CN-120943943-B - Anti-parvovirus monoclonal antibodies from canine sources

Abstract

The invention discloses a canine parvovirus monoclonal antibody, belonging to the field of biological medicine, which is a canine parvovirus monoclonal antibody screened by high-throughput sequencing, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID No. 2. The monoclonal antibody can react with canine parvovirus particle structural protein, is favorable for detection and identification of canine parvovirus, can neutralize each subtype strain of canine parvovirus, and has important significance for diagnosis and treatment of canine parvovirus diseases.

Inventors

• ZHOU HONGBO
• LI YING
• WANG ZHIHAO
• ZOU JIAHUI
• LIN MINGQIN
• WANG SHENG
• ZHANG CHENGGUANG
• ZHAO LING

Assignees

• 华中农业大学

Dates

Publication Date

20260505

Application Date

20250915

Claims (8)

1. 1. The canine parvovirus resistant monoclonal antibody is characterized in that the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID No. 2.
2. 2. The monoclonal antibody according to claim 1, wherein the heavy chain constant region amino acid sequence of the monoclonal antibody is shown in SEQ ID No.3 and the light chain constant region amino acid sequence of the monoclonal antibody is shown in SEQ ID No. 4.
3. 3. A DNA fragment encoding the monoclonal antibody of claim 1 or 2.
4. 4. A vector or engineering bacterium comprising the DNA fragment of claim 3.
5. 5. Use of the monoclonal antibody of claim 1 or 2 in the preparation of a canine parvovirus diagnostic kit.
6. 6. Use of the monoclonal antibody of claim 1 or 2 in the preparation of an anti-canine parvovirus agent.
7. 7. A medicament containing the monoclonal antibody of claim 1 or 2.
8. 8. The medicament of claim 7, further comprising a carrier for monoclonal antibody delivery.

Description

Anti-parvovirus monoclonal antibodies from canine sources Technical Field The invention belongs to the field of biological medicine, and relates to a canine parvovirus broad-spectrum neutralization monoclonal antibody obtained by high-throughput sequencing and application thereof. Background The treatment of canine parvovirus disease mainly depends on symptomatic support therapy, and the latest veterinary clinical data so far show that the cure rate can be increased to 82% by using monoclonal antibody drugs in combination with symptomatic treatment strategies. The core breakthrough of this combination therapy is the use of CPV monoclonal antibodies, which specifically bind canine parvovirus, preventing viral adsorption and entry. In a word, the antibody treatment has great significance in the treatment of canine parvovirus disease, and is expected to further improve the cure rate and improve the prognosis of dogs through continuous research and clinical practice. Disclosure of Invention The invention aims to provide a canine anti-parvovirus monoclonal antibody with broad-spectrum neutralization effect. The canine anti-parvovirus monoclonal antibody has the technical scheme that the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID No. 2. Further, the heavy chain constant region amino acid sequence of the monoclonal antibody is shown as SEQ ID No.3, and the light chain constant region amino acid sequence of the monoclonal antibody is shown as SEQ ID No. 4. A DNA fragment encoding the above monoclonal antibody. A vector or an engineering bacterium containing the DNA fragment. The monoclonal antibody is applied to preparing a canine parvovirus diagnostic kit. The monoclonal antibody is applied to preparation of an anti-canine parvovirus reagent or medicament. A medicament comprising the monoclonal antibody described above. Further, vectors for monoclonal antibody delivery are also included. Compared with the prior art, the invention has the following beneficial effects: 1. The invention can rapidly obtain a large number of naturally paired antibody sequences by screening antigen-specific B cells and high-throughput sequencing, thereby greatly shortening the development period of the antibody. 2. The canine monoclonal antibody prepared by the invention does not need to carry out caninization transformation, and ensures the natural originality of antibody conformation to a great extent. 3. Compared with the mouse or rabbit monoclonal antibodies prepared by the traditional technology, the monoclonal antibodies prepared by the invention have smaller immune rejection. 4. The monoclonal antibody prepared by the invention has good neutralizing activity on various strains of CPV, the neutralizing titer of the antibody is 1.22-1.92 mug/mL, and the monoclonal antibody has high application value in the aspects of CPV diagnosis and treatment. Drawings FIG. 1 shows the results of biotin labeling of canine parvovirus particles. FIG. 2 results of flow-sorting antigen-specific B cells. FIG. 3 SDS-PAGE results of successful expression of monoclonal antibodies, wherein E9-RE is a denatured band and E9-NR is an undenatured band. FIG. 4 shows the results of monoclonal antibody reactivity detection (ELISA). FIG. 5 shows the results of the reactivity test (IFA) of the monoclonal antibodies. Detailed Description The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below, unless otherwise specified, were purchased from commercial sources. EXAMPLE 1 preparation of canine parvovirus monoclonal antibody 1. Immunization of animals 3 Healthy dogs of 1 month old were purchased, immunized three times with CPV-2b attenuated vaccine, and boosted 2 times with CPV-2c inactivated vaccine, with a 14 day interval between each two immunizations. 2. Specific B cell isolation in peripheral blood (1) Biotin labelling of antigen CPV-2c strain was propagated in large amounts on F81 cells (cat kidney cells). The specific method comprises culturing passage F81 cells and synchronously inoculating, repeatedly freezing and thawing 3-4days after inoculating, collecting cell supernatant, concentrating supernatant with PEG6000, centrifuging to purify virus particles with sucrose density gradient, observing the virus particles to be complete by transmission electron microscopy, labeling the purified virus particles with Biotin, detecting Biotin by streptavidin antibody, and marking on the virus particles as CPV-2c-Biotin, wherein the result is shown in figure 1. (2) Sorting of antigen-specific B cells The immunized dogs were collected by jugular vein, diluted with PBS, and peripheral blood mononuclear cells were isolated with lymphocyte separation solution (Tianjin, ocean organism). Cells were re