A specific monoclonal antibody against Cap protein of pigeon circovirus and its application

Abstract

The present invention discloses a specific monoclonal antibody against the Cap protein of pigeon circovirus and its application. The monoclonal antibody of the present invention comprises a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region; the CDR of the heavy chain variable region comprises a CDR1 as shown in SEQ ID No.3, a CDR2 as shown in SEQ ID No.4 and a CDR3 as shown in SEQ ID No.5 in terms of amino acid sequence; the CDR of the light chain variable region comprises a CDR1 as shown in SEQ ID No.6, a CDR2 as shown in SEQ ID No.7 and a CDR3 as shown in SEQ ID No.8 in terms of amino acid sequence. The present invention obtains a pigeon circovirus blocking ELISA detection kit and a blocking ELISA detection method, which are simple to operate, have good specificity, high sensitivity, short time consumption, and a large application space, and can accurately and quickly determine PiCV specific antibodies in samples.

Description

Technical Field

[0001] The invention relates to the fields of molecular virology and immunotechnology, and in particular to a specific monoclonal antibody against Cap protein of pigeon circovirus and application thereof. Background Art

[0002] Pigeon circovirus (PiCV) infection can lead to the occurrence of young pigeon disease syndrome (YPDS), causing immunosuppression, seriously endangering the meat pigeon industry and the racing pigeon industry, and causing serious economic losses. Clinically, pigeons infected with PiCV often show atypical symptoms, such as lethargy, depression, diarrhea, crop effusion, vomiting, etc., and are more susceptible to infection with other viruses and bacteria. Under laboratory conditions, pigeons infected only with PiCV often do not show clinical symptoms. Due to the special habits of pigeons, the virus spreads widely and quickly.

[0003] PiCV belongs to the Circovirus genus of the Circoviridae family. The Circovirus genus includes pigeon circovirus, porcine circovirus (PCV), psittacine beak and feather disease virus (BFDV), goose circovirus (GoCV) and duck circovirus (DuCV). PiCV is a non-enveloped icosahedral particle with a diameter of 15-18nm, and its genome is a single-stranded circular DNA. The length of the complete PiCV genome reported so far varies slightly between different isolates, with a size of about 2kb. The PiCV genome contains 5 open reading frames, including V1, C1, C2, 3' intergenic region and 5' intergenic region.

[0004] As the only structural protein of the virus, the capsid protein (Cap) of pigeon circovirus has good immunogenicity and can induce the body to produce neutralizing antibodies. The Cap protein is used as a coating antigen in ELISA due to its antigenic activity and can be recognized by PiCV-specific antibodies. The Cap protein contains neutralizing antibody epitopes and can be considered as a potential target in vaccine development.

[0005] There is no in vitro cell culture system for PiCV, and there is also a lack of vaccines for immune prevention. The disease is usually diagnosed in the laboratory by real-time fluorescence quantitative PCR method. There is no commercial PCR diagnostic kit, let alone commercial serological diagnostic kit. Therefore, efficient, accurate and rapid determination of the antibody content of PiCV in pigeons is of great significance for the development and diagnosis of PiCV vaccines. Summary of the invention

[0006] The invention provides a specific monoclonal antibody against pigeon circovirus Cap protein and application thereof, which solves the problems existing in the prior art.

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0008] A specific monoclonal antibody against the Cap protein of pigeon circovirus, the monoclonal antibody comprising a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region; the CDR of the heavy chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID No.3, a CDR2 with an amino acid sequence as shown in SEQ ID No.4 and a CDR3 with an amino acid sequence as shown in SEQ ID No.5;

[0009] The light chain variable region CDR includes a CDR1 with an amino acid sequence as shown in SEQ ID No.6, a CDR2 with an amino acid sequence as shown in SEQ ID No.7, and a CDR3 with an amino acid sequence as shown in SEQ ID No.8.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.9, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.10.

[0011] Furthermore, the nucleotide sequence of the pigeon circovirus Cap protein is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0012] Furthermore, the gene sequence encoding the heavy chain variable region is shown as SEQ ID No.11, and the gene sequence encoding the light chain variable region is shown as SEQ ID No.12.

[0013] The use of the above-mentioned specific monoclonal antibody against the Cap protein of pigeon circovirus in the preparation of reagents, reagent strips, kits or biochips for detecting pigeon circovirus. In specific applications, the detection method can be serological detection, antibody-coupled nucleic acid detection, etc.

[0014] A pigeon circovirus blocking ELISA detection kit, the kit comprising the above-mentioned specific monoclonal antibody against pigeon circovirus Cap protein.

[0015] Furthermore, the kit also includes an ELISA plate, a blocking solution, a diluent, a washing solution, a color developer, and a stop solution.

[0016] The use of the above-mentioned specific monoclonal antibody against the Cap protein of pigeon circovirus in detecting pigeon circovirus antibodies in samples for non-diagnostic purposes. The detection sample is a virus antibody serum sample.

