Phage Displayed Peptides to Avian H5N1 Virus Distinguished the Virus from Other Viruses
Citation: Wu D, Li G, Qin C, Ren X (
Phage Displayed Peptides to Avian H5N1 Virus Distinguished the Virus from Other Viruses
Dan Wu 0
Guangxing Li 0
Chengfeng Qin 0
Xiaofeng Ren 0
Paul Digard, University of Cambridge, United Kingdom
0 1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University , Harbin , China , 2 Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University , Harbin , China , 3 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing , China
The purpose of the current study was to identify potential ligands and develop a novel diagnostic test to highly pathogenic avian influenza A virus (HPAI), subtype H5N1 viruses using phage display technology. The H5N1 viruses were used as an immobilized target in a biopanning process using a 12-mer phage display random peptide library. After five rounds of panning, three phages expressing peptides HAWDPIPARDPF, AAWHLIVALAPN or ATSHLHVRLPSK had a specific binding activity to H5N1 viruses were isolated. Putative binding motifs to H5N1 viruses were identified by DNA sequencing. In terms of the minimum quantity of viruses, the phage-based ELISA was better than antiserum-based ELISA and a manual, semiquantitative endpoint RT-PCR for detecting H5N1 viruses. More importantly, the selected phages bearing the specific peptides to H5N1 viruses were capable of differentiating this virus from other avian viruses in enzyme-linked immunosorbent assays.
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Funding: This work was supported, in part, by the National 973 Plan of China (2010CB534002). We also acknowledge National Natural Science Foundation of
China (30972195), the Program for New Century Excellent Talents in Heilongjiang Provincial University (1155-NCET-005) and Heilongjiang Provincial Science and
Technology Department, China (ZJN0702-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Since the first evidence regarding direct transmission of highly
pathogenic avian influenza A virus (HPAI), subtype H5N1 from
poultry to human in 1997 and resulted in the death of 6 of the 18
infected individuals [13]. The HPAI H5N1 has become one of
the most important public health concerns worldwide. At present,
the virus has spread to many countries in Europe, Asia and Africa
[4]. In 2009, an identified fatal influenza (H5N1) infection in a
human was reported on January 17, 2009 [5]. Increased
geographical distribution and continued evolution of H5N1
viruses as well as an immunologically nave human population
has maintained the pandemic potential of these viruses [68].
In addition to vaccination and administration of antiviral drugs
against H5N1 viruses, development of effective detection
approaches is required to manage and control the deadly disease.
Phage display is a recently developed technology and phage
random peptide library consists of a pool of billions of
heterologous peptides that can be produced by the fusion of
random nucleic acid sequences to the N terminus of one of the
capsid protein genes of a filamentous bacteriophage [9]. Phage
display peptide library is a powerful tool to identify specific ligands
of a target protein by a biopanning process. This technology has
been applied successfully in numerous aspects, including antibody
engineering [10], peptide and protein drug discovery and
manufacture [11], diagnostic analysis [12] and vaccine
development [13]. Herein we identified three phage clones that specifically
binding to the HAPI H5N1 viruses using a 12-mer random phage
library. The binding peptides of the phages were sequenced. More
importantly, these identified phages were able to distinguish HAPI
H5N1 from other avian viruses.
Materials and Methods
Cell and virus
Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas,
VA) were grown in Dulbeccos MEM with 1 mM L-glutamine and
10% fetal bovine serum at 37uC and 5% CO2 in air. HPAI H5N1
strain A/goose/Jilin/hb/2003 were propagated in the MDCK
cells in the absence of serum and purified by differential
centrifugation conventionally. The concentration of the purified
viruses diluted in PBS was measured by Thermo Scientific
NANODROP 2000 Spectrophotometer ((NanoDrop
Technologies, Thermo Fisher Scientific, Wilmington, DE) and calculated by
the molar absorbance coefficient A260/A280 according to the
manufacturers instructions.
Biopanning and enrichment analysis
Phage display was done according to the manufacturers
instructions (New England Biolabs) with minor modifications.
For the first round of panning, 96-well plates were coated with the
H5N1 viruses at a concentration of 14 mg/well in 0.1 M NaHCO3
(pH 8.6) buffer overnight at 4uC. The next day, the plates were
blocked for 1 h at 4uC (...truncated)