A monoclonal antibody against PCV2, a hybridoma cell line and their applications

Abstract

The present invention relates to the field of veterinary biomedical technology, and particularly relates to a monoclonal antibody against PCV2, a hybridoma cell line and their applications. The hybridoma cell line provided by the present invention and the monoclonal antibodies 5A8 and 4C2 secreted by it can specifically recognize different genotype strains of PCV2. When used in combination, they can distinguish PCV2d genotype strains from PCV2a and PCV2b genotype strains. The specificity experiment of the monoclonal antibody shows that the monoclonal antibody provided by the present invention has no reactivity to other common swine-origin viruses, such as classical swine fever virus, African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, rotavirus, etc., and has good sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biomedicine, and particularly relates to a monoclonal antibody against PCV2, a hybridoma cell line and their applications. Background Art

[0002] Porcine circovirus (PCV) is a member of the genus Circovirus in the family Circoviridae. The virus particles have no envelope and show an icosahedral symmetric structure. It contains a single-stranded circular negative-stranded DNA of 1.76 kb to 2.3 kb, with a diameter of about 20 nm, and is the smallest animal virus found so far that can replicate autonomously in mammalian cells. Early studies believed that there were two serotypes of PCV. Among them, porcine circovirus type 1 (PCV1) widely exists in pig populations and is non-pathogenic. The other serotype, porcine circovirus type 2 (PCV2), has a nucleotide sequence homology of 68% - 76% with PCV1 and is pathogenic. The genome of PCV2 includes two main open reading frames (ORFs). Among them, ORF2 encodes the only structural protein Cap protein of the virus, which is the main immunogenic protein of PCV2, can induce the body to produce neutralizing antibodies, and is closely related to the pathogenicity of the virus. PCV2 mainly invades the immune system of pigs, resulting in immunosuppression, reducing the body's resistance, and thus secondary to other diseases. The genotypes of PCV2 prevalent in China include PCV2a, PCV2b, and PCV2d. With the spread of the virus, it has been found that there are antigenic variabilities among multiple genotypes of PCV2. And clinical cases of PCV2 infection are often accompanied by various complications such as porcine reproductive and respiratory syndrome, porcine parvovirus infection, Mycoplasma pneumoniae infection, and Haemophilus parasuis infection.

[0003] Effective guarantee of laboratory diagnostic techniques plays a crucial role in the detection of PCV2 infection in pig herds and the prevention and control of diseases. The diagnosis of PCV2 relies on virus isolation and identification, molecular biology techniques, and immunological diagnostic methods. PCV2 can proliferate and be passaged in PK-15 cells and ST cells. However, since no obvious cytopathic effect is produced, subsequent identification requires the application of specifically recognizing antibodies. Polyclonal antibodies can be obtained relatively quickly by preparing immune sera from pigs or mice, but misjudgments often occur due to complex serum backgrounds, limited antibody titers, and weak specificity. Therefore, researchers tend to use monoclonal antibodies with strong specificity and high sensitivity. By preparing hybridoma cell lines that can secrete monoclonal antibodies specifically recognizing PCV2, the monoclonal antibodies expressed by them can be used to identify PCV2 antigens in cells or determine virus titers through indirect immunofluorescence assay (IFA) or peroxidase monolayer assay (IPMA). Molecular biology techniques, namely detection methods targeting the nucleic acid components of pathogens in the tested samples, currently mainly include polymerase chain reaction, gene chip detection, nucleic acid hybridization detection techniques, etc. These techniques have high requirements for technology and equipment and are not suitable for actual production and breeding. Immunological diagnostic methods include immunohistochemical staining (IHC), enzyme-linked immunosorbent assay (ELISA), etc. The above technical methods have high requirements for monoclonal antibodies that specifically recognize PCV2 strains. Screening monoclonal antibodies that can accurately recognize different PCV2 genotypes is of great significance for in-depth understanding of the virus and improvement of disease diagnostic techniques. Summary of the Invention

[0004] To solve the above problems, the present invention provides an anti-PCV2 monoclonal antibody, a hybridoma cell line, and their applications. The anti-PCV2 monoclonal antibody provided by the present invention can specifically recognize different genotype strains of PCV2. When used in combination, it can distinguish PCV2d genotype strains from PCV2a and PCV2b genotype strains.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an anti-PCV2 monoclonal antibody, including monoclonal antibody 5A8 and / or monoclonal antibody 4C2; the amino acid sequence of the heavy chain variable region of monoclonal antibody 5A8 is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of monoclonal antibody 4C2 is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:7.

