Anti-PCV2 monoclonal antibody, hybridoma cell strain and application thereof

Abstract

The invention relates to the technical field of veterinary biomedicine, in particular to an anti-PCV2 monoclonal antibody, a hybridoma cell strain and application thereof. According to the hybridoma cell strain and the monoclonal antibody 5A8 and the monoclonal antibody 4C2 secreted by the hybridoma cell strain, different genotype strains of PCV2 can be specifically recognized, and the PCV2d genotype strain can be identified from PCV2a and PCV2b genotype strains when the hybridoma cell strain and the monoclonal antibody 5A8 and the monoclonal antibody 4C2 are combined for use. Specificity experiments of the monoclonal antibody show that the monoclonal antibody provided by the invention has no reactivity to other common swine viruses, such as hog cholera virus, African hog cholera virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, rotavirus and the like, and has good sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biomedicine, and in particular to an anti-PCV2 monoclonal antibody, a hybridoma cell line and applications thereof. Background Art

[0002] Porcine circovirus type (PCV) is a member of the circovirus genus of the Circoviridae family. The virus particles are non-enveloped and have an icosahedral symmetric structure. They contain 1.76kb to 2.3kb of single-stranded circular negative-strand DNA and are about 20nm in diameter. They are the smallest animal viruses that can replicate autonomously in mammalian cells. Early studies believed that PCV has two serotypes. Among them, porcine circovirus type 1 (PCV1) is widely present in pigs and is non-pathogenic. The other serotype, porcine circovirus type 2 (PCV2), has a nucleotide sequence homology of 68% to 76% with PCV1 and is pathogenic. The genome of PCV2 includes two main open reading frames (ORFs). ORF2 encodes the only structural protein of the virus, Cap protein, which is the main immunogenic protein of PCV2 and can induce the body to produce neutralizing antibodies. It is closely related to the pathogenicity of the virus. PCV2 mainly invades the immune system of pigs, leading to immunosuppression and reducing the body's resistance, which in turn leads to other diseases. The genotypes of PCV2 prevalent in China include PCV2a, PCV2b, and PCV2d. As the virus spreads, studies have found that there is antigenic variability among multiple genotypes of PCV2. Clinical cases of PCV2 infection are often accompanied by multiple complications such as porcine reproductive and respiratory syndrome, porcine parvovirus infection, Mycoplasma pneumoniae infection, and Haemophilus parasuis infection.

[0003] The effective guarantee of laboratory diagnostic technology plays a vital role in the infection of PCV2 in pig herds and the prevention and control of diseases. The diagnosis of PCV2 depends on the isolation and identification of the virus, molecular biology technology, and immunological diagnostic methods. PCV2 can proliferate and propagate in PK-15 cells and ST cells, but since it does not produce obvious cytopathic effects, subsequent identification requires the application of specific recognition antibodies. Polyclonal antibodies can be quickly obtained by homemade pig or mouse immune serum, but they often lead to misjudgment due to complex serum background, limited antibody titer, and weak specificity. Therefore, researchers prefer to use monoclonal antibodies with strong specificity and high sensitivity. By preparing hybridoma cell lines that can secrete monoclonal antibodies that specifically recognize PCV2, the monoclonal antibodies expressed by them can be used to identify PCV2 antigens in cells or determine the toxicity through indirect immunofluorescence assay (IFA) or peroxidase monolayer assay (IPMA). Molecular biology technology, that is, the detection method for the nucleic acid components of pathogens in the sample being tested, currently mainly includes polymerase chain reaction, gene chip detection, nucleic acid hybridization detection technology, etc., which have high requirements for technology and equipment and are not suitable for actual production and breeding links. Immunological diagnostic methods include immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), etc. The above technical methods have high requirements for monoclonal antibodies that specifically recognize PCV2 strains, and screening monoclonal antibodies that can accurately identify different PCV2 genotypes is of great significance for the in-depth understanding of the virus and the improvement of disease diagnostic technology. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an anti-PCV2 monoclonal antibody, a hybridoma cell line and its application. The anti-PCV2 monoclonal antibody provided by the present invention can specifically identify different genotypes of PCV2 strains, and when used in combination, it can distinguish PCV2d genotype strains from PCV2a and PCV2b genotype strains.

[0005] In order 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 the monoclonal antibody 5A8 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4C2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7.

[0007] Preferably, 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.

