Open Access
Research
(Published
online:
13-02-2016)
9.
Expression of biologically active bovine interleukin 7 and
evaluating the activity
in vitro -
J. Lijo, N. Vijay, H. J. Dechamma and G. R. Reddy
Veterinary World, 9(2): 160-165
doi:
10.14202/vetworld.2016.160-165
J. Lijo:
FMD Research Laboratory, Indian Veterinary Research Institute,
Hebbal, Bengaluru, Karnataka, India; lijo1john@gmail.com
N. Vijay:
FMD Research Laboratory, Indian Veterinary Research Institute,
Hebbal, Bengaluru, Karnataka, India; vijvetco@gmail.com
H. J. Dechamma:
FMD Research Laboratory, Indian Veterinary Research Institute,
Hebbal, Bengaluru, Karnataka, India; dechammahj@yahoo.com
G. R. Reddy:
FMD Research Laboratory, Indian Veterinary Research Institute,
Hebbal, Bengaluru, Karnataka, India; drreddygr@gmail.com
Received: 28-09-2015, Revised: 26-12-2015, Accepted: 31-12-2015,
Published online: 13-02-2016
Corresponding author:
G. R. Reddy, e-mail: drreddygr@gmail.com
Citation:
Lijo J, Vijay N, Dechamma HJ, Reddy GR (2016) Expression of
biologically active bovine interleukin 7 and evaluating the
activity
in vitro,
Veterinary World 9(2);
160-165.
Abstract
Aim:
Interleukin 7 (IL-7) is a
ϒc
family cytokine involved in the homeostatic proliferation and
maintenance of immune cells. In the present study, we report the
expression of bovine IL-7 (bIL-7) in
Escherichia coli
and evaluated for its biological activity.
Materials and Methods:
The sequence coding for bIL-7 (mature protein) was amplified
from primary bovine kidney cell culture and cloned into pET28-a
vector and expressed in
E.coli
(BL 21 DE3). The expressed protein was purified by nickel-nitrilotriacetatechromatography,
and the reactivity of the protein was confirmed by western
blotting using monoclonal antibodies raised against human IL-7.
The biological activity of expressed bIL-7 was evaluated by
analyzing its effect on the expression of a nuclear factor for
activated T-cells c1 (NFATc1), B-cell lymphoma 2 (Bcl2),
suppressor of cytokine signaling 3 (SOCS3) molecules in bovine
peripheral blood mononuclear cells (PBMCs) by quantitative
polymerase chain reaction. Ability of the expressed protein was
also analyzed by its effect on phosphorylating signal transducer
and activator 3 (STAT3) molecule by immunostaining in human
embryonic kidney cells 293 (HEK293) cells.
Results:
The bIL-7 was able to induce the expression of Bcl2 and
NFATc1expression in bovine PBMCs by 7 and 5-folds, respectively,
whereas a 2-fold decrease was observed in the case of SOCS3
expression. Immunostaining studies in HEK293 cells using
antihuman phospho-STAT3 showed activation and nuclear
translocation of STAT3 molecule on bIL-7 treatment.
Conclusion:
bIL-7 gene was successfully amplified, cloned, and expressed in
a prokaryotic expression system. The biological activity study
showed that the
E.coli
expressed bIL-7 protein is biologically active. Considering the
role of IL-7 in T-cell homeostasis and memory cell generation,
this molecule can be used for enhancing the vaccine response and
that has to be proved by further experiments.
Keywords:
B-cell lymphoma 2, nuclear factor for activated T-cells c1,
recombinant bovine interleukin 7, signal transducer and
activator 3.
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