[0007] Preferably, the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody 5A8 is as shown in SEQ ID NO: 2, and the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody 5A8 is as shown in SEQ ID NO: 4; the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody 4C2 is as shown in SEQ ID NO: 6, and the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody 4C2 is as shown in SEQ ID NO: 8.

[0008] Preferably, the subtype of the monoclonal antibody 5A8 is IgG2α, and the light chain is of the Kappa type; the subtype of the monoclonal antibody 4C2 is IgG1, and the light chain is of the Kappa type.

[0009] The present invention provides hybridoma cell lines secreting the anti-PCV2 monoclonal antibodies described in the above technical solutions. The preservation number of the hybridoma cell line secreting the monoclonal antibody 5A8 is CGMCC NO. 46256; the preservation number of the hybridoma cell line secreting the monoclonal antibody 4C2 is CGMCC NO. 46257.

[0010] The present invention provides the application of the anti-PCV2 monoclonal antibody described in the above technical solution or the hybridoma cell line described in the above technical solution in detecting PCV2 and / or PCV2 Cap protein, and the direct purpose of the application is non-diagnostic and non-therapeutic.

[0011] The present invention provides the application of the anti-PCV2 monoclonal antibody described in the above technical solution or the hybridoma cell line described in the above technical solution in preparing products for detecting PCV2 and / or PCV2 Cap protein.

[0012] Preferably, the products include products for identifying different PCV2 genotypes; the different PCV2 genotypes include PCV2a, PCV2b, and PCV2d.

[0013] Preferably, the products include reagents or kits.

[0014] The present invention provides a kit for detecting PCV2 and / or PCV2 Cap protein, which includes the anti-PCV2 monoclonal antibody described in the above technical solution or the hybridoma cell line described in the above technical solution.

[0015] Preferably, the kit further includes a fluorescently labeled anti-IgG antibody.

[0016] Beneficial effects:

[0017] The present invention provides an anti-PCV2 monoclonal antibody, including monoclonal antibody 5A8 and / or monoclonal antibody 4C2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5A8 is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4C2 is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:7. The monoclonal antibodies 5A8 and 4C2 in the anti-PCV2 monoclonal antibody provided by the present invention can specifically recognize different genotype strains of PCV2. When used in combination, they can distinguish PCV2d genotype strains from PCV2a and PCV2b genotype strains. The specificity experiment of the monoclonal antibody shows that the monoclonal antibody provided by the present invention has no cross-reactivity with other common porcine viruses, such as classical swine fever virus, African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, rotavirus, etc., and has good sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0019] Figure 1 It is the protein electrophoresis pattern of monoclonal antibody 5A8;

[0020] Figure 2 It is the protein electrophoresis pattern of monoclonal antibody 4C2;

[0021] Figure 3 It is the detection result of monoclonal antibody 5A8 for PCV2a, PCV2b and PCV2d;

[0022] Figure 4 It is the detection result of monoclonal antibody 4C2 for PCV2a, PCV2b and PCV2d;

[0023] Figure 5 It is the sensitivity detection result of monoclonal antibody 5A8 and monoclonal antibody 4C2;

[0024] Figure 6 It is the detection result of monoclonal antibody 5A8 for PK15 cells infected with porcine circovirus type 2 (200×);

[0025] Figure 7 It is the detection result of monoclonal antibody 5A8 for PK15 cells infected with porcine pseudorabies virus;

[0026] Figure 8 It is the detection result of monoclonal antibody 5A8 for MARC145 cells infected with porcine reproductive and respiratory syndrome virus;

[0027] Figure 9Detection results of monoclonal antibody 5A8 on Vero cells infected with porcine epidemic diarrhea virus;

[0028] Figure 10 Detection results of monoclonal antibody 5A8 on PK15 cells infected with transmissible gastroenteritis virus of swine;

[0029] Figure 11 Detection results of monoclonal antibody 5A8 on MA104 cells infected with porcine rotavirus;

[0030] Figure 12 Detection results of monoclonal antibody 5A8 on PK15 cells infected with porcine parvovirus;

[0031] Figure 13 Detection results of monoclonal antibody 5A8 on BHK21 cells infected with Japanese encephalitis virus of swine;

[0032] Among them, Figures 7 to 13 under the microscope, it is 200×. The left figure is the bright field of view, and the right figure is the detection result of blue excitation light.

