Biology Reference
In-Depth Information
showed a signifi cant NA-P1 interaction which is almost 100 times higher than the
control. So, NA-P1 interaction cannot be simply ignored and further investigations are
required to analyze the kind of interaction between the NA glycoproteins and peptide
P1. But, the HA and P1 interaction has been clearly demonstrated without any doubt
in all the performed experiments.
Taking all together, this study has identifi ed a novel antiviral molecule which in-
hibits the avian infl uenza virus infection by interacting with the surface glycoprotein
HA and preventing its attachment to the host cell. To our knowledge, the selected
peptide is the only antiviral peptide among the currently identifi ed anti-viral peptides
with 7 or 9 amino acids in length. This short sequence will be an added advantage for
commercialization purpose as it can greatly reduce the cost of production. However,
additional studies are required to defi ne the broad-spectrum activity of the peptide
against various strains including the currently circulating potential pandemic strains
such as H1N1 and H5N1 as well as its diagnostic potential.
MATERIAL AND METHODS
Viruses, Cells, and Viral Purification
Avian influenza A/Chicken/Iran/16/2000(H9N2), a low pathogenic avian influenza vi-
rus and Newcastle disease virus (NDV) strain AF2240 was kindly provided by Abdul
Rahman Omar. Viruses were propagated in 9-day old specific pathogen free embryo-
nated chicken eggs. The allantoic fluid was clarified and the viruses were purified and
concentrated as explained previously [25]. The virus titer was determined by hemag-
glutination test (HA) and the protein concentration of the purified virus was deter-
mined by Bradford assay [37].
Selection of Peptides Against AIV Sub-type H9N2
The virus (15 μg/ml; 100 μl) was coated onto a microtiter plate well with NaHCO
3
(0.1
M, pH 8.6) buffer overnight at 4°C. Streptavidin (0.1 mg/ml; 100 μl) was also coated
and used as positive control. Phages from a disulfide constrained 7-mer phage display
random peptide library (New England Biolabs, USA) were biopanned as explained by
the manufacturer. The amplified phages from the first round of biopanning were used
for the second round of biopanning. Totally four rounds of biopanning were carried
out. Phage titration was carried out according to the method described by Sambrook et
al. [38]. Phages were propagated in
Escherichia coli
(
E. coli
) host cells grown in LB
broth (1 L). The phage particles were precipitated by PEG and purified through cesium
chloride density gradient centrifugation as descried by Smith and Scott [39].
Sequence Analysis of Phagemids
The nucleotide sequence encoding the hypervariable heptapeptide region of pIII coat
protein of M13 phage was sequenced by 1st Base Laboratories Sdn Bhd, Kuala
Lumpur, with the -96 gIII sequencing primer 5′ CCC TCA TAG TTA GCG TAA CG
3′. Sequence analyses such as comparison with wild type M13 phage pIII coat protein
and prediction of amino acid sequences were performed with the free bioinformatics
software package, SDSC Biology Workbench 3.2.
