Biology Reference
In-Depth Information
In
Vitro
Study of Protein-protein Interactions
Construction of Recombinant Pc-Hat, Pc-NA, and Pc-P1 and In Vitro
Transcription and Translation
The
HA
t
and
NA
gene of AIV strain H9N2 as well as the recombinant peptide gene
P1
was amplified from pY-HA, pY-NA and pH-P1 respectively as templates using the
primers pC-HA-F and R, pC-NA-F and R and pC-P1-F and R respectively (Table 5)
and cloned into the pCITE2a vector. The
in vitro
transcription and translation was per-
formed in a single tube in a reaction mixture (15 μl) containing circular recombinant
plasmid (1 μg), TNT
®
Quick Master Mix (12 μl; Promega, USA), Methionine (0.3 μl,
1 mM; Promega, USA). The above mixture was incubated at 30°C for 90 min. The
translated products (3 μl) were electrophoresed on 15% SDS-PAGE and then trans-
ferred by electrophoresis for 1 hr onto a nitrocellulose membrane. They were detected
with anti-His antibody for P1 protein and HAt/NA proteins were detected with the
polyclonal antibodies raised against the AIV sub-type H9N2 in rabbit.
Co-immunoprecipitation
Co-immunoprecipitation was performed using the Pierce
®
Co-IP kit (Thermo Scien-
tific, USA) as per the instructions given by the manufacturer. Briefly, the bait and pray
complex was prepared separately by mixing the HA
t
or NA with His-conjugated P1
peptide. The complex was precipitated using purified anti-AIV polyclonal antibodies,
which were immobilised on antibody coupling resin. The peptide P1 in the eluted co-
immunoprecipitated complex was analyzed by Western blotting using anti-His mono-
clonal antibodies (Novagen, USA) and detected with Amersham
®
ECL
®
Western blot-
ting detection reagents (GE Healthcare, USA).
Statistical Analysis
All experiments were carried out in triplicate and are representative of at least three
separate experiments. The results represent the means ± standard deviations or stan-
dard error means of triplicate determinations. Statistical significance of the data was
determined by independent t test or one-way ANOVA method using SPSS software.
KEYWORDS
•
Co-immunoprecipitation
•
Hemagglutination
•
Hydrophobicity
AUTHORS' CONTRIBUTIONS
Mohamed Rajik designed the study, carried out all of the experiments and drafted the
manuscript. Fatemeh Jahanshiri participated in the design of yeast two hybrid assay
experiments. Abdul Rahman Omar, Aini Ideris, and Sharifah Syed Hassan participated
in the design of avian influenza virus related experiments. Khatijah Yusoff conceived
the study, participated in its design and co-ordination and helped to draft the manu-
script. All authors read and approved the final manuscript.