[0033] Biological deposit description

[0034] The hybridoma cell line PCV2-5A8, scientifically described as a PCV2 monoclonal antibody hybridoma cell, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 13, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 46256.

[0035] The hybridoma cell line PCV2-4C2, scientifically described as a hybridoma cell, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 13, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 462567. Detailed implementation manners

[0036] The present invention provides an anti-PCV2 monoclonal antibody, including monoclonal antibody 5A8 and / or monoclonal antibody 4C2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5A8 is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4C2 is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 7.

[0037] As an implementation manner, the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody 5A8 is as shown in SEQ ID NO: 2, and the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody 5A8 is as shown in SEQ ID NO: 4; the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody 4C2 is as shown in SEQ ID NO: 6, and the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody 4C2 is as shown in SEQ ID NO: 8.

[0038] As an implementation manner, the subtype of the monoclonal antibody 5A8 is IgG2α, and the light chain is of the Kappa type; the subtype of the monoclonal antibody 4C2 is IgG1, and the light chain is of the Kappa type.

[0039] The monoclonal antibodies 5A8 and 4C2 in the anti-PCV2 monoclonal antibodies provided by the present invention can specifically recognize different genotype strains of PCV2. When used in combination, they can distinguish between PCV2d genotype strains and PCV2a and PCV2b genotype strains. The determination criterion is as follows: when the detection results of both monoclonal antibodies 5A8 and 4C2 are positive, it indicates that the infected strain is one of PCV2a, PCV2b, and PCV2d; when the detection result of monoclonal antibody 5A8 is positive and the detection result of monoclonal antibody 4C2 is negative, it indicates that the infected strain type is not PCV2d but one of PCV2a or PCV2b. The specificity experiment of the monoclonal antibody shows that the monoclonal antibodies provided by the present invention have no cross-reactivity with other common porcine viruses, such as classical swine fever virus, African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, rotavirus, etc., and have good sensitivity and specificity. They can be used to detect PCV2 and / or PCV2 Cap protein, or to prepare products for detecting PCV2 and / or PCV2 Cap protein.

[0040] The present invention also provides hybridoma cell lines secreting the anti-PCV2 monoclonal antibodies described in the above technical solution. The preservation number of the hybridoma cell line secreting the monoclonal antibody 5A8 is CGMCC NO. 46256; the preservation number of the hybridoma cell line secreting the monoclonal antibody 4C2 is CGMCC NO. 46257.

[0041] Based on the above advantages, the present invention provides the application of the anti-PCV2 monoclonal antibody or the hybridoma cell line described in the above technical solution in detecting PCV2 and / or PCV2 Cap protein, and the direct purpose of the application is non-diagnostic and non-therapeutic. The anti-PCV2 monoclonal antibody or hybridoma cell line provided by the present invention can be used to detect whether a test sample contains the intermediate value of detecting PCV2 and / or PCV2 Cap protein, and the test sample can be food.

[0042] Based on the above advantages, the present invention provides the application of the anti-PCV2 monoclonal antibody described in the above technical solution or the hybridoma cell line described in the above technical solution in the preparation of products for detecting PCV2 and / or PCV2 Cap protein.

[0043] As an embodiment, the product includes products that recognize different PCV2 genotypes; the different PCV2 genotypes include PCV2a, PCV2b, and PCV2d.

[0044] As an embodiment, the product includes a reagent or a kit. As another embodiment, the reagent or kit can be a reagent or kit for indirect immunofluorescence detection.

[0045] Based on the above advantages, the present invention provides a kit for detecting PCV2 and / or PCV2 Cap protein, including the anti-PCV2 monoclonal antibody described in the above technical solution or the hybridoma cell line described in the above technical solution.

[0046] As an embodiment, the kit further includes a fluorescently labeled anti-IgG antibody.

