Monoclonal antibody 3E1 of anti-PGPV envelope protein and application method thereof

Abstract

The invention relates to the technical field of biology, and aims to provide a monoclonal antibody 3E1 for resisting PGPV envelope protein and an application method of the monoclonal antibody 3E1. A heavy chain and a light chain of the monoclonal antibody respectively have three CDR subregions forming antigen binding sites, amino acid sequences of CDR1, CDR2 and CDR3 in a heavy chain variable region are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and amino acid sequences of CDR1, CDR2 and CDR3 in a light chain variable region are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The monoclonal antibody 3E1 provided by the invention is high in affinity, strong in immunocompetence and good in specificity and uniformity, and has the advantages of high stability, high activity, strong affinity and the like; the monoclonal antibody is high in sensitivity and strong in specificity, and can be used for a PGPV diagnostic kit; a new technical support can be provided for rapid detection of the PGPV antibody, and the method has important significance in promoting healthy development of the pigeon industry and preventing, controlling and purifying PGPV epidemic situations.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to a monoclonal antibody 3E1 against the envelope protein of PGPV and its application. Background Art

[0002] Pigeonpox virus (PGPV) belongs to the family Poxvirinae, subfamily Chordopoxvirinae (ChPV), genus Avipoxvirus (APV), and has the typical morphology of poxviruses. Pigeonpox, a viral infectious disease it causes, is also known as avian diphtheria and infectious dermatoma. Pigeonpox has a relatively high incidence in tropical and subtropical regions, such as regions with humid and warm climates like Brazil, Colombia, and the southeastern United States. Pigeonpox is prevalent in summer and autumn, and is transmitted through contact between healthy pigeons and diseased pigeons or by mosquito bites. Pigeons of all breeds, ages, and genders can be infected, which can lead to a decrease in the survival ability of pigeons, making the affected birds vulnerable to predation and secondary infections, resulting in high mortality and morbidity. The condition of young pigeons is more severe, and the case fatality rate is higher. The prevention and control of pigeonpox mainly rely on immunoprophylaxis. Once infected, recovery depends on the pigeon's own immunity, and there is no specific medicine.

[0003] Currently, the pigeon industries such as meat pigeons, carrier pigeons, and racing pigeons are booming in China. Among them, the meat pigeon breeding industry is the fourth largest poultry breeding industry after chickens, ducks, and geese, and has important economic value and public health significance. Currently, pigeonpox is one of the most common infectious diseases in the pigeon breeding industry in China, seriously affecting the health of pigeons and the quality of carcasses. In southern China, such as Sichuan, it is a high-incidence area.

[0004] Therefore, if new diagnostic techniques can be found, it is of great significance for the prevention, control, and purification of PGPV epidemics and the healthy development of the pigeon industrial chain. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a monoclonal antibody 3E1 against the envelope protein of PGPV and its application method.

[0006] To solve the technical problem, the solution of the present invention is:

[0007] Provided is a monoclonal antibody 3E1 against the PGPV envelope protein. Each of the heavy chain and the light chain of the monoclonal antibody has three CDR sub-regions constituting the antigen-binding site, wherein: the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.

[0008] As a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:8.

[0009] In the present invention, the heavy chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown as SEQ ID NO:9, and the light chain variable region is encoded by the nucleic acid sequence shown as SEQ ID NO:10.

[0010] In the present invention, the constant region of the heavy chain is of the IgG1 type, and the constant region of the light chain is a κ chain.

[0011] In the present invention, the monoclonal antibody is prepared using the vaccinia virus p35 protein as the target protein, and the recognition site of the monoclonal antibody is in the 47aa - 53aa interval of the p35 protein.

[0012] The present invention further provides a method for using the aforementioned pigeonpox virus monoclonal antibody, which is to use the monoclonal antibody to prepare a kit for detecting pigeonpox virus.

[0013] The present invention also provides a kit for detecting pigeonpox virus, which includes any one of the aforementioned pigeonpox virus monoclonal antibodies.

[0014] As a preferred embodiment of the present invention, the kit is an ELISA detection kit, or any one of an immunochromatographic diagnostic kit using an immunochromatographic test strip and an immunohistochemistry kit; when using the kit, the detection method is serological detection. That is, the kit is used in a serological detection manner, and the sample to be detected taken is venous blood.

