Canine anti-parvovirus monoclonal antibody

Abstract

This invention discloses a canine parvovirus monoclonal antibody, belonging to the field of biomedicine. It is a canine parvovirus monoclonal antibody screened using high-throughput sequencing. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2. This monoclonal antibody can react with the structural proteins of canine parvovirus particles, which is helpful for the detection and identification of canine parvovirus. Simultaneously, this monoclonal antibody can neutralize various subtypes of canine parvovirus, which is of great significance for the diagnosis and treatment of canine parvovirus disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a broad-spectrum neutralizing monoclonal antibody against canine parvovirus obtained by high-throughput sequencing and its application. Background Technology

[0002] Treatment for canine parvovirus disease primarily relies on symptomatic and supportive therapy. The latest veterinary clinical data shows that a strategy combining monoclonal antibody drugs with symptomatic treatment can increase the cure rate to 82%. The core breakthrough of this combination therapy lies in the application of CPV monoclonal antibodies, which can specifically bind to canine parvovirus, preventing viral adsorption and entry. In conclusion, antibody therapy plays a crucial role in the treatment of canine parvovirus disease, and through continuous research and clinical practice, it is hoped that the cure rate can be further improved, and the prognosis for dogs can be better. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a canine anti-parvovirus monoclonal antibody with broad-spectrum neutralizing effect.

[0004] The technical solution of the present invention is: a canine anti-parvovirus monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2.

[0005] Furthermore, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID No. 3, and the amino acid sequence of the light chain constant region of the monoclonal antibody is shown in SEQ ID No. 4.

[0006] The DNA fragment encoding the monoclonal antibody described above.

[0007] Vectors or engineered bacteria containing the DNA fragments described above.

[0008] The application of the monoclonal antibody described above in the preparation of a canine parvovirus diagnostic kit.

[0009] The above-described monoclonal antibody is used in the preparation of reagents or drugs for the treatment of canine parvovirus.

[0010] A drug containing the monoclonal antibody described above.

[0011] Furthermore, it also includes vectors for the delivery of monoclonal antibodies.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. This invention can rapidly obtain a large number of naturally paired antibody sequences by screening antigen-specific B cells and high-throughput sequencing, which greatly shortens the antibody development cycle.

[0014] 2. The monoclonal antibody prepared by this invention is a canine monoclonal antibody, which does not require canine-derived modification and largely ensures the natural origin of the antibody conformation.

[0015] 3. The monoclonal antibodies prepared by this invention have lower immune rejection compared to mouse or rabbit monoclonal antibodies prepared by traditional techniques.

[0016] 4. The monoclonal antibodies prepared in this invention exhibit good neutralizing activity against all CPV strains, with antibody neutralizing titers of 1.22-1.92 μg / mL; they have high application value in the diagnosis and treatment of CPV. Attached Figure Description

[0017] Figure 1 Biotin labeling results of canine parvovirus particles.

[0018] Figure 2 Flow cytometry results of antigen-specific B cells.

[0019] Figure 3 SDS-PAGE results of successful monoclonal antibody expression, where E9-RE is the denatured band and E9-NR is the undenatured band.

[0020] Figure 4 : Results of reactivity assay for monoclonal antibodies (ELISA).

[0021] Figure 5 : The result of the reactivity assay (IFA) of the monoclonal antibody. Detailed Implementation

[0022] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from commercial channels.

[0023] Example 1: Preparation of canine parvovirus monoclonal antibody

[0024] 1. Animal immunization

[0025] Purchase three healthy 1-month-old puppies, vaccinate them three times with CPV-2b attenuated live vaccine, and then vaccinate them twice with CPV-2c inactivated vaccine, with an interval of 14 days between each two vaccinations.

[0026] 2. Isolation of specific B cells from peripheral blood

[0027] (1) Biotin labeling of the antigen: The CPV-2c strain was extensively proliferated on F81 cells (cat kidney cells). The specific method was as follows: F81 cells were cultured and passaged and simultaneously inoculated with the virus. After inoculation, the cells were repeatedly frozen and thawed for 3-4 days, and the cell supernatant was collected. The supernatant was concentrated with PEG6000, and the virus particles were purified by sucrose density gradient centrifugation. Transmission electron microscopy showed that the virus particles were morphologically intact. The biotin-labeled purified virus particles were successfully labeled with biotin using streptavidin antibody detection, and the label was denoted as CPV-2c-Biotin. The results are as follows: Figure 1 As shown.

[0028] (2) Sorting of antigen-specific B cells

[0029] Blood was collected from the jugular vein of immunized dogs. Peripheral blood mononuclear cells were isolated using lymphocyte separation medium (Tianjin Haoyang Biotechnology) after dilution with PBS. Cells were resuspended in PBS and stained: Anti-dog-IgG-FITC (Bio-Rad), Anti-canine-IgM-PE (Bio-Rad), and CPV-2c-Biotin were added, and the cells were incubated at 4°C for 30 min. After washing three times, Anti-Biotin-APC (Miltenyi) was added to the cell suspension as a secondary antibody, and the cells were incubated at 4°C for 30 min. To better distinguish antigen-specific B cells, an FMO control was also set up (other antibodies besides the antigen were incubated in the same way). Subsequently, antigen-specific single B cells were sorted using BD FACS Aria II flow cytometry. The results are shown below. Figure 2 As shown.

