 16μg of recombinant baculovirus plasmid to one tube and gently mix with a pipette. Add 8μl of LipoInsect™ (Beyotime Biotechnology) transfection reagent to the other tube and gently mix with a pipette. Incubate the diluted plasmid and transfection reagent at room temperature for approximately 2-5 minutes. Then, gently add the diluted plasmid to the diluted LipoInsect™ transfection reagent using a pipette, gently invert the centrifuge tube to mix, and incubate at room temperature for 20 minutes. Finally, add 200μl of the LipoInsect™ transfection reagent-plasmid mixture to each well, gently mix, and incubate at 27°C for 4 hours. After 4 hours of incubation, the culture medium was discarded and replaced with fresh complete culture medium. The culture was then continued at 27°C for approximately 120 hours. The culture medium from each well was collected, centrifuged at 500g for 5 minutes, and the supernatant obtained was the P1 generation baculovirus. The collected P1 generation virus was supplemented with fetal bovine serum to a final concentration of 2% and stored at 4°C in the dark.

[0048] (4) Amplify the P1 virus strain using suspended SF9 cells. The density for good suspension culture is 5 × 10⁶ cells / year. 5 In dense sf9 cells, an appropriate amount of P1 virus strain was inoculated, and the cells were cultured in suspension for 5-7 days. The supernatant was collected, which was the P2 virus strain, and the virus titer was determined. A suitable density for suspension culture was 5 × 10⁶ cells / year. 5 In high-density High Five cells, an appropriate amount of P2 virus strain was inoculated, and the cells were cultured in suspension for 5-7 days. The supernatant and cells were collected as the expressed Cap antigen for subsequent purification.

[0049] 3. Purification

[0050] (1) The collected High Five suspension expression Cap protein samples were placed on an ice bath and ultrasonically disrupted using an ultrasonic cell disruptor at 30% power for 3 seconds, 3 seconds off, and 20 minutes. After disruption, the samples were centrifuged at 10,000g at 4℃ for 20 minutes. The supernatant was collected after centrifugation and used as the sample before loading.

[0051] (2) Before purification, add 3 to 5 column volumes of distilled water to the sample column to wash the Ni-NTA packing. Then add 3 to 5 column volumes of PBS buffer to equilibrate. Add the supernatant collected by sonication and centrifugation to the sample column containing 5 mL of Ni-NTA packing. Shake gently at room temperature for 1 h.

[0052] (3) After incubation, allow the supernatant to flow slowly out of the column at a rate of 0.5 ml / min and collect it as flowthrough. Store the sample at 4°C for later sample analysis.

[0053] (4) Add 3 column volumes of low-concentration imidazole washing buffer (NaCl 500mM, Na2HPO4 100mM, Imidazole 10mM, pH 8.0) to elute the impurities bound to the packing material. Collect 1 mL of the eluted sample for analysis when the last column volume of elution is used.

[0054] (5) Although, add 3 times the column volume of high concentration imidazole washing buffer (NaCl 500mM, Na2HPO4 100mM, Imidazole 20, 50, 250, 500mM, pH 8.0) to elute the target protein bound to the packing material. Collect the eluent of different concentrations of imidazole in a 1.5mL centrifuge tube for subsequent identification of protein purification status. Store the eluted protein sample at 4℃.

[0055] (6) After elution, add 2-3 column volumes of PBS buffer to wash the packing material, so as to wash away the proteins bound to the packing material and facilitate the recycling and reuse of the packing material.

[0056] (7) Finally, add 5 column volumes of distilled water to wash the packing material. After it flows out, add 5 column volumes of 20% ethanol to thoroughly wash the packing material. After completion, store the packing material in 20% ethanol.

[0057] (8) All collected samples were added to (5×SDS-PAGE sample buffer: Tris-HCl 225mM, glycerol 50%, SDS 5%, bromophenol blue 0.05%), boiled at 100℃ for 10 min, and then analyzed by SDS-PAGE.