[0015] As a preferred embodiment of the present invention, the kit includes: an enzyme-linked immunosorbent assay (ELISA) plate coated with PGPV P35 protein, the aforementioned monoclonal antibody labeled with HRP, positive control serum and negative control serum, PBS buffer, PBST buffer, blocking solution, antibody diluent, coating solution, and termination solution.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. The monoclonal antibody 3E1 provided by the present invention has high affinity, strong immune ability, good specificity and homogeneity, and has the advantages of stability, high activity and strong affinity.

[0018] 2. The monoclonal antibody provided by the present invention has high sensitivity and strong specificity, and can be used in a PGPV diagnostic kit; it can provide new technical support for the rapid detection of PGPV antibodies, and is of great significance for promoting the healthy development of the pigeon industry, the prevention and control of PGPV epidemic situations and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the result of the serum antibody titer after immunizing mice with PGPV p35 protein in Example 1.

[0020] Figure 2 It is the Western blot result of the specificity analysis of the monoclonal antibody in Example 2.

[0021] Figure 3 It is the indirect immunofluorescence result of the specificity analysis of the monoclonal antibody in Example 2.

[0022] Figure 4 It is the virus verification of the monoclonal antibody in Example 2.

[0023] Figure 5 It is the confirmation of the recognition site of the monoclonal antibody in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0024] Vaccinia virus, as the prototype virus of poxviruses, has relatively clear relevant research results. Vaccinia virus P35 protein is an immunodominant antigen protein on the surface of the envelope of intracellular mature virus particles, and is the main protein inducing antibody production, especially playing an important role in generating the secondary immune response, and is also involved in the virus adsorbing host cells, and also plays a role in aspects such as binding, assembling mature virus particles, virus virulence and immunogenicity. However, there is currently no publicly reported research on the function of pigeonpox virus P35 protein.

[0025] The R & D team of the applicant found in the long-term in-depth research process that PGPV P35 protein is a pigeonpox virus envelope protein, homologous to a bovinepox virus p35 (encoded by H3L), an immunodominant membrane protein, and the lack of H3L in vaccinia virus will lead to a reduction in its replication or cell-to-cell spread. Therefore, the applicant took this as the starting point of the innovative design plan, selected this protein as the target protein to prepare monoclonal antibodies. The monoclonal antibodies obtained on this basis have good specificity, good homogeneity, and can be supplied indefinitely, and can provide new technical support for the rapid detection of PGPV.

[0026] To better illustrate the technical solution of the present invention, the following will describe the technical solution in combination with embodiments and accompanying drawings, but not limited thereto.

[0027] Example 1 Preparation of Hybridoma Cells and Screening of Monoclonal Antibodies

[0028] 1.1 Animal Immunization

[0029] In the present invention, the purified pigeonpox virus pET-30a-p35 protein is used as an antigen. The sequence of this protein is obtained by comparing PGPV with FPV140 and is sent to a biological company for synthesis.

[0030] After emulsifying the pET-30a-p35 protein with an equal volume of Freund's (in)complete adjuvant, 6-8-week-old SPF-grade female BALB / c mice are immunized by intraperitoneal and subcutaneous multi-point injection. The immunization process is shown in Table 1.

[0031] Table 1 Immunization Process

[0032]

[0033] Figure 1 For the results of the serum antibody titers of mice immunized with the PGPV p35 protein in Example 1, 1-4 are serum samples of 4 different mice.

