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Conserved neutralizing epitope mimic peptide of H5 subtype avian influenza viruses and use thereof

A bird flu virus, epitope mimic peptide technology, applied in antiviral agents, antiviral immunoglobulins, peptides, etc.

Inactive Publication Date: 2011-02-23
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it should be avoided to use a monoclonal antibody to screen a random peptide library, so that although an epitope mimic peptide (mimotope) with the same antigenicity as the target protein can be obtained, the epitope mimic peptide may also bind to a different CDR in the same antibody Area

Method used

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  • Conserved neutralizing epitope mimic peptide of H5 subtype avian influenza viruses and use thereof
  • Conserved neutralizing epitope mimic peptide of H5 subtype avian influenza viruses and use thereof
  • Conserved neutralizing epitope mimic peptide of H5 subtype avian influenza viruses and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Screening of short peptides that mimic 13D4 recognition epitopes from phage 12 peptide library, heptad library and cyclic heptad library

[0096] The 12-peptide phage display library (ph.D.12peptide library), the heptapeptide phage display library (ph.D.7peptide library) and the cyclic heptapeptide phage display library (ph.D.C7C peptide library) from New England Biolabs were selected for screening The mimetic peptide combined with the monoclonal antibody 13D4 was screened according to the operating instructions.

[0097] Pipette 50 μl of Protein A-agarose medium (50% aqueous suspension) into a microcentrifuge tube, and add 1 ml of TBS+0.1% Tween (TBST) solution. Resuspend the medium by flicking the tube wall or gently vortexing. Centrifuge at low speed for 30 seconds to pellet the medium and carefully aspirate the supernatant. The medium was resuspended in 1ml blocking buffer, and reacted at 4°C for 60 minutes, mixing occasionally. During this time, 2 × 10...

Embodiment 2

[0105] Example 2. Grouped immunization of phage peptides and Dot blot detection results of antisera to viruses

[0106] Each of the above phage peptides was amplified in large quantities. Each phage spot was picked into 4ml of ER2738 in the logarithmic phase, 4.5ml was shaken at 37°C, and then transferred to 100mL of ER2738 host bacterial fluid in the early logarithmic growth stage, and shaken at 37°C for 4.5h. Transfer the bacterial solution into a 50mL centrifuge tube and centrifuge at 10000rpm for 10min. Aspirate the upper 80% supernatant, add 1 / 6 volume of PEG / NaCl (20% PEG-8000, 2.5M NaCl) solution, and let it stand overnight at 4°C. Centrifuge at 10000rpm at 4°C for 15min. Pour off the supernatant and dissolve the pelleted phage with 10 mL PBS. Centrifuge at 4°C for 5min, absorb the supernatant, add 1 / 6 volume of PEG / NaCl solution, and let stand at 4°C for 1h. Centrifuge at 10000rpm at 4°C for 15min. Pour off the supernatant, dissolve the precipitated phage with 1mL...

Embodiment 3

[0114] Example 3 Synthetic Peptide Activity Detection

[0115] Competitive ELISA Experiment of Synthetic Peptide and Avian Influenza Virus

[0116] Mix and coat avian influenza H5 subtype monoclonal antibodies 2F2 and 3G4 at 200 ng / well each; incubate with 1:50 diluted H5N1 virus Ck / HKYU22 / 02 at 37°C for 1 hour, discard unbound virus supernatant; dilute 1:1000 Diluted 13D4mAb / HRP was mixed with 50ug, 25ug, and 12.5ug of synthetic peptides respectively, added to the wells and incubated at 37°C for 0.5h, and the irrelevant 12 peptide P 35 was used as a negative control; The 13D4mAb / HRP readout was used as a control to investigate the competition between the synthetic peptide and the virus.

[0117] Synthetic peptide blocking HI experiment

[0118] Adjust the virus Ck / HKYU22 / 02 to an appropriate titer according to the conventional HI method; take 25ug of synthetic peptide and dilute it to 1 / 2 8 , each dilution gradient was mixed with 1:500 diluted 13D4 ascites, and the two wel...

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Abstract

The invention provides a hemagglutinin (HA) protein neutralizing epitope mimic peptide of H5 subtype avian influenza viruses, a conserved derivative or active fragment or mutant sequence thereof, related coding sequence thereof and the use of the mimic peptide or the conserved derivative or active fragment or mutant sequence thereof in prevention and diagnosis.

Description

field of invention [0001] The present invention relates to H5 subtype avian influenza virus neutralizing epitope mimetic peptides, conservative variants or active fragments thereof, or related coding sequences of mimetic peptides and analogues thereof, and the application of the mimetic peptides or analogues in prevention or diagnosis the use of. Background technique [0002] Since H5 avian influenza first broke out in geese on farms in Guangdong Province in my country in 1996 (Xu X et al., 1999, Virology), another strain of H5 virus derived from this virus has spread in poultry farms in Hong Kong ( April 1997) and the market (November 1997) broke out, causing the first time in history that the avian influenza virus was directly transmitted from birds to humans, and 6 people died in a total of 18 confirmed cases. Since 2003, H5 virus strains have broken out successively throughout East Asia and Southeast Asian countries. WHO and influenza experts predict that H5 avian influe...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/08C07K19/00C07K16/10C07K17/00C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/145A61K47/48A61P31/16G01N33/569A61K47/68
Inventor 罗文新陈毅歆宋慧娟陈瑛炜陈鸿霖张军夏宁邵
Owner XIAMEN UNIV
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