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

Abstract

The invention 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 as shown in SEQ ID No. 11; the heavy chain variable region of the monoclonal antibody comprises CDR1-3 as shown in SEQ ID No.1 to SEQ ID No.3; the light chain variable region comprises CDR1-3 as shown in SEQ ID No. 4 to 6; the monoclonal antibody does not react with a plurality of other swine viruses such as PCV2, PCV3, PEDV, PRRSV and the like, and has good specificity; the antibody can be obtained by biological means such as conventional genetic engineering, and the problem that the genetic sequence of the antibody is easy to lose due to long-term cryopreservation of traditional hybridoma cells is avoided; optimization of the antibody on the gene or protein level is facilitated, and the specificity and affinity of the antibody are further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology and in vitro diagnosis, and specifically relates to a porcine circovirus disease type 4 Cap protein antigen epitope peptide and a monoclonal antibody and application thereof. Background Art

[0002] Porcine circovirus (PCV) is a member of the genus circovirus and is the smallest non-enveloped DNA virus known to date. It is characterized by non-enveloped virus particles with icosahedral symmetry and a diameter of 13nm to 25nm. The virus particles are composed of 60 capsid protein subunits arranged in twelve pentamer units. According to the difference in antigenicity and genotype, porcine circovirus is now divided into four serotypes: PCV1, PCV2, PCV3 and PCV4. PCV1 was first reported in porcine kidney cells (PK-15) in 1974, but it is generally believed that PCV1 is not pathogenic to pigs. PCV2 was first discovered in pigs with Porcine Multi systemic Wasting Syndrome (PMWS) between 1997 and 1998, and PCV2 is associated with the occurrence of clinical symptoms of PMWS. In 2016, clinical symptoms of porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders appeared in a sow farm in the United States, and metagenomic sequencing technology confirmed that it was caused by PCV3. Since then, PCV3-positive infections have been reported in the Americas, Asia, and Europe. In 2019, PCV4 was first discovered in a pig farm in Hunan Province, my country, and then PCV4 was found in Hunan Province, Henan Province, Shanxi Province, Jiangsu Province, Guangxi Zhuang Autonomous Region, Inner Mongolia Autonomous Region, and South Korea. PCV4 infection may be associated with a variety of clinical symptoms such as respiratory diseases, nervous system 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 replicative DNA double strand of porcine circovirus and encodes the Rep protein. ORF2 is located on the negative strand of the replicative viral DNA double strand of the porcine circovirus genome and encodes the Cap protein, the only structural protein in the porcine circovirus particle. Cap protein has strong immunogenicity and is a key target protein for PCV virus diagnosis and vaccine development.

[0003] Currently, PCV4 continues to spread around the world, but the PCV4 virus has not yet been successfully isolated, nor has there been any commercialized PCV4 monoclonal antibody. Research on its dominant antigenic epitopes is also insufficient, which has severely restricted basic theoretical research on PCV4 pathogenicity and immunity and the development of effective prevention and control products. Summary of the invention

[0004] In view of the above technical problems, the object of the present invention is to provide a porcine circovirus disease type 4 Cap protein antigen epitope peptide and its monoclonal antibody and application.

[0005] In a first aspect, the present invention provides an antigenic epitope peptide of the Cap protein of porcine circovirus type 4, 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 antigen epitope peptide described in the first aspect in the preparation of monoclonal antibodies against porcine circovirus type 4 Ca p protein.

[0007] In a third aspect, the present invention provides the use of the antigen epitope peptide described in the first aspect in the preparation of a porcine circovirus epitope vaccine.

[0008] In a fourth aspect, the present invention provides the use of the antigenic epitope peptide described in the first aspect above in the preparation of a multivalent epitope vaccine by expressing it in tandem with other dominant antigenic epitopes of porcine-derived viruses.

[0009] In a fifth aspect, 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 CDR1 shown in the amino acid sequence of 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 CDR1 shown in the amino acid sequence of 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 as SEQ ID No.7, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID No.8.

[0013] In a sixth aspect, the present invention provides a nucleic acid encoding 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 as SEQ ID No.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown as SEQ ID No.10.

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

[0016] In an eighth aspect, the present invention provides the use of the monoclonal antibody described in the fifth aspect in the preparation of a reagent, a test strip or a kit 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 in the preparation of a reagent for detecting porcine circovirus type 4.

