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Research
(Published online:
10-02-2015)
4. Construction and characterization of
recombinant human adenovirus type 5 expressing foot-and-mouth
disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75
- Ramesh Kumar, B. P. Sreenivasa and R.
P. Tamilselvan
Veterinary World, 8(2): 147-155
doi:
10.14202/vetworld.2015.147-155
Ramesh
Kumar:
FMD Research Centre, Indian Veterinary Research Institute,
Bangalore - 560 024, Karnataka, India; drrkvet13@gmail.com
B. P.
Sreenivasa:
FMD Research Centre, Indian Veterinary Research Institute,
Bangalore - 560 024, Karnataka, India; bpsrini@gmail.com
R. P.
Tamilselvan: FMD Research Centre, Indian Veterinary Research
Institute, Bangalore - 560 024, Karnataka, India;
mukthitamil@gmail.com
Received: 20-10-2014, Revised: 18-12-2014, Accepted: 27-12-2014,
Published online: 10-02-2015
Corresponding author:
B. P. Sreenivasa, e-mail: bpsrini@gmail.com
Citation:
Kumar R,
Sreenivasa BP, Tamilselvan RP (2015) Construction and
characterization of recombinant human adenovirus type 5 expressing
foot-and-mouth disease virus capsid proteins of Indian vaccine
strain, O/IND/R2/75, Veterinary World, 8(2):
147-155.
Abstract
Aim:
Generation of recombinant human adenovirus type 5 expressing
foot-and-mouth disease virus (FMDV) capsid protein genes along
with full-length 2B, 3B and 3C pro and its
characterization.
Materials and Methods: FMD viral RNA isolation, cDNA
synthesis, and polymerase chain reaction were performed to
synthesize expression cassettes (P1-2AB3BC wt
and P1-2AB3BCm) followed by cloning in
pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were
transformed with the recombinant pShuttle-CMV to produce
recombinant adenoviral plasmids. HEK-293 cells were transfected
with the recombinant adenoviral plasmids to generate recombinant
adenoviruses (hAd5/P1-2AB3BCwt and
hAd5/P1-2AB3BCm). Expression of the target
proteins was analyzed by sandwich ELISA and indirect
immunofluorescence assay. The recombinant adenoviruses were
purified and concentrated by CsCl density gradient
ultracentrifugation. Growth kinetics and thermostability of the
recombinant adenoviruses were compared with that of
non-recombinant replication-defective adenovirus (dAd5).
Results: The recombinant adenoviruses containing capsid
protein genes of the FMDV O/IND/R2/75 were generated and amplified
in HEK-293 cells. The titer of the recombinant adenoviruses was
approximately 10 8, 109.5
and 1011 TCID50/ml
in supernatant media, cell lysate and CsCl purified preparation,
respectively. Expression of the FMDV capsid protein was detectable
in sandwich ELISA and confirmed by immunofluorescence assay.
Growth kinetics of the recombinant adenoviruses did not reveal a
significant difference when compared with that of dAd5. A
decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded
in the virus titers during 60 h incubation period and found to be
statistically significant (p<0.01).
Conclusion: Recombinant adenoviruses expressing capsid
proteins of the FMDV O/IND/R2/75 were constructed and produced in
high titers. In vitro expression of the target proteins in
the adenovirus vector system was detected by sandwich ELISA and
immunofluorescence assay.
Keywords: foot-and-mouth disease, growth
kinetics, recombinant hAd5, thermostability, virus-like particles.
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