[0058] PCV2 Cap protein was successfully purified by SDS-PAGE analysis using affinity chromatography. The purified protein size was consistent with the expected molecular weight. (See attached figure.) Figure 1 It is the PCV2d genotype, 235aa, 28kDa, isoelectric point: 10.75, and its amino acid sequence is shown in SEQ ID No. 11.

[0059] Sequence 11: Amino acid sequence of PCV2-Cap protein

[0060] MTYPRRRRFRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPPLTVPFEYYRIRKVKVEFWPCSPITQGDRG VGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDGTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK

[0061] Example 2: Assembly of PCV 2 Cap virus-like particles

[0062] After SDS-PAGE analysis of the affinity-purified protein prepared in Example 1, the identified high-concentration and high-purity proteins with good expression were added to a 7000D dialysis bag, sealed at both ends with dialysis bag clamps, and placed in the assembly buffer (Tris-HCl 50mM, NaCl 500mM, pH 7.0) used in this patent, and dialyzed at 4°C for 48 h. After completion, the sample was collected and concentrated using a Millipore ultrafiltration tube. The concentrated sample was centrifuged at 4000 r / min for 30 min to concentrate the sample 5-10 times. After completion, the sample was collected, negatively stained with 1% phosphotungstic acid, and the morphology and size of virus-like particles were observed under a transmission electron microscope.

[0063] Electron microscopy results as follows Figure 2 As shown, the sample can form uniform and intact VLPs, which can be used as standard samples for subsequent monoclonal antibody preparation and sandwich ELISA.

[0064] Example 3: Preparation of Polyclonal and Monoclonal Antibodies

[0065] 1. Preparation of polyclonal antibodies

[0066] Healthy New Zealand white rabbits were selected. The purified PCV2 Cap protein, at a ratio of 500 μg / rabbit, was mixed with Freund's complete adjuvant at a 1:1 ratio and used as the initial immunization antigen. Two weeks after the initial immunization, the rabbits were immunized again with a mixture of PCV2 Cap protein and Freund's incomplete adjuvant at a 1:1 ratio. A total of four immunizations were performed, with extensive blood collection. Whole blood was collected, incubated at 4°C for 4 hours, and then centrifuged at 1000g for 10 minutes. The supernatant serum was collected.

[0067] 2. Establishment of monoclonal antibody cell lines

[0068] (1) PCV2 Cap Immunization

[0069] Five SPF-grade 5-6 week old BALB / c mice were selected. The purified PCV2 Cap protein was mixed with Freund's adjuvant at a ratio of 1:1 and administered via intraperitoneal injection at a dose of 100 ng / mouse. After five immunizations, blood was collected from the tails of the mice, and serum was obtained to determine the antibody titer.

[0070] (2) Evaluation of serum titers in BALB / c mice by indirect ELISA

[0071] Dilute PCV2 Cap to 2 μg / ml with PBS at pH 7.4, add 100 μl / well to a 96-well plate, and coat overnight at 4°C. Wash off the coating solution with PBST, and add 100 μl of 3% BSA, then block at 37°C for 1 h. Mouse serum was serially diluted with PBST at 1000, 2000, 4000, 8000, 16000, 32000, 64000, and 128000 times, and 100 μl / well was added to sealed 96-well plates and incubated at 37°C for 1 h. The liquid in the wells was washed away with PBST. HRP-labeled goat anti-mouse enzyme-labeled secondary antibody (purchased from Thermo Fisher) was diluted 1:5000 with PBST and added at 100 μl / well, and incubated at 37°C for 1 h. The liquid in the wells was washed away with PBST. 50 μl of TMB chromogenic solution was added and incubated at 37°C in the dark for 15 min. Immediately afterwards, 50 μl of 2M H2SO4 was added to stop the reaction. The chromogenic reaction was measured at OD600 using a microplate reader. 450nm Take the reading at the location.

[0072] like Figure 3 As shown, the serum antibody levels of five mice after five immunizations were evaluated, and it was found that the serum titers of the five mice were greater than 128,000, which was qualified for post-fusion.