[0034] 1.2 Preparation of Hybridoma Cells

[0035] Resuscitate SP2 / 0 cells about one month before fusion and observe the cell state one day before fusion. On the day of fusion, select the mouse with the best immunization effect, collect blood, decapitate it to death, soak it in 75% ethanol for disinfection, and separate the mouse spleen cells for fusion with SP2 / 0 cells. Add the washed SP2 / 0 and spleen cells to a 15 mL centrifuge tube at a ratio of 1:10, add 1640 culture medium to 10 ml, pipette and mix well, centrifuge at 1000·rpm for 5 min, and discard the supernatant. Gently flick the tube wall with your finger to disperse the cell pellet, then place the cells in a 37 °C water bath in a beaker, slowly add 1 mL of pre-warmed PEG cell fusion agent at 37 °C, add it evenly within 1 min, shake the centrifuge tube and pipette while adding, and then let it stand for 90 s. Subsequently, add 1 mL of pre-warmed complete medium, add it within 1 min while shaking, let it stand for one minute, repeat three times, after a 10 min water bath at 37 degrees, centrifuge at 1000 rpm for 10 min and discard the supernatant, resuspend with 10 mL of 1640 medium, centrifuge at 800·rpm for 10 min, discard the supernatant, resuspend the pellet with HAT complete medium, first resuspend with a small dose and gently disperse it, and then make up to 100 ml, mix well. Spread it into a 96-well cell culture plate containing feeder cells, 200 ul per well, change the medium after 7 days, and observe the cell growth status.

[0036] 1.3 Screening of Hybridoma Cells

[0037] About ten days after cell fusion, when the cell mass grew to about 1 / 10 of the well area of the culture plate under the microscope, 160 μL of the cell culture supernatant was taken for ELISA detection. Positive cell wells were picked out, and the cells were serially diluted according to the following steps to screen positive monoclonal hybridoma cells.

[0038] (1) Take a 96-well cell culture plate and add 100 μL of HAT cell culture medium to each well, with 150 μL added to well A1; take a 48-well cell culture plate and add 1 mL of HAT cell culture medium to each well according to the number of positive cell wells.

[0039] (2) According to the iELISA detection results, observe and mark the positive cell wells under the microscope, discard the supernatant in the wells, wash twice with PBS, add 200 μL of cell culture medium, and gently blow down the cells at the bottom of the well; take 50 μL of the cell suspension and add it to well A1 of the above 96-well cell culture plate, and mix gently. The remaining suspension is added to the above 48-well cell culture plate, and subcultured in sequence to a 6-well cell culture plate and then cryopreserved for standby to prevent the loss of positive cells during the screening process.

[0040] (3) Take 100 μL of the cell suspension from well A1 above and add it to well B1. After mixing, add it to wells C1 - H1 in sequence for 2-fold serial dilution, and make up the culture medium to 200 μL in each well. Use an 8-channel pipette to respectively aspirate 100 μL of the cell suspension from wells A1 - H1 in the first row and add it to the second row. After mixing, add it to rows 3 - 12 in sequence for 2-fold serial dilution, and make up the culture medium to 200 μL in each well. Culture under the conditions of 37 °C and 5% CO2.

[0041] (4) After culturing for 3 days, mark the monoclonal cell wells (only one cell mass in each well) under an inverted microscope. Discard 100 μL of the cell culture supernatant from each well, and add an equal amount of HT cell culture medium for half-medium change. After 6 days, take 110 μL of the cell culture supernatant for standby and make up with 120 μL of HT culture medium. The cell culture supernatant is subjected to iELISA detection, and the second detection is performed after 2 days.

[0042] (5) Take the monoclonal cell wells with positive results in both detections, higher OD450 values, and good cell growth status, and perform the second subcloning according to the above steps. Each positive hybridoma cell is subcloned 3 - 4 times until all the cell wells in the plate are positive and in a stable state; the screened hybridoma cells are successively subcultured from the 96-well cell culture plate to 48-well, 24-well, 12-well, and 6-well plates and then transferred to a cell culture dish for expansion culture. During this period, the HT culture medium is gradually replaced with 20% FBS cell culture medium, and the cells are cryopreserved after the state is stable.

[0043] Example 2 Specificity Analysis of Monoclonal Antibodies

[0044] 2.1 Western Blot Analysis of Monoclonal Antibodies (WB)

[0045] (1) Sample Preparation: Take the pET-30a-p35 and pET-30a empty vector protein solutions, add 4xSDS-PAGE Loading Buffer and mix well, and incubate in a 100°C water bath for 10 min for later use.

[0046] (2) Electrophoresis: Perform 12% SDS-PAGE gel electrophoresis with a loading volume of 10 μL per well.

[0047] (3) Membrane Transfer: After electrophoresis, transfer the gel to a PVDF membrane.