[0017] A blocking ELISA method for detecting pigeon circovirus antibodies for non-diagnostic purposes, the method comprising: coating pigeon circovirus Cap protein onto an ELISA plate, blocking, adding a sample to be tested and incubating, washing the ELISA plate, adding the enzyme-labeled monoclonal antibody to react, adding a color developer to develop color after washing, adding a stop solution to stop, reading the absorbance at 450nm, and determining the detection result according to the PI value.

[0018] Furthermore, the above-mentioned blocking ELISA method for detecting pigeon circovirus antibodies for non-diagnostic purposes includes the following steps:

[0019] (1) Coating: Dilute the pigeon circovirus Cap protein with pH 9.6 coating buffer to a concentration of 0.5 ng / μl, add 100 μl per well to a 96-well plate, coat overnight at 4°C, and wash with 200 μl TBST;

[0020] (2) Blocking: Add 100 μl of 10% skim milk to each well, block at 37°C for 2 h, and wash with 200 μl of TBST;

[0021] (3) Adding the samples to be tested: dilute the samples to be tested and the standard with TBS at a ratio of 1:8, add 100 μl / well to a 96-well plate, incubate at 37°C for 60 min, and wash with 200 μl TBST;

[0022] (4) Adding monoclonal antibodies: dilute the monoclonal antibodies of the above sequences with TBS 1:8000, add them to the wells, react at 37°C for 40 min, and wash with 200 μl TBST;

[0023] (5) Add enzyme-labeled secondary antibody: dilute HRP-labeled goat anti-mouse secondary antibody with TBS 1:10000, add to each well, react at 37°C for 45 min, and wash with 200 μl TBST;

[0024] (6) Color development: Add 100 μl TMB and develop color for 15 min at room temperature in the dark;

[0025] (7) Termination: Add 50 μl 2M H2SO4 to terminate the reaction. OD 450nm reading.

[0026] Furthermore, the blocking ELISA method for detecting pigeon circovirus antibodies for non-diagnostic purposes also includes the steps of: (8) result calculation and determination: subtract the OD value of the negative control from the OD value of the standard and divide the result by the OD value of the negative control to obtain a percentage. If the percentage is greater than or equal to 28.83%, it is positive; if the percentage is less than or equal to 21.71%, it is negative.

[0027] Use of the above-mentioned specific monoclonal antibody against the Cap protein of pigeon circovirus in the preparation of a drug for preventing and / or treating pigeon circovirus.

[0028] Beneficial effects of the present invention:

[0029] 1. The specific monoclonal antibody against the pigeon circovirus Cap protein of the present invention can specifically bind to the pigeon circovirus Cap protein and has the application potential of being developed into a pigeon circovirus antibody detection kit.

[0030] 2. The present invention uses anti-pigeon circovirus Cap protein monoclonal antibody as a competitive antibody to establish a blocking ELISA method for detecting pigeon circovirus antibodies based on specific monoclonal antibodies. The detection method is simple to operate, has good specificity, high sensitivity, short time consumption, and a large application space. It can accurately and quickly determine PiCV-specific antibodies in samples, complete the determination test and data collation and judgment results within 5 hours, and provide data reference for the development of commercial serological detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of SDS-PAGE protein electrophoresis identification and His tag affinity purification results of PiCV Cap protein;

[0032] Figure 2 This is a schematic diagram of the ELISA evaluation results of mouse serum antibody levels after four immunizations;

[0033] Figure 3 This is a schematic diagram of the results of western blot screening of hybridoma cells;

[0034] Figure 4 This is a schematic diagram of the results of IFA detection of good binding between PiCV Cap monoclonal antibody 1G6-4C4 and eukaryotic expressed Cap protein;

[0035] Figure 5 This is a schematic diagram of the sensitivity test results of the monoclonal antibody 1G6-4C4 determined by ELISA;

[0036] Figure 6 To block the specific detection results of ELISA detection method;

[0037] Figure 7The results are for the sensitivity test of the blocking ELISA method.

[0038] in, Figure 1 In the middle, M: protein molecular weight standard; 1-4: purified PiCV Cap protein; 5: recombinant plasmid prokaryotic expression whole bacteria ultrasonically broken; 6: empty plasmid prokaryotic expression whole bacteria ultrasonically broken;

[0039] Figure 6 In the figure, A is the OD of different virus serum 450nm ; B is the serum PI of different viruses. DETAILED DESCRIPTION

[0040] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings. The scope of the present invention is not limited to the following embodiments. Professionals in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0041] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0042] The experimental animals and cells used in the following examples were purchased from:

[0043] BALB / c mice were purchased from Chongqing Tengxin Biotechnology Co., Ltd. Sp2 / 0, HEK293T, and BHK-21 cells were purchased from ATCC, USA.