[0018] In a tenth aspect, the present invention provides the use of the monoclonal antibody described in the fifth aspect in detecting the infectivity of porcine circovirus type 4 virus for non-disease diagnosis purposes, or in studying the function of porcine circovirus type 4 Cap protein.

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

[0020] Preferably, the kit further comprises an ELISA plate, a blocking solution, a diluent, a washing solution, a color developer, and a stop solution.

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

[0022] The beneficial effects of the present invention are:

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

[0024] (2) Using the Cap protein as an immune source to immunize mice, a monoclonal antibody against the PCV4 Cap protein was successfully obtained through cell fusion and subcellular screening;

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

[0026] (4) The present invention expresses PCV4 ORF2 gene gradient truncations, and finds that the region where the antibody can specifically recognize and bind to PCV4 Cap protein is 179-185aa, a total of 7 amino acids, providing a reliable research tool for exploring the function of PCV4 Cap, PCV4 diagnostic reagents, and the development of tandem multivalent vaccines based on dominant immune epitopes;

[0027] (5) The monoclonal antibodies provided by the present invention can be obtained by conventional genetic engineering or protein engineering methods, thereby avoiding the loss of antibodies during long-term cryopreservation of hybridoma cells. It is also beneficial to optimize the antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

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

[0030] Figure 2 The results of serum titer test of mice immunized with PCV4 Cap protein;

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

[0032] Figure 4 The titer identification results of the prepared PCV4 Cap protein monoclonal antibody;

[0033] Figure 5 WB identified the reactivity of monoclonal antibodies with eukaryotically expressed PCV4 Cap protein;

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

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

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

[0037] Fig. 9 Identification results of the sites of PCV4 Cap protein recognized by monoclonal antibodies. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below by specific embodiments. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate and explain the present invention and should not be considered as limiting the scope of protection of the present invention. In addition, those who do not indicate specific technical operation steps or conditions in the embodiments are all carried out according to the technology or conditions described in the general literature in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained by commercial purchase.

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

[0040] The PCV4 ORF2 gene was optimized for Escherichia coli. The optimized nucleotide sequence is shown in SEQ ID No. 12. The sequence contains a signal peptide sequence and does not need to be removed. The optimized sequence was amplified and connected to 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] PCV4 ORF2 amplification primers are shown in Table 1, where the underlined sequences correspond to restriction sites. After the primers were designed, they were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0042] Table 1 PCV4 ORF2 amplification primers

[0043]

[0044] 1.2 PCR amplification

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

[0046] Table 2 Target gene PCR amplification system

[0047]

[0048] Table 3 Reaction procedure

[0049]

[0050] 1.3 Construction and identification of recombinant vectors

[0051] The target gene amplified above and the pET28a empty vector stored in the laboratory were digested with two enzymes, and then the target gene and the enzyme-cut vector were connected and transformed into DH5a competent cells. The selected clones were subjected to colony PCR using the primers in Table 1, and the positive bacteria were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmids extracted from the bacterial solution with correct sequencing were frozen for later use.

[0052] The sequencing results showed that the recombinant plasmid sequence was consistent with the expected sequence. The recombinant plasmid was then digested with two restriction endonucleases, NdeⅠ and XhoⅠ, for restriction endonuclease identification, which was consistent with the expected sequence.

[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), and a single clone colony was picked and placed in 1 mL of liquid LB medium containing kanamycin resistance and shaken for overnight culture; the next day, the bacterial solution was inoculated into 10 mL of fresh culture medium at a ratio of 1:100, and cultured at 37°C, 220 r / min, and shaken; after two hours of culture, the OD of the bacterial solution was measured every 30 min. 600 Value, until OD 600 When the value was between 0.6 and 0.8, 1 mmol / L IPTG was added to induce expression; after 6 hours of induction, the bacterial solution was collected, centrifuged at 8000 r / min for 5 minutes, and the precipitate was collected; the precipitate was resuspended in 10 mL PBS, the bacteria were ultrasonically disrupted, and the precipitate and supernatant were collected separately after centrifugation; the supernatant was directly added to 2× loading buffer, the precipitate was dissolved with 8 M urea solution and then added to 2× loading buffer, the sample was identified by SDS-PAGE and stained with Coomassie Brilliant Blue.