[0073] (3) Hybridoma cell screening

[0074] Spleens from mice with serum titers meeting requirements were aseptically removed, ground, and prepared into a spleen lymphocyte suspension. Mouse myeloma cells (SP2 / 0) were fused with the lymphocytes using PEG. The suspension was evenly seeded into 96-well plates, and the culture medium was gradually changed from HAT to HT. Cell supernatants from different wells were analyzed using indirect ELISA to screen for positive wells. A control group was also included to exclude cells from recognizing the His tag. Cells from the positive wells were subcloned using limiting dilution. After the subcloned cells grew, the cell supernatants from different wells were analyzed using indirect ELISA.

[0075] The test results are shown in Table 1. Cell supernatants from 18 wells of the 96-well plate were all positive, indicating successful hybridoma cell fusion. The generated antibodies did not react with the His tag protein. After three subcloning cycles, positive monoclonal cell lines were collected and amplified, then cryopreserved in liquid nitrogen. This positive monoclonal cell line, 5C4, was named: Mouse Hybridoma Monoclonal Cell Line 5C4.

[0076] Table 1. Indirect ELISA screening of hybridoma cells

[0077]

[0078] (4) Western blot identification

[0079] PCV2 Cap and total protein from uninfected High Five cells infected with recombinant baculovirus were subjected to SDS-PAGE, followed by Western blot to transfer the protein onto a PVDF membrane. After completion, the membrane was blocked overnight at 4°C with 5% skim milk. The supernatant from positive monoclonal cell lines was diluted with 1% BSA and incubated at room temperature for 1 hour. HRP-labeled goat anti-mouse enzyme-labeled secondary antibody was added and incubated at room temperature for 1 hour. Finally, chromogenic buffer was added, and results were obtained using a gel imaging analyzer.

[0080] like Figure 4 As shown, in the Western Blot system, the prepared monoclonal antibody can effectively recognize the PCV2d Cap protein expressed in High Five cells, and the reaction is strongly positive.

[0081] (5) IFA identification

[0082] PK15 cells were seeded into 96-well plates. When the cells reached 60%–70% confluence, PCV2d virus was added and incubated for 1 hour. The cells were then washed with PBS, replaced with fresh culture medium, and cultured in a cell culture incubator at 37°C and 5% CO2 for approximately 120 hours. After 120 hours, the culture medium was discarded, and the cells were washed three times with PBS. 100 μl of 4% paraformaldehyde was added to each well for fixation. 100 μl of 0.1% Triton 100 was added to perforate the cell membrane, and the cells were blocked with 3% BSA for 1 hour. The cell membrane was then incubated with diluted supernatant from a positive monoclonal cell line for 1 hour. Finally, FITC-labeled goat anti-mouse fluorescent secondary antibody (purchased from Thermo Fisher Scientific) was added and the cells were incubated in the dark for 1 hour. The results were then observed under a fluorescence microscope.

[0083] like Figure 5 As shown, after PCV2d virus infection of PK15 cells, the monoclonal antibody 5C4 was identified by IFA as being able to bind to PCV2d virus.

[0084] 3. Monoclonal antibody preparation and purification

[0085] One week prior to injection of the monoclonal cell line, sterile liquid paraffin was injected intraperitoneally into each BALB / c mouse (500 μL). Immunoreactive hybridoma monoclonal cells were collected, counted, and adjusted to a cell density of 1 × 10⁻⁶. 6 Cells / ml. Subsequently, the cell suspension was injected into the peritoneal cavity of BALB / c mice, and ascites fluid was collected multiple times over 10–14 days.

[0086] Mouse ascites fluid was centrifuged (12000 rpm, 10 min), filtered to remove impurities such as lipids and tissue debris, and clarified ascites fluid was obtained. Antibodies were purified by affinity chromatography using Proteinlso® Protein A / G Resin (TransGen Biotech, catalog number DP501-01) as the affinity chromatography medium. The purification method is as follows:

[0087] (1) Column packing: Resuspend the medium, add an appropriate amount of medium to the chromatography column and let it stand.

[0088] (2) Equilibration: Equilibrate the chromatography column with 5-10 times the column volume of equilibration buffer (20mM PB, 0.15M KCl, pH7.0) until the pH of the effluent remains constant.

