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Recombinant baculovirus for expressing spike protein of chicken infectious bronchitis virus and application thereof

A technology of recombinant baculovirus and bronchitis, applied in the biological field, can solve problems such as not suitable for large-scale quarantine and grass-roots use, and easy to disperse viruses

Active Publication Date: 2011-01-19
国家兽用生物制品工程技术研究中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country has established methods such as indirect ELISA (I-ELSIA) with whole virus as antigen and hemagglutination inhibition test to detect chicken IBV antibody, but these methods are not suitable for large-scale quarantine and grassroots use, and the use of live virus as antigen is easy to disperse the virus

Method used

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  • Recombinant baculovirus for expressing spike protein of chicken infectious bronchitis virus and application thereof
  • Recombinant baculovirus for expressing spike protein of chicken infectious bronchitis virus and application thereof
  • Recombinant baculovirus for expressing spike protein of chicken infectious bronchitis virus and application thereof

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Embodiment 1

[0022] Example 1 Preparation of Recombinant Baculovirus Expressing Chicken Infectious Bronchitis Virus Spike Protein

[0023] (1) Source of material

[0024] Restriction enzymes Sal I, BamH I, T 4 DNA ligase, Ex-Tag DNA polymerase, and pMD18 were all purchased from Bao Biotechnology Co., Ltd.

[0025] In the present embodiment, the nephrophilic chicken infectious bronchitis virus virulent vaccine strain is used as a template to amplify its spike protein gene (S gene). The microorganism deposit number is: CGMCC No.3957.

[0026] Preservation information is as follows:

[0027] Biological material (virus strain): Ck / Jiangsu / DS10 / 2008

[0028] Taxonomic designation: chicken infectious bronchitis virus

[0029] Latin name: Infectious Bronchitis Virus (IBV)

[0030] Deposit unit: General Microbiology Center of China Committee for Microorganism Culture Collection (CGMCC)

[0031] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West...

Embodiment 2

[0051] Example 2 Application of recombinant baculovirus vAcIBV-S to detect chicken infectious bronchitis virus antibody

[0052] Preparation of coated antigens (vAcIBV-S, vAc): Inoculate Sf9 insect cells with recombinant baculovirus vAcIBV-S at an inoculation volume of 5-10 MOI (multiplicity of infection), and harvest virus vAcIBV-S after culturing at 27°C for 5 days. The virus-infected cells were scraped off, centrifuged for 10 minutes (10000g), and the precipitate was collected and suspended in the cell lysate (20mmol / L Tris-HCl, 150mmol / L NaCl, pH7.4) of 1 / 20 of the original culture volume, Concentrate 20 times to prepare concentrated vAcIBV-S antigen. Concentrated vAc antigen was prepared as a blank control in the same way.

[0053] Coating solution: carbonate buffer solution (pH 9.6) with a concentration of 0.05M.

[0054] Washing solution: PBS buffer containing 10mL / L Tween-20.

[0055] Diluent: PBS buffer containing 10g / L skimmed milk powder and 10mL / L Tween-20.

[...

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Abstract

The invention relate to a recombinant baculovirus for expressing spike protein of chicken infectious bronchitis virus and application thereof, belonging to the biotechnical field. The recombinant baculovirus is prepared by a method comprising the following steps: obtaining S genes; building and indentifying a recombinant transfer vector pFastBacI-S; building and indentifying a recombinant shuttle vector AcIBV-SBacmid which infects Sf9 inset cells; and obtaining the recombinant baculovirus vAcIBV-S for expressing the spike protein of the chicken infectious bronchitis virus. The invention further provides the application of the recombinant baculovirus in the aspect of detecting chicken infectious bronchitis virus antibodies. The recombinant baculovirus can be used for detecting the chicken infectious bronchitis virus antibodies, thereby avoiding the risk of easy toxin diffusion when live virus serves as an antigen. The recombinant baculovirus can be used for detecting the chicken infectious bronchitis virus antibodies, can be used for quarantine on a large scale, and is especially suitable for use of quarantine departments at the basic level.

Description

technical field [0001] The invention relates to a recombinant baculovirus expressing chicken infectious bronchitis virus spike protein and application thereof, belonging to the field of biotechnology. Background technique [0002] Chicken infectious bronchitis (infectious bronchitis, IB) is an acute, highly contagious disease in chickens caused by infectious bronchitis virus (IBV) of the family Coronavirndae and the genus Coronavirus. Infectious diseases mainly attack the respiratory system, digestive system and genitourinary system of chickens, causing symptoms such as coughing, sneezing, tracheal rales, dyspnea, enteritis, and decreased egg production and quality. Chickens of various ages and sexes are susceptible to infection, but it mainly affects 1-4W chicks, and the mortality rate is as high as 40%-90%. The disease causes a reduction in feed remuneration, an increase in the death rate, and the loss caused by low feeding efficiency is even greater than the death loss o...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/50C12N15/866G01N33/569
Inventor 吴培培唐应华于漾揭鸿英侯继波何家惠
Owner 国家兽用生物制品工程技术研究中心