[0048] (4) Blocking: Block with 5% skim milk powder for 2 h, and wash the membrane 3 times with TBST, 10 min each time.

[0049] (5) Primary Antibody: Dilute the hybridoma cell supernatant 1000-fold with TBST, incubate with the PVDF membrane at room temperature for 1 h, and wash the membrane 3 times with TBST, 10 min each time.

[0050] (6) Secondary Antibody: Dilute the enzyme-labeled secondary antibody 5000-fold, incubate with the PVDF membrane at room temperature for 1 h, and wash the membrane 3 times with TBST, 10 min each time.

[0051] (7) Exposure: Drop an appropriate amount of ECL chemiluminescent substrate on the membrane, and perform exposure analysis using a gel imaging system.

[0052] Figure 2 The Western blot results for the specificity analysis of the monoclonal antibody in Example 2. Lane 1 is the pET-30a empty vector, and lane 2 is the pET-30a-p35 recombinant protein.

[0053] 2.2 Indirect Immunofluorescence Analysis of Monoclonal Antibodies (IFA)

[0054] The PGPV P35 protein was constructed into the p3×FLAG-CMV7.1 vector and named FLAG-PGPV P35.

[0055] (1) Transfection: Seed 293T cells in a 24-well plate. When they reach 60-70% confluence, transfection can begin. Prepare 50 μL of jet PRIME buffer, 0.5 μg of FLAG-PGPV P35 plasmid, and 1 μL of jet PRIME reagent solution according to the kit. Mix well and let stand for 10 min, then slowly add the mixture to the corresponding wells. After thorough mixing, incubate in a 37°C incubator.

[0056] (2) Fixation: After 24 hours of transfection, discard the culture medium, add 200 μL of 4% paraformaldehyde, and after 30 minutes at room temperature, wash 3 times with PBS for 5 minutes each time;

[0057] (3) Permeabilization: Permeabilize the fixed cells with 0.5% Triton X-100, and after 10 minutes at room temperature, wash 3 times with PBS for 10 minutes each time;

[0058] (4) Blocking: Add blocking solution and block at 37 °C for 30 minutes, then wash 3 times with PBS for 5 minutes each time;

[0059] (5) Incubation with primary antibody: Dilute the monoclonal antibody at 1:200 and add it to the wells, and incubate for 1 hour at 37 °C. At the same time, set up a positive control (FLAG antibody) and a negative control (p3×FLAG-CMV7.1 empty vector);

[0060] (6) Incubation with secondary antibody: Discard the primary antibody and wash 3 times with PBST. Dilute FITC-labeled goat anti-mouse IgG at 1:2000, add 200 μL to each well, and incubate for 1 hour at 37 °C;

[0061] (7) DAPI staining: Add 200 μL of DAPI staining solution and stain at room temperature for 10 minutes, discard the DAPI staining solution, and wash 3 times with PBST for 5 minutes each time;

[0062] Figure 3 It is the indirect immunofluorescence result for the specific analysis of the monoclonal antibody in Example 2.

[0063] 2.3 Verification of the monoclonal antibody against the virus

[0064] Determine whether the monoclonal antibody can specifically recognize the PGPV virus. Inoculate 10- to 11-day-old SPF chicken embryos with PGPV virus and 1×PBS respectively. After the chicken embryos are sealed with wax and incubated in a 37 °C constant temperature incubator for 120 hours, the allantoic membrane in the inoculated group shows swelling and pox spots, while the mock group does not show such symptoms. Take 0.2 g of the allantoic membrane from each group, add 500 μL of PBS, homogenize, and take the supernatant for sample preparation for Western blotting verification. The primary antibody used is the purified monoclonal antibody.

[0065] Figure 4 It is the verification of the monoclonal antibody against the virus in Example 2. 1 is the mock group and 2 is the inoculated group.