[0089] (3) Sample loading: Add the clear ascites fluid into the chromatography column. If conditions permit, incubate at 4°C for 2 hours.

[0090] (4) Washing: After sample loading, wash the chromatography column with 5-10 column volumes of equilibration buffer and collect the eluent.

[0091] (5) Elution: Elute with 5-10 times the amount of elution buffer (20mM citric acid, pH 3.0-4.0) and collect the eluent. After elution, immediately neutralize the acidic environment of the eluent with alkaline buffer (1M Tris-HCl, pH 9.0).

[0092] (6) Column washing: Rinse the column with 5-10 column volumes of pure water, then immediately rinse with 5 volumes of equilibration buffer to neutralize the column. Affinity chromatography media are stored in 20% ethanol.

[0093] (7) SDS-PAGE, the results are as follows Figure 6 As shown, heavy and light chain bands of the antibody were observed at 50 kDa and 25 kDa, respectively, indicating good purification results.

[0094] 4. Determination of the variable region of monoclonal antibodies

[0095] Cells were collected using monoclonal antibody 5C4, centrifuged at 1500 rpm for 10 min, and RNA was extracted using the TRIZOL method. Subsequently, the obtained RNA template was reverse transcribed into cDNA using random primers.

[0096] Using the prepared cDNA as a template, the gene fragment was amplified with Prime STAR Max Premix high-fidelity polymerase. The system consisted of 25 μL Prime STAR Max Premix (2×), 1 μL each of Forward and Reverse, 1 μL cDNA, and ddH2O added to a final volume of 50 μL.

[0097] The amplification conditions were: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles; and a final extension at 72℃ for 10 min. After the PCR reaction, the samples were electrophoresed on a 1% agarose gel to detect the results.

[0098] The heavy chain and light chain genes were cloned into the pMD18-T vector, respectively. White colonies were picked and cultured, and the plasmids that tested positive by PCR were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and analyzed with the antibody gene library (IMGT).

[0099] Table 2 VH Primer Sequence List

[0100]

[0101] Table 3 VL Primer Sequence List

[0102]

[0103] Note: I=inosine

[0104] R=A or G; W=A or T; Y=C or T; V=A or C or G; D=A or G or T; K =G or T; H=Aor C or T; S =C or G; M =A or C; B=C or G or T

[0105] The primers numbered VH F1-15 are upstream primers for VH, the primers numbered VL F1-16 are upstream primers for VL, the primers numbered VH R are downstream primers for VH, and the primers numbered VL R are downstream primers for VL.

[0106] The sequencing results were compared with the antibody gene library (IMGT). The complementarity-determining region (CDR) sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody against porcine circovirus type 2 Cap protein prepared in this embodiment are shown in Table 4.

[0107] Table 4 Antibody variable region sequences

[0108]

[0109] The amino acid sequence of the heavy chain variable region is: EVQLQQSGPEVVEPGASVKISCKTSGYTFS DYTMHWVRQSRGKSLEWIGGLYPKNGGAMHDQKFRGKATLTVDQSSRTAYMEFRGLTSEDSAVYYCARTYASHRYDGGFAYWGHGTTLTI;

[0110] The gene sequence encoding the heavy chain variable region is: GAGGTCCAGCTGCAGCAGTCTGGACCTGAGGTGGTGGAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCTCTGACTACACCATGCACTGGGTGAGGCAGAGCCGTGGAAAGAGCCTTGAGTGGATTGGAGGTCTTTATCCTAAGAATGGTGGTGC TATGCACGACCAGAAGTTCAGGGGCAAGGCCACATTGACTGTAGACCAGTCGTCCAGGACAGCCTACATGGAGTTCCGTGGCCTGACATCTGAGGATTCTGCGGTCTATTACTGTGCACGAACTTACGCTTCCCATAGGTACGACGGGGGGTTTGCCTACTGGGGCCACGGCACCACGCTCACAATC;

[0111] The amino acid sequence of the light chain variable region is: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK*;

[0112] The gene sequence encoding the variable region of the light chain is: GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGACCAAGCTGGAAATAA;

[0113] 5. Identification of monoclonal antibody subtypes

[0114] The subtypes of monoclonal antibody 5C4 in ascites fluid and positive hybridoma cell supernatant were identified using a mouse monoclonal antibody subtype identification kit (company: Proteintech; Catalog No. PK20002). The method of administration is as follows:

[0115] (1) Take out the monoclonal antibody subtype identification kit at 4°C and equilibrate at room temperature for 30 minutes.

