Biology Reference
In-Depth Information
In
Vivo
Study of Protein-Protein Interactions: Yeast Two-hybrid Assay
Cloning of Ha
t
, NA snd P1 Genes Into Pyestrp2 and Phyblex/Zeo Vectors
The NA and truncated HA protein (
HA
t
) genes of AIV sub-type H9N2 were ampli-
fied by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) from the viral
RNA using the primers pY-HA
t
-F and R and pY-NA-F and R, mentioned in Table 5.
The
NA
gene carried the recognition sites for EcoRI and XhoI whereas the
HA
t
gene
carried the recognition sites KpnI and XhoI restriction enzymes in their forward and
reverse primers respectively. The peptide gene (
P1
) was amplified including the N1
domain of the P3 protein of the recombinant phage using the primer pH-P1-F and R
(Table 5) from the ssDNA genome of the phage as the peptide is displayed as a fusion
protein to this domain of the P3 protein. The
P1
gene carried the recognition sites for
EcoRI and XhoI restriction enzymes in its forward and reverse primers respectively.
The amplified
HA
t
and
NA
genes were ligated into pYESTrp2 vectors separately (In-
vitrogen, USA) and the
P1
gene was cloned into pHybLex/Zeo (Invitrogen, USA)
vector. The resultant clones were named as pY-HA, pY-NA and pH-P1 respectively.
The constructs were sequenced using the primers pYESTrp2-F and R and pHybLex/
Zeo-F and R (Table 5) to check the reading frame and for the absence of mutations.
The
Saccharomyces cerevisiae
strain L40 was then co-transformed with the recombi-
nant plasmids using lithium acetate method and the transformants were analyzed for
their β-galactosidase activity as explained in Ausubel et al. [41].
Table 5.
Oligonucleotides used to amplify the
NA
,
HA
t
and
P1
genes.
Primers
Sequence
pY-NA-F
a
5' CAT AGAA TTCGCAAAAGCAGGAGT 3'
pY-NA-R
5' TATCGCTCGAGAGT AGAAACAAGGAG 3'
pY-HA-F
5' ATTTAAGGTACCGACAGCCATGGA 3'
pY-HAt-R
5' ATGCTGCTCGAGTATACAAATGTTGC 3'
pH-PI-F
5' AGCCTGGAATTCATGAAAAAATTA 3'
pH-PI-R
5' ATCGAACTCGAGA TTTTCAGGGAT 3'
pHyblex/Zeo-F
5' AGGGCTGGCGGTTGGGGGTT A TTCGC 3'
pHyblex/Zeo-R
5' GAGTCACTTTAAAATTTGTATACAC 3'
pYESTrp2-F
5' GATGTTAACGATACCAGCC 3'
pYESTrp2-R
5' GCGTGAA TGT AAGCGTGAC 3'
pC-HA-F
5'A TTT AAGGATCCGAGAGCCATGGA 3'
pC-HA-R
5'ATGCTGCTCGAGTTATATACAAA TGTTGC 3'
pC-NA-F
5'CATAGAA TTCGCAAAAGCAGGAGT 3'
pC-NA-R
5'T A TCGCTCGAGAGT AGAAACAAGGAG 3'
pC-PI-FP
5'AGCCTGGAA TTCATGAAAAAATT A 3'
pC-PI-RP
5'CTCACTCGAGACATTTTCAGGGA 3'
• In all of the above mentioned oligonucleotides, the suffi xes F and R refers Forward and Reverse primers respectively