[0044] The main reagents used in the following examples were purchased from:

[0045] BamH I, EcoR I, Not I endonucleases, and Turbofect transfection reagent were purchased from Thermo Fisher Scientific, USA; T4 DNA ligase was purchased from TaKaRa; gel recovery kit and plasmid extraction kit were purchased from OMEGA; Max Super-Fidelity DNA Polymerase was purchased from Nanjing Novozymes Biotech Co., Ltd.; DH5α and BL21 competent cells were purchased from Beijing Solebold Technology Co., Ltd.; Ni-NTA filler was purchased from Changzhou Tiandirenhe Biotech Co., Ltd.; ampicillin, kanamycin, His tag purification resin and affinity chromatography column, and BCA protein concentration determination kit were purchased from Solebold Biotech Co., Ltd.; mouse monoclonal antibody subtype identification kit was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0046] Example 1 Preparation, expression and purification of PiCV Cap protein

[0047] 1. Preparation of PiCV Cap Protein

[0048] The pET-30a-Cap recombinant plasmid was constructed by conventional methods. The PiCV Cap target fragment was amplified by PCR, and BamH I and Not I restriction sites were added at 5' and 3', respectively. The amplified target fragment with the expected size was double-digested with the pET-30a vector using BamH I and Not I endonucleases. The digested products were connected at 16°C for 30 minutes using T4 DNA ligase. The ligation products were transformed into BL21 competent cells, cultured at 37°C air shaker at 200rpm for 45 minutes, spread on LB medium plates containing 100μg / mL kanamycin, and inverted at 37°C for 12 hours. 5-8 white monoclonal colonies were randomly picked and identified by PCR. The positive plasmids were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0049] 2. Expression

[0050] (1) The constructed pET-30a-Cap recombinant plasmid was transformed into BL21 competent cells, cultured at 37°C with an air shaker at 200 rpm for 45 min, spread on LB medium plates containing 30 μg / ml kanamycin, and cultured at 37°C with an inverted incubator for 12 h. Single clones were picked into 5 mL of kanamycin-resistant LB liquid medium and cultured at 37°C with an air shaker at 200 rpm for 12 h.

[0051] (2) Add the bacterial suspension to kanamycin-resistant LB liquid culture medium at a ratio of 1:100 and culture at 37°C in an air shaker at 200 rpm for about 4 h.

[0052] (3) Add the inducer IPTG to the bacterial solution to a final concentration of 0.1 mmol / L and culture at 30°C and 200 rpm in an air shaker for 6 h.

[0053] (4) Centrifuge the bacterial solution at 6000 rpm for 10 min, discard the supernatant, add lysis buffer (Lysis Buffer: NaH2PO4 20 mM, NaCl 500 mM, pH 8.0) to the precipitate at a ratio of 10:1, resuspend and add PMSF to make the final concentration of 1 mmol / L.

[0054] The bacterial solution was placed on ice and ultrasonically disrupted at 30% power, working for 3s, with an interval of 3s, until the bacterial solution was basically transparent. After ultrasonic disruption, the sample was centrifuged at 6000rpm for 10min at 4°C, and the supernatant was collected as the pre-loading sample.

[0055] 3. Purification

[0056] (1) Add 1.5 ml of Ni-NTA to the sample column, and then add 3 to 5 times the column volume of Lysis Buffer for equilibration.

[0057] (2) Add the supernatant collected by centrifugation after ultrasound to a sample column containing 1.5 ml of Ni-NTA filler and incubate at 4°C on a shaker for 1 h; collect the supernatant that flows out of the column and add it to the column again, repeating this process three times.

[0058] (3) After the incubation, the supernatant was allowed to flow out of the column and collected as the flow through; the sample was stored at 4°C for later sample analysis.

[0059] (4) Add 3 times the column volume of low-concentration imidazole washing buffer (Wash Buffer: NaH2PO4 20mM, NaCl 500mM, imidazole 5mM, pH 8.0) to elute the impurities bound to the filler and collect the washing buffer that flows out each time for subsequent sample analysis.

[0060] (5) Add 5 ml of high-concentration imidazole elution buffer (Elution buffer: NaH2PO4 20 mM, NaCl 500 mM, imidazole 250 mM, pH 8.0) to elute the target protein bound to the filler. Repeat 4 times and collect the eluate flowing out each time for subsequent identification of protein purification. Store the eluted protein sample at 4°C.

[0061] (8) 100 μl of all collected samples were added to 25 μl (5× SDS-PAGE loading buffer: Tris-HCl 250 mM, SDS 10%, BPB 0.5%, glycerol 50%, β-mercaptoethanol 5%), boiled at 100°C for 10 min, and subjected to SDS-PAGE analysis.

[0062] According to the His tag affinity purification method, PiCV Cap protein was successfully purified by SDS-PAGE analysis, and the size of the purified protein was consistent with the expected protein molecular weight. The results are shown in Figure 1 , is PiCV gene group C, 273aa, 32kDa, isoelectric point: 10.23, its nucleotide sequence is shown in SEQ ID No.1, and its amino acid sequence is shown in SEQ ID No.2.

