Single clone antibody aiming at H3N2 dog flu virus HA2 protein

A monoclonal antibody, canine influenza virus technology, applied in the direction of antiviral immunoglobulins, antibodies, antiviral agents, etc., to achieve good protection, improve safety, and reduce risks.

Active Publication Date: 2015-01-21
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, due to the high variability in the amino acid sequence of HA1, the neutralizing activity of monoclonal antibodies against HA1 is limited [de Jong JC, Palache AM, Beyer W, et al. Haemagglutination-inhibiting antibody to influenza virus. Dev Biol (Basel) 2003, 115 :63-73.]; and HA2 is located in the stem of HA, which is the conserved region of all subtypes of influenza virus HA, and plays a key role in the adhesion of the virus and the invasion of the host cell membrane by the virus

Method used

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  • Single clone antibody aiming at H3N2 dog flu virus HA2 protein
  • Single clone antibody aiming at H3N2 dog flu virus HA2 protein
  • Single clone antibody aiming at H3N2 dog flu virus HA2 protein

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Embodiment Construction

[0035] Below in conjunction with specific embodiment. The experimental method that does not indicate specific condition in the embodiment, generally adopts routine condition, for example " Molecular Cloning Laboratory Handbook " (New York: Cold Spring Harbor Laboratory Press, 1989) (Sambrook et al.) described condition, or according to The method recommended by the manufacturer. For standard hybridoma technology, refer to [Vareckova E, Betakova T, Mucha V, et al. Preparation of monoclonal antibodies for the diagnosis of influenza A infection using different immunization protocols. J Immunol Methods 1995, 180: 107-116].

[0036] (1) Preparation of monoclonal antibody to H3N2 canine influenza virus HA2 protein

[0037] 1. Selection of standard strains

[0038] The standard strain used in the present invention is canine influenza virus strain A / Canine / Jiangsu / 06 / 2010 (H3N2) (abbreviated as JS / 10), whose GenBank sequence numbers are from JN247615 to JN247623. The strain and its...

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Abstract

The invention provides a single clone antibody aiming at H3N2 dog flu virus HA2 protein, belonging to the technical field of biologics. The single clone antibody is prepared by inoculating an H3N2 dog flu virus Jiangsu separated strain JS / 10 separated in 2010 with SPF chicken embryos of 10 days old, collecting allantoic fluid of which the blood clotting titer is greater than or equal to 6, purifying virus inoculation MDCK cells, performing intracutaneous multipoint injection to inoculate mice, after cell fusion, performing three times of ELISA to detect cell supernate that cell colonies are generated, selecting an anti-H3 type hybridization tumor cell strain mAbD7, and recognizing and identifying the antigen so as to verify that the antibody aims at conservative H2A protein, wherein the neutralizing antibody titer is up to 1:12800, and the antibody subclass is IgG2b. The single clone antibody aiming at H3N2 dog flu virus and HA2 protein, which is provided by the invention, provides an important immunological preparation for treating H3N2 dog flu virus infection and has a potential application prospect.

Description

technical field [0001] The invention relates to a monoclonal antibody against H3N2 canine influenza virus HA2 protein, belonging to the field of biotechnology. Standard hybridoma technology, using a H3N2 canine influenza virus strain JS / 10 as the test strain, and finally obtained the monoclonal antibody D7 targeting the conserved protein HA2 through hemagglutination inhibition, neutralization test and Western blotting screening, which is H3N2 canine influenza virus Treatment of infection provides important immune agents. Background technique [0002] The principle of hybridoma technology is to use polyethylene glycol (PEG) as a cell fusion agent to fuse the spleen cells of immunized mice with mouse myeloma cells with long-term reproductive ability in vitro. The effect of HAT selective medium Under this condition, only hybridoma cells that have successfully fused are allowed to grow, and after repeated immunological testing, screening and single cell culture (cloning), a hyb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10A61K39/42A61P31/16
Inventor 刘永杰谢星庞茂达林焱赵艳兵梁珊陆承平
Owner NANJING AGRICULTURAL UNIVERSITY
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