[0066] Identification of hybridoma cell 3E1 in Example 3

[0067] 3.1 Determination of the antibody variable region sequence

[0068] Collect cultured cells, add 1 mL of Trizol reagent, repeatedly pipette to lyse the cells, and then extract total RNA according to the Trizol reagent instruction manual. Use a multifunctional microplate reader to detect the concentration and purity of the extracted RNA. Take 1 μg of RNA and perform reverse transcription using HISCRIPT III All-in-one RT SuperMix Perfect for qPCR from Novoprotein. Take 2 μL of cDNA as a template and perform PCR amplification using a high-fidelity enzyme (2xKOD plus Neo) to obtain the target gene fragment. The primer sequences are shown in Table 2 below. PCR reaction conditions: After pre-denaturation at 98°C for 3 min, perform 30 cycles of amplification. The conditions for each cycle are: denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 68°C for 5 s, and finally thorough extension at 68°C for 5 min. Take the PCR product for 1% agarose gel electrophoresis.

[0069] Purify the PCR product of about 300 bp in size according to the agarose gel recovery / PCR purification kit instruction manual. Use pMD18-T as a cloning vector and ligate the target genes of the heavy and light chain variable regions with pMD18-T respectively according to the pMD18-T Vector Cloning Kit instruction manual for TA cloning. Transform the recombinant product into DH5α competent cells, and then spread them on an LB plate containing 100 μg / ml ampicillin, and incubate overnight at 37°C. Use fresh sterilized pipette tips to pick 5 - 10 single colonies of the heavy and light chains respectively from the plate and inoculate them into 1 mL of LB medium (containing 50 μg / mL ampicillin), and culture at 37°C with 300 rpm for 8 - 12 h, then send the bacterial liquid for sequencing using the universal primers M13F and M13R. TM The nucleotide sequences of the heavy chain variable region SEQ ID NO:9 and the light chain variable region SEQ ID NO:10 encoding the monoclonal antibody 3E1 were obtained by sequencing.

[0070] Table 2 Primer Sequence Table

[0071]

[0072]

[0073] The nucleotide sequences of the heavy chain variable region SEQ ID NO:9 and the light chain variable region SEQ ID NO:10 encoding the monoclonal antibody 3E1 were obtained by sequencing.

[0074] 3.2 Identification of the linear epitope recognized by the antibody

[0075] To accurately determine the epitope region recognized by the monoclonal antibody for the p35 protein, the p35 protein was first truncated in segments based on pcold-PGPV-T and cloned into the pET-30a vector. The first round of truncation constructs was divided into 4 segments, which were respectively constructed on the pET-30a vector and named JD1 (1aa - 50aa), pcold-PGPV-T (35aa - 77aa), JD2 (63aa - 182aa), and JD3 (168aa - 333aa). After successful construction, they were transformed into Rosetta competent cells, induced for expression, and after sample preparation, PAGE verification and Western blot verification with the monoclonal antibody as the primary antibody were respectively performed to identify the region where the site targeted by the antibody is located. After preliminary verification, the region targeted by 3E1 was 50aa - 67aa.

[0076] To further verify the specific sites, JD1 (1aa - 50aa), pcold-PGPV-T (35aa - 77aa), JD2 (63aa - 182aa), JD3 (168aa - 333aa), JD4 (51aa - 183aa), JD5 (45aa - 182aa), JD6 (46aa - 182aa), JD7 (47aa - 182aaa), JD8 (48aa - 182aa), JD9 (1aa - 56aa), JD10 (1aa - 53aa), and JD11 (1aa - 52aa) were respectively constructed.

[0077] The verification results showed that the monoclonal antibody could recognize JD7 and JD10, but could not recognize JD8 and JD11, that is, the recognition site of the 3E1 strain was 47aa - 53aa.

[0078] Figure 5 It is the confirmation of the recognition site of the monoclonal antibody in Example 3. In this figure:

[0079] M: Marker; 1: 30a + full-length P35; 2: JD1 (1aa - 50aa); 3: pcold-PGPV-T (35aa - 77aa); 4: JD2 (63aa - 182aa); 5: JD3 (168aa - 333aa); 6: JD4 (51aa - 183aa); 7: JD5 (45aa - 182aa); 8: JD6 (46aa - 182aa); 9: JD7 (47aa - 182aaa); 10: JD8 (48aa - 182aa); 11: JD9 (1aa - 56aa); 12: JD10 (1aa - 53aa); 13: JD11 (1aa - 52aa).

[0080] Example 4 Kit and Application Method

[0081] 4.1 Prepare the reagents used in the ELISA kit according to the following formula:

[0082] (1) 10×PBS buffer: 80 g sodium chloride, 2 g potassium chloride, 14.4 g disodium hydrogen phosphate heptahydrate, 2.4 g potassium dihydrogen phosphate, made up to 1 L with ddH2O (pH = 7.4);

[0083] (2) 10×PBST buffer: 80 g sodium chloride, 2 g potassium chloride, 14.4 g disodium hydrogen phosphate heptahydrate, 2.4 g potassium dihydrogen phosphate, made up to 1 L with ddH2O (pH = 7.4);

[0084] (3) Antibody diluent, 1×PBST: 100 ml of 10×PBST buffer, 900 ml of ddH20

[0085] (4) Blocking solution, 5% non-fat milk powder: 2.5 g non-fat milk powder, 50 ml of 1×PBST buffer;

[0086] (5) Coating solution: 0.75 g sodium carbonate, 1.46 g sodium bicarbonate, 500 mL of ddH2O, completely dissolved;

[0087] (6) Stop solution: Slowly add 54 mL of 98% sulfuric acid to 446 mL of ddH2O.

[0088] 4.2 Use the ELISA kit according to the following method:

[0089] (1) Coating; Dilute the PGPV P35 protein with the coating solution to 250 ng / wells and incubate overnight at 4°C for 20 h.

[0090] (2) Blocking; Add 300 μl of blocking solution to each well and block at 37°C for 2 h.

[0091] (3) Washing; Add 300 μl of 1×PBST to each well, wash with shaking for 10 min, and repeat 3 times.

[0092] (4) Primary antibody binding; Mix the test serum diluted 1:5 with the HRP-labeled monoclonal antibody diluted 1:3200 at a ratio of 1:1, add 100 μl to each well, incubate at 37°C for 1.5 h, and wash after discarding the primary antibody binding solution.

[0093] (6) TMB color development; Add TMB color development solution, 100 μL / well, incubate at 37°C in the dark for 10 min

[0094] (7) Stopping; Add the reaction stop solution, 50 μL / well, mix well and immediately measure the OD 450 nm value.

[0095] The competition ELISA system established above was used for detection. When the PI% was less than 10.64%, it was judged as negative; when the PI% was greater than 31.73%, it was judged as positive; when the PI% was between 10.64% and 31.73%, it was judged as suspicious.

[0096] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

[0097]

[0098]

[0099]

[0100]

Claims

1. A monoclonal antibody 3E1 against PGPV envelope protein, characterized in that, The heavy chain and light chain of the monoclonal antibody each have three CDR sub-regions that constitute the antigen-binding site, where: the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.

2. The monoclonal antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The monoclonal antibody according to claim 2, wherein The heavy chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown in SEQ ID NO:9, and the light chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO:

10.

4. The monoclonal antibody according to claim 1 or 2, characterized in that, The constant region of the heavy chain is of the IgG1 type, and the constant region of the light chain is the κ chain.

5. The monoclonal antibody according to claim 1 or 2, characterized in that, This monoclonal antibody is prepared using the vaccinia virus p35 protein as the target protein, and the recognition site of this monoclonal antibody is in the 47aa - 53aa interval of the p35 protein.

6. The application method of any monoclonal antibody according to claim 1 or 2, characterized in that It is to use the monoclonal antibody to prepare a kit for detecting pigeonpox virus.

7. A kit for detecting pigeon pox virus, characterized in that, This kit includes any one of the monoclonal antibodies against the PGPV envelope protein described in claim 1 or 2.

8. The kit according to claim 7, characterized in that, This kit is an ELISA detection kit, or any one of an immunochromatographic diagnostic kit using an immunochromatographic test strip and an immunohistochemistry kit; when using the kit, the detection method is serological detection.

9. The kit according to claim 7, wherein This kit includes: an enzyme-labeled plate coated with PGPV P35 protein, the monoclonal antibody described in claim 1 or 2 labeled with HRP, positive control serum and negative control serum, PBS buffer, PBST buffer, blocking solution, antibody diluent, coating solution, and termination solution.

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