[0063] SEQ ID No.1:

[0064] atgagaaggcggagattctaccgccgtaggagggcgcccattcgccgacgcaggatcaggagacggaggacacgcctccgcaggcgtggccaccgatcagccggccggatatactacttccgcctacgtcgcaaggacaagataaccctgcacacgaatgcaaacgacttcaaattcggaacggggatatttacattcaaactggcggatgtactaaccgttggcctcaacgcaccaactctgaaagtcccatttgaggactatcagattgcattggtaaaggtggaaatgaggccaattggggccgagataacaacatggaaaggattcgggcacaccgtgcccctttatgacgccaggctaaaaacatttcagcaggtaacggaccgaggcgacgaccccctcatggactttgacggcgccagaaaatgggatctgagaaaggggtttaaacgcctaataaggccaaagccccaactaacaatttcagatttagccacggcaaaccagacggcagcaacctggttctctaaccaaagaaaccagtggataccgctgcagacctcaggtaccagcttggaccccacgaaggtgatctactacgggatggcattcagttacctccaccctgcccctgacccaatgtattatgagtgtgaagttacatactatgtcaagttcaggcagttcgcttggacggccctcaatgtccccccgacccccaatgttgagggcatggaattaatgcatatttgtaacggggattgtaaccagtgttttgctgatgcattggacccagactcagctgttgattctgaataa

[0065] SEQ ID No.2:

[0066] MRRRRFYRRRRAPIRRRRIRRRRRTRLRRRGHRSAGRIYYFRLRRKDKITLHTNANDFKFGTGIFTFKLADVLTVGLNAPTLKVPFEDYQIALVKVEMRPIGAEITTWKGFGHTVPLYDARLKTFQQVTDRGDDPLMD FDGARKWDLRKGFKRLIRPKPQLTISDLATANQTAATWFSNQRNQWIPLQTSGTSLDPTKVIYYGMAFSYLHPAPDPMYYECEVTYYVKFRQFAWTALNVPPTPNVEGMELMHICNGDCNQCFADALDPDSAVDSE*.

[0067] The last "*" of SEQ ID No. 2 represents a stop codon, which is not shown in the corresponding sequence shown in the sequence listing.

[0068] Example 2 Preparation of monoclonal antibodies

[0069] 1. PiCV Cap Immunization

[0070] Five SPF-grade 6-week-old BALB / c female mice were selected, and the immunization dose for each mouse was 50 ng. For the first immunization, the purified PiCV Cap protein was mixed with equal volumes of Freund's complete adjuvant and emulsified, and injected subcutaneously in the abdomen. For the second immunization, the protein was mixed with equal volumes of Freund's incomplete adjuvant and emulsified, and injected subcutaneously in the abdomen. For the third and fourth immunizations, no adjuvant was used and intraperitoneal injection was used. After the immunization was completed, blood was collected from the retro-orbital venous plexus of the mice, and serum was separated to determine the antibody titer.

[0071] 2. Evaluation of serum titer of BALB / c mice by indirect ELISA

[0072] PiCV Cap was diluted with coating buffer (Na2CO3 15mM, NaHCO3 35mM, pH 9.6) and added to a 96-well plate at a concentration of 100μl / well, and coated overnight at 4°C. The coating solution was washed off with TBST (Tris-HCl 20mM, NaCl 150mM, Triton X-100 0.05%, pH 7.4), and 100μl of 10% skim milk was added to block at 37°C for 2h. Mouse serum was serially diluted with TBS (Tris-HCl 20 mM, NaCl 150 mM, pH 7.4) at 5000-fold, 10000-fold, 20000-fold, 40000-fold, 80000-fold, 160000-fold, 320000-fold, and 640000-fold, respectively, and added to a sealed 96-well plate at 100 μl / well for reaction at 37°C for 1 h. The liquid in the wells was washed away with TBST. HRP-labeled goat anti-mouse enzyme-labeled secondary antibody (purchased from Abcam) was diluted 1:10000 with TBS, added at 100 μl / well, and reacted at 37°C for 1 h. The liquid in the wells was washed away with TBST, 100 μl TMB colorimetric solution was added, and color was developed at room temperature in the dark for 15 min, followed by the immediate addition of 50 μl 2M The reaction was terminated with H2SO4 and read at OD450nm in a microplate reader.

[0073] like Figure 2 As shown, the serum antibody levels of 5 mice after four immunizations were evaluated, and it was found that the serum antibody titers of 4 mice were greater than 320,000, and the serum antibody titer of 1 mouse was greater than 160,000. The serum titers were all qualified and could be used for post-fusion.

[0074] 3. Hybridoma cell screening

[0075] The spleen of mice with serum titers that meet the requirements is taken out under sterile conditions, ground and prepared into a spleen lymphocyte suspension, and mouse myeloma cells (Sp2 / 0) are fused with lymphocytes under the action of PEG. Evenly spread into a 96-well plate, and the culture medium is gradually replaced from HAT to HT. The supernatants of cells cultured in different wells are detected by indirect ELISA to screen out positive wells. The cells in the positive wells are subcloned by limiting dilution method, and after the subcloned cells grow, different cell supernatants are detected by indirect ELISA.

