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)
coding sequences which could also be the reason
for these lower expression levels.
Purification of rHA and rNA gene in E.coli:
The rHA and rNA proteins were cloned in such a
way that the expressed gene will have a Nterminus
His-tag so as to enable the purification
simpler and quicker. The cells expressing rHA
and rNA were scaled up in a 2 liter flasks and the
rHA and the rNA proteins were purified through
Ni-NTA chromatography. The elution fractions
were analyzed on SDS-PAGE and confirmed by
western blot which showed a 66 kDa rHA and
50kDa rNA protein (Fig. 3). The fractions
containing purified proteins were pooled. The
purified HA and NA proteins were dialyzed
against 50mM sodium phosphate buffer to
remove the imidazole and were adjusted to a final
concentration of 1mg/ml before storing at -80ºC
for further analysis.
Fig.3. Purification of rHA and rNA from E.coli. The
clones expressing rHA and rNA were induced with
0.5mM IPTG and the cells were harvested 4 hours
post induction. The rHA and rNA were purified through
Ni-NTA column chromatography. The purified
fractions were analyzed in a 12% SDS-PAGE and
western blot. M-Marker; 1-SDS-PAGE showing
purified rHA; 2-western blot showing purified rHA; 3-
SDS-PAGE showing purified rNA; 4-western blot
showing purified rNA
226
Haemagglutination assay : The purified rHA
protein was adjusted to a final concentration of
1mg/ml and the haemagglutination activity was
analyzed using various RBCs. The guinea pig
RBC showed the maximum haemagglutination
titer followed by chicken and horse. The rHA
protein showed a HA titer of 32 with chicken RBC
(Fig. 4), 64 with guinea pigs RBC, 4 with horse
RBC and no agglutination with sheep RBC. This
clearly demonstrates the use of bacterial
expressed rHA in diagnostic assays and as a
candidate for subunit vaccine.
Fig. 4. Haemagglutination activity of purified rHA. The
purified rHA protein was adjusted to a final
concentration of 1mg/ml and the haemagglutination
activity was analyzed using various RBCs. The guinea
pig RBC showed the maximum haemagglutination
titer followed by chicken and horse. The rHA protein
showed a HA titer of 32 with chicken RBC, 64 with
guinea pigs RBC, 4 with horse RBC and no
agglutination with sheep RBC.
Conclusion
A variety of reports support the role of
rHA and rNA as potential vaccine and diagnostic
tools for avian influenza. Although, both HA and
NA antigens are known to elicit neutralizing
antibody response in animals (15, 16, 17),
antibodies to HA is known to play a major role in
virus control while antibodies to NA helps in partial
not complete protection. Even though HA alone
can provide maximum protection in animals from
Cloning, Expression and purification of H5N1 in E. coli