[0047] To further illustrate the present invention, the following describes in detail an anti-PCV2 monoclonal antibody, a hybridoma cell line, and their applications provided by the present invention in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0048] Example 1 Preparation of PCV2 Monoclonal Antibody Hybridoma Cell Line

[0049] (1) Animal immunization:

[0050] Take 5 pregnant BALB / C female mice. After mixing and emulsifying PCV2 Cap protein (purchased from Yangzhou Youbang Biopharmaceutical Co., Ltd.) with an equal volume of Freund's complete adjuvant, inject it intradermally at multiple points on the back of the mice at a dose of 100 μg / mouse; after 14 days, use PCV2 Cap protein mixed and emulsified with an equal amount of Freund's incomplete adjuvant, and inject it intradermally at multiple points on the back of the mice at a dose of 100 μg / mouse for booster immunization; after 14 days, boost immunize once again according to the above protocol. After 14 days, collect blood from the orbital cavity, separate the serum, and measure the antibody titer of the immunized mice by the indirect ELISA method. Select the mouse with the highest antibody titer and inject PCV2 Cap protein into the abdominal cavity at a dose of 100 μg / mouse. After 4 days, decapitate and sacrifice the immunized mice, aseptically remove the spleens, grind and filter them through 150-mesh and 200-mesh copper meshes respectively to obtain spleen cells for cell fusion.

[0051] (2) Cell fusion:

[0052] One day before cell fusion, mouse peritoneal macrophages were prepared and seeded in a 96-well cell culture plate. On the day of fusion, the splenocytes obtained in step (1) were mixed with SP2 / 0 cells in the logarithmic growth phase. The ratio of the number of splenocytes to SP2 / 0 cells was 5:1. After centrifugation, the supernatant was discarded. 1 mL of 50% polyethylene glycol (PEG) was slowly added to the cell mixture within 1 min, and then 10 mL of DMEM medium was added step by step to terminate the reaction. After centrifugation, the cells were resuspended in DMEM medium containing 1% HAT culture solution and 15% fetal bovine serum, and seeded in the 96-well cell culture plate containing mouse peritoneal macrophages. The cells were cultured at 37 °C in 5% CO2 for one week to obtain fused cells.

[0053] (3) Screening and subcloning of hybridoma cells:

[0054] The PCV2 virus culture solution was inoculated into a 96-well PK15 cell plate. After culturing at 37 °C in 5% CO2 for 96 h, the culture solution was discarded and the cells were fixed for screening of the fused cells.

[0055] The supernatant of the fused cells obtained in step (2) was added to the fixed PK15 cell plate. The reaction was carried out at 37 °C for 1 h, followed by rinsing 3 times with PBS. Then, FITC-labeled goat anti-mouse secondary antibody was added and the reaction was carried out at 37 °C for 1 h. After rinsing 3 times with PBS, the cells were observed under an inverted fluorescence microscope. The hybridoma cells corresponding to the positive wells showing specific intracellular staining were subcloned by the limiting dilution method. The above screening method was repeated for 3 - 4 rounds, and finally two hybridoma cell lines stably secreting PCV2 Cap protein monoclonal antibodies were obtained, named PCV2-5A8 and PCV2-4C2 respectively.

[0056] The hybridoma cell lines PCV2-5A8 and PCV2-4C2 were deposited with the China General Microbiological Culture Collection Center (CGMCC), and the deposit numbers were CGMCC NO.46256 and CGMCC NO.46257 respectively.

[0057] Example 2 Preparation of monoclonal antibody

[0058] 1. Preparation of monoclonal antibody ascites:

[0059] Two pregnant BALB / C mice were intraperitoneally injected with sterile liquid paraffin, 0.5 mL / mouse. One week later, they were intraperitoneally injected with the hybridoma cells PCV2-5A8 and PCV2-4C2 obtained in Example 1, 1 - 2×10 5 cells / mL, 0.5 mL / mouse. When the mouse abdomen swelled and showed an obvious sense of fluctuation, ascites was aspirated with a syringe. The aspirated ascites was centrifuged at 10000 r / min for 10 min, and the supernatant was stored at -20 °C.

[0060] 2. Purification of monoclonal antibody:

[0061] Centrifuge the collected ascites of monoclonal antibody at 10,000 r / min for 10 min, take the supernatant, add 40 μL of 10% dextran sulfate solution and 1 mL of CaCl2 solution to each milliliter of ascites. After mixing at room temperature for 15 min, centrifuge at 10,000 r / min for 10 min, and take the supernatant. Perform desalting treatment with a Sephadex G-50 chromatography column, and purify the collected protein according to the instructions of the HiTrap Protein G HP affinity chromatography column, and collect the target protein peak.