[0076] The test results are shown in Table 1. The cell supernatants of 13 wells in the 96-well plate were all positive, indicating that the hybridoma cells were successfully fused. After two subclones, positive monoclonal cell lines were screened and collected. Positive monoclonal cell lines were screened and obtained, and then amplified and frozen in a liquid nitrogen tank.

[0077] Table 1 Indirect ELISA screening of hybridoma cells

[0078] 1 2 3 4 5 6 7 8 9 10 11 12 A 1.508 1.236 2.994 1.710 1.347 1.716 0.609 1.663 1.457 0.775 0.865 1.268 B 1.243 1.477 1.376 2.121 3.883 1.048 0.783 1.862 1.034 1.314 1.301 0.334 C 0.880 0.713 0.513 0.779 0.538 0.546 1.312 1.452 2.935 1.090 1.270 0.842 D 1.132 2.086 0.646 0.757 0.912 2.072 1.682 0.785 0.685 0.915 1.045 1.442 E 1.880 0.396 1.956 0.638 1.178 3.777 1.369 2.213 2.376 1.111 1.219 2.783 F 1.814 1.391 1.942 0.399 0.863 1.759 1.056 1.054 0.598 1.573 0.810 0.855 G 1.951 2.696 0.525 0.844 1.311 2.743 0.947 0.599 1.343 0.595 0.808 1.030 H 2.627 0.721 0.820 0.503 0.928 1.113 1.767 1.449 0.810 0.925 1.007 0.121

[0079] 4. Western blot identification

[0080] After SDS-PAGE of total protein of HEK293T cells transfected with Cap gene, Western blot was performed and the protein was transferred to PVDF membrane. After completion, it was blocked with 10% skim milk at 4℃ overnight, the supernatant of positive monoclonal cell line culture was diluted with TBS and incubated at room temperature for 1h, HRP-labeled goat anti-mouse enzyme-labeled secondary antibody was added and incubated at room temperature for 1h, and the color developing solution was added to obtain the results in gel imaging analyzer. Figure 3 As shown in the figure, in the Western blot system, the prepared monoclonal antibody can effectively recognize the PiCV Cap protein expressed by HEK293T cells, and the reaction is strongly positive. After subcloning the hybridoma cells with positive reaction, subsequent identification is performed.

[0081] 5. IFA Identification

[0082] BHK-21 cells were plated in 48-well plates. When the cells grew to 60% to 70%, 0.6 μg of plasmid pcDNA3.1-2×FLAG-Cap was transfected and cultured in a cell culture incubator at 37°C and 5% CO2 for about 36 hours. After 36 hours, the culture medium was discarded, the cells were washed three times with PBS, 100 μl of 4% paraformaldehyde was added to each well for fixation for 25 minutes, 100 μl of 0.1% Triton-100 was added to perforate the cell membrane for 10 minutes, PBS was washed three times, and 100 μl of 1% BSA was used to block the cell membrane at 37°C for 2 hours. After washing three times with PBS, the diluted supernatant of the positive monoclonal cell line was added and incubated at 37°C for 2 hours, PBS was washed three times, and goat anti-mouse (Alexa Fluor) was added. After incubation with 594) fluorescent secondary antibody (purchased from Abcam) in the dark for 30 min, the results were observed under a fluorescence microscope.

[0083] like Figure 4 As shown, after plasmid pcDNA3.1-2×FLAG-Cap was transfected into BHK-21 cells, the monoclonal antibody 1G6-4C4 was identified by IFA to bind to PiCV Cap. The positive monoclonal cell line was named mouse hybridoma monoclonal cell line 1G6-4C4.

[0084] 6. Monoclonal Antibody Preparation

[0085] One week before the injection of monoclonal cell line 1G6-4C4, each BALB / c mouse was intraperitoneally injected with 500 μl of sterilized liquid paraffin. Hybridoma monoclonal cells with good immunoreactivity were collected and intraperitoneally injected with 1×106 cells, and ascites was collected several times within 10 to 14 days after injection.

[0086] 7. Determination of the variable region of monoclonal antibodies

[0087] The 1G6-4C4 monoclonal cell line was collected, centrifuged at 1000 rpm for 3 min, and RNA was extracted using the TRIZOL method. Subsequently, the obtained RNA template was reverse transcribed into cDNA using random primers.

[0088] Using the prepared cDNA as template, Max Super-Fidelity DNA Polymerase high-fidelity polymerase was used to amplify the gene fragment. The system was 2×Phanta Max Buffer 25μl, Phanta Max Super-Fidelity DNA Polymerase 1μl, dNTP Mix Buffer 1μl, Forward and Reverse 1μl each, cDNA 1μl, and ddH2O was added to 50μl.