[0056] 2.2 Purification of PCV4 Cap protein

[0057] PCV4 Cap protein is expressed in both the supernatant and the precipitate. The supernatant is selected for further purification. The purification method is to purify it in a gravity column through His tag affinity chromatography resin (Ni-NTA Resin), and finally obtain PCV 4 Cap protein with high purity. The specific steps are as follows:

[0058] (1) After the bacterial solution has been ultrasonically disrupted, centrifuge it, take the supernatant, and filter it through a 0.45 μm filter for later use;

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

[0060] (3) After the incubation is completed, the incubation liquid is poured into the gravity column, the effluent is collected, and the target protein is eluted with an imidazole gradient of 10 mM, 50 mM, 100 mM, 250 mM, 500 mM, 1 M and 2 M in sequence;

[0061] (4) Sampling the effluent and eluates with different imidazole concentrations for SDS-PAGE identification;

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

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

[0064] The results of PCV4 Cap protein expression, purification and concentration were as follows: Figure 1 As shown, PCV4 Cap protein was successfully expressed and purified, with a size consistent with expectations and a high degree of 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 to 8 weeks were selected, and the PCV4 Cap recombinant protein obtained above was immunized four times according to the immunization cycle. For the first immunization, the PCV4 Cap recombinant protein was mixed with complete Freund's adjuvant in a ratio of 1:1, and the mice were subcutaneously immunized at multiple points at a dose of 50 μg / mouse; the second immunization was performed 14 days later, and the PCV4 Cap recombinant protein was mixed with incomplete Freund's adjuvant in a ratio of 1:1; 100 μg / mouse was immunized; similarly, the third and fourth immunizations were performed 14 days later. 14 days after the four immunizations, blood was collected from the mice's eyeballs, and serum was taken to determine the antibody titer. Before fusion, a booster immunization was performed, and the PC V4 Cap recombinant protein was injected intraperitoneally at a dose of 30 μg / mouse into the mice to be subjected to cell fusion for sensitization.

[0068] (2) Cell fusion: Resuscitate SP2 / 0 myeloma cells, subculture them three times to restore cell viability, and expand the cells until they reach fusion conditions. One day in advance, take peritoneal macrophages from 6-8 week old NC mice as feeder cells and spread them into 5 96-well cell plates. Select mice with high serum titers for B cell isolation, remove the spleen of the mice aseptically, and repeatedly inject and rinse with 1640 culture medium containing 1% penicillin-streptomycin-amphotericin (triple antibody) until the internal B cells are flushed into the culture medium; then place the spleen on a cell sieve and grind it with a syringe piston to obtain mouse B cells. Take the SP2 / 0 myeloma cells prepared in the early stage, discard the original culture medium, add 1640 culture medium, take a picture of the cells and transfer them to the prepared B cells for cell fusion.

[0069] (3) Screening and determination of monoclonal hybridoma cells: The PCV4 Cap recombinant protein prepared above is coated on an ELISA reaction plate, and the positive wells with hybridoma cells are selected and the supernatant is collected and added to the ELISA reaction plate for titer determination. The wells with higher reactivity are selected for subcloning, and subcloning is performed three times in succession until monoclonal hybridoma cells with good reactivity are screened. Monoclonal hybridoma cells with high titer and good reactivity are expanded and frozen.

[0070] ELISA titer test results Figure 2 As shown, the sera of the five mice after immunization all had high levels of titer, among which the titer of mouse No. 5 was the highest and was used for subsequent hybridoma cell fusion.

[0071] (4) Preparation of ascites: 10-12 week old female BALB / c mice were sensitized by intraperitoneal injection of 0.4 mL of Freund's incomplete adjuvant. Within 7-14 days, the selected monoclonal hybridoma cell lines were immunized into the mice, with approximately 1×10 6 7 days after the injection of hybridoma cells, the mice were observed once in the morning and evening every day. When the back hair of the mice became rough and messy, and it was difficult to move, and the abdominal cavity bulged and had obvious fluctuations when touched, the ascites was collected and frozen.

[0072] (5) Purification of ascites: The ascites was purified according to the instructions of ProteinIso Protein G Resin kit of Quanshijin Company, and the purified samples were identified by SDS-PAGE. The obtained PCV4 Cap protein-specific monoclonal antibody was named 2D3.

[0073] The purified ascites was identified by SDS-PAGE. Figure 3 As shown, two distinct light chain and heavy chain bands are visible, and their sizes are consistent with expectations.