[0062] Take a sample of the purified antibody for protein electrophoresis analysis, and the results are as Figure 1 and Figure 2 shown. The results show that there are only two bands of heavy chain and light chain in the purified antibody, and the purification effect is good.

[0063] 3. Identification of monoclonal antibody:

[0064] Entrust Beijing BGI Protein R & D Center Co., Ltd. to identify the two monoclonal antibodies, and the results are as follows:

[0065] The affinity constant K values of monoclonal antibody 5A8 secreted by hybridoma cell PCV2-5A8 and monoclonal antibody 4C2 secreted by hybridoma cell PCV2-4C2 are 1.09×10 -9 mol / L and 5.2×10 -9 mol / L respectively. The subtype of monoclonal antibody 5A8 is IgG2α, and the light chain is Kappa type; the subtype of monoclonal antibody 4C2 is IgG1, and the light chain is Kappa type.

[0066] The amino acid sequence of the heavy chain of monoclonal antibody 5A8 is as shown in SEQ ID NO:1, specifically as follows:

[0067] DVQLVESGGGLVQPGGSRKLSCAASGFTFRSFGMHWVRQAPGKGLEWV AYISSGSSASISYADTVKGRFTISRDNPRDTLFLQMTSLRSEDTAMYYCARFPD YYGGLYAMDYWGHGTSVTVSS;

[0068] The nucleotide sequence of the heavy chain of monoclonal antibody 5A8 is as shown in SEQ ID NO:2, specifically as follows:

[0069] 5'-GACGTGCAGCTGGTGGAATCAGGAGGTGGACTGGTGCAGCCAGGA GGCAGTAGAAAGCTGTCATGCGCTGCATCTGGCTTTACCTTCAGGAGCTTCGGAATGCATTGGGTCCGGCAGGCTCCAGGAAAAGGACTCGAATGGGTGGCCTACATCTCTAGTGGCTCTAGCGCCAGCATCAGCTACGCCGATACCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACCCAAGAGACACACTGTTCCTCCAGATGACCAGCCTGAGAAGCGAGGACACAGCCATGTACTATTGCGCTAGATTCCCAGACTACTACGGCGGACTGTACGCCATGGACTATTGGGGCCACGGAACCAGCGTCACAGTGTCTAGC-3';

[0070] The amino acid sequence of the light chain of monoclonal antibody 5A8 is shown in SEQ ID NO:3, as follows:

[0071] DIQMTQSPASLSVSVGETVTITCRASENIYSNLVWYQQKQGKSPQLLVYG ATSLADGVPSRFSGSGSGAQYSLKIDSLQSGDFGNYYCQHFWGTLFTFGSGT KLEIKR;

[0072] The nucleotide sequence of the light chain of monoclonal antibody 5A8 is shown in SEQ ID NO:4, as follows:

[0073] 5'-GACATCCAGATGACCCAGAGCCCAGCTTCACTGTCTGTGTCCGTGG GAGAGACAGTGACAATCACTTGCAGAGCCAGCGAGAACATCTACAGCAACCTCGTCTGGTACCAGCAGAAGCAGGGCAAGTCTCCTCAGCTCCTGGTGTACGGAGCTACATCTCTGGCAGACGGAGTGCCTAGCAGATTCAGCGGCAGCGGTTCAGGAGCTCAGTACAGCCTGAAGATCGACAGCCTGCAGAGCGGCGACTTCGGCAACTACTACTGCCAGCACTTTTGGGGCACACTGTTCACCTTCGGAAGCGGTACCAAGCTGGAGATCAAGAGA-3';

[0074] The amino acid sequence of the heavy chain of monoclonal antibody 4C2 is shown in SEQ ID NO:5 as follows:

[0075] QVQLQQSGPELVGPGVSVKISCTGSGYTFTDRAVHWVKQSHAKSLEWIG VFSTYYGNTNYNQKFQGKATMTVDKSSNTVYMELARLTSEDSAIYYCAREGI YYYGSSPHWYFDVWGAGTTVTVSS;

[0076] The nucleotide sequence of the heavy chain of monoclonal antibody 4C2 is shown in SEQ ID NO:6 as follows:

[0077] 5'-CAGGTGCAACTGCAACAGTCAGGACCAGAACTGGTCGGACCAGGA GTGTCCGTGAAGATCTCTTGCACCGGCAGCGGCTACACCTTTACCGACAGAGCCGTGCATTGGGTGAAGCAGAGCCACGCTAAGAGCCTGGAGTGGATTGGCGTGTTCAGCACCTACTACGGCAACACCAACTACAACCAGAAGTTCCAGGGCAAGGCCACCATGACCGTGGACAAGAGCAGCAACACCGTGTACATGGAGCTGGCCAGACTGACCAGCGAGGATTCAGCCATCTACTATTGCGCCAGGGAGGGCATCTACTACTACGGCAGCAGCCCCCATTGGTACTTCGACGTCTGGGGAGCCGGTACCACAGTGACCGTGTCTAGC-3';

[0078] The amino acid sequence of the light chain of monoclonal antibody 4C2 is shown in SEQ ID NO:7 as follows:

[0079] QIVLTQSPTIMSTSPGEKVTMTCSASLSVSYIHWYQQKPGSSPKPWIYATS NLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSKSWTFGGGTKLE IKR;

[0080] The nucleotide sequence of the light chain of monoclonal antibody 4C2 is shown in SEQ ID NO:8 as follows:

[0081] 5'-CAGATTGTGCTGACCCAGAGCCCTACCATCATGTCTACCAGCCCAG GCGAGAAAGTGACCATGACTTGTAGCGCTAGTCTGAGCGTGTCCTACATCCATTGGTACCAGCAGAAGCCCGGAAGCAGCCCTAAGCCTTGGATCTACGCTACCAGCAATCTGGCCAGCGGAGTGCCAGCTAGATTCAGTGGTAGCGGATCAGGCACAAGCTACAGCCTGACCATCAGCAGAGTGGAGGCTGAAGACGCAGCCACCTACTATTGCCAACAGTGGAGTAGCAAGTCTTGGACATTCGGTGGCGGAACCAAGCTGGAGATCAAGAGA-3'.

[0082] Example 3

[0083] The monoclonal antibodies 5A8 and 4C2 in Example 2 were used to detect PK15 cells infected with three genotypes of PCV2 virus (PCV2a, PCV2b, PCV2d) respectively. The steps were as follows:

[0084] (1) Dilute 10×PBS to 1×PBS as the working concentration.

[0085] (2) Take the monolayer of PK15 cells to be tested, discard the culture medium, and wash twice with PBS. Each time when washing, add 300 μL of PBS to each well and discard the PBS. The monolayer of PK15 cells was infected with three genotypes of PCV2 virus respectively.

[0086] (3) Add 100 μL of 80% cold acetone solution to each well, fix at 2 - 8 °C for 30 min, then discard the acetone and air dry naturally.

[0087] (4) Wash the cell surface once with PBS, then add 50 μL of each of the monoclonal antibodies 5A8 and 4C2 (both at a concentration of 0.5 μg / mL) to the wells infected with different genotypes of PCV2, and incubate in a humid box at 37 °C for 1 h.

[0088] (5) Repeat step (2).

[0089] (6) Discard the washing solution, add FITC-labeled goat anti-mouse IgG (diluted 1:500) at a dose of 50 μL / well, and incubate in a humid box at 37 °C for 1 h.

[0090] (7) Repeat step (2).

[0091] (8) Observe under an inverted microscope with blue excitation light (wavelength 490 nm) at magnifications of 100× and 200×.

[0092] Result judgment criteria: If specific yellow-green fluorescence appears in the nuclei of the cells to be tested, it is judged as positive; if no specific yellow-green fluorescence appears in the nuclei of the cells to be tested, it is judged as negative.

[0093] The results are shown in Figures 3 to 4 . It can be seen from the results that both monoclonal antibodies 5A8 and 4C2 can recognize PCV2a or PCV2b strains, and monoclonal antibody 4C2 cannot recognize the PCV2d strain.

[0094] According to the above results, monoclonal antibodies 5A8 and 4C2 can be used in combination for detection, and the judgment criteria are as follows: When both monoclonal antibodies 5A8 and 4C2 show intracellular yellow-green fluorescence in the detection, it indicates that the infected strain is PCV2a or PCV2b; when monoclonal antibody 5A8 shows intracellular yellow-green fluorescence in the detection and monoclonal antibody 4C2 shows no fluorescence, it indicates that the infected strain is PCV2d.