[0116] (2) ELISA strips: Select the number of ELISA strips according to the number of samples (this experiment only detects antibody subtypes in ascites and hybridoma cells, and the number of samples is 2). (One ELISA strip is required to measure 1 sample.)

[0117] (3) Prepare 1× sheep anti-mouse IgM+IgG-HRP in advance: 10uL 100× sheep anti-mouse IgM+IgG-HRP+990uL 1×PBST.

[0118] (4) Dilute the ascites fluid with PBST to a ratio of 1:100000. First, add 1 mL of 1×PBST (1:1000) to 1 μL of ascites fluid, then add 10 μL of the mixture to 990 μL of 1×PBST. Then add the diluted sample to the well of the strip sample, 50 μL / well, without incubation.

[0119] (5) Add 1× goat anti-mouse IgM+IgG-HRP to the sample wells, 50 uL / well, and shake for 1 min to mix. Cover with sealing film and incubate at room temperature for 1 h.

[0120] (6) Discard the liquid in the well, wash the plate 3 times with 1×PBST, and pat dry on absorbent paper.

[0121] (7) Add the freshly prepared colorimetric solution to the wells, 100 uL / well. Note: The colorimetric solution should be prepared and used immediately. The formula is: A solution: B solution = 1:100, that is, 10 uL of A solution and 1 mL of B solution are mixed and used immediately after mixing.

[0122] (8) After developing color at room temperature in the dark for 10-20 min, add stop solution, 100 uL / well.

[0123] (9) Result interpretation: By reading the OD450nm value of the microplate reader, the well with the highest value corresponds to the corresponding subtype.

[0124] Based on the readings, the results are shown in Table 5. The prepared PCV2 Cap monoclonal antibody 5C4 heavy chain type is IgG 2a, and the light chain type is κ.

[0125] Table 5. Identification results of monoclonal antibody subtypes

[0126]

[0127] 6. Determination of purified antibody concentration using the BCA method

[0128] The concentration of the purified antibody was determined using a BCA protein quantification kit (Yaxin, ZJ101L), as follows:

[0129] (1) Diluting BSA standards: Take 120 μL of protein standards (which need to be completely dissolved) and dilute them to 300 μL with a diluent (PBS buffer) that is consistent with the protein sample to be tested, so that the final concentration is 2 mg / mL. Then serially dilute to make the final concentrations of each standard 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL, 1500 μg / mL, and 2000 μg / mL.

[0130] (2) Prepare the color developing solution

[0131] a. Calculate the total volume of the colorimetric working solution: Total volume of working solution = (Number of BSA standard samples + Number of samples to be tested) × Number of replicates × Volume of colorimetric working solution for each sample; This experiment requires 6 mL of colorimetric solution, but there is some loss during sample addition, so 7 mL of colorimetric solution is prepared.

[0132] b. Based on the calculated required amount of colorimetric working solution, prepare the colorimetric working solution by mixing reagent A and reagent B in a volume ratio of 50:1 and mix thoroughly.

[0133] (3) Protein quantification detection

[0134] a. Add the diluted standard to each well of a 96-well plate; 20 μL per well.

[0135] b. Dilute the test sample (purified antibody) appropriately (e.g., 2x, 4x or 8x dilution; this experiment tested the original concentration and the 4x dilution concentration) and add 20 μL to each well of the 96-well plate.

[0136] c. Add 200 μL of colorimetric working solution to each well, mix thoroughly, cover with the 96-well plate cap, incubate at 37°C for 30 min, and cool to room temperature.