[0030] 3. High-throughput sequencing enables the acquisition of variable region genes in antibody heavy and light chains.

[0031] Primers for amplifying the antibody heavy chain variable region and light chain variable region were designed based on the usage preference of antibody variable region genes. After mixing the sorted antigen-specific B cells with a single barcode, high-throughput sequencing was performed to obtain the antibody heavy chain variable region gene sequence and the light chain variable region gene sequence.

[0032] 4. Eukaryotic expression and purification of antibodies

[0033] The antibody heavy and light chain variable region genes obtained from sequencing were constructed into the eukaryotic expression vector pcDNA3.4 containing the antibody constant region sequence. The antibody was expressed via plasmid co-transfection using Zhuhai Kairui's serum-free suspension culture expression system. After 5 days of suspension culture, cell supernatant was collected, and antibody was purified using a protein A pre-packed column. Denaturing / non-denaturing SDS-PAGE electrophoresis was used to identify a portion of the purified antibody. Under non-denaturing conditions, the antibody molecular weight was approximately 170 kDa. After denaturation, the disulfide bonds between the heavy and light chains were broken, and the antibody separated into heavy chains (approximately 55 kDa) and light chains (approximately 30 kDa), indicating that the canine CPV monoclonal antibody E9 was correctly expressed and assembled. The results are as follows: Figure 3 As shown.

[0034] Example 2: Reactivity of canine monoclonal antibodies

[0035] 1. Enzyme-linked immunosorbent assay (ELISA)

[0036] The purified CPV was added to 100 μL / well (the same amount of antibody was added subsequently) and incubated overnight at 4°C. After washing 5 times with PBST, 5% skim milk was added and the plate was blocked at 37°C for 2 h. After washing 5 times with PBST, 2-fold serially diluted antibody E9 was added, with PBS as a control, and the plate was incubated at 37°C for 1.5 h. After washing 5 times with PBST, 1:5000 diluted Anti-dog-IgG-HRP antibody was added and the plate was incubated at 37°C for 0.5 h. After washing 5 times with PBST, chromogenic solution was added and the plate was incubated at 37°C for 10-15 min. The OD values ​​were then read using a microplate reader. 630 nm The value, the result is as follows Figure 4 As shown.

[0037] 2. Indirect immunofluorescence assay (IFA)

[0038] Different canine parvoviruses (CPV-2, CPV-2a, CPV-2b, CPV-2c) were inoculated into 24-well plates containing F81 cells. After 48 hours of inoculation, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton-100 for 10 minutes, washed three times with PBS, and then 200 μL / well of 10 μg / mL antibody was added and incubated at 37°C for 1.5 hours. After washing three times with PBS, 1:200 diluted anti-canine IgG-FITC antibody was added and incubated at 37°C in the dark for 0.5 hours. After washing three times with PBS, 1 mL of PBS was added, and the cells were observed and photographed under a fluorescence inverted microscope. The results are shown below. Figure 5 As shown.

[0039] Example 3: Neutralizing activity of canine monoclonal antibodies

[0040] Serially diluted antibody with CPV (200 TCID) 50Mix well and incubate at 37°C for 1 hour. Then, add the digested F81 cells to a 96-well plate containing the virus-antibody mixture and incubate the plate at 37°C with 5% CO2 for 4 days. Finally, determine the IC50 of the antibody using immunofluorescence. 50 The neutralizing activity of the antibody was assessed using a value, and the results are shown in Table 1.

[0041] Table 1 Neutralizing titers of monoclonal antibodies

[0042] CPV-2 CPV-2a CPV-2b CPV-2c E9 1.92 μg / mL 1.51 μg / mL 1.56 μg / mL 1.22 μg / mL

[0043] As shown in Table 1, the monoclonal antibodies prepared in this invention exhibit good neutralizing activity against all CPV strains, with antibody neutralizing titers ranging from 1.22 to 1.92 μg / mL.

[0044] Example 4: Application of Monoclonal Antibodies

[0045] The monoclonal antibody of this invention has high application value in the diagnosis and treatment of CPV.

[0046] This monoclonal antibody can be used to prepare drugs for the treatment or prevention of canine parvovirus, and the drugs may also contain a carrier for delivering the drug.

[0047] This monoclonal antibody can also be used for the diagnosis of parvovirus, and it can be prepared into a canine parvovirus diagnostic kit.

Claims

1. A canine anti-parvovirus monoclonal antibody, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID No. 3, and the amino acid sequence of the light chain constant region of the monoclonal antibody is shown in SEQ ID No.

4.

3. A DNA fragment encoding the monoclonal antibody of claim 1 or 2.

4. A vector or engineered bacteria containing the DNA fragment described in claim 3.

5. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a canine parvovirus diagnostic kit.

6. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of reagents for treating canine parvovirus.

7. A drug comprising the monoclonal antibody as described in claim 1 or 2.

8. The medicament according to claim 7, characterized in that, It also includes vectors for monoclonal antibody delivery.

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