d(GC) - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 6 (2) 222-228 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)
and the reaction volume made to 50µl using
nuclease free water. A PCR reaction with an initial
denaturation at 98 o C for 2 min; denaturation at
98 o C for 20 sec, annealing at 55 o C for 30
seconds, extension at 72 o C for 60 sec (25 cycles)
and the final extension for 72 o C for 10 min was
performed to amplify the HA and NA gene. The
PCR products were analyzed on 1% agarose gel
electrophoresis and the PCR products were
purified using GenElute Extraction Kit (Sigma,
USA). The purified HA and NA amplicon were
cloned into pRSET ‘A’ (Invitrogen, USA) in-frame
with the N-terminus his-tag after digestion with
NheI and Hind III (New England biolabs, USA)
and the transformed E.coli cells were plated on
LB agar containing 100µg/ml of ampicillin. The
resulted colonies were screened by restriction
digestion with NheI and HindIII. The clones
releasing HA and NA genes were analyzed by
DNA sequencing to confirm the clone and the
sequence.
Expression of HA and NA gene in E.coli: The
positive clones of HA and NA were transformed
into BL21 (DE3) pLysS cells and grown to an
optical density 0.6 at 600nm. The cells were
induced with 0.5mM of Isopropyl β-D-1thiogalactopyranoside
(IPTG) for four hours. The
cells were harvested four hours post induction
and the expression was analyzed in SDS-PAGE
and western blot using HisProbe-HRPO.
Purification of rHA and rNA using Ni-NTA
chromatography: The clones expressing the HA
and NA were scaled up to 2 liter and the cells
were induced as described above. After four
hours, the cells were harvested by centrifuging
at 5000rpm for 30 minutes. The supernatant was
discarded and the pellet was resuspended in
50mM sodium phosphate buffer (pH-8)
containing 300mM NaCl. The cells were lysed
by sonication (40% amplitude, 9 sec pulse on
and 5 sec pulse off). The lysate was filtered
through a 0.22µM filter and the rHA and rNA was
purified from the filtered lysate by Ni-NTA
chromatography under native conditions. The
lysate was passed through a pre-equilibrated Ni-
NTA Superflow resin (Qiagen, USA) at a speed
224
of 1ml / min. The column was washed with 50mM
sodium phosphate buffer containing 300mM
NaCl, 30mM imidazole (Sigma, USA) to remove
the unbound protein and the bound protein was
eluted with 300mM imidazole in 50mM sodium
phosphate buffer containing 300mM NaCl. All the
fractions were analyzed on SDS-PAGE and the
fractions containing target protein were pooled.
The pooled fractions were dialyzed against 50mM
sodium phosphate buffer containing 200mM
NaCl and the protein concentration was
determined using BCA protein assay reagent
(Pierce, Rockford, IL). The protein was
concentrated through a vivaspin20 (10,000
MWCO) and analyzed on SDS-PAGE and the
final protein concentration was estimated using
BCA reagent.
Haemagglutination assay: Different erythrocyte
species such as 1% horse, 0.5% chicken and
0.75% guinea pig were used to analyze the
haemagglutination activity of the rHA as
described previously (14). Briefly, the erythrocyte
species were suspended in phosphate buffered
saline (PBS), pH 7.4. The rHA was adjusted to a
protein concentration of 1mg/ml. In a ‘U’
bottomed 96 well plate, 50µl of rHA antigen was
serially (two fold) diluted from 1/2 and proceeded
till 1/1024 and 50µl of different erythrocyte
species were added. The reaction mixture was
incubated at room temperature for 45minutes.
The HA titer was defined as the highest antigen
dilution that yielded complete haemagglutination
of the test erythrocytes.
Results and Discussion
In the present study the haemagglutinin and
neuraminidase gene of highly pathogenic avian
influenza was cloned into E.coli expression vector
and expressed in E.coli. The E.coli expression
platform is an easy, cheaper yet efficient
expression system for the expression of
heterologous protein. Many such proteins
expressed in E.coli are reported to be useful for
wide range of applications such as diagnostic
assays and vaccines. In an effort to develop a
quicker and cheaper subunit vaccine and
diagnostic tool for avian influenza, the HA and
Cloning, Expression and purification of H5N1 in E. coli