Porcine circovirus disease type 4 cap protein antigen epitope peptide, monoclonal antibody thereof and application

Abstract

The application discloses a porcine circovirus type 4 Cap protein antigen epitope peptide and a monoclonal antibody thereof. The amino acid sequence of the antigen epitope peptide is shown as SEQ ID No. 11; the monoclonal antibody heavy chain variable region comprises CDR1-3 shown as SEQ ID No. 1-3; the light chain variable region comprises CDR1-3 shown as SEQ ID No. 4-6; the monoclonal antibody has no reaction with PCV2, PCV3, PEDV, PRRSV and other various other pig-derived viruses, has good specificity; and the monoclonal antibody can be obtained by using conventional genetic engineering and other biological means, avoids the problem that an antibody genetic sequence is easy to be lost due to long-term storage of traditional hybridoma cells, is beneficial to optimization of the antibody at a gene or protein level, and further improves the specificity and affinity of the antibody.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and in vitro diagnostic technology, specifically relating to a porcine circovirus type 4 Cap protein antigen epitope peptide and its monoclonal antibody and applications. Background Technology

[0002] Porcine circovirus (PCV) is a member of the genus Porcine circovirus and is the smallest known non-enveloped DNA virus. It is characterized by non-enveloped viral particles with icosahedral symmetry and a diameter of 13 nm–25 nm. The viral particles consist of 60 capsid protein subunits arranged in twelve pentamer units. Based on antigenicity and genotype, porcine circovirus is now classified into four serotypes: PCV1, PCV2, PCV3, and PCV4. PCV1 was first reported in porcine kidney cells (PK-15) in 1974, but it is generally considered non-pathogenic to pigs. PCV2 was first discovered in pigs suffering from Porcine Multisystemic Wasting Syndrome (PMWS) between 1997 and 1998, and PCV2 is associated with the occurrence of clinical symptoms of PMWS. In 2016, a sow farm in the United States reported clinical symptoms of porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, which were confirmed to be caused by PCV3 using metagenomic sequencing. Subsequently, reports of PCV3-positive infections have emerged in the Americas, Asia, and Europe. In 2019, PCV4 was first discovered at a pig farm in Hunan Province, my country. Subsequently, PCV4 was found in Hunan, Henan, Shanxi, Jiangsu, Guangxi Zhuang Autonomous Region, Inner Mongolia Autonomous Region, and South Korea, among other regions. PCV4 infection may be associated with various clinical symptoms, including respiratory diseases, neurological diseases, diarrhea, and reproductive disorders, and can infect pigs of different ages. PCV4 mainly contains two large open reading frames (ORFs), ORF1 and ORF2. ORF1 is located on the positive strand of the replicating double-stranded DNA of porcine circovirus and encodes the Rep protein. ORF2 is located on the negative strand of the replicating viral DNA of the porcine circovirus genome and encodes the Cap protein, the only structural protein in the porcine circovirus particle. Cap protein is highly immunogenic and is a key target protein for PCV virus diagnosis and vaccine development.

[0003] Currently, PCV4 continues to spread globally, but no PCV4 virus has been successfully isolated, nor are there any commercially available PCV4 monoclonal antibodies. Research on its dominant antigenic epitopes is also insufficient, which severely limits the research on the basic theories of PCV4 pathogenesis and immunity, as well as the development of effective prevention and control products. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a porcine circovirus type 4 Cap protein antigen epitope peptide, its monoclonal antibody, and its applications.

[0005] In a first aspect, the present invention provides a porcine circovirus type 4 Cap protein antigenic epitope peptide, wherein the antigenic epitope peptide is located at 179-185aa of the PCV4 Cap protein, and its amino acid sequence is shown in SEQ ID No. 11.

[0006] In a second aspect, the present invention provides the use of the antigenic epitope peptide described in the first aspect above in the preparation of monoclonal antibodies against porcine circovirus type 4 Cap protein.

[0007] Thirdly, the present invention provides the application of the antigenic epitope peptide described in the first aspect above in the preparation of porcine circovirus epitope vaccines.

[0008] Fourthly, the present invention provides the application of tandemly expressing the antigenic epitope peptide described in the first aspect with other porcine viral dominant antigenic epitopes in the preparation of multivalent epitope vaccines.

[0009] Fifthly, the present invention provides a monoclonal antibody against porcine circovirus type 4 Cap protein, wherein the monoclonal antibody comprises an antibody heavy chain and an antibody light chain.

[0010] The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3;

[0011] The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No. 6.

[0012] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No. 8.

[0013] In a sixth aspect, the present invention provides a nucleic acid that encodes the antibody heavy chain and antibody light chain of the monoclonal antibody described in the fifth aspect above.

[0014] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID No. 9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID No. 10.

[0015] In a seventh aspect, the present invention provides an expression cassette, expression vector, and recombinant bacteria containing the nucleic acid described in the sixth aspect above.

[0016] Eighthly, the present invention provides the use of the monoclonal antibody described in the fifth aspect above in the preparation of reagents, test strips or kits for detecting porcine circovirus type 4.

[0017] In a ninth aspect, the present invention provides the use of the monoclonal antibody described in the fifth aspect above in the preparation of a reagent for detecting porcine circovirus type 4.

[0018] In a tenth aspect, the present invention provides the application of the monoclonal antibody described in the fifth aspect above in the detection of porcine circovirus type 4 infectivity for non-disease diagnosis purposes, or in the study of the function of the cap protein of porcine circovirus type 4.

[0019] In one aspect, the present invention provides a detection kit for porcine circovirus type 4, the kit comprising the monoclonal antibody described in the fifth aspect above.

[0020] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

[0021] In a twelfth aspect, the present invention provides a method for preparing the monoclonal antibody described in the fifth aspect above. The method comprises: cloning the porcine circovirus type 4 ORF2 gene into the pET-28a vector to construct a prokaryotic expression vector, inducing expression, and purifying the protein by elution with different concentrations of imidazole; immunizing Balb / c mice with the purified PCV4 Cap recombinant protein as an antigen, fusing mouse spleen cells with myeloma cells SP2 / 0 to prepare hybridoma cells; verifying the cell supernatant by indirect ELISA and Western Blot, and screening for positive clones; after three subcloning processes, injecting the hybridoma cells into mice to prepare ascites fluid, and finally purifying the obtained ascites fluid to obtain a monoclonal antibody against porcine circovirus type 4 Cap protein.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) The present invention uses a prokaryotic expression system to express and purify recombinant PCV4 Cap protein without removing the signal peptide sequence, and expresses it in the full-length form of the ORF2 gene.

[0024] (2) Mice were immunized with Cap protein as an immunogen, and a monoclonal antibody against PCV4 Cap protein was successfully obtained through cell fusion and subcellular screening.

[0025] (3) The prepared monoclonal antibody does not react with unrelated porcine viruses such as PCV2, PCV3, PRRSV and PEDV, but reacts with both prokaryotically expressed PCV4 Cap and eukaryotically expressed PCV4 Cap, showing good specificity.

[0026] (4) This invention expresses the PCV4 ORF2 gene in a gradient truncated manner and found that the antibody can specifically recognize and bind to the PCV4Cap protein in the region of 179-185aa, a total of 7 amino acids. This provides a reliable research tool for exploring the function of PCV4 Cap, PCV4 diagnostic reagents, and the development of tandem multivalent vaccines based on dominant immunotopes.

[0027] (5) The monoclonal antibody provided by the present invention can be obtained by conventional genetic engineering or protein engineering methods, avoiding the loss of antibody during long-term cryopreservation of hybridoma cells. It is also beneficial to optimize the antibody at the gene and protein level, thereby improving the specificity and affinity of the antibody. Attached Figure Description

[0028] 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.

[0029] Figure 1 PCV4 Cap protein expression form, purification and concentration identification results;

[0030] Figure 2 Results of serum titer assay in mice immunized with PCV4 Cap protein;

[0031] Figure 3 Results of the purification effect of PCV4 Cap protein monoclonal antibody on ascites fluid;

[0032] Figure 4 The titer of the prepared PCV4 Cap protein monoclonal antibody was determined.

[0033] Figure 5 Western blot analysis results of the reactivity of the monoclonal antibody with the eukaryotically expressed PCV4 Cap protein;

[0034] Figure 6 Specificity identification results of PCV4 Cap protein monoclonal antibody;

[0035] Figure 7 Results of subtype classification and identification of the prepared monoclonal antibodies;

[0036] Figure 8Schematic diagram of the construction of the truncated PCV4 Cap protein;

[0037] Figure 9 Identification results of the PCV4 Cap protein site recognized by monoclonal antibody. Detailed Implementation

[0038] The present invention will be further described in detail below through specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustration and explanation of the present invention and should not be regarded as limiting the scope of protection of the present invention. In addition, where specific technical operation steps or conditions are not specified in the embodiments, they are all carried out in accordance with the techniques or conditions described in general literature in the art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0039] Example 1: Construction and identification of the pET28a-PCV4 ORF2 recombinant plasmid

[0040] The PCV4 ORF2 gene was optimized for *E. coli*, and the optimized nucleotide sequence is shown in SEQ ID No. 12. This sequence contains the signal peptide sequence and does not need to be removed. The optimized sequence was amplified and ligated into the pET28a prokaryotic expression vector to construct the pET28a-PCV4 ORF2 recombinant plasmid. The recombinant plasmid was identified by double enzyme digestion. The specific operations are as follows: 1.1 Primer design

[0041] The PCV4 ORF2 amplification primers are shown in Table 1. The underlined sequences correspond to enzyme restriction sites. After the primers were designed, they were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0042] Table 1. Primers for PCV4 ORF2 amplification

[0043]

[0044] 1.2 PCR amplification

[0045] Using the optimized pUC57-PCV4-ORF2 plasmid as a template, the target gene was amplified by PCR using the primers described above. The PCR amplification system and reaction procedure are shown in Tables 2-3.

[0046] Table 2 PCR amplification system for the target gene

[0047]

[0048] Table 3 Reaction Procedure

[0049]

[0050] 1.3 Construction and Identification of Recombinant Vectors

[0051] The amplified target gene and the laboratory-stored pET28a empty vector were digested with two enzymes. The target gene was then ligated to the digested vector and transformed into DH5α competent cells. Clones were selected and colony PCR was performed using the primers in Table 1. Positive bacteria were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Plasmids were extracted from correctly sequenced bacterial cultures and cryopreserved for later use.

[0052] Sequencing results showed that the recombinant plasmid sequence was consistent with expectations. Further digestion of the recombinant plasmid with both NdeI and XhoI restriction endonucleases confirmed the sequence was as expected.

[0053] Example 2: Expression and purification of PCV4 Cap protein

[0054] 2.1 Expression of PCV4 Cap protein

[0055] The pET-28a-PCV4 ORF2 plasmid constructed in Example 1 was transformed into the prokaryotic expression strain BL21(DE3). A single colony was picked and cultured overnight in 1 mL of LB liquid medium containing kanamycin resistance with shaking. The next day, the bacterial culture was inoculated into 10 mL of fresh medium at a ratio of 1:100 and cultured at 37°C with shaking at 220 rpm. After two hours of culture, the OD of the bacterial culture was measured every 30 minutes. 600 Value, up to OD 600 When the value is between 0.6 and 0.8, 1 mmol / L IPTG is added to induce expression; after 6 h of induction, the bacterial culture is collected, centrifuged at 8000 r / min for 5 minutes, and the precipitate is collected; the precipitate is resuspended in 10 mL PBS, the bacterial cells are sonicated and the precipitate and supernatant are collected separately after centrifugation; 2× loading buffer is added directly to the supernatant, and 2× loading buffer is added after dissolving the precipitate in 8M urea solution. The samples are identified by SDS-PAGE and stained with Coomassie Brilliant Blue.

[0056] 2.2 Purification of PCV4 Cap protein

[0057] PCV4 Cap protein was expressed in both the supernatant and the precipitate. The supernatant was selected for further purification using His-tagged affinity chromatography resin (Ni-NTA Resin) in a gravity column, ultimately yielding highly purified PCV4 Cap protein. The specific steps are as follows:

[0058] (1) After centrifuging the ultrasonically disrupted bacterial solution, take its supernatant and filter it with a 0.45μm filter for later use;

[0059] (2) Add 2 mL of His affinity chromatography resin to the purification column, elute the nickel column with 10 mL of equilibration buffer, pour in the filtered supernatant and mix well, and incubate the mixed liquid in a 50 mL centrifuge tube at 4 °C overnight.

[0060] (3) After incubation, pour the incubation liquid into the gravity column, collect the effluent, and elute the target protein sequentially with an imidazole gradient of 10mM, 50mM, 100mM, 250mM, 500mM, 1M and 2M.

