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R esearch
(Published online:
12-03-2015)
8.
Expression of bovine interleukin 15 and
evaluation of its biological activity
in vitro -
N. Vijay, J. Lijo, H.
J. Dechamma, V. Bhanuprakash, B. Suresh, K. Ganesh and G. R. Reddy
Veterinary World, 8(3): 295-300
doi:
10.14202/vetworld.2015.295-300
N.
Vijay:
FMD Research Laboratory, Indian Veterinary Research Institute,
Hebbal, Bangalore, Karnataka, India; vijvetco@gmail.com
J. Lijo:
FMD Research Laboratory, Indian Veterinary Research Institute,
Hebbal, Bangalore, Karnataka, India; lijo1john@gmail.com
H. J.
Dechamma:
FMD Research Laboratory, Indian Veterinary Research Institute,
Hebbal, Bangalore, Karnataka, India; dechammahj@yahoo.com
V.
Bhanuprakash:
FMD QC/QA Lab, Indian Veterinary Research Institute, Hebbal,
Bangalore, Karnataka, India; bhanu6467@gmail.com
B.
Suresh:
FMD Vaccine Production Unit, Indian Veterinary Research Institute,
Hebbal, Bangalore, Karnataka, India; surbs2001@gmail.com
K.
Ganesh:
FMD QC/QA Lab, Indian Veterinary Research Institute, Hebbal,
Bangalore, Karnataka, India; kondabattula@gmail.com
G. R.
Reddy: FMD Research Laboratory, Indian Veterinary Research
Institute, Hebbal, Bangalore, Karnataka, India;
drreddygr@gmail.com
Received: 06-11-2015, Revised: 14-01-2015, Accepted: 21-01-2015,
Published online: 12-03-2015
Corresponding author:
G. R. Reddy, e-mail: drreddygr@gmail.com
Citation:
Vijay N, Lijo J,
Dechamma HJ, Bhanuprakash V, Suresh B, Ganesh K, Reddy GR (2015)
Expression of bovine interleukin 15 and evaluation of its
biological activity in vitro, Veterinary World 8(3):295-300.
Abstract
Background/Aim: Recent studies have shown that interleukin-15
(IL-15)is a critical factor for the development and proliferation
of CD8 + memory T cells. The aim of present
study is to study the role bovine IL-15 (bIL-15)in activation
pathway of bovine CD8+ T cells if any, which
will be useful in designing the adjuvant to increase the duration
of immunity of the vaccine preparations.
Materials and Methods: Coding region of bIL-15 (489) was
amplified from cDNA of lipopolysaccharide-induced bovine
peripheral blood mononuclear cells (PBMCs) using gene specific
primers and cloned into pcDNA3.1 +.
Mature length of bIL-15 was amplified using gene specific primers
and cloned into pET32a for expression studies. Expressed fusion
protein was purified using Ni-Nitrilotriacetic acid agarose
affinity chromatography and analyzed by SDS-Polyacryamide gel
electrophoresis (PAGE) and western blotting. Biological activity
of purified protein was analyzed by quantitative Polymerase Chain
Reaction (qPCR) for an increase in levels of Bcl2, STAT3 and
STAT5a using cDNA synthesized from RNA of PBMCs induced with
different concentrations of purified bIL-15. Role of IL-15 in
inducing memory CD8+ T cells was analyzed by
qPCR for increase in the level of Carnitine Palmitoyl Transferase
1a (CPT1a) using cDNA synthesized from RNA of PBMCs induced with
different concentrations of purified bIL-15.
Results: Bovine IL-15 was amplified and
analyzed by agarose gel electrophoresis, which showed a specific
product of ~490bp, mature sequence was amplified using full-length
as a template to get a product of ~350bp. The protein was
expressed, purified and analyzed by SDS-PAGE and Western blotting,
which showed a specific product of 32kDa. Biological activity of
purified bIL-15 fusion protein showed an increase in levels of
Bcl2, STAT3 and STAT5a with 5 fold, 9 fold, and 10 fold increases
as analyzed by qPCR, respectively. Role of IL-15 in inducing
memory T cells showed an increase in expression level of CPT1a at
2.5 fold increase as compared to control cells.
Conclusion: Bovine IL-15 has been successfully cloned and
expressed in our work, and the biological activity shows that the
purified fusion protein is biologically active. As there is an
increase in levels of CPT1a an enzyme critical for survival of
memory T cells, IL-15 can be used for increase in the memory
response, which can be used as an adjuvant with viral vaccines for
increasing the immunity.
Keywords: Bovine interleukin 15, Carnitine
Palmitoyl Transferase 1a, Memory CD8+T
cells.
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