[0089] The amplification conditions were 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 30 s, 35 cycles, and 72°C final extension for 5 min. After the PCR reaction was completed, the samples were electrophoresed on 1% agarose gel to detect the results.

[0090] The heavy chain gene and light chain gene were cloned into the pMD19-T vector respectively, and a single colony was selected for culture. The positive plasmid was identified by PCR and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the antibody gene library (IMGT).

[0091] Table 2 VH primer sequence list

[0092] Primers Sequence (5'-3') VH-F TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC VH-R SAGGTGCAGYTGKTGSMGTCTGG

[0093] Table 3 VL primer sequence list

[0094] Primers Sequence (5'-3') VL-F CCGTTTYAKYTCCARCTTKGTSCC VL-R GACATTGAGCTCACCCAGTCTCCA

[0095] R=Aor G; Y=C or T; K=G or T; S=C or G; M=Aor C.

[0096] The sequencing results were compared with the antibody gene library (IMGT) for analysis. The complementarity determining region (CDR) sequences of the heavy chain variable region and the light chain variable region of the anti-pigeon circovirus Cap protein monoclonal antibody prepared in this example are shown in Table 4.

[0097] Table 4 Antibody variable region sequences

[0098]

[0099] Among them, the amino acid sequence of the heavy chain variable region is:

[0100] RCKLVESGGGLVQPGGSLRLSCATSGFTFTDHYMTWVRQPPGKALEYLGFI

[0101] RNKTYGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARSGA WFAYWGQGTTVTVSS(SEQ ID No.9)

[0102] The gene sequence encoding the heavy chain variable region is:

[0103] AGGTGCAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGCT

[0104] CTCTGAGACTCTCCTGCGCAACTTCTGGTTTCACCTTCACTGATCACTACA

[0105] TGACCTGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTACTTGGGTTTT

[0106] ATTAGAAACAAAACTTATGGGTTACACAACAGAGTACAGTGCATCTGTGAA

[0107] GGGTCGGTTCACCATCTCCAGAGATAATTCCCAAAGCATTCTCTATCTTCA

[0108] AATGAACACCCTGAGAACTGAGGACAGCGCCACTTATTACTGTGCAAGA

[0109] TCTGGCGCCTGGTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID No.11)

[0110] The amino acid sequence of the light chain variable region is:

[0111] DIELTQSPSSLAVSVGEKVSMNCKSSQTLLYTINQKNYLAWYQEKPGQSPKL

[0112] LIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYFSYPLTFG AGTKLELKR(SEQID No.10)

[0113] The gene sequence encoding the light chain variable region is:

[0114] GACATTGAGCTCACCCAGTCTCCATCCTCCCTAGCTGTGTGTCAGTTGGAGA

[0115] GAAGGTTTCTATGAATTGCAAGTCCAGTCAGACCCTTTTATATACTATCAA

[0116] TCAAAAGAACTACTTGGCCTGGTACCAGGAGAAACCAGGGCAGTCTCCT

[0117] AAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCG

[0118] CTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTG

[0119] TGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTTTAGCTATCCGCTCACGTTCGGTGCTGGGACCAAGTTGGAGCTGAAACGG (SEQ ID No. 12)

[0120] 8. Identification of monoclonal antibody subtypes

[0121] The subtype identification kit for mouse monoclonal antibody (Company: Proteintech; Catalog No. PK20003) was used to identify the subtype of ascites monoclonal antibody 1G6-4C4. The method of use is as follows:

[0122] (1) Take out the monoclonal antibody subtype identification kit at 4°C and equilibrate at room temperature for 30 minutes;

[0123] (2) ELISA strips: Select the number of ELISA strips based on the number of samples (this test only detects ascites antibody subtypes, and the number of samples is 1);

[0124] (3) Prepare 1× goat anti-mouse IgM + IgG-HRP in advance: 10 μL 100× goat anti-mouse IgM + IgG-HRP + 990 μL 1× PBST;

[0125] (4) Dilute the ascites with PBST to 1:100000. First, take 1 μL of ascites + 1 ml 1×PBST (1:1000), and then take 10 μL of the above mixture + 990 μL 1×PBST; then add the diluted sample to be tested into the sample wells of the strip, 50 μL / well, without incubation;

[0126] (5) Add 1× goat anti-mouse IgM + IgG-HRP into the sample wells, 50 μL / well, and shake for 1 min to mix.

[0127] Cover with sealing film and incubate at room temperature for 1 h;

[0128] (6) Discard the liquid in the wells, wash the plate three times with 1× PBST, and pat dry on absorbent paper;

[0129] (7) Add the prepared colorimetric solution to the wells, 100 μL / well; Note: The colorimetric solution should be prepared and used immediately, the formula is: solution A: solution B = 1:100, that is, 10 μL solution A is mixed with 1 ml solution B, and use immediately after mixing;

[0130] (8) After color development at room temperature in the dark for 10 to 20 min, add stop solution (100 μL / well);

[0131] (9) Result interpretation: Read the OD value of the microplate reader 450nm , the hole with the highest value corresponds to the corresponding subtype.