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

[0009] The present invention provides a hybridoma cell strain secreting the anti-PCV2 monoclonal antibody described in the above technical solution. The deposit number of the hybridoma cell strain secreting the monoclonal antibody 5A8 is CGMCCNO.46256; the deposit number of the hybridoma cell strain secreting the monoclonal antibody 4C2 is CGMCCNO.46257.

[0010] The present invention provides the use 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, wherein the direct purpose of the use is non-diagnosis and non-treatment.

[0011] The present invention provides the use 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 a product for detecting PCV2 and / or PCV2 Cap protein.

[0012] Preferably, the product comprises a product for identifying different PCV2 genotypes; the different PCV2 genotypes comprise PCV2a, PCV2b and PCV2d.

[0013] Preferably, the product comprises a reagent or a kit.

[0014] The present invention provides a kit for detecting PCV2 and / or PCV2 Cap protein, comprising 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 comprises 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 shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4C2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7. The monoclonal antibody 5A8 and the monoclonal antibody 4C2 in the anti-PCV2 monoclonal antibody provided by the present invention can specifically identify different genotype strains of PCV2, and when used in combination, the PCV2d genotype strain can be distinguished from the 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 to other common swine-derived viruses, such as 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 embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

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

[0020] Figure 2 This is the protein electrophoresis diagram of monoclonal antibody 4C2;

[0021] Figure 3 The results of the detection of monoclonal antibody 5A8 against PCV2a, PCV2b and PCV2d;

[0022] Figure 4 The results of the monoclonal antibody 4C2 against PCV2a, PCV2b and PCV2d;

[0023] Figure 5 These are the sensitivity test results of mAb 5A8 and mAb 4C2;

[0024] Figure 6 The results of the detection of monoclonal antibody 5A8 on PK15 cells infected with porcine circovirus type 2 (200×);

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

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

[0027] Fig. 9This is the detection result of monoclonal antibody 5A8 on Vero cells infected with porcine epidemic diarrhea virus;

[0028] Fig.10 The results of the monoclonal antibody 5A8 on PK15 cells infected with porcine transmissible gastroenteritis virus;

[0029] Fig.11 This is the detection result of monoclonal antibody 5A8 on MA104 cells infected with porcine rotavirus;

[0030] Fig.12 This is the detection result of monoclonal antibody 5A8 on PK15 cells infected with porcine parvovirus;

[0031] Fig.13 The results of the monoclonal antibody 5A8 on BHK21 cells infected with porcine Japanese encephalitis virus;

[0032] in, Figures 7 to 13 In the figure, the microscope is 200×, the left picture is the bright field, and the right picture is the blue excitation light detection result.

[0033] Biological Deposit Description

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

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

[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 shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4C2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7.

[0037] As an embodiment, 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.

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

[0039] The monoclonal antibody 5A8 and the monoclonal antibody 4C2 in the anti-PCV2 monoclonal antibody provided by the present invention can specifically identify different genotype strains of PCV2. The combination of the two can distinguish the PCV2d genotype strain from the PCV2a and PCV2b genotype strains. The judgment standard is: when the test results of monoclonal antibodies 5A8 and 4C2 are both positive, it means that the infected strain is one of PCV2a, PCV2b, and PCV2d; when the test result of monoclonal antibody 5A8 is positive and the test result of monoclonal antibody 4C2 is negative, it means 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 antibody provided by the present invention has no cross-reactivity to other common swine-derived viruses, such as swine fever virus, African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, rotavirus, etc., has good sensitivity and specificity, and 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 a hybridoma cell strain secreting the anti-PCV2 monoclonal antibody described in the above technical solution. The deposit number of the hybridoma cell strain secreting the monoclonal antibody 5A8 is CGMCC NO.46256; the deposit number of the hybridoma cell strain secreting the monoclonal antibody 4C2 is CGMCC NO.46257.

[0041] Based on the above advantages, the present invention provides the use of the anti-PCV2 monoclonal antibody or 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-diagnosis and non-treatment. The anti-PCV2 monoclonal antibody or hybridoma cell line provided by the present invention can be used to detect whether the sample to be tested contains the intermediate value of detecting PCV2 and / or PCV2 Cap protein, and the sample to be tested can be food.

[0042] Based on the above advantages, the present invention provides the use 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 a product for detecting PCV2 and / or PCV2 Cap protein.

[0043] As an embodiment, the product includes a product that identifies 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 the kit can be a reagent or a 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, comprising 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 comprises a fluorescently labeled anti-IgG antibody.

