April - June 2007 - Kasetsart University

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proliferative signals through the proto-oncogenes
c-myc and c-fos, in combination with antiapoptotic
signals through an essentially identical
receptor (Ma, 2000; Carson et al., 1997; Giri et
al., 1994; Giri et al., 1995). Interleukin-2 promotes
production of NK-derived cytokines such as TNF·,
and granulocyte macrophage colony stimulating
factor (GMCSF). Furthermore, IL-2 acts
synergistically to enhance NK cytotoxic activity
(Khatri, 1998). A number of functions for IL-2 in
B cells have been identified, mostly pertaining to
antibody secretion. In IgM expressing B cells, IL-
2 (in synergy with IL-5) upregulates expression
of heavy and light chain genes as well as inducing
de novo synthesis of the immunoglobulin J chain
gene (Blackman et al., 1986). The latter is required
for oligomerization of the IgM pentamer, and
represents a tightly controlled stage in B cells
activation (Koshland, 1985). As in T cell, IL-2
increases expression of IL-2Rα in B cells, thus
enhancing their responsiveness to IL-2 (Gaffen et
al., 1996). Therefore, IL-2 is one of the key
cytokines in immunology-base studies.
Measurement of IL-2 by ELISA method has been
widely used in clinical investigations and research.
There are quite a number of commercially
available IL-2 ELISA kits which are very
expensive. Production of the ELISA system is
necessary for our use to investigate the effect of
plant extract on mouse immune response. The
objectives of this study were, therefore, to produce
mouse recombinant IL-2 (rmIL-2) by cloning its
gene into pDrive cloning vector and express the
rmIL-2 in pQE30 expression vector to investigate
its biological activities in vitro for the use in
immunization.
MATERIALS AND METHODS
Media and reagents
Complete culture medium was RPMI
1640 (Hyclone, Utah, USA) supplemented with 2
mM L-glutamine, 0.05 mM 2-mercaptoethanol
Kasetsart J. (Nat. Sci.) 41(2)
and 10% fetal calf serum (Hyclone, Utah, USA),
100 U/ml penicillin and 100 µg/ml streptomycin
(Sigma-Aldrich, St. Louis, USA). Concanavalin
A (Con A) (Sigma-Aldrich, St. Louis, USA) was
used for stimulation of splenocytes.
Preparation of mouse splenocytes
A BALB/c male mouse weighing 25-30
g, 12 weeks of age was purchased from the
National Laboratory Animal Centre, Mahidol
University, Nakhon Pathom, Thailand and used
in the experiments. Mouse spleen was removed
aseptically, homogenated and cells were washed
with cold RPMI 1640 medium (HyClone, Utah,
USA) and resuspended in complete medium.
Viability and number of splenocytes were
determined microscopically by staining with
trypan blue (Gibco, New York, USA). Splenocytes
(5 × 10 6 cell/ml) were activated with Con A
(2 µg/ml) in complete medium and transferred into
24 well, flat-blottom tissue culture plate (Costar,
New York, USA). Cells were incubated for 36 h
at 37°C in 5% CO 2 and were harvested for mRNA
extraction.
Primers design
The PCR primers were designed
specifically to mouse IL-2 gene by the FastPCR
program. Briefly, primers for the amplification of
IL-2 mRNA were selected from the conserved
nucleotide sequences of the five strains of mice
(GeneBank accession numbers AY147902.1,
MMU41494, MMU41504, MMU41505,
MMU41506). Additionally, primer sequences for
the detection of mouse β-actin gene were taken
from the literature (Deng et al., 2000) (Table 1).
RNA extraction and cDNA library synthesis
Splenocytes were harvested after the
incubation period (36 h). Total RNA was extracted
from mitogen-stimulated cells (1 × 10 7 cells) and
non-stimulated controls according to the methods
of RNA purification kit (Epicentre, Madison,