[0137] d. Use an ELISA reader to measure the absorbance of each sample and BSA standard at A562, or other wavelengths between 540 and 590 nm. Note that the absorbance of the blank control (diluent + working solution) should be subtracted.

[0138] e. Plot a standard curve and calculate the antibody concentration based on the OD value.

[0139] Based on the standard curve, the concentration of the purified monoclonal antibody was calculated to be 2660 μg / mL.

[0140] 7. Monoclonal antibody sensitivity test

[0141] (1) The purified PCV2 Cap protein was expressed and coated onto an ELISA plate at a concentration of 200-300 ng / well (the protein was diluted with phosphate buffer at pH 9.6 – 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4), 100 μL per well, and incubated overnight at 4°C.

[0142] (2) Add 100 μl of 3% BSA to each well, block at 37°C for 1 h, discard the blocking solution in the plate, wash 3 times with 300 μl of PBST, discard the liquid after the last wash, and gently pat dry the remaining liquid on absorbent paper.

[0143] (3) Dilute the antibody in a gradient of 2000, 4000, 8000, 16000, 32000, 64000, 128000, and 256000, add 100 μl / well to a 96-well plate, incubate at 37°C for 60 min, discard the liquid in the plate, add 300 μl of PBST to each well, wash 3 times, and gently pat dry the remaining liquid on absorbent paper;

[0144] (4) The HRP-labeled goat anti-mouse enzyme-labeled secondary antibody was diluted at 1:5000 and reacted at 37℃ for 30 min. The liquid in the plate was discarded, and 300 μl of PBST was added to each well. The plate was washed 3 times and the remaining liquid was gently patted dry on absorbent paper.

[0145] (5) Add 50 μl TMB and develop color at room temperature in the dark for 15 min;

[0146] (6) Add 50 μl of 1.5 M H2SO4 to terminate the reaction, and take an OD reading of 450 nm.

[0147] The results are as follows Figure 7 As shown, the OD value is linearly related to the antibody concentration, indicating that the antibody has good sensitivity to PCV2 Cap protein.

[0148] 8. Monoclonal antibody specificity test

[0149] (1) The purified PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein, PRRSV N protein, and PEDV N protein were expressed and purified and coated onto ELISA plates at a concentration of 200-300 ng / well (the protein was diluted with phosphate buffer (pH 9.6, 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4) and phosphate buffer (pH 9.6, negative control), 100 μL per well, and incubated overnight at 4°C.

[0150] (2) Add 100 μl of 3% BSA to each well, block at 37°C for 1 h, discard the blocking solution in the plate, wash 3 times with 300 μl of PBST, discard the liquid after the last wash, and gently pat dry the remaining liquid on absorbent paper.

[0151] (3) Dilute the antibody 1:10000, add 100 μl / well to a 96-well plate, incubate at 37°C for 60 min, discard the liquid in the plate, add 300 μl PBST to each well, wash 3 times, and gently pat dry the remaining liquid on absorbent paper.

[0152] (4) The HRP-labeled goat anti-mouse enzyme-labeled secondary antibody was diluted at 1:5000 and reacted at 37℃ for 30 min. The liquid in the plate was discarded, and 300 μl of PBST was added to each well. The plate was washed 3 times and the remaining liquid was gently patted dry on absorbent paper.

[0153] (5) Add 50 μl TMB and develop color at room temperature in the dark for 15 min;

[0154] (6) Add 50 μl of 1.5 M H2SO4 to terminate the reaction, and take an OD reading of 450 nm.

[0155] The results are as follows Figure 8 As shown, the monoclonal antibody exhibits good reactivity with the PCV2 Cap protein and shows no cross-reactivity with other proteins.

[0156] Example 4: Establishment of a quantitative sandwich ELISA method for determining Cap protein content

[0157] 1. Detailed operating steps of sandwich ELISA

[0158] (1) Coating: Dilute the antibody with phosphate buffer (pH 9.6) (135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 8mM K2HPO4). Dilute the rabbit polyclonal antibody that can effectively recognize the amino acid sequence of PCV2 Cap protein with phosphate buffer at a ratio of 1:10000. Add 100 μl to each well of a 96-well plate and coat at 4°C for 12 h. Discard the liquid in the plate and wash three times with 300 μl of PBST (135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 8mM K2HPO4, 0.1% Tween-20). Discard the liquid after the last wash and gently pat dry the remaining liquid on absorbent paper.