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

[0048] Example 1 Preparation of PCV2 monoclonal antibody hybridoma cell line

[0049] (1) Animal immunization:

[0050] Five BALB / C female mice were selected. PCV2 Cap protein (purchased from Yangzhou Youbang Biopharmaceutical Co., Ltd.) was mixed and emulsified with an equal volume of Freund's complete adjuvant, and then injected at multiple points on the back of the mice at a dose of 100 μg / mouse. After 14 days, PCV2 Cap protein was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and then injected at multiple points on the back of the mice at a dose of 100 μg / mouse for booster immunization. After 14 days, booster immunization was performed once more according to the above scheme. After 14 days, blood was collected from the eye sockets, serum was separated, and the antibody titer of the immunized mice was determined by the indirect ELISA method. The mice with the highest antibody titer were selected and intraperitoneally injected with PCV2 Cap protein at a dose of 100 μg / mouse. After 4 days, the immunized mice were killed by cervical dislocation, and the spleens were aseptically removed, ground and filtered with 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 plated in a 96-well cell culture plate. On the day of fusion, the spleen cells obtained in step (1) were mixed with SP2 / 0 cells in the logarithmic growth phase, with the ratio of the number of spleen cells to SP2 / 0 cells being 5:1. The supernatant was discarded after centrifugation, and 1 mL of 50% polyethylene glycol (PEG) was slowly added to the cell mixture within 1 min, followed by stepwise addition of 10 mL of DMEM culture medium to terminate the process. After centrifugation, the cells were resuspended in DMEM culture medium containing 1% HAT culture medium and 15% fetal bovine serum, and plated in a 96-well cell culture plate cultured with mouse peritoneal macrophages, and incubated at 37°C with 5% CO 2 After culturing for one week, fusion cells were obtained.

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

[0054] The PCV2 virus culture medium was inoculated into a 96-well PK15 cell plate and incubated at 37°C with 5% CO 2 After 96 h of culture, the culture medium was discarded and the cells were fixed for screening of fusion cells.

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

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

[0057] Example 2 Preparation of monoclonal antibodies

[0058] 1. Preparation of monoclonal antibody ascites:

[0059] Two BALB / C mice were intraperitoneally injected with sterile liquid paraffin (0.5 mL / mouse). One week later, the hybridoma cells PCV2-5A8 and PCV2-4C2 obtained in Example 1 were intraperitoneally injected with 1-2×10 5 / mL, 0.5mL / mouse. When the abdominal cavity of the mouse swells and shows obvious fluctuation, use a syringe to extract the ascites. Centrifuge the extracted ascites at 10000r / min for 10min, separate the supernatant and store it at -20℃.

[0060] 2. Purification of monoclonal antibodies:

[0061] The collected monoclonal antibody ascites was centrifuged at 10000r / min for 10min, the supernatant was taken, and 40μL 10% dextran sulfate solution and 1mL CaCl were added per mL of ascites. 2 Solution. After mixing at room temperature for 15 minutes, centrifuge at 10000r / min for 10 minutes and collect the supernatant. Desalt the mixture using a dextran G-50 column, purify the collected protein according to the instructions of the HiTrap Protein G HP affinity column, and collect the target protein peak.

[0062] The purified antibody was sampled for protein electrophoresis analysis, and the results were as follows Figure 1 and Figure 2 The results showed that the purified antibody had only two bands, heavy chain and light chain, and the purification effect was good.

[0063] 3. Identification of monoclonal antibodies:

[0064] Beijing BGI Protein Research and Development Center Co., Ltd. was commissioned to identify the two monoclonal antibodies, and the results are as follows:

[0065] The affinity constants K values ​​of monoclonal antibody 5A8 secreted by hybridoma cell PCV2-5A8 and monoclonal antibody 4C2 secreted by hybridoma cell PCV2-4C2 were 1.09×10 -9 mol / L and 5.2×10 -9 mol / L, the subtype of monoclonal antibody 5A8 is IgG2αa, 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 shown in SEQ ID NO: 1, and is as follows:

[0067] DVQLVESGGGLVQPGGSRKLSCAASGFTFRSFGMHWVRQAPGKGLEWV AYISSGSSASISYADTVKGRFTISRDNPRDTLFLQMTSLRSEDTAMYYCARFPD YYGGLYAMDYWGHGTSVTVSS;