[0061] (4) Samples of the effluent and imidazole eluents of different concentrations were taken and identified by SDS-PAGE.

[0062] (5) After identification by SDS-PAGE, the eluent with a large amount of protein and high purity was concentrated using an ultrafiltration tube and filtered with a 0.45 μm filter.

[0063] (6) Take a small amount of protein, mix it with G250 solution, and measure its OD. 595nm And calculate the protein concentration;

[0064] PCV4 Cap protein expression form, purification, and concentration identification results are as follows: Figure 1 As shown, the PCV4 Cap protein was successfully expressed and purified, with the expected size and high purity.

[0065] Example 3: Preparation, reactivity, and variable region sequence determination of monoclonal antibodies

[0066] 3.1 Preparation of Monoclonal Antibodies

[0067] (1) Animal Immunization: Five female BALB / c mice aged 6-8 weeks were selected, and the PCV4 Cap recombinant protein obtained above was used to immunize the mice four times according to the immunization cycle. For the first immunization, the PCV4 Cap recombinant protein was mixed with complete Freund's adjuvant at a 1:1 ratio and administered subcutaneously at multiple sites at a dose of 50 μg / mouse. After a 14-day interval, a second immunization was performed, in which the PCV4 Cap recombinant protein was mixed with incomplete Freund's adjuvant at a 1:1 ratio and administered at 100 μg / mouse. Similarly, a third and fourth immunization were performed 14 days later. Blood was collected from the eyes of the mice 14 days after the four immunizations, and serum was collected to determine the antibody titer. Before fusion, a booster immunization was performed by intraperitoneal injection of PCV4 Cap recombinant protein at a dose of 30 μg / mouse into the mice to be subjected to cell fusion.

[0068] (2) Cell fusion: SP2 / 0 myeloma cells were revived and passaged three times to restore cell viability, and the cells were proliferated to meet the fusion conditions. One day in advance, peritoneal macrophages were taken from 6-8 week old NC mice as feeder cells and seeded into five 96-well cell plates. Mice with high serum titers were selected for B cell isolation. The spleen of the mice was aseptically removed and repeatedly injected with 1640 medium containing 1% penicillin-streptomycin-amphotericidal (triple antibody) until the internal B cells were flushed out into the medium. The spleen was then placed on a cell sieve and ground through the sieve using a syringe plunger to obtain mouse B cells. The previously prepared SP2 / 0 myeloma cells were taken, the original culture medium was discarded, 1640 medium was added, the cells were tapped, and then transferred to the prepared B cells for cell fusion.

[0069] (3) Screening and identification of monoclonal hybridoma cells: The PCV4 Cap recombinant protein prepared above was coated onto an ELISA plate. The supernatant from positive wells containing hybridoma cells was collected and added to the ELISA plate for titer determination. Wells with high reactivity were selected for subcloning. Subcloning was performed three times consecutively until well-reactive monoclonal hybridoma cells were selected. High-titer, well-reactive monoclonal hybridoma cells were proliferated and cryopreserved.

[0070] ELISA potency assay results as follows Figure 2 As shown, the serum of all five mice after immunization had high titers, with mouse number 5 having the highest titer, which was used for subsequent hybridoma cell fusion.

[0071] (4) Preparation of ascites fluid: 10-12 week old BALB / c female mice were sensitized by intraperitoneal injection of 0.4 mL Freund's incomplete adjuvant. Within 7-14 days, the selected monoclonal hybridoma cell line was immunized into the mice, approximately 1 × 10⁻⁶ cells. 6 One cell per mouse. Seven days after injection of hybridoma cells, mice were observed twice a day, morning and evening. When the mice's back fur became rough and disordered, and they had difficulty moving, and their abdominal cavity swelled and showed obvious fluctuations upon touch, ascites fluid was collected and cryopreserved.

[0072] (5) Purification of ascites fluid: The obtained ascites fluid was purified according to the instructions of the TransGen ProteinIso Protein G Resin Kit. The purified sample was identified by SDS-PAGE. The obtained PCV4 Cap protein-specific monoclonal antibody was named 2D3.

[0073] The purified ascites fluid was identified using SDS-PAGE, and the results are as follows: Figure 3 As shown, two distinct bands, one light chain and one heavy chain, are visible, and their sizes are as expected.