[0159] (2) Blocking: Add 100 μl of 3% BSA to each well and block at 37°C for 1 h. Discard the blocking solution in the plate and wash 3 times with 300 μl of PBST. Discard the liquid after the last wash and gently pat dry the remaining liquid on absorbent paper.

[0160] (3) Add the test sample: Add the test sample (10, 100, 1000 times serial dilution) and standard to a 96-well plate at 100 μl / well, incubate at 37°C for 60 min, discard the liquid in the plate, add 300 μl PBST to each well, wash 3 times, and gently pat dry the remaining liquid on absorbent paper.

[0161] (4) Washing: The monoclonal antibody 5C4, which showed a strong positive reaction with PCV Cap, was diluted 1:7500 with phosphate buffer (135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 8mM K2HPO4) at pH 7.2. The solution was added to the wells and reacted at 37°C for 60 min. The liquid in the plate was discarded, and the plate was washed 3 times with 300 μl PBST. The remaining liquid was then patted dry.

[0162] (5) Add enzyme-labeled secondary antibody: dilute HRP-labeled goat anti-mouse enzyme-labeled secondary antibody at 1:5000 and react at 37℃ for 30 min. Discard the liquid in the plate, add 300 μl PBST to each well, wash 3 times, and gently pat dry the remaining liquid on absorbent paper.

[0163] (6) Color development: Add 50 μl TMB and develop color for 15 min at room temperature in the dark;

[0164] (7) Termination: The reaction was terminated by adding 50 μl of 1.5 M H2SO4, OD 450nm reading.

[0165] (8) Result Calculation and Judgment: Subtract the OD value of the blank control from the value of the standard, input the result into Excel for curve fitting, and obtain the corresponding concentration-OD value conversion formula. Substitute the OD value of the test sample minus the blank control into the formula to calculate the Cap protein concentration of the test sample.

[0166] 2. Establish a standard curve

[0167] PCV2 Cap protein, concentrated by affinity chromatography and dialysis, was used as a standard sample. The PCV2 Cap protein concentration was adjusted to 70 μg / mL using the BCA method, followed by dilution to obtain PCV2 Cap standards of different concentrations. This involved serial dilution of PCV2 Cap to final concentrations of 224 ng / mL, 112 ng / mL, 56 ng / mL, 28 ng / mL, 14 ng / mL, 7 ng / mL, 3.5 ng / mL, and 0 ng / mL. 100 μl was added to each well to establish a standard curve for PCV2 quantification. The OD values ​​corresponding to different PCV2 Cap concentrations were input into GraphPad Prism for curve fitting, yielding the corresponding concentration-OD value conversion formula.

[0168] The results are as follows Figure 9 As shown, PCV2 Cap was used as a standard sample, and a standard curve was established by serially diluting it. The background-removed OD values ​​and their corresponding concentrations were statistically analyzed, and the corresponding formula was obtained. It was found that the Cap concentration showed a good linear relationship in the range of 0-200 ng / ml. The obtained formula was y = 0.01110x + 0.24, with a correlation coefficient R0. 2 The value is 0.9909, and the detection limit of Cap concentration in the sample to be tested can reach 7 ng / ml.

[0169] 3. Comparison experiment of different quantitative methods

[0170] After purification, PCV2 Cap at different concentrations was tested using a BCA protein concentration assay kit, and then detected using a sandwich ELISA. Taking three samples with BCA quantification concentrations of 50 μg / ml, 100 μg / ml, and 200 μg / ml as examples, the concentration of PCV2 Cap was calculated based on the obtained sample OD values ​​and the corresponding standard curves, and the differences between the two concentration quantification methods were analyzed and statistically analyzed.

