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