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Competitive ELISA antibody detection kit for infectious bovine rhinotracheitis virus and application thereof

Active Publication Date: 2021-10-01
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report on the competitive ELISA antibody detection method for bovine infectious rhinotracheitis virus

Method used

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  • Competitive ELISA antibody detection kit for infectious bovine rhinotracheitis virus and application thereof
  • Competitive ELISA antibody detection kit for infectious bovine rhinotracheitis virus and application thereof
  • Competitive ELISA antibody detection kit for infectious bovine rhinotracheitis virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1. Design and expression of IBRV recombinant gB protein

[0058] 1. Sequence optimization and primer synthesis

[0059] Download the IBRV gB gene sequence (GeneID: 4783419) from the GenBank database, analyze the sequence using the online analysis website (http: / / www.cbs.dtu.dk / services / TMHMM / ), and design a truncated gB gene sequence ( SEQ ID NO: 1), which encodes a truncated gB protein (SEQ ID NO: 2). In order to secrete and express the truncated gB protein in insect cells, the codon optimization of the truncated gB gene sequence was performed according to insect cell preference to obtain the target gene sequence (SEQ ID NO: 3). Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the target gene. According to the base sequence of the baculovirus expression vector and the target gene sequence, a pair of homology arm amplification primers were designed, and restriction sites NdeI (CATATG) and XhoI (CTCGAG) were added to the upstream and downstre...

Embodiment 2

[0079] Example 2. Preparation and screening of anti-IBRV monoclonal antibodies

[0080] 1. Animal Immunization

[0081] First immunization: After mixing the purified IBRV virus liquid (Bartha Nu / 67 standard strain) with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, product number F5881) and emulsifying completely, immunize 6-8 week-old SPF grade female BALB / c mice, the immunization dose was 50 μg / mouse. Two weeks after the first immunization, an equal volume of IBRV virus fluid (Bartha Nu / 67 standard strain) was fully emulsified with Freund's incomplete adjuvant (Sigma-Aldrich, product number F5506), and the second, third and third times were carried out. 4 times of immunization, the immunization dose was 50μg / monkey, and the interval between each immunization was 2 weeks.

[0082] 2. Serum titer determination

[0083] The indirect ELISA method for hybridoma screening was established by using the positive serum of the mice after three immunizations, and the...

Embodiment 3

[0107] Example 3. Establishment of IBRV competition ELISA antibody detection method

[0108] 1. Horseradish peroxidase-labeled monoclonal antibody

[0109] The 1F24 monoclonal antibody was labeled with horseradish peroxidase (HRP) by the sodium periodate method, and the steps were as follows:

[0110] (1) Weigh 5 mg of HRP (Sigma-Aldrich, SRE0082) and dissolve it in 1000 μL of double-distilled water, and continue to add 500 μL of newly prepared 0.1M NaIO 4 , mix well, and incubate at 4°C in the dark for 30min.

[0111] (2) Add 500 μL of freshly prepared 0.2M ethylene glycol (Sigma-Aldrich, 324558) solution, mix well, and incubate at 4°C in the dark for 30 minutes.

[0112] (3) Add 5 mg of purified 1F24 monoclonal antibody to the above solution, mix well and put it into a pre-treated dialysis bag (Solarbio, MW14000), dialyze overnight in 0.1M carbonate buffer solution with pH 9.6, and dialyze The medium was changed three times during this period.

[0113] (4) Pour the dialy...

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Abstract

The invention relates to antibody detection of infectious bovine rhinotracheitis virus, in particular to a competitive ELISA antibody detection kit for the infectious bovine rhinotracheitis virus and the application of the competitive ELISA antibody detection kit. The invention provides the monoclonal antibody for resisting infectious bovine rhinotracheitis virus. The monoclonal antibody is secreted by hybridoma cells with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.23005. The invention further provides an antibody detection kit for the infectious bovine rhinotracheitis virus, and the antibody detection kit comprises the monoclonal antibody. Based on the monoclonal antibody and the truncated infectious bovine rhinotracheitis virus gB protein designed and expressed in the invention, an antibody detection method of the infectious bovine rhinotracheitis virus is established. The method has the advantages of high specificity, high sensitivity, simplicity and convenience in operation and rapidness.

Description

technical field [0001] The invention relates to antibody detection of bovine infectious rhinotracheitis virus, in particular to a competitive ELISA antibody detection kit of bovine infectious rhinotracheitis virus and application thereof. Background technique [0002] Infectious bovine rhinotracheitis virus (IBRV), also known as bovine herpesvirus-1 (BHV-1), belongs to the herpesviridae family and is an important pathogen of cattle. Infectious Bovine Rhinotracheitis (IBR). Bovine infectious rhinotracheitis is a febrile, acute and highly latent infectious disease. Since Miller first reported the disease in the United States in 1995, IBR has shown a global epidemic trend, which has caused huge economic losses to the world cattle industry. The main hazard of IBRV is that after the virus invades the host, it can latently infect the ganglion of the host, causing the sick cattle to be infected for life, which brings a huge challenge to the prevention and control of the disease. ...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N5/20G01N33/577G01N33/569G01N33/68G01N33/58G01N33/543
CPCC07K16/085G01N33/577G01N33/56994G01N33/6854G01N33/581G01N33/54393C07K2317/76G01N2469/20
Inventor 刘文晓李永清程晶江波江岳王晓玥段景龙
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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