[0171] The results showed that when PCV2 Cap samples of different concentrations were quantified using BCA, taking samples of 50 μg / ml, 100 μg / ml and 200 μg / ml as examples, the sample concentrations obtained after detection by sandwich ELISA were all within a range of less than 5% after calculation based on the standard curve.

[0172] In summary, this invention provides a specific monoclonal antibody against the Cap protein of porcine circovirus type 2 (PCV2). Using a rabbit polyclonal antibody against PCV2 Cap protein as the capture antibody and a monoclonal antibody against PCV2 Cap protein as the detection antibody, this invention establishes a sandwich ELISA method for detecting PCV2 based on a polyclonal antibody and a specific monoclonal antibody. This detection method is simple to operate, has good specificity and sensitivity, is time-efficient, and has broad application potential. This invention can accurately and rapidly determine the PCV2 Cap antigen content in multiple samples simultaneously, completing the Cap protein antigen content determination experiment, data processing, and result judgment within 4 hours. This effectively ensures vaccine quality, reduces testing costs and labor intensity, improves detection efficiency, and enables high-throughput vaccine efficacy testing.

Claims

1. A specific monoclonal antibody against the Cap protein of porcine circovirus type 2, characterized in that, The monoclonal antibody includes a heavy chain constant region and a light chain constant region, a heavy chain variable region and a light chain variable region. The CDR of the heavy chain variable region of the monoclonal antibody includes CDR1 with the amino acid sequence shown in SEQ ID No. 1, CDR2 with the amino acid sequence shown in SEQ ID No. 2 and CDR3 with the amino acid sequence shown in SEQ ID No.

3. The CDR of the light chain variable region of the monoclonal antibody includes CDR1 with the amino acid sequence shown in SEQ ID No. 4, CDR2 with the amino acid sequence shown in SEQ ID No. 5 and CDR3 with the amino acid sequence shown in SEQ ID No.

6.

2. The monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

8.

3. A gene fragment encoding the monoclonal antibody of claim 2, characterized in that, The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 9, and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

10.

4. An expression carrier, characterized in that, The expression vector contains the gene fragment as described in claim 3.

5. A host cell, characterized in that, The host cell contains the expression vector of claim 4, or the gene fragment of claim 3 is integrated into its genome.

6. The use of the monoclonal antibody as described in claim 1 or 2 in the preparation of reagents, test strips, or kits for detecting porcine circovirus type 2.

7. A type 2 porcine circovirus double antibody sandwich ELISA detection kit, characterized in that, The kit includes the monoclonal antibody as described in claim 1 or 2.

8. The type 2 porcine circovirus double antibody sandwich ELISA detection kit as described in claim 7, characterized in that, The kit also includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

9. A double-antibody sandwich ELISA method for detecting porcine circovirus type 2 antigen for non-diagnostic purposes, characterized in that, The method includes the following steps: (1) Coating: The rabbit polyclonal antibody against porcine circovirus type 2 Cap protein was diluted with phosphate buffer at pH 9.6 at a ratio of 1:10000. 100 μl of the solution was added to each well of a 96-well plate and coated at 4°C for 12 h. The plate was then washed with 300 μl of PBST. (2) Blocking: Add 100 μl of 3% BSA to each well, block at 37℃ for 1 h, and wash with 300 μl of PBST; (3) Add the test sample: Add the test sample and standard to the 96-well plate at 100 μl / well and incubate at 37°C for 60 min; (4) Washing: The monoclonal antibody described in claim 1 was diluted 1:7500 with phosphate buffer at pH 7.2 and added to the wells respectively. The mixture was reacted at 37°C for 60 min and then washed with 300 μl PBST. (5) Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse enzyme-labeled secondary antibody 1:5000 with phosphate buffer at pH 7.2, add it to each well, react at 37℃ for 30 min, and add 300 μl PBST to each well; (6) Color development: Add 50 μl TMB and develop color for 15 min at room temperature in the dark; (7) Termination: The reaction was terminated by adding 50 μl of 1.5 M H2SO4, OD 450nm Read the value and determine the test result based on the P / N ratio.

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