[0132] The results are shown in Table 5. The heavy chain type of the prepared PiCV Cap monoclonal antibody 1G6-4C4 is IgG1, and the light chain type is Kappa.

[0133] Table 5 Identification results of monoclonal antibody subtypes

[0134] ascites IgG1 2.038 IgG2a 0.281 IgG2b 0.427 IgG3 0.128 IgA 0.184 IgM 0.199 Kappa 1.168 Lambda 0.133

[0135] 9. Monoclonal antibody sensitivity test

[0136] (1) The expressed and purified PiCV Cap protein was coated on the ELISA coated plate at a concentration of 100 ng / well (the protein was diluted with pH 9.6 coating buffer), 100 μl per well, and incubated overnight at 4°C;

[0137] (2) Add 100 μl of 10% skim milk to each well and block at 37°C for 2 h. Discard the blocking solution in the plate and wash three times with 200 μl of TBST. Discard the liquid in the last wash and gently pat dry the remaining liquid on absorbent paper.

[0138] (3) The antibody was diluted in a gradient of 4000, 8000, 16000, 32000, 64000, 128000, and 256000, and added to a 96-well plate at 100 μl / well. The plate was incubated at 37°C for 1 h, and the liquid in the plate was discarded. 200 μl of TBST was added to each well, and the plate was washed three times. The remaining liquid was gently patted dry on absorbent paper.

[0139] (4) HRP-labeled goat anti-mouse enzyme-labeled secondary antibody was diluted 1:10,000 and reacted at 37°C for 1 h. The liquid in the plate was discarded, 200 μl TBST was added to each well, and the plate was washed three times. The remaining liquid was gently patted dry on absorbent paper;

[0140] (5) Add 100 μl of TMB colorimetric solution and color for 15 min at room temperature in the dark;

[0141] (6) Add 50 μl 2M H2SO4 to terminate the reaction. 450nm reading.

[0142] The results are as follows Figure 5 As shown, there is a linear relationship between the OD value and the antibody concentration, and the sensitivity of the antibody to the PiCV Cap protein is good.

[0143] Example 3 Establishment of a blocking ELISA assay for detecting PiCV antibodies

[0144] Blocking ELISA specific steps:

[0145] (1) Coating: Dilute the pigeon circovirus Cap protein with pH 9.6 coating buffer to a concentration of 0.5 ng / μl, add 100 μl per well to a 96-well plate, coat overnight at 4°C, wash three times with 200 μl TBST, discard the liquid for the last time, and gently pat dry the remaining liquid on absorbent paper;

[0146] (2) Blocking: Add 100 μl of 10% skim milk to each well, block at 37°C for 2 h, wash three times with 200 μl of TBST, and discard the remaining liquid;

[0147] (3) Adding the samples to be tested: dilute the samples to be tested and the standard with TBS at a ratio of 1:8, add 100 μl / well to a 96-well plate, incubate at 37°C for 60 min, wash three times with 200 μl TBST, and discard the remaining liquid;

[0148] (4) Adding monoclonal antibody: dilute the monoclonal antibody obtained in step 7 of Example 2 with TBS 1:8000, add it to each well, react at 37°C for 40 min, wash three times with 200 μl TBST, discard the solution and pat dry the remaining liquid;

[0149] (5) Adding enzyme-labeled secondary antibody: HRP-labeled goat anti-mouse secondary antibody was diluted with TBS at 1:10,000 and added to each well. The wells were reacted at 37°C for 45 min, and the wells were washed three times with 200 μl TBST. The remaining liquid was discarded and patted dry.

[0150] (6) Color development: Add 100 μl TMB and develop color for 15 min at room temperature in the dark;

[0151] (7) Termination: Add 50 μl 2M H2SO4 to terminate the reaction. OD 450nm reading;

[0152] (8) Result calculation and judgment: Subtract the OD value of the negative control from the OD value of the standard and divide the result by the OD value of the negative control to obtain a percentage. If the percentage is greater than or equal to 28.83%, it is positive; if the percentage is less than or equal to 21.71%, it is negative; and if the percentage is between the two, it is suspicious.

[0153] The established blocking ELISA method was used to detect the positive sera for antibodies to pigeon Newcastle disease virus (PPMV-1), avian influenza virus (H9N2), rotavirus (RV) and fowl adenovirus (FAdV-4), with PiCV antibody positive and negative sera as controls. Figure 6 As shown, the above serum PIs were all lower than the cut-off value, indicating that the established blocking ELISA detection method had good specificity.

[0154] The PiCV antibody-positive serum was diluted in a gradient of 4, 8, 16, 32, 64, and 128 and tested using the established blocking ELISA method. Figure 7 As shown, when the serum was diluted 1:32, the PI was still greater than the cut-off value, indicating that the established blocking ELISA detection method had good sensitivity.

