d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
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 />
vaccine development targets the two major<br />
structural antigens namely hemagglutinin (HA)<br />
<strong>and</strong> neuraminidase (NA). Antibodies to HA seem<br />
to play a major role in controlling the disease<br />
while NA is reported to assist in controlling virus<br />
replication but, it does not protect an animal from<br />
being infected (5,6). On the other h<strong>and</strong>, there<br />
are 16 HA subtypes <strong>and</strong> 9 NA subtypes have<br />
already been reported. The newer avian influenza<br />
virus can posses one among many probabilities<br />
<strong>of</strong> HA <strong>and</strong> NA (7).<br />
There are several alternative strategies<br />
have been developed to produce p<strong>and</strong>emic <strong>and</strong><br />
seasonal influenza vaccines which involves<br />
mammalian cell culture <strong>and</strong> baculovirus<br />
expression systems. Several studies have<br />
demonstrated the use <strong>of</strong> HA <strong>and</strong> NA either in the<br />
form <strong>of</strong> whole virus or expressed in various<br />
platform such as baculovirus <strong>and</strong> DNA vaccine<br />
expressing HA <strong>and</strong> NA aid partial protection in<br />
animals. None the less, the development <strong>of</strong> large<br />
quantities <strong>of</strong> such vaccines in mammalian cell<br />
culture <strong>and</strong> baculovirus may not fulfill the<br />
necessity <strong>of</strong> a p<strong>and</strong>emic as millions <strong>and</strong> millions<br />
<strong>of</strong> vaccine doses would be required. Few reports<br />
demonstrated the use <strong>of</strong> yeast as an expression<br />
system for producing avian influenza antigens<br />
(8,9,10,11) which can be used as potential<br />
p<strong>and</strong>emic vaccine c<strong>and</strong>idates. However,<br />
expression in yeast based system consumes<br />
considerable time for producing bulk vaccines.<br />
Hence, development <strong>of</strong> such vaccine in a<br />
bacterial expression system may be an attractive<br />
alternative to produce faster, cheaper <strong>and</strong> bulk<br />
vaccines. Development <strong>of</strong> avian influenza HA in<br />
E.coli expression system has already been<br />
described (12) yet, not many reports describe<br />
the development <strong>of</strong> such cheaper <strong>and</strong> quick<br />
vaccine against avian influenza. Furthermore, the<br />
recombinant vaccines produced in bacteria will<br />
not only avoid the use <strong>of</strong> live virus but also<br />
expected to help in reducing complications<br />
associated with the whole virus vaccines (13).<br />
In an attempt to produce a faster <strong>and</strong> safer<br />
vaccine against avian influenza, the aim <strong>of</strong> this<br />
study was to examine the feasibility <strong>of</strong> E.coli<br />
Subathra et al<br />
223<br />
expressed avian influenza HA <strong>and</strong> NA as a<br />
subunit vaccine against potential influenza<br />
p<strong>and</strong>emic. The HA <strong>and</strong> NA gene <strong>of</strong> H5N1 strain<br />
(A/Hatay/2004/H5N1) was cloned into E.coli<br />
expression vector, the protein was expressed <strong>and</strong><br />
purified from E.coli. The possibility <strong>of</strong> using the<br />
rHA <strong>and</strong> rNA as a vaccine c<strong>and</strong>idates discussed.<br />
Material <strong>and</strong> methods<br />
Strains, Plasmids <strong>and</strong> culture conditions : The<br />
cDNA <strong>of</strong> HA <strong>and</strong> NA gene <strong>of</strong> Avian Influenza<br />
virus H5N1 strain (A/Hatay/2004/H5N1) was<br />
obtained from the International Centre for Genetic<br />
Engineering <strong>and</strong> <strong>Biotechnology</strong> (ICGEB), New<br />
Delhi, India. Escherichia coli TOP10 (F– mcrA<br />
Δ(mrr-hsdRMS-mcrBC) Δ80lacZΔM15 ΔlacX74<br />
recA1 araD139 Ä(ara leu) 7697 galU galK rpsL<br />
(StrR) endA1 nupG) cells were used for all the<br />
cloning <strong>and</strong> plasmid propagation work <strong>and</strong><br />
BL21(DE3) pLysS (F– ompT hsdSB(rB–, mB–)<br />
gal dcm (DE3) pLysS (CamR) was used for the<br />
expression <strong>of</strong> rHA <strong>and</strong> rNA genes <strong>and</strong> the<br />
bacterial cells were grown <strong>and</strong> maintained in LB<br />
broth <strong>and</strong>/or LB agar containing appropriate<br />
antibiotics.<br />
Cloning <strong>of</strong> HA <strong>and</strong> NA into pRSET A vector:<br />
The HA <strong>and</strong> NA gene <strong>of</strong> avian influenza H5N1<br />
was PCR amplified from the cDNA using specific<br />
primers employing st<strong>and</strong>ard PCR protocols. The<br />
gene encoding NA was amplified using forward<br />
primer (5’- ATCG<strong>GC</strong>TA<strong>GC</strong>ATGA ATCC<br />
AAATCAG AAGATAATAA-3’) <strong>and</strong> a reverse<br />
primer (5’- ATCGAA<strong>GC</strong>TTCT ACTTGTCAA<br />
TGGTGAATG-3’) consisting NheI <strong>and</strong> HindIII<br />
restriction endonuclease sites respectively. The<br />
gene encoding HA was amplified using forward<br />
primer (5’- ATCG<strong>GC</strong>TA<strong>GC</strong>AT GGAGAAAATA<br />
GT<strong>GC</strong>TTCT-3’) <strong>and</strong> a reverse primer (5’-<br />
ATCGAA<strong>GC</strong>TTTTAAAT<strong>GC</strong>AAATTCT<strong>GC</strong>ATTG-<br />
3’) consisting NheI <strong>and</strong> HindIII restriction<br />
endonuclease sites (underlined) respectively. A<br />
PCR reaction mixture containing 50 ng <strong>of</strong> cDNA,<br />
1xPCR reaction buffer, 200µM <strong>of</strong> each dNTPs,<br />
25 picomoles <strong>of</strong> each forward <strong>and</strong> reverse<br />
primers specific for HA <strong>and</strong> NA, 0.5 units <strong>of</strong><br />
Phusion DNA polymerase enzyme was prepared