[0074] 3.2 Identification of the reactivity of monoclonal antibodies

[0075] (1) ELISA to identify the titer of monoclonal antibodies

[0076] The PCV4 Cap protein obtained above was coated into ELISA plates at a rate of 100 ng / well. The plates were incubated at 37°C for 2 hours or overnight at 4°C. After washing with PBS, 5% skim milk powder was added for blocking. The prepared PCV4 Cap protein monoclonal antibody 2D3 was serially diluted and added to the coated plates. The plates were incubated at 37°C for 1 hour, followed by 4 washes with PBS. HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added, and the plates were incubated at 37°C for 1 hour, followed by 4 washes with PBS. TMB was added for 15 minutes for color development, and then stop buffer was added. The plates were then used to measure OD using a microplate reader. 450 Numerical value.

[0077] ELISA results as follows Figure 4 As shown, the monoclonal antibody 2D3 prepared by this invention can react with PCV4 Cap protein and has a high titer.

[0078] (2) Western blotting to identify the reaction between the monoclonal antibody and the eukaryotically expressed PCV4 Cap protein.

[0079] Given that no PCV4 has been successfully isolated to date, and there are no commercially available PCV4 Cap antibodies and PCV4 Cap proteins with good reactivity, to fully verify the reactivity of the PCV4 Cap monoclonal antibody prepared in this invention, a eukaryotic expression vector CMV-Flag-PCV4-ORF2 expressing PCV4 Cap was further constructed. This vector was transfected into HEK-293T cells, and samples were collected. Western blotting was used to verify the reactivity of the prepared monoclonal antibody with the eukaryotically expressed PCV4 Cap protein. The primary antibodies were... Anti-Flag Mouse Monoclonal Antibody and PCV4 Cap monoclonal antibody, the secondary antibody is Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP.

[0080] Reactivity test results as follows Figure 5 As shown in the Western blot results, the monoclonal antibody 2D3 prepared in this invention reacts not only with PCV4 Cap protein expressed in prokaryotes, but also with PCV4 Cap protein expressed in eukaryotes without optimized sequence, while showing no reaction with 293 cells or cells transfected with the empty Flag vector. This further verifies the good reactivity of the monoclonal antibody prepared in this invention with the porcine circovirus type 4 Cap protein.

[0081] (3) ELISA was used to identify the specificity of the prepared PCV4 Cap protein monoclonal antibody.

[0082] To fully identify the specificity of the PCV4 Cap protein monoclonal antibody prepared in this invention, ELISA assays were performed using unrelated porcine viral target proteins such as PCV2-Cap, PCV3-Cap, PEDV-N, PRRSV-GP5, and PRRSV-N, with the PCV4 Cap protein monoclonal antibody as the primary antibody, to identify its reactivity.

[0083] The results are as follows Figure 6 As shown, the monoclonal antibody 2D3 prepared in this invention reacts only with PCV4 Cap and has no cross-reaction with unrelated porcine viruses such as PCV2, PCV3, PEDV, and PRRSV, indicating that the monoclonal antibody prepared in this invention has good specificity.

[0084] (4) Identification of monoclonal antibody subtypes

[0085] The obtained PCV4 Cap protein monoclonal antibody was identified according to the instructions of the Proteintech Mouse Monoclonal Antibody Isotyping Kit.

[0086] The results are as follows Figure 7 As shown, the monoclonal antibody 2D3 prepared in this invention has a heavy chain constant region of IgG2b type and a light chain constant region of Kappa type.

[0087] (5) Identification of protein epitopes bound by monoclonal antibodies: The PCV4 ORF2 gene was truncated in a gradient and ligated into a prokaryotic expression vector to construct a recombinant plasmid. The corresponding protein was then expressed. Western blot reactions were performed on the truncated protein samples using the prepared PCV4 Cap protein monoclonal antibody. The protein was gradually truncated until the key antigenic epitopes bound to the protein were finally identified. The gradient truncation construction strategy is as follows: Figure 8 As shown.

[0088] Epitope identification results of monoclonal antibody 2D3 are as follows Figure 9 As shown, the key region where the monoclonal antibody 2D3 prepared in this invention specifically binds to the PCV4 Cap protein is C13, i.e., 179-185aa. 179 WWISMAD 185 It contains 7 amino acids. The specific binding site sequence is shown in SEQ ID No. 11.