[0095] Example 4 Sensitivity Detection

[0096] Monoclonal antibodies 5A8 and 4C2 were respectively diluted to three concentrations of 0.5 μg / mL, 0.05 μg / mL, and 0.005 μg / mL with 1×PBS. Referring to the method of Example 3, PK15 cells infected with PCV2a virus were detected using different concentrations of monoclonal antibody 5A8. The detection results are shown in Figure 5 .

[0097] It can be Figure 5 seen that monoclonal antibodies 5A8 and 4C2 have good sensitivity.

[0098] Example 5 Specificity Detection

[0099] Monoclonal antibody 5A8 in Example 2 was used to detect cells infected with porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus of swine (TGEV), rotavirus of swine (RV), porcine parvovirus (PPV), and Japanese encephalitis virus of swine (JEV). The detection method referred to Example 3. Among them, PCV2, PRV, TGEV, and PPV infected PK15 cells, PRRSV infected MARC145 cells, PEDV infected Vero cells, RV infected MA104 cells, and JEV infected BHK21 cells.

[0100] The results are shown in Figures 6 to 13The results showed that when the monoclonal antibody 5A8 was used to detect the positive cell fixing plates infected with porcine circovirus type 2, specific yellow-green fluorescence in the cell nuclei appeared in the wells of the cells to be tested, indicating a positive result. When the monoclonal antibody 5A8 was used to detect the positive cell fixing plates infected with porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus of swine, porcine rotavirus, porcine parvovirus, and porcine Japanese encephalitis virus, no specific yellow-green fluorescence in the cell nuclei appeared in the wells of the cells to be tested, all indicating negative results. It can be seen that the monoclonal antibody 5A8 of the present invention has good specificity for porcine circovirus type 2.

[0101] In summary, the hybridoma cell line provided by the present invention, the monoclonal antibody 5A8 and the monoclonal antibody 4C2 secreted by it can specifically recognize different genotype strains of PCV2. When used in combination, they can distinguish PCV2d genotype strains from PCV2a and PCV2b genotype strains. The specificity experiment of the monoclonal antibody showed that the monoclonal antibody provided by the present invention has no reactivity to other common porcine viruses, such as classical swine fever virus, African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, rotavirus, etc., and has good sensitivity and specificity.

[0102] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against PCV2, characterized in that, It includes monoclonal antibody 5A8 and / or monoclonal antibody 4C2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5A8 is shown as SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4C2 is shown as SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

7.

2. The anti-PCV2 monoclonal antibody according to claim 1, wherein The nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody 5A8 is shown as SEQ ID NO:2, and the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody 5A8 is shown as SEQ ID NO:4; the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody 4C2 is shown as SEQ ID NO:6, and the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody 4C2 is shown as SEQ ID NO:

8.

3. The anti-PCV2 monoclonal antibody according to claim 1 or 2, characterized in that, The subtype of the monoclonal antibody 5A8 is IgG2α, and the light chain is of the Kappa type; the subtype of the monoclonal antibody 4C2 is IgG1, and the light chain is of the Kappa type.

4. A hybridoma cell line secreting the anti-PCV2 monoclonal antibody according to any one of claims 1 to 3, characterized in that, The preservation number of the hybridoma cell line secreting the monoclonal antibody 5A8 is CGMCC No.46256; the preservation number of the hybridoma cell line secreting the monoclonal antibody 4C2 is CGMCC No.46257.

5. The application of the anti-PCV2 monoclonal antibody according to any one of claims 1 to 3 or the hybridoma cell line according to claim 4 in detecting PCV2 and / or PCV2 Cap protein, and the direct purpose of the application is non-diagnostic and non-therapeutic.

6. The application of the anti-PCV2 monoclonal antibody according to any one of claims 1 to 3 or the hybridoma cell line according to claim 4 in preparing a product for detecting PCV2 and / or PCV2 Cap protein.

7. The application according to claim 6, characterized in that, The product includes products for identifying different PCV2 genotypes; the different PCV2 genotypes include PCV2a, PCV2b and PCV2d.

8. The application according to claim 6 or 7, characterized in that, The product includes reagents or reagent kits.

9. A kit for detecting PCV2 and / or PCV2 Cap protein, characterized in that, It includes the anti-PCV2 monoclonal antibody according to any one of claims 1 to 3 or the hybridoma cell line according to claim 4.

10. The kit according to claim 9, wherein The reagent kit further includes a fluorescently labeled anti-IgG antibody.

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