[0155] For 3 PiCV antibody positive sera and 3 negative sera, intra-batch repeatability test was performed using 3 ELISA plates coated with the same batch according to the established blocking ELISA detection method; inter-batch repeatability test was performed using 3 ELISA plates coated with different batches. The results are shown in Tables 6 and 7. The coefficients of variation of intra-batch repeat and inter-batch repeat were less than 8%, indicating that the established blocking ELISA detection method has good repeatability.

[0156] Table 6 Intra-batch repeatability test

[0157]

[0158] Table 7 Batch-to-batch repeatability test

[0159]

[0160] In summary, the present invention obtains a specific monoclonal antibody against the Cap protein of pigeon circovirus. The present invention uses the Cap protein of pigeon circovirus as an antigen and the monoclonal antibody against the Cap protein of pigeon circovirus as a competitive antibody to establish a blocking ELISA method for detecting pigeon circovirus antibodies based on specific monoclonal antibodies. The detection method is simple to operate, has good specificity, high sensitivity, short time consumption, and a large application space. It can accurately and quickly determine the PiCV specific antibodies in the sample, complete the determination test and data collation and judgment results within 5 hours, and provide data reference for the development of commercial serological detection kits.

[0161] The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation manner of the present invention falls within the protection scope of the present invention.

[0162] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A specific monoclonal antibody against the Cap protein of pigeon circovirus, the monoclonal antibody comprising a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region, characterized in that: The CDRs of the heavy chain variable region are CDR1 with an amino acid sequence as shown in SEQ ID No. 3, CDR2 with an amino acid sequence as shown in SEQ ID No. 4, and CDR3 with an amino acid sequence as shown in SEQ ID No. 5; The light chain variable region CDRs are CDR1 with an amino acid sequence as shown in SEQ ID No. 6, CDR2 with an amino acid sequence as shown in SEQ ID No. 7, and CDR3 with an amino acid sequence as shown in SEQ ID No.

8.

2. The specific monoclonal antibody against the Cap protein of pigeon circovirus according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

10.

3. The specific monoclonal antibody against the Cap protein of pigeon circovirus according to claim 1, characterized in that: The nucleotide sequence of the pigeon circovirus Cap protein is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.

2.

4. A gene fragment encoding a monoclonal antibody against the Cap protein of pigeon circovirus according to claim 1 or 2, characterized in that: in, The gene sequence encoding the heavy chain variable region is shown in SEQ ID No. 11, and the gene sequence encoding the light chain variable region is shown in SEQ ID No.

12.

5. Use of the specific monoclonal antibody against pigeon circovirus Cap protein as claimed in claim 1 or 2 in the preparation of a reagent, a reagent strip, a kit or a biochip for detecting pigeon circovirus antibodies.

6. A pigeon circovirus blocking ELISA detection kit, characterized in that, The kit comprises the specific monoclonal antibody against the Cap protein of pigeon circovirus as claimed in claim 1 or 2.

7. Use of the specific monoclonal antibody against pigeon circovirus Cap protein as claimed in claim 1 or 2 in detecting pigeon circovirus antibodies in samples for non-diagnostic purposes.

8. The use according to claim 7, characterized in that: The invention relates to a blocking ELISA method for detecting pigeon circovirus antibodies in a sample, which comprises: coating the pigeon circovirus Cap protein onto an ELISA plate, blocking, adding a sample to be tested and incubating, washing the ELISA plate, adding the enzyme-labeled monoclonal antibody as claimed in claim 1 or claim 2 for reaction, adding a color developer for color development after washing, adding a stop solution for termination, reading the absorbance at 450 nm, and determining the detection result according to the PI value.

9. The use according to claim 8, characterized in that: The blocking ELISA method comprises the following steps: (1) Coating: Dilute the pigeon circovirus Cap protein with pH 9.6 coating buffer to a concentration of 0.5 ng / μl, add 100 μl per well to a 96-well plate, coat overnight at 4°C, and wash with 200 μl TBST; (2) Blocking: Add 100 μl of 10% skim milk to each well, block at 37°C for 2 h, and wash with 200 μl of TBST; (3) Add the sample to be tested: dilute the sample to be tested and the standard with TBS at a ratio of 1:8, add 100 μl / well to a 96-well plate, incubate at 37°C for 60 min, and wash with 200 μl TBST; (4) Adding monoclonal antibody: dilute the monoclonal antibody described in claim 1 or 2 with TBS at a ratio of 1:8000, add it to the wells, react at 37°C for 40 min, and wash with 200 μl TBST; (5) Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse secondary antibody with TBS at 1:10,000, add to each well, react at 37°C for 45 min, and wash with 200 μl TBST. (6) Color development: Add 100 μl TMB and develop color for 15 min at room temperature in the dark. (7) Termination: Add 50 μl 2 M H2SO4 to terminate the reaction. 450nm reading.

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