Monoclonal antibody and antibody composition for neutralizing canine distemper virus (CDV)

A canine distemper virus, monoclonal antibody technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulins, etc., can solve the loss of neutralization ability of virus variants, easy transmission of blood-borne diseases, and neutralization titer. The problem of unevenness, etc., achieves the effect of a wide range of clinical applications, improved treatment effect, and strong virus neutralization ability.

Active Publication Date: 2013-04-24
JIANGSU ACAD OF AGRI SCI
View PDF2 Cites 20 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Canine distemper hyperimmune serum has problems such as high production cost, easy transmission of blood-borne diseases, uneven neutralization potency, and difficulty in standardization. Monoclonal antibodies can make up for the above defects, but the monoclonal antibody recognizes a single antigenic site , using a monoclonal antibody to treat canine distemper, may lose its neutralizing capacity against emerging canine distemper virus variants

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Establishment of Hybridoma Cell Line Secreting Monoclonal Antibody

[0027] 1. Preparation of CDV Antigen Inoculate CDV NJ01 virulent strain (Bi Zhenwei et al., Sequence Analysis of Atypical Canine Distemper Virus Nucleocapsid Protein Gene and Its Expression in Escherichia coli. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(8): 625-629.) In Vero cells, cell lesions (CPE) occurred in 3 days, and the poison was collected after 5 days. Lesioned cell cultures were repeatedly frozen and thawed three times, and cell debris were removed after centrifugation at 4000×g. After the virus supernatant was precipitated by 1M zinc acetate solution, the precipitate was resuspended with saturated EDTA solution, centrifuged at 49000×g for 60 min at 4°C, and the precipitate was fully suspended with NTE. Add 20%, 30%, 40%, 50% and 60% (mass / volume) density gradient sucrose solutions, and centrifuge at 61000×g for 90 min. Aspirate the virus band, observe the v...

Embodiment 2

[0036] Example 2: Cloning and sequence determination of the variable region gene of the monoclonal antibody 2G3

[0037] 1. RNA Extraction from Hybridoma Cells

[0038] The hybridoma cell line 2G3 secreting monoclonal antibody 2G3 was cultured in RPMI 1640 containing 15% FCS (product of Lanzhou Minhai Company) at 37°C, 5% CO 2 Cultured in the incubator until the logarithmic growth phase, the cells were blown down with an appropriate amount of PBS, and placed in a -20°C refrigerator to freeze and thaw three times. Total RNA was extracted according to the method used by TRIZOL® Reagent (InvitrogenTM Life technologies).

[0039] 2. RT-PCR method to amplify the VL and VH genes of monoclonal antibodies

[0040] Two pairs of primers, VL F and VL B for the variable region of the light chain of the monoclonal antibody, and VH F and VH B for the variable region of the heavy chain were designed and sent to Shanghai Invitrogen Company for synthesis. The primer sequences are as ...

Embodiment 3

[0049] Example 3: Biological properties of monoclonal antibody 2G3

[0050] 1. Identification of Monoclonal Antibody Specificity

[0051] Indirect immunofluorescence test was used to verify whether monoclonal antibody 2G3 reacted with Vero cells. Infect Vero cells with CDV, and after culturing for 72 hours, discard the cell culture medium, wash twice with serum-free culture medium, then add -20°C pre-cooled absolute ethanol 1mL / well to the cell culture wells, and fix at 4°C 30 min, wash with PBS 3 times, pat dry; add monoclonal antibody 2G3, incubate at 37°C for 1 h, wash 3 times with PBS, pat dry; add 200 times diluted FITC-labeled goat anti-mouse IgG antibody (purchased from Wuhan Boster Bioengineering Co., Ltd.), incubated at 37°C for 1 h, washed 5 times with PBS, and observed under a fluorescent microscope. Monoclonal antibody 2G3 reacted with CDV-infected Vero cells and produced fluorescence, but had no fluorescence with normal Vero cells. Use an indirect ELISA met...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a monoclonal antibody and an antibody composition for neutralizing canine distemper virus (CDV), belonging to the technical field of biology. The monoclonal antibody 2G3 disclosed by the invention has high activity for neutralizing CDV and is used for identifying epitopes differently acting on CDV with a monoclonal antibody for neutralizing CDV prepared from another screened hybridoma cell 1D7 strain. Two monoclonal antibodies are prepared into the antibody composition, the neutralizing capability on CDV of which is obviously prior to the single monoclonal antibodies 2G3 and 1D7 in the composition; after being combined, the two monoclonal antibodies can generate antiviral synergistic enhancement action; and both the range and the capability for neutralizing CDV are increased. The monoclonal antibody disclosed by the invention is used for preparing the antibody composition for treating clinical canine distemper paroxysm animal; the virus neutralizing capability is stronger; failure of the monoclonal antibody caused by CDV heteromorphosis can be avoided; and the clinical application range is wider.

Description

technical field [0001] The invention relates to neutralizing canine distemper virus (CDV) monoclonal antibody 2G3 and an antibody composition, belongs to the field of biotechnology, and relates to antibody engineering technology. Specifically, the monoclonal antibody 2G3 has high neutralizing activity against CDV, and the combination with another neutralizing monoclonal antibody 1D7 acting on different epitopes of CDV produces synergistic antiviral enhanced effect, the neutralizing ability is improved, and CDV is neutralized The range of virus strains is wide, and it can be applied to the clinical treatment of canine distemper (CD) diseased animals. Background technique [0002] Canine distemper virus (CDV) is the pathogen that causes canine distemper (CD) in many animals such as dogs, foxes, and minks. It can infect various cells and tissues in animals, and lymphocytes and epithelial cells have the strongest affinity. It is a pantropic virus that is extremely harmful to an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/11A61K39/42A61P31/14
Inventor 毕振威夏兴霞王永山潘群兴诸玉梅董晨红欧阳伟王晓丽徐立波
Owner JIANGSU ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap