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Chicken infectious bursal disease virus ibdv VP2 protein ligand-binding epitope polypeptide and application thereof

A technology of epitope polypeptide and bursal disease, applied in the field of molecular pathology and immunology

Inactive Publication Date: 2011-12-07
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Preliminary research results have obtained some information on IBDV receptors and ligands. For example, the receptor is a glycoprotein, which is related to the surface molecule of SIgM-positive B cells, and may also be chicken heat shock protein 90; the ligand protein that binds to the IBDV receptor is IBDV VP2, but IBDV ligand protein VP2 and receptor-binding polypeptide epitopes have not been reported

Method used

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  • Chicken infectious bursal disease virus ibdv VP2 protein ligand-binding epitope polypeptide and application thereof
  • Chicken infectious bursal disease virus ibdv VP2 protein ligand-binding epitope polypeptide and application thereof
  • Chicken infectious bursal disease virus ibdv VP2 protein ligand-binding epitope polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Design and synthesis of IBDV VP2 polypeptide, Sulfo-NHS-Biotin labeled polypeptide and coupling of the polypeptide to mouse IgG (moIgG)

[0064] 1.1 Design of peptides

[0065] Using DNASIS (Ver. 2.5, Hitachi) software to analyze the amino acid sequence of IBDV (P15480), and referring to the crystal structure of IBDV VP2 (Fasséli Coulibaly et al., 2005), design an IBDV VP2 polypeptide P22, located in the D-E of the S region loop, and Cys is added to the N-terminus of the polypeptide for carrier protein coupling.

[0066] Synthesis of Peptides

[0067] Use the Symphony 12-channel peptide synthesizer to synthesize peptides on Fmoc-Amino Acids attached to Wang Resin (Shanghai Jier) by solid-phase peptide synthesis (solid-phase peptide synthesis), and the peptides are synthesized according to standard operating procedures conduct. The basic synthesis process is: Execute the Std_sw program to swell the C-terminal amino acid king resin → execute the Std ...

Embodiment 2

[0089] Example 2 Cell immunochemical staining method to detect the binding test of Biotin-P22 and CEF

[0090]⑴ Prepare CEF, culture at 37°C for 24 hours, wait until the cells cover the 96-well cell culture plate;

[0091] (2) Pour off the supernatant, wash the cultured monolayer cells with pre-cooled PBS for 3 times, and add the concentration of 0.2mmol / L, 0.1mmol / L, 0.05mmol / L to 0.025mmol / L respectively on the culture plate. Biotin-labeled polypeptide Biotin-P22, 100 μl / well, make 3 replicate wells for each dilution, combine at 4°C for 1 hour to fully combine cells with Biotin-P22 polypeptide, set 6 wells of normal cells without Biotin-labeled polypeptide P22 As a control, the cells added with Biotin-BSA in 6 wells were used as a control.

[0092] (3) Gently wash the cells 3 times with pre-cooled PBS to wash away unbound peptides and stop the peptide binding test;

[0093] ⑷ Fix, add 0.3% H 2 o 2 Methanol solution 50 μl / well, fixed at 4°C for 10 min, poured off the su...

Embodiment 3

[0098] Example 3 Flow Cytometry Analysis Polypeptide Binding Test and Virus Blocking Test

[0099] The following tests were performed simultaneously with CEF, respectively. The test was divided into three groups, test group A: P22-moIgG combined with CEF test; test group B: moIgG combined with CEF test, as a negative control; test group C: after IBDV blocked the CEF binding site, then performed P22-moIgG combined with CEF test , as a positive control. The specific test steps are as follows:

[0100] ⑴ Prepare CEF according to the conventional method. After culturing the cells and cell culture flasks for 24 hours, wait for the cells to grow into a monolayer;

[0101] (2) Wash 3 times with pre-cooled PBS, digest the cells with trypsin for 10 minutes, blow the cells from the cell bottle with a 10ml pipette, mix well to reduce the large cell clumps, centrifuge at 1500r / m for 5 minutes, discard the cell supernatant .

[0102] (3) Add new pre-cooled PBS, resuspend the cells, a...

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Abstract

The present invention relates to the linear ligand epitope of Bursal bursal virus and its specific application. The polypeptide P22 on the IBDVVP2 protein is synthesized, and the CEF is used as the target cell. Through the infection and harvest of IBDV, the virulence and half tissue culture of IBDV are measured. Infection dose and infection ratio, the combination test of synthetic polypeptides and target cells, the virus blocking test and the test of polypeptide blocking IBDV infection target cells, the screened specific binding target cells can inhibit virus infection ligand epitope polypeptides, and The ligand epitope of IBDV was precisely mapped. The polypeptide P22 of the present invention can inhibit IBDV from infecting CEF, and with the increase of polypeptide concentration, the infection rate of CEF decreases in a dose-dependent manner. When the polypeptide concentration is 0.2 mmol / L, it can completely inhibit IBDV from infecting CEF target cells. The invention provides a theoretical basis for further research on the virus ligand epitope from the aspect of the interaction between the virus ligand and the virus receptor.

Description

technical field [0001] The invention belongs to the technical field of molecular pathology and immunology, and specifically relates to a binding epitope polypeptide of a chicken infectious bursal disease virus IBDV VP2 protein ligand and an application thereof. Background technique [0002] IBD is a highly contagious infectious disease of chickens and turkeys caused by IBDV. The virus multiplies rapidly in the B lymphocytes of the bursa and damages the B lymphocytes of the bursa, causing severe immunosuppression and often feeding Chicken industry brings bigger economic loss. In recent years, the continuous emergence of super-virulent strains of IBDV has increased the difficulty in the prevention and control of the disease. In the process of virus infection of cells, virus adsorption to cells is the first and critical step, which involves the interaction between virus-binding receptors on cells and ligands on cells and viruses. Viruses must first bind to the surface of targe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12Q1/02C12Q1/70G01N1/30A61K38/16A61P31/14C12R1/93
Inventor 王选年朱艳平宁红梅银梅朱彦彩岳锋贾文科张艳芳李鹏孙国鹏
Owner XINXIANG UNIV
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