Bacteriophage containing short-peptide specifically combined with avian infectious bronchitis virus and use thereof

A bronchitis and chicken infectious technology, applied in the direction of hybrid peptides, specific peptides, peptides, etc., can solve problems that have not been discovered, and achieve the effect of simple operation and short process cycle

Inactive Publication Date: 2006-01-18
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

After searching the PubMed database, so far no relevant reports have been found on the use of pha

Method used

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  • Bacteriophage containing short-peptide specifically combined with avian infectious bronchitis virus and use thereof
  • Bacteriophage containing short-peptide specifically combined with avian infectious bronchitis virus and use thereof
  • Bacteriophage containing short-peptide specifically combined with avian infectious bronchitis virus and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0079] Example 1 Panning and identification of phages containing short peptides that specifically bind to chicken infectious bronchitis virus

[0080] 1.1 Purification of IBV virus

[0081] The H52 strain (Cheng Liqin et al., Cloning and sequence analysis of the S gene of the H52 strain of chicken infectious bronchitis virus, Journal of Zhejiang University. 2002, 28(3): 303-306) allantoic fluid was diluted 10 times and filtered. 1mL for sterility test. The specific method is: add 1 mL of allantoic fluid to 25 mL of medium, and incubate with shaking at 37°C for 3 hours. If it does not become turbid, it proves to be sterile.

[0082] The allantoic fluid was inoculated with 10-day-old chicken embryos, and the allantoic fluid was collected after 96 hours of moisturizing and culturing at 37° C. The allantoic fluid was frozen and thawed three times, centrifuged at a low speed of 3000 rpm for 10 minutes, and the supernatant was taken, PEG6000 (NaCl 2.33%, PEG600010.00%) was The pre...

Example Embodiment

[0112] The application of embodiment 2 positive clone

[0113] 2.1 Amplification of positive phage clones: Take 5 μL of the positive phage clone supernatant obtained in step 1.4 and add it to 20 mL of early logarithmic growth (OD). 600 ~0.3) host strain ER2738 (Ph.D.-12 TM Phage Display Peptide Library Kit, purchased from New England Biolabs), amplified for 4 hours, centrifuged at 10,000 rpm for 10 minutes, took the supernatant, precipitated twice with PEG / NaCl, and finally dissolved with 200 μL TBS to obtain amplified positive phage clones , and then calculate its titer (required reagent formula is the same as step 3).

[0114] 2.2 Hemagglutination inhibition test: The pure virus obtained in step 1.1 was treated with 1% trypsin and then double-diluted from 100× on the hemagglutination plate, and 50 μL of 0.5% fresh chicken erythrocytes were added to each well, and the reaction was carried out at 37°C for 1 Hour, record the coagulation unit. Take 25 μL and mix it with an e...

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Abstract

The present invention belongs to the field of animal virology technology, and is especially one kind of short peptide capable of combining specifically with chicken infectious bronchitis virus (IBV) H52, bacteriophage containing the short peptide and its preparation process. The short peptide has the amino acid sequence of GSHHRHVHSPFV, and may combine with chicken infectious bronchitis virus H52 specifically after passing through the substitution, deletion or addition of one or several amino acid residues. The present invention also includes the application of the bacteriophage in preparing IBV identifying and diagnosing kit, and the IBV identifying and diagnosing kit is used in the preventing and treating of chicken IBV.

Description

technical field [0001] The present invention relates to the technical field of animal virology, in particular to the technical field of phage display. Phage display technology is used to screen phages that can specifically bind to avian infectious bronchitis virus short peptides on the surface, and animal virology technology is used to identify and apply the screened phages. Background technique [0002] Infectious bronchitis of chickens (Infections Bronchitis of chickens, IB) is an acute high-contact infectious disease of chickens, and its pathogen is Infections Bronchitis of chickens Virus (IBV, hereinafter referred to as IBV). IBV causes severe chicken respiratory disease. It is a positive-strand RNA virus belonging to the order Nidovirales and Coronavirdae. Its envelope protein (S) is the receptor-binding protein (Casais) of the virus. R, Recombinant AvianInfectious Bronchitis Virus Expressing a Heterologous Spike Gene Demonstrates that the SpikeProtein Is a Determinant...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K19/00C12Q1/04G01N33/569
Inventor 郭爱珍彭博陈焕春陈汉阳金梅林王冲
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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