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

[0077] 5'-CAGGTGCAACTGCAACAGTCAGGACCAGAACTGGTCGGACCAGGA GTGTCCGTGAAGATCTCTTGCACCGGCAGCGGCTACACCTTTACCGACAGAGCCGTGCATTGGGTGAAGCAGAGCCACGCTAAGAGCCTGGAGTGGATTGGCGTGTTCAGCACCTACTACGGCAACACCAACTACAACCAGAAGTTCCAGGGCAAGGCCACCATGAC CGTGGACAAGAGCAGCAACACCGTGTACATGGAGCTGGCCAGACTGACCAGCGAGGATTCAGCCATCTACTATTGCGCCAGGGAGGGCATCTACTACTACGGCAGCAGCCCCATTGGTACTTCGACGTCTGGGGAGCCGGTACCACAGTGACCGTGTCTAGC-3';

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

[0081] 5'-CAGATTGTGCTGACCCAGAGCCCTACCATCATGTCTACCAGCCCAG GCGAGAAAGTGACCATGACTTGTAGCGCTAGTCTGAGCGTGTCCTACATCCATTGGTACCAGCAGAAGCCCGGAAGCAGCCCTAAGCCTTGGATCTACGCTACCAGCAATCTGGCCAGCGGAGTGCCAGCTAGATTCAG TGGTAGCCGGATCAGGCACAAGCTACAGCCTGACCATCAGCAGAGTGGAGGCTGAAGACGCAGCCACCTACTATTGCCAACAGTGGAGTAGCAAGTCTTGGACATTCGGTGGCGGAACCAAGCTGGAGATCAAGAGA-3'.

[0082] Example 3

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

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

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

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

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

[0088] (5) Repeat (2).

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

[0090] (7) Repeat (2).

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

[0092] Result judgment criteria: If the specific yellow-green fluorescence in the nucleus of the cell well to be tested appears, it is judged as positive; if the specific yellow-green fluorescence in the nucleus of the cell well to be tested does not appear, it is judged as negative.

[0093] Results Figures 3-4 The results showed that both monoclonal antibodies 5A8 and 4C2 could identify PCV2a or PCV2b strains, but monoclonal antibody 4C2 could not identify PCV2d strains.

[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 monoclonal antibody 5A8 and 4C2 both show intracellular yellow-green fluorescence in the cells, it indicates that the virus is infected with PCV2a or PCV2b strains; when monoclonal antibody 5A8 shows intracellular yellow-green fluorescence and monoclonal antibody 4C2 shows no fluorescence, it indicates that the virus is infected with PCV2d strain.

[0095] Example 4 Sensitivity Detection

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

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

[0098] Example 5 Specificity Detection

[0099] The monoclonal antibody 5A8 in Example 2 was used to detect cells infected with porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (RV), porcine parvovirus (PPV), and porcine Japanese encephalitis virus (JEV), respectively. The detection method was referenced to Example 3, wherein 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] Results Figure 6 to Figure 13. The results show that monoclonal antibody 5A8 detects the positive cell fixed plate infected with porcine circovirus type 2, and the cell wells to be tested show specific yellow-green fluorescence in the nucleus, which is positive; monoclonal antibody 5A8 detects the positive cell fixed plate infected with pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine parvovirus and porcine Japanese encephalitis virus, and no specific yellow-green fluorescence in the nucleus appears in the cell wells to be tested, which are all negative. 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 and the monoclonal antibody 5A8 and monoclonal antibody 4C2 secreted therefrom can specifically identify different genotypes of PCV2 strains, and when used in combination, 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 had no reactivity to other common swine-derived viruses, such as swine fever virus, African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, rotavirus, etc., and had good sensitivity and specificity.

[0102] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An anti-PCV2 monoclonal antibody, characterized in that: 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 shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4C2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7.

2. The anti-PCV2 monoclonal antibody according to claim 1, characterized in that: 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 Kappa type; the subtype of the monoclonal antibody 4C2 is IgG1, and the light chain is 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 deposit number of the hybridoma cell line secreting the monoclonal antibody 5A8 is CGMCC NO.46256; the deposit number of the hybridoma cell line secreting the monoclonal antibody 4C2 is CGMCC NO.46257.

5. Use 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, wherein the direct purpose of the use is non-diagnostic and non-therapeutic.

6. Use 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 the preparation of a product for detecting PCV2 and / or PCV2 Cap protein.

7. The use according to claim 6, characterized in that: The products include products that identify different PCV2 genotypes; the different PCV2 genotypes include PCV2a, PCV2b and PCV2d.

8. The use according to claim 6 or 7, characterized in that: The product comprises a reagent or a kit.

9. A kit for detecting PCV2 and / or PCV2 Cap protein, characterized in that: It comprises 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, characterized in that The kit also includes a fluorescently labeled anti-IgG antibody.

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