[0074] 3.2 Identification of monoclonal antibody reactivity

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

[0076] The PCV4 Cap protein obtained above was coated into the ELISA reaction plate at 100 ng / well, coated at 37℃ for 2h or 4℃ overnight, washed with PBS'T and blocked with 5% skim milk powder. The PCV4 Cap protein monoclonal antibody 2D3 prepared above was graded diluted, added to the coated plate, reacted at 37℃ for 1h, washed 4 times with PBS'T; HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added, reacted at 37℃ for 1h, washed 4 times with PBS'T; TMB color development was performed for 15min, then the stop solution was added, and the OD was measured on an ELISA reader. 450 Numeric value.

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

[0078] (2) Western blot analysis of the reaction between monoclonal antibodies and eukaryotically expressed PCV4 Cap protein

[0079] In view of the fact that PCV4 has not been successfully isolated and there are no commercially available PCV4 Cap antibodies and PCV4 Cap proteins with good reactivity, in order to fully verify the reactivity of the PCV4 Cap monoclonal antibody prepared by the present invention, a eukaryotic expression vector CMV-Flag-PCV4-ORF2 expressing PCV4 Cap was further constructed, which was transfected into HEK-293T cells for expression and then samples were collected. The reactivity of the prepared monoclonal antibody with the eukaryotic expressed PCV4 Cap protein was verified by Western Blot. 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] The results of the reactivity test were as follows Figure 5 As shown, the Western Blot results show that the monoclonal antibody 2D3 prepared by the present invention can react with the PCV4 Cap protein expressed in prokaryotes, as well as the PCV4 Cap protein expressed in eukaryotes without optimized sequences, but has no reaction with 293 cells and Flag-transfected empty cells. This further verifies the good reactivity of the monoclonal antibody prepared by the present invention with the Cap protein of porcine circovirus disease type 4.

[0081] (3) ELISA to identify the specificity of the monoclonal antibody against PCV4 Cap protein

[0082] In order to fully identify the specificity of the PCV4 Cap protein monoclonal antibody prepared by the present invention, an ELISA plate was coated with unrelated porcine virus target proteins such as PCV2-Cap, PCV3-Cap, PEDV-N, PRRSV-GP5, and PRRSV-N, and an ELISA test was performed using the monoclonal antibody of the PCV4 Cap protein as the primary antibody to identify its reactivity.

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

[0084] (4) Identification of monoclonal antibody subtypes

[0085] According to the operating instructions of the Mouse Monoclonal Antibody Lsotyping Kit of Proteintech Company, the subtype of the obtained PCV4 Cap protein monoclonal antibody was identified.

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

[0087] (5) Identification of protein epitopes bound by monoclonal antibodies: PCV4 ORF2 gene was truncated and connected to a prokaryotic expression vector to construct a recombinant plasmid, and the corresponding protein was induced to express. The prepared PCV4 Cap protein monoclonal antibody was used to perform Western blot reaction with the truncated protein samples, and the protein epitopes were gradually shortened until the key antigen epitopes to which it binds were finally identified. The gradient truncation construction strategy is as follows: Figure 8 shown.

[0088] The epitope identification results of monoclonal antibody 2D3 are as follows Fig. 9 As shown, the key region of the monoclonal antibody 2D3 prepared by the present invention that specifically binds to the PCV4 Cap protein is C13, i.e., 179-185aa. 179 WWISMAD 185 , a total of 7 amino acids. The specific binding site sequence is shown in SEQ ID No.11.

[0089] 3.3 Determination of monoclonal antibody variable region gene sequence

[0090] The frozen monoclonal hybridoma cells 2D3 were revived, genomic RNA was extracted using the Trizol method, and RNA was reversed into cDNA using the reverse transcription kit HiScript IIQ RT SuperMix for qPCR produced by Nanjing Novozymes.

[0091] Nested PCR was used to amplify the variable region genes of antibodies. The above cDNA was used as a template to amplify the variable region genes of antibodies using the first round of mouse antibody IgG and κ light chain primers, and then the first round product was used as a template to amplify the variable region genes of antibodies using the second round of mouse antibody IgG and κ light chain primers. The primer synthesis references (von Boehmer L, Liu C, Ackerman S, Gitlin 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 2016 Sep 15. PMID: 27658009.).

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

[0093] The sequencing results showed that the amplified sequences were the complementarity determining region (CDR) sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody as shown in Table 4.

