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