[0089] 3.3 Monoclonal antibody variable region gene sequencing

[0090] The frozen monoclonal hybridoma cells 2D3 were revived, genomic RNA was extracted using the Trizol method, and the RNA was reverse-transcribed into cDNA using the HiScript IIQ RT SuperMix for qPCR kit from Nanjing Novizan Pharmaceutical Co., Ltd.

[0091] Nested PCR was used to amplify the antibody variable region gene. Using the above cDNA as a template, the antibody variable region gene was amplified using the first round of mouse antibody IgG and κ light chain primers. Then, using the first round product as a template, the antibody variable region gene was amplified using the second round of mouse antibody IgG and κ light chain primers. Primer synthesis reference (von Boehmer L, Liu C, Ackerman S, Git lin AD, Wang Q, Gazumyan A, Nussenzweig MC. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc. 2016 Oct; 11(10):1908-1923. doi:10.1038 / nprot.2016.102. Epub 2016Sep 15.PMID:27658009.).

[0092] After amplification, the target fragment was ligated into the pMD-19T vector to construct a sequencing plasmid, which was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using the NCBI and IMGT gene libraries to identify the antibody variable region sequence.

[0093] The amino acid sequence of the heavy chain variable region is: AVKLVDSGGGLVKPGGSLKLSCAASGFTFSSYAM SWVRQTPEKRLEWVASISYDASTFYPDSVKGRFTISRDIVRNIVYLQMNNLRSEDTAMYYCTRGLPSPYWGQGTLVTVS;

[0094] The amino acid sequence of the light chain variable region is: DIVMSQSPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNYL AWYQQKPGQSPEMMIYWTSTWESGVPDRFTGSGSGTDFTLTISGVQAEDLAIYFCHQSYNL GRSVEAPSCKSN;

[0095] The gene sequence encoding the heavy chain variable region is: GCAGTGAAACTGGTGGACTCTGGGGGAGGCTTAGT GAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTTCCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAATGGGTCGCGTCCATTAGTTATGATGCTAGTACCTTCTATCCAGACAGTG TGAAGGGCCGATTCACCATCTCCAGAGATATTGTCAGGAACATTGTTTACCTGCAAATGAACAATCTGAGGTCTGAGGACACGGCCATGTATTACTGTACAAGAGGCCTCCCCTCGCCTTACTGGGGCCAGGGGGACTCTGGTCACTGTCTCT;

[0096] The gene sequence encoding the light chain variable region is: GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCT GTGTCAGCAGGAGAGAAGGTCACTTTGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGGAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCCCCTGAAATGATGATCTACTGGACATCCACTTGGGAATCT GGGGTCCCTGATCGCTTCACGGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAATTTATTTCTGCCACCAATCTTATAATCTTGGACGTTCAGTGGAGGCACCAAGCTGTAAATCAAAC

[0097] The PCV4 Cap protein monoclonal antibody 2D3 can specifically recognize the WWISMAD region of the porcine circovirus type 4 Cap protein.

[0098] In summary, this invention provides a monoclonal antibody against porcine circovirus type 4 (PCV4) Cap protein. This monoclonal antibody specifically recognizes and binds to 179-185 aa of the Cap protein and does not react with other porcine viruses such as PCV2, PCV3, PEDV, and PRRSV. It reacts simultaneously with both prokaryotic and eukaryotic PCV4 Cap expressions, exhibiting good specificity. The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. The monoclonal antibody prepared by this invention provides a reliable research tool for exploring the function of PCV4 Cap, developing PCV4 diagnostic reagents, and developing tandem multivalent vaccines based on dominant immunoepitaphs.

[0099] The embodiments described above are only some embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the patent claims of the present invention should be included within the scope of the patent claims of the present invention.

Claims

1. A monoclonal antibody specific to the Cap protein of porcine circovirus type 4, characterized in that, The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No.

8.

2. A nucleic acid, characterized in that, The nucleic acid encodes the antibody heavy chain and antibody light chain of the monoclonal antibody of claim 1.

3. The nucleic acid as described in claim 2, characterized in that, The nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID No. 9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID No.

10.

4. The use of the monoclonal antibody as described in claim 1 in the preparation of a reagent for detecting porcine circovirus type 4.

5. The application of the monoclonal antibody as described in claim 1 in the detection of porcine circovirus type 4 infectivity for non-disease diagnosis purposes.

6. A porcine circovirus type 4 detection kit, characterized in that, The kit includes the monoclonal antibody as described in claim 1.

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