[0094] Table 4 Antibody variable region sequences

[0095]

[0096] Among them, the amino acid sequence of the heavy chain variable region is: AVKLVDSGGGLVKPGGSLKLSCAASGFTFSSYAM SWVRQTPEKRLEWVASISYDASTFYPDSVKGRFTISRDIVRNIVYLQMNNLRSEDTAMYYCTRGLPSPYWGQGTLVTVS;

[0097] The amino acid sequence of the light chain variable region is: DIVMSQSPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNYL AWYQQKPGQSPEMMIYWTSTWESGVPDRFTGSGSGTDFTLTISGVQAEDLAIYFCHQSYNL GRSVEAPSCKSN;

[0098] The gene sequence encoding the heavy chain variable region is: GCAGTGAAACTGGTGGACTCTGGGGGAGGCTTAGT GAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTTCCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAATGGGTCGCGTCCATTAGTTATGATGCTAGTACCTTCTATCCAGACAGTG TGAAGGGCCGATTCACCATCTCCAGAGATATTGTCAGGAACATTGTTTACCTGCAAATGAACAATCTGAGGTCTGAGGACACGGCCATGTATTACTGTACAAGAGGCCTCCCCTCGCCTTACTGGGGCCAGGGGGACTCTGGTCACTGTCTCT;

[0099] The gene sequence encoding the light chain variable region is: GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCT GTGTCAGCAGGAGAGAAGGTCACTTTGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGGAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCCCCTGAAATGATGATCTACTGGACATCCACTTGGGAATCT GGGGTCCCTGATCGCTTCACGGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAATTTATTTCTGCCACCAATCTTATAATCTTGGACGTTCAGTGGAGGCACCAAGCTGTAAATCAAAC

[0100] PCV4 Cap protein monoclonal antibody 2D3 can specifically recognize the region of porcine circovirus type 4 Cap protein: WWISMAD.

[0101] In summary, the present invention provides a monoclonal antibody against porcine circovirus type 4 Cap protein, which can specifically recognize and bind to 179-185aa of the Cap protein, and does not react with other porcine viruses such as PCV2, PCV3, PEDV and PRRSV, and can react with prokaryotically expressed PCV4 Cap and eukaryotically expressed PCV4 Cap at the same time, and has good specificity. The monoclonal antibody provided by the present invention can be obtained by conventional genetic engineering or protein engineering methods, avoiding the loss of antibodies during long-term cryopreservation of hybridoma cells, and is also conducive to optimizing antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibodies. The monoclonal antibody prepared by the present invention 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 immune epitopes.

[0102] The embodiments described above are only partial embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the patent application scope of the present invention.

Claims

1. A dominant antigen epitope peptide of porcine circovirus type 4 Cap protein, characterized in that: The antigen epitope peptide is located at 179-185aa of PCV4 Cap protein, and its amino acid sequence is shown in SEQ ID No.

11.

2. Use of the antigen epitope peptide according to claim 1 in the preparation of monoclonal antibodies against porcine circovirus type 4 Cap protein.

3. Use of the antigen epitope peptide as claimed in claim 1 in the preparation of porcine circovirus epitope vaccine.

4. Use of the antigenic epitope peptide according to claim 1 in the preparation of a multivalent epitope vaccine by expressing it in series with other dominant antigenic epitopes of porcine viruses.

5. A porcine circovirus type 4 Cap protein-specific monoclonal antibody, characterized in that: The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; The variable region CDR of the antibody light chain includes CDR1 shown in the amino acid sequence of SEQ ID No.4, CDR2 shown in SEQ ID No.5 and CDR3 shown in SEQ ID No.

6.

6. The monoclonal antibody according to claim 5, characterized in that 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.

7. A nucleic acid, characterized in that The nucleic acid encodes the antibody heavy chain and antibody light chain of the monoclonal antibody according to claim 5 or 6.

8. The nucleic acid according to claim 7, wherein 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.

9. Use of the monoclonal antibody according to claim 5 or 6 in the preparation of a reagent for detecting porcine circovirus type 4, or in detecting porcine circovirus type 4 virus infectivity for purposes other than disease diagnosis, or in the study of porcine circovirus type 4 Cap protein function.

10. A porcine circovirus type 4 detection kit, characterized in that: The kit comprises the monoclonal antibody according to claim 5 or 6.

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