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A kind of monoclonal antibody hybridoma cell line 2g8d10 of orfv086 protein of sheep oral ulcer virus and its monoclonal antibody

A technology of ORFV086, hybridoma cell line, applied in antiviral immunoglobulins, microorganisms, biochemical equipment and methods, etc., to achieve high specificity and sensitivity, and reduce application costs.

Active Publication Date: 2016-06-29
GUANGZHOU HONGXIANG BIOMEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no commercial ORFV086 antibody at home and abroad, and its monoclonal antibody subtype is IgG2b, which can be used for ELISA, western-blot, immunofluorescence and immunohistochemical detection, so as to verify the protein as a clinical target and its function. Research lays a solid foundation

Method used

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  • A kind of monoclonal antibody hybridoma cell line 2g8d10 of orfv086 protein of sheep oral ulcer virus and its monoclonal antibody
  • A kind of monoclonal antibody hybridoma cell line 2g8d10 of orfv086 protein of sheep oral ulcer virus and its monoclonal antibody
  • A kind of monoclonal antibody hybridoma cell line 2g8d10 of orfv086 protein of sheep oral ulcer virus and its monoclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 Preparation and identification of monoclonal antibody of the present invention

[0036] 1. Preparation of monoclonal antibody 2G8D10

[0037] 1) Preparation of recombinant ORFV086 antigen

[0038] Using the aphthous ulcer virus genome as a template, the full-length ORFV086 gene was amplified by PCR, and the prokaryotic recombinant expression vector PET33b(+) / ORFV086 was constructed to express in Escherichia coli BL21. Urea was used to open the inclusion bodies, and gel was tapped for purification after gel electrophoresis. The recombinant ORFV086 protein with a purity of more than 90% is obtained.

[0039] 2) Immune mice

[0040] 6-8 week old female BALB / c mice were immunized with purified recombinant antigen.

[0041] The first immunization: After mixing the recombinant antigen with an equal volume of Bentonite adjuvant, inject BALB / c mice intraperitoneally at 50 μg / 500 μL each;

[0042] The second immunization: After 2 weeks, take the recombinant anti...

Embodiment 2

[0053] Example 2, Characterization of Monoclonal Antibodies

[0054] 1) Determination of antibody concentration: the ascitic fluid prepared by hybridoma cells CCTCCNO:C201471 was purified to obtain ORFV086 monoclonal antibody 2G8D10, and the concentration was 0.51mg / ml respectively using the SmartSpecplus nucleic acid and protein analyzer produced by BIO-RAD Company. .

[0055] 2) Antibody subtype identification: use the Roche company's mouse monoclonal antibody subtype identification kit to identify the subtype of the hybridoma cell line, the subtype of the antibody secreted by 2G8D10 is IgG2b type, and the light chains are all κ chains, such as figure 1 shown.

[0056] 3) Identification of the titer of the purified antibody: 50 μg of recombinant ORFV086 antigen was dissolved in 10 ml of 0.05 M carbonate coating buffer at pH 9.6, added to 100 μL per well of a 96-well plate, and left overnight at 4°C. Wash the plate three times with PBS (containing 0.05% (V / V) Tween-20), use...

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Abstract

The invention discloses an orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and a monoclonal antibody thereof. A collection number of the hybridoma cell strain 2G8D10 is CCTCC NO: C201471. The monoclonal antibody is secreted from the hybridoma cell strain 2G8D10 with the collection number of CCTCC NO: C201471. The monoclonal antibody can carry out high-sensitivity specific antigen-antibody reaction with the orf virus protein ORFV086, and can be effectively used for carrying out biological diagnosis including ELISA (enzyme-linked immuno sorbent assay), western-blot, immunofluorescence and immunohistochemical detection onto the orf virus protein ORFV086.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a hybridoma cell line 2G8D10 and a monoclonal antibody of the ORFV086 monoclonal antibody of the oral ulcer virus protein. Background technique [0002] Ovine infectious impetigo (Orf), also known as ovine infectious impetigo, is an acute, contagious disease of goats and sheep caused by oral ulcer virus (ORFV). The lesion is characterized by the formation of erythema, papules, abscesses, ulcers and verrucous scabs on the skin and mucous membranes of the affected sheep. Lambs are very susceptible and often occur in groups. The disease occurs all over the world, and it is also common in the main sheep-raising areas of my country. It is one of the main diseases that harm sheep. The virus has strong resistance, and once the flock is infected, it is not easy to get rid of it. It can continue to harm the flock for many years and cause major economic losses to the animal husb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/08C12R1/91
Inventor 罗树红成敏王勇
Owner GUANGZHOU HONGXIANG BIOMEDICAL TECH
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