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Cytokine 32 (2005) 219e225

Expression of nine-banded armadillo (Dasypus novemcinctus)

interleukin-2 in E. coli
J.E. Adams, M.T. Peña, T.P. Gillis, D.L. Williams, L.B. Adams, R.W. Truman*
Laboratory Research Branch National Hansen’s Disease Programs, Louisiana State University,
School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70803, USA
Received 15 June 2005; received in revised form 26 August 2005; accepted 1 September 2005

Abstract

The nine-banded armadillo (Dasypus novemcinctus) is the only immunologically intact animal that regularly develops lepromatous-type lep-
rosy when inoculated with Mycobacterium leprae. However, the ability to exploit this model for understanding the pathogenesis of leprosy has
been limited by a lack of suitable immunological reagents. Recently, efforts began to sequence the entire armadillo genome, and this sequence
information will help make possible the development of a wide array of new immunological reagents suitable for use with armadillos. Using the
available sequence data, a region of high homology to interleukin-2 of other mammals was identified. Primers were designed to amplify the
coding region corresponding to the mature peptide and its exact sequence was confirmed. cDNA was made from ConA-stimulated armadillo
PBMC. The amplified coding region was sub-cloned into a pET expression vector and transformed into Escherichia coli for over-expression.
The subsequent product was characterized by SDS-PAGE and bioassays. Tritiated thymidine incorporation by CTLL-2 and armadillo lympho-
blasts confirmed functionality of the recombinant product. The advent of the D. novemcinctus genome sequence and subsequent generation of
immunological tools will assist in advancing the armadillo as a translational model for leprosy.
Published by Elsevier Ltd.

Keywords: Dasypus novemcinctus; Interleukin-2; Leprosy; Lymphocyte proliferation; Mycobacterium leprae; Nine-banded armadillo; Recombinant

1. Introduction over a broad clinical spectrum of immunological and histo-

Nine-banded armadillos, Dasypus novemcinctus, are the
most abundant living members of the mammalian order
Xenarthra. These exotic-looking cat-sized animals range
throughout North and South America and are especially abun-
dant in the southern United States. Armadillos in Texas and
Louisiana are known to harbor Mycobacterium leprae, the
etiological agent for leprosy [1e3]. This bacterium cannot
be cultivated on artificial laboratory media and armadillos
have developed into the hosts of choice for in vivo propagation
of leprosy bacilli [4,5].
Nine-banded armadillos are the only immunologically
intact animal species that exhibit high susceptibility to
M. leprae. Like man, leprosy in the armadillo can manifest
* Corresponding author. Tel.: þ1 225 578 9860; fax: þ1 225 578 9856.
E-mail address: rtruma1@lsu.edu (R.W. Truman).
pathological responses classifiable as Lepromatous or Tuber-
culoid and the associated borderline forms [6]. Owing to
their unique susceptibility, armadillos have been considered
potentially valuable models for leprosy pathogenesis, under-
standing factors underlying susceptibility and resistance to
the infection, and for development of new diagnostics and vac-
cines [5]. Unfortunately, owing to their exotic nature and scant
commercial value, relatively few armadillo-specific immuno-
logical reagents have been generated. As a result, few transla-
tional benefits have been realized from work with this model.
Resistance to M. leprae is mediated through cellular immune
responses and involves a complex interplay of cytokines and
chemokines. It has not been possible to monitor the cytokine re-
sponse of armadillos. IL-2 is an essential driver of any T-cell re-
sponse [7] and the IL-2 of many mammals have already been
cloned and overexpressed in Escherichia coli [8e13]. This
protein often exhibits functional cross reactivity between

1043-4666/$ - see front matter Published by Elsevier Ltd.

doi:10.1016/j.cyto.2005.09.011
220 J.E. Adams et al. / Cytokine 32 (2005) 219e225
mammals, but the nucleic acid sequence appears to be only par-
tially conserved. Monoclonal antibodies and primer sequences
prepared for mouse or human proteins seldom react with arma-
dillos (Promega, Madison, WI; R&D, Minneapolis, MN).
Owing to the armadillo’s evolutionary and medical signifi-
cance, The Human Genome Consortium recently began se-
quencing the Dasypus novemcinctus genome. Trace sequence
reads for D. novemcinctus are already available (http://
www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml) and annotation
of the genome is now underway. We searched this sequence
data for regions homologous to IL-2 and report here the
identified sequence, cloning, over expression and biological
activity of recombinant Dasypus novemcinctus IL-2.
2. Materials and methods
2.1. Database searches
tBLASTn was employed by using the amino acid sequence of
Mus musculus IL-2 (GI: 7110653) as a query sequence to search
for homologous translated sequences in the high throughput ge-
nomic sequences (htgs) database. The putative genomic se-
quence of DnIL-2 (GI:33620804) was found at NISC (NIH
Intramural Sequencing Center) comparative sequencing initia-
tive (http://www.nisc.nih.gov/). The genomic sequence was sub-
mitted to FGENESH (http://www.softberry.com) to derive
a putative cDNA and a corresponding translation for the putative
amino acid sequence. The cDNA and the amino acid sequence
were submitted to BLAST (http://www.ncbi.nlm.nih.gov/
BLAST/) to verify homology to other IL-2’s [14].
2.2. Generation of D. novemcinctus cDNA
Armadillo peripheral blood mononuclear cells (PBMC) were
harvested from whole blood and treated with ConA (Sigma-
Aldrich, St. Louis, MO). Aliquots of the ConA stimulated
cells (5 mg/mL for 4 h) were washed 3
in cold PBS, harvested,
snap frozen in liquid nitrogen, and stored at 70  C until RNA
purification. Total RNA was purified using the FASTRNAÔ kit
(Qbiogen, Carlsbad, CA) and the FASTPrep fpizo 120Ô instru-
ment (Qbiogen, Carlsbad, CA) according to the manufacturer’s
protocol. cDNA was generated from 1 mg RNA and the Advan-
tage RT-for-PCR kit with random hexamers (BD Biosciences
Clonetech, Palo Alto, CA) in a final volume of 50 mL according
to the manufacturer’s recommendations.
2.3. Plasmid construction, expression
and purification of DnIL-2
Primers designed to amplify the coding region corresponding
to the mature peptide (the protein without the signal peptide) of
DnIL-2 (MIL2cDNAf: 5#-TGCACCTACTTCAAGCTCTA-
CAAA-3# and MIL2cDNAr: 5#-TAGCAAACCATACATC-
CAAAAATA-3#) (BIOMEDD, Baton Rouge, LA) were used
with high fidelity polymerase, Pfu (Strategene, La Jolla, CA),
to generate the fragment to be subcloned. This fragment was
purified using QIAquick columns (QIAgen, Valencia, CA),
verified by automated DNA sequencing using an ABI prism
377 DNA sequencer (Applied Biosystems, Foster City, CA) (BI-
OMMED, Baton Rouge, LA) then reamplified using a primer
containing the topo sequence (CACC) on the 5# terminus
(MIL2-TOPO: 5#-CACCGCACCTACTTCAAGCTCTAC-3#).
This topo fragment was subcloned into pET 200D/topo vector
(Invitrogen, Carlsbad, CA) and transformed into E. coli
BL21star (Invitrogen, Carlsbad, CA) according to manufactur-
er’s recommendations and verified by PCR and subsequent au-
tomated DNA sequencing. These cultures were grown to an
OD600 of 0.6 and induced with a final concentration of 1 mM iso-
propyl b-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St.
Louis, MO). The overexpressed protein was then purified using
a Ni-NTA Purification system using the recommended, denatur-
ing regimen (Invitrogen Carlsbad, CA). The resulting product
was concentrated using a Vivaspin 15 mL concentrator with
a 10 KDD molecular weight cut-off (Vivascience, Hanover,
Germany). The over expressed product was separated via
SDS-PAGE (4% to 20% gradient polyacrylamide gel) and
stained with coomassie brilliant blue (Biorad, Hercules, CA),
compared against Kaleidoscope pre-stained standard (Biorad,
Hercules, CA), and a western blot using the Anti-xpressÔ anti-
body (Invitrogen Carlsbad, CA) was used to verify the size and
presence of the polyhistidine epitope of the recombinant product.
2.4. Lymphocyte isolation from D. novemcinctus
and mitogen induction
To avoid sacrificing animals and to permit re-examination
of individual hosts, lymphocytes were isolated from armadillo
peripheral blood. PBMC were purified from 14 mL peripheral
blood collected in Vacutainer tubes containing 150 U of hepa-
rin. Blood was diluted in 2
volumes of cold Hanks’ Balanced
Salt Solution (HBSS) and lymphocytes were isolated by den-
sity gradient centrifugation after layering over Ficoll-PaqueÔ
Plus (Pharmacia Biotech, Piscataway, NJ) in a 50 mL tube and
centrifuged at 400
g for 45 min at 18  C. The lymphocyte
layer was removed using a sterile pipette and washed 3

with cold PBS. The suspension was centrifuged at 400
g
for 10 min at 4  C and resuspended in 1 mL of RPMI 1640
media (RPMI) with 5% fetal bovine serum (FBS). Cell viabil-
ity was determined using trypan blue exclusion. This cell sus-
pension was seeded into 24 well culture plates (1.5 mL/well)
at a concentration of 2
106 cells/mL. ConA was added to
a final volume of 5 mg/mL. This concentration was found to
be optimal by a titration assay where ConA was used at 5,
10 and 20 mg/mL (data not shown). The plates were incubated
for 0, 4, 8, and 24 h [15] at 37  C, 5% CO2. After incubation,
supernatants were harvested and clarified at 9300
g for
10 min and stored at 70  C until used.
2.5. CTLL-2 bioassay
Briefly, the murine IL-2 dependent cell line CTLL-2
(ATCC# TIB-214) was obtained from the American Type Cul-
ture Collection (ATCC, Manassas, VA) and maintained in com-
plete growth medium consisting of RPMI 1640 with 2 mM
 J.E. Adams et al. / Cytokine 32 (2005) 219e225
L-glutamine and supplemented with 10% rat T-STIM contain-
ing ConA (rat IL-2 culture supplement) (Becton-Dickson, Bed-
ford, MA) and 10% fetal bovine serum (FBS). The biological
activity of rDnIL-2 was determined in a cell proliferation assay
[16]. The CTLL-2 cells were harvested in active log-phase and
washed with RPMI media three times to remove residual rat IL-
2. Recombinant human IL-2 (rHuIL-2) (R&D Systems, Inc)
was used a positive control for the assays. rHuIL-2 (1883 ng/
mL) recombinant DnIL-2 (rDnIL-2) (770 ng/mL), and superna-
tants were two-fold serially diluted in RPMI 10% FBS and
50 mL of each dilution was added to a 96-well microtiter plate.
CTLL-2 cells were distributed in 50 mL RPMI medium at an
optimal density (determined by titration) of 8
105 cells/mL
to a 96-well microtiter plate containing 50 mL of the serially di-
luted recombinant IL-2’s or supernatants from the cells cultured
for different time intervals. The cells were cultured for 20 h at
37  C with 5% CO2. After incubation, plates were pulsed with
10 mL tritiated thymidine (3[H]dTr) (Perkin-Elmer, Boston,
MA) (10 mCi) per well and incubated for 16 h. The plates
were harvested using a PHD Cell harvester (Cambridge Tech-
nologies, Inc., Cambridge, MA) and the incorporation of
3
[H]dTr was determined by liquid scintillation counting in
cpm using a LS 6500 multi purpose scintillation counter
(Beckman-Coulter, Fullerton, CA).
2.6. D. novemcinctus lymphocyte blasts
We also assessed the ability of rDnIL-2 and D. novemcinc-
tus PBMC ConA stimulated supernatants to maintain prolifer-
ation of D. novemcinctus lymphoblasts. Lymphoblasts were
obtained after isolation of PBMCs from whole blood using
Ficoll-Paque gradient. The cells were adjusted to 5
106
cells/ml in culture medium (DMEM with 10% FBS) and plat-
ed in 24 well tissue culture plates (1 ml aliquots) [12]. After
the addition of Con A (5 mg/ml) the plates were incubated
for 5 days. The cells were harvested and overlayed onto
10 ml of Ficoll-Paque and centrifuged at 700
g for
20 min. rDnIL-2, rHuIL-2, and supernatants were serially di-
luted with growth medium Dulbecco’s modified eagle medium
(DMEM) and 50 mL was added to each well of a 96 well tissue
culture plate. 50 mL of lymphoblast cells (5
105 cells/mL)
was added and the plate was incubated for 24 h at 37  C or
33  C with 5% CO2. Proliferation of lymphoblasts was deter-
mined using thymidine incorporation as described with the
CTLL-2 bioassay.
2.7. Protein quantification
Protein concentrations were determined colorimetrically
using the BCA protein assay kit (Pierce, Rockford, IL) on
a Biorad plate reader (Hercules, CA) according to manufacturer’s
recommendation.
2.8. Statistical analyses
The means of the groups were compared using ANOVA and
TukeyeKramer test was used to make multiple comparisons
221
between the different groups (GraphPad InStat software,
GraphPad Software, Inc, San Diego, CA).
3. Results
3.1. Sequences
We found the nucleic acid sequence for the putative DnIL-2
(Fig. 1) at NISC using BLAST with the amino acid sequence
of Mus musculus IL-2. The resulting 5979 bp genomic frag-
ment was then submitted to FGNESH to map the coding
region of DnIL-2 (Fig. 2). The genomic region contains four
exons (Fig. 2) that compose a 468 bp predicted coding region.
We verified this by sequencing cDNA from ConA stimulated
PBMC. DnIL-2 showed the greatest homology after the first
20 amino acids when compared to other mammalian IL-2 amino
acid sequences (Fig. 3) suggesting that these were part of
the signal peptide. The entire DnIL-2 amino acid sequence
(with signal peptide) was most homologous to that of Macaca
mulatta (rhesus monkey) IL-2 (E-value: 1ee45 (84%)) and
other mammalian IL-2’s: Cercocebus torquatus (red-crowned
mangabey) (E-value: 1ee45 (83%)), Homo sapiens (E-value:
4ee45 (84%)), Papio hamadryas (hamadryas baboon) (E-value:
1ee45 (84%)), and Saimiri sciureus (common squirrel mon-
key) (E-value: 3ee45 (84%)). A ClustalW multiple alignment
(http://searchlauncher.bcm.tmc.edu/multi-align/Options/clustalw.
html) was done to examine similarities and differences between
these IL-2’s and DnIL-2 (Fig. 4). Additionally, rat (Rattus
norvegicus) and human (Homo sapiens) IL-2 was also examined
for similarities and differences to DnIL-2 as rat T-STIM was
used to maintain the CTLL-2 cells and rHuIL-2 was used as the
positive control in bioassays.
3.2. Purification
We found rDnIL-2 in the insoluble fraction (data not
shown) of the E. coli lysate as has been reported with other
mammalian recombinant IL-2’s [12,13]. The predicted
rDnIL-2 fusion protein had a molecular weight of 19.7 KDD
using ExPASY (http://us.expasy.org/tools/pi_tool.html). The
high molecular weight was due to the inclusion of the his-
tag and enterokinase recognition site (M R G S H H H H H
H G M A S M T G G Q Q M G R D LY D D D D K D H
P F T) added to the mature polypeptide sequence starting at
residue A-21 (Fig. 4) in the recombinant product. We
confirmed its size and purity by coomassie blue staining
and a western blot using an antibody against the poly-
histidine tag on the n-terminus of the recombinant product
(Fig. 5).
3.3. Bioassays
Multiple assays demonstrated rDnIL-2 and D. novemcinc-
tus supernatants were capable of sustaining proliferation of
CTLL-2 cells and D. novemcinctus lymphoblasts (Fig. 6a,b).
Significant proliferation was seen when CTLL-2 cells or
D. novemcinctus lymphoblasts were cultured with rDnIL-2
222 J.E. Adams et al. / Cytokine 32 (2005) 219e225

1 aagtaatact ttttgccaca caggtagagc ctttgaaaat atgtgtaata tgtaaaaCAT Tttgtttgac acccccaTAA TAttttttcc agaattaaca
101 gtataaattg cttctcttgt tcaagagctt catatcaacc tctctaatca ctactcacag taacctcaac tcctgccaca ATGTACAAGA TGCAACTCGT
201 GGCTTGCATT GCATTAAGTC TTGTACTCAT CACAAACAGT GCACCTACTT CAAGCTCTAC AAAGGAAACC CAGCAACAAC TGGAGCAATT ACTACTGGAT
301 TTAAAGATGC TGTCCAAGAT GGTTAATgta agtatattcc ctttttaact aaaattatta cacttaattt ttctagctgg agtttgattg taaataacaa
401 tgtgctattc tttctcagAA TAAGGATCTC AAACTCCCAA GGATGCTCAC ATTTAAATTT TACATGCCTA AGAGGgtaag tacaattttc tatgttccaa
501 ttatgtgtta aaattcaaag aaacatgaaa tttttaacat gttgacttaa gagcatctca tctgaaggaa aagaattagt aaatgcacta ttttctaaaa
601 tccaagttag ttaatagagt ttctattaaa atggcttggc ctatactgtt tagatttact tgggtgtatt ttcgggatca gggggaaagc tgtaagaaaa
701 gaaaacaaga ataatattct ctttattgct gggacaattg taaaagaaga ttttgttgaa ttcctgcttt gtttaaaaat gcaaaactct agagatattt
801 gaatttatta ttgtatcatg caacatgtca aaattgagtt cagaggagca cactttgaaa catcctttat tgtttgtgga tagccacaac ttctagagaa
901 agtttatttg aaccaaaatc ccagctctga catgtgctaa ctttcatcat ctttttatgt gaataataat tttatttact ttatatagtt cttgtgacaa
1001 ttagtgagta aatgtagagt actcaaaaca gtgtctggca catggtaaac accaaataag ccttagcttt tattagtagt aattaataca gaattctcac
1101 tgaggagtat gaagtaaaat tttggactta cacatctctt atgtcaatag acctggagat ttatggtgct atgtgattta tttccaggag taaaatttca
1201 tttgattgct tgggaggcca gatagttaga taagcacagc tcattcaagc tcctagaact acaacaaaga tgtaaagttt cctagtagta gtgccagccc
1301 gcaaggatgg agcaaagctt tgatgctaga cagaacgtgg cccagggtga aaccaagtcc agaaaacaat gggatttaag ggtacagaga tataatcctg
1401 ttgatttcct tctattagac tgagaatttt gtttcaaagt actgttttga aagacaaaaa gaaaatagct tagagactcc tccaatttaa ccaacgctac
1501 ataaccctta cctccaatca acctaagaga cactagactg tttttggagg aaaatagttc caagttgagc atgccgaaaa cttaagcctg gaactagtag
1601 tagtaagttg aaagggcagt aaatggaaaa gtcaggagat gcataatgat tagtcctacc tttctctcta aaaattgtta gaaagataac atgagattat
1701 gtgtataatt gggggtggag gctagagaat tctttgaaat tgaatagttt ctaaatgaca actcaaattc tatttgaaac caaaagtaat gtggtaaact
1801 ccattcactt gttcatttta ccaccattcc acatttgcaa aagattttag aaagtcatta aaaatttaaa gagatgctct gttaatgcta taaagtgaat
1901 taaaatttaa tcccggctct acctactcat gtggccttgg ggaaggcccc ttacttttga gattcagttt ccttatctgc aaaatggata tagcaatact
2001 gtactgccaa ccaccaaggg tggctgtgac acttaaaagg gcttagtgaa actataatgt accatagaag tacaagtatt atcattatta ataaaaatgc
2101 atggctagtt atctgcagca tctagatcag gaggcctgag gaaaaaaaaa aatccttcaa atattggaac cttatatttg gtttggacaa ataataaaga
2201 aatgcatctt aatgttgtaa aaacttaaga cacagcttta tgacttgctc cagactatat tgaatgtcat tattatagct accattatct gatctgtaaa
2301 atgttgggac tgtgataaat ggtgtccata gcagtcccta gcagtctaag attttcaaaa tcaaagctta gaattgcaga atgtgatttc tttttttcca
2401 cattctggct gctctggttc taatgatttc tatgtaggtt agaagaaata gcctatatat ctgtgatttg tatttgggtg cagaaatgct gactttttac
2501 aaaggcaaac taacatgaat gagttcttag tcattaacag ttaatgctat ttatggttaa ttgatctcca gttttttcag tcttataagg taagctatta
2601 cactggtcca tctttatagc aatgctgaaa acagggagaa aacaggtttc taaaatctat tgtagattaa tttctgattc tgatcttcat ttgagggtaa
2701 attgagatga taaatattat cattattcta gGTCACAGAG TTGAAACATC TTCAGTGTCT AGTAGAAGAA CTCAAACCTT TGGAGAATGT GCTAAATTTA
2801 GCTCAAAGCC AAATGTCTCA ACTGGAACAT AATGGGGACT TAATCAGTAA TATCAATATA ACCGTTCTGG AGCTAAAGgt aagccattac tttatttgct
2901 ctcctagaaa taaaataaat gcaaggggtg aaatattgta tttaaagttt tataatattt ttggtatttt gtaaaatatc catgtacttg ataagctaat
3001 attttaagcc cactgtaaac accaagtatt tttaatgtta gatgatatgt tattgagcta agaagtagaa gcttgaggtt tctaactaca gtctacccag
3101 ggacaagtcc cagcctctat atgaataatc accttcccct aatcagccac atctagtccc tcagcagtgg gagaaggcct tgaactgaat ttcacagaaa
3201 gcagagtttt tcttcagatc atttaccagg atagttaata cagattacct gtgaggcttt ctaaatggca tttccctatc atttggtaga aggactgatc
3301 agaaatgatt tgtctcaaat aatggctgtc attaaataaa atggccaatg gaataaataa ctaactcttt atttagagga aatgcccact gtcaggactg
3401 tatttttaag tgcacaggaa gtattaaaat atataaaggt ctcacatgat gtgaaagaga ttcaaggtgc tcttttatca tcctccctgc caaagtacaa
3501 aattcataag ttaataggta tcctacataa gcagtataat tttaactaca gtagaaccaa gcttctaaaa atacataaat tttagttgtt tacaagttag
3601 ttattttagc ggaatttttt tcagcctaat ttaaaaaaaa caatacatca acattgagac cactgttatc tagagcccta taactgttaa gagatttgct
3701 ataaaatctt tgaccaatat cagaaaactc atgctaaatc tctgatgcaa ggttaaaata aagttatgtg aaagtcagca aaatagggtt caaatgccaa
3801 agactctttt taacttaaac aaacaaacaa aaacagagcc caagaacagg gaattaatgt ttacaatata tagagtttct atttggaatg atggaaaagt
3901 tttcgtaatg aacaatggtg aattaaatat ttgaatggta aaaggggaaa ttttaggtat atatgttact ttaaggataa agtatttttt aaaaattcat
4001 ggaacaacac agtgaaccct gttaaaccat gcactatagt tattagtaca attataaaaa tgtacttcta ttgattttaa caaatgtacc acaccaatgc
4101 aaggtgttca taacagggta gtatacagga acattgcatt ttatgcatga tttttctgta aacccactac tgtctaataa aaaaaaaaga ttataatact
4201 taaataattg tgtaagagga tacataatac tatagcagct ttttgtttgt ttgtttgttt gttttttcag ggaatacttt acgctacctc actttttttg
4301 tgttcccaag ataggaatta cttcaaattg gaaaaagtta tcttttgtgt ttcactatta taaaaatatt tgtttcttag ctagtcaggt agaggaagag
4401 gtgaaatggc tcctatcatc aaaaaaagat taatttccaa aaaggcaaca gagaaaaatg gctaatgttt aacttttaaa agttgggata cttgcttgca
4501 tagatactgg gataaagggg atgggtatga gcagttatga agatgttcat ataaaccttc cccaaacaaa agcttgtcct aactttctct gggcaagttc
4601 tatcctcgga ttcactcaaa tagaatatta agtgtctctt aatgaaggac agaaaagaaa gtaaaatgtg catgccactg catacatgta acctacatag
4701 gtatatagat atacatacct agataaacac agagaggaaa agagatcaaa taacttcata tttgatgcag tatttaatga agtatttgtc tagtattact
4801 taaaatggtt acttgactac aatccaggaa aacatcttgt gttcattgag actgacagaa aaattaacag atgaagaaaa tctcaggtta tatgctagta
4901 tcattgatat cataaatata attttgacac attttaatat tttgatcatt tcattttaga agatatgaat gactaatatg tgtaacatgc ttgaaaataa
5001 aggtcatgtg cctataaaac ttcaactgag aataattaca taaaaggcaa actaccctaa actaaaaaaa attaaaattt tcttttatag GGATCTGAAA
5101 CAACATTCAT GTGTGACTAT GATGATGAGG CAGCAACCAT TGTAGAATTT CTGAACAAAT GGATTATCTT TTGTCAAAGC ATCATCTCAA AAAGACTTGA
5201 TAATTAAgtg ccttccattc aaaatacatc tatttattta aatatttaaa ttttataatt tatttttgga tgtatggttt gctacttttt gtaactatta
5301 tacttagatg atgaatatgg atcttttaag attctttttg taggccctag gggctctaaa atgctttcac tttaaattat tttatctcaa agtatttatt
5401 atattgaatt gttacatata atgtctatat aggtcaatta ataaaattgt ttaataaact tgatgaatgg ttatttggga acagcacaga ggaagtacta
5501 aaatattgtg aattatttat gtgaattcta agatggttaa aatgcttaac aaaagtcact tttcccatag agaggtatgt agaacaggga aatgggcttt
5601 tcaaagctct tgctttctct ttccaagagt tgatcagatc cttgatatct tagttctggt ctgagaaaac tacctcataa aactatactc ttttccttcc
5701 tttgatcatg ccttaaattt aaacaaatgt gaaacatatt cttgaaggtc ttcatacaac cggtgtgcct ctctatccac cctttaccat cttactgctt
5801 acccaccacc cctcaggagg ctaagctata acaaaaccct cccttgtctt ttggctttgg ctttccattg ggtcttttga ttggggagcc ctgttaagag
5901 acttaaggga cacagaggag attctcctga ctccctccta gcaatattac atctggatgg cgatacctct tgaccaaag

Fig. 1. The genomic sequence of DnIL-2 as obtained from NISC. The putative CATT box and TAATA box and coding region are indicated by uppercase bold
letters.

or rHuIL-2. Supernatants derived from Con-A stimulated
PBMC also drove proliferation of both CTLL-2 cells and
D. novemcinctus lymphoblasts. Similar to what others have
observed, greater proliferation was seen with supernatants
that had been stimulated for 24 h than for shorter time periods
[15]. Armadillo lymphoblasts incubated at 33  C showed
a similar proliferative response (data not shown).
A representative dose response of CTLL-2 cells prolifera-
tion to different concentrations of rDnIL-2 and rHuIL-2 is
shown in Fig. 7a. The 50% effective dose (ED 50) on CTLL-
2 proliferation was achieved at 0.023 pmol of rHuIL-2 and
0.19 pmol of our rDnIL-2 (Fig. 7a). Fig. 7b also shows a similar
dose response on D. novemcinctus lymphoblasts prolifera-
tion with different concentrations of rDnIL-2 and rHuIL-2.

TATAA box
Polyadenylation Signal
Exon I
Exon II Exon III Exon IV

1 Kb

Fig. 2. Map of genomic DnIL-2. The 5979 bp region shown is predicted to contain the four exons that code for IL-2. Exon I (147 bp), Exon II (57 bp), Exon III
(147 bp), and Exon IV (117 bp) are indicated by arrows. The putative TATAA box and putative polyadenylation signal are also indicated.
 J.E. Adams et al. / Cytokine 32 (2005) 219e225 223

atg tac aag atg caa ctc gtg gct tgc att gca tta agt ctt gta ctc atc aca
M Y K M Q L V A C I A L S L V L I T
aac agt gca cct act tca agc tct aca aag gaa acc cag caa caa ctg gag caa
N S A P T S S S T K E T Q Q Q L E Q
tta cta ctg gat tta aag atg ctg tcc aag atg gtt aat aat aag gat ctc aaa
L L L D L K M L S K M V N N K D L K
ctc cca agg atg ctc aca ttt aaa ttt tac atg cct aag agg gtc aca gag ttg
L P R M L T F K F Y M P K R V T E L
aaa cat ctt cag tgt cta gta gaa gaa ctc aaa cct ttg gag aat gtg cta aat
K H L Q C L V E E L K P L E N V L N
tta gct caa agc caa atg tct caa ctg gaa cat aat ggg gac tta atc agt aat
L A Q S Q M S Q L E H N G D L I S N
atc aat ata acc gtt ctg gag cta aag gga tct gaa aca aca ttc atg tgt gac
I N I T V L E L K G S E T T F M C D
tat gat gat gag gca gca acc att gta gaa ttt ctg aac aaa tgg att atc ttt
Y D D E A A T I V E F L N K W I I F
tgt caa agc atc atc tca aaa aga ctt gat aat taa
C Q S I I S K R L D N stop

Fig. 3. The predicted coding region of 468 bp and resulting amino acid sequence of 155 aa of DnIL-2. The signal peptide is indicated in italics.

The ED 50 of D. novemcinctus lymphoblasts proliferation was
achieved with 0.0013 pmol of rHuIL-2 and 0.028 pmol of
rDnIL-2.
4. Discussion
This report demonstrates that the coding region for the ma-
ture DnIL-2 was successfully amplified, subcloned, and over-
expressed. DnIL-2 has high homology at the amino acid level
to other mammalian IL-2’s and the recombinant protein ap-
peared to be biologically adequate for sustaining proliferation
of both CTLL-2 and armadillo lymphoblasts.
The in silico predicted DnIL-2 had low Expect (E) values
to other mammalian amino acid sequences of IL-2 (<4ee
45) indicating that the DnIL-2 was a close match [14]. The
deduced protein showed high amino acid sequence homology
especially to the IL-2’s of Macaca mulatta as well as some
other mammals (Fig. 4). It has been shown previously that the
residue, D-40, in HuIL-2 is important for inducing proliferative
activity of CTLL-2 cells and is responsible for high affinity
receptor (IL-2R) binding [17]. Amino acid residues Y-64 and
D-130 have been implicated in internal stabilization of
HuIL-2 [17]. Residues 38e41 cannot be deleted without mod-
ifying the structure and function of murine IL-2 because they
are related to the last turn of helix A and the first residue of the
loop connecting a-helices A and B [18]. Residues K-55, R-58,
F-62, and K-63 are located in the B a-helix of HuIL-2 and
have been implicated as important residues for the binding
of HuIL-2 to the low affinity (IL-2R) [19]. The translated
cDNA sequence shows that all of these residues are conserved

10 20 30 40 50 60
Ph MYRMQLLSCI ALSLALITNS APTSSSTKKT QLQLEHLLLD LQMLLNGINN YKNPKLTRML
Ss MYRMQLLSCI ALSLALITNS APTSSSTKKT QLQLEHLLLD LQMLLNGINN YKNPKLTRML
Mm MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML
Ct MYRMQLLSCI ALSLALVTNS APTSRSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML
Hs MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML
Dn MYKMQLVACI ALSLVLITNS APTSSSTKET QQQLEQLLLD LKMLSKMVNN -KDLKLPRML
Rn MYSMQLASCV ALTLVLLVNS APTSSPAKET QQHLEQLLLD LQVLLRGIDN YKNLKLPMML
** *** :*: **:*.*:.** **** .:*:* * :**:**** *::: . :: *: **. **
70 80 90 100 110 120
Ph TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RDTRDIISNI NVLVLELKGS
Ss TFKFYLPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RDTRDIISNI NVLVLELKGS
Mm TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RDTKDLISNI NVIVLELKGS
Ct TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RDTKDLISNI NVIVLELKGS
Hs TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RP-RDLISNI NVIVLELKGS
Dn TFKFYMPKRV TELKHLQCLV EELKPLENVL NLAQSQMSQL EHNGDLISNI NITVLELKGS
Rn TFKFYLPKQA TELKHLQCLE NELGALQRVL DLTQSKSFHL EDAGNFISNI RVTVVKLKGS
*****:**:. ********* :** .*:.** :*:**: :* . ::**** .: *::****
130 140 150
Ph ETTFTCEYDD DTATIIEFLN GWITFCQSII STLT--
Ss ETTFTCEYDD DTATIIEFLN GWITFCQSII STLT--
Mm ETTLMCEYAD ETATIVEFLN RWITFCQSII STLT--
Ct ETTLMCEYAD ETATIVEFLN RWITFCQSII STLT--
Hs ETTFMCEYAD ETATIVEFLN RWITFCQSII STLT--
Dn ETTFMCDYDD EAATIVEFLN KWIIFCQSII SKRLDN
Rn ENKFECQFDD EPATVVEFLR RWIAICQSII STMTQ-
*..: *:: *:.**::***. ** :****** .

Fig. 4. ClustalW alignment of closest matches in NCBI nr database using BLAST where Ph- Papio hamadryas; Ss- Saimiri sciureus; Mm- Macaca mulatta; Ct-
Cercocebus torquatus; Hs- Homo sapiens; Dn- Dasypus novemcinctus (bold-faced); and Rn- Rattus norvegicus. The signal peptide is indicated in italics. ‘‘*’’ in-
dicates positions which have a single, fully conserved residue. ‘‘:’’ indicates that one of the following ‘‘strong’’ groups is fully conserved: STA NEQK NHQK
NDEQ QHRK MILV MILF HY FYW. ‘‘.’’ indicates that one of the following ‘‘weaker’’ groups is fully conserved: CSA ATV SAG STNK STPA SGND SNDEQK
NDEQHK NEQHRK FVLIH FYM.
224 J.E. Adams et al. / Cytokine 32 (2005) 219e225
Fig. 5. Purification of rDnIL-2. (A) A coomassie stained gel with uninduced E.
coli transformed with pET D topo þ DnIL-2 plasmid (U), E. coli transformed
with pET D topo þ DnIL-2 plasmid induced with IPTG (I), and the purified
rDnIL-2 (P); and, (B) a western blot using Anti-xpressÔ showing uninduced
E. coli transformed with pET D topo þ DnIL-2 plasmid (U), E. coli trans-
formed with pET D topo þ DnIL-2 plasmid induced with IPTG (I), and the
purified rDnIL-2 (P). rDnIL-2 is indicated by arrows.
in DnIL-2, and it possesses similar functionality. The two a-
helices on the N-terminus of the protein that are responsible
for binding to the IL-2 high affinity and low affinity receptors
may be preferred targets to DnIL-2 for future attempts to raise
monoclonal antibodies.
Fig. 6. (a) CTLL-2 proliferation (in cpm) after incubation with media, super-
natants generated by induction of D. novemcinctus PBMC with ConA at 0 h,
4 h, 8 h, and 24 h, and recombinants (recombinant D. novemcinctus IL-2
(rDn) and recombinant human IL-2 (rHu)). Media is cells with no recombinant
IL-2 or supernatant and used as a negative control. Error bars represent
mean  SD of triplicate wells. (b) Armadillo blast proliferation (in cpm) after
incubation with media, supernatants generated by induction of D. novemcinc-
tus PBMC with ConA at 0 h, 4 h, 8 h, and 24 h, and recombinants (recombi-
nant D. novemcinctus IL-2 (rDn) and recombinant human IL-2 (rHu)). Media
indicates cells with no recombinant IL-2 or supernatant and used as a negative
control. Error bars represent mean  SD of triplicate wells.
Fig. 7. (a) Relative efficacy of recombinant IL-2’s to induce CTLL-2 prolifer-
ation on a per pmol basis. (C) represents the recombinant human IL-2
(rHuIL-2) and (:) represents recombinant D. novemcinctus IL-2 (rDnIL-2).
Each point represents the mean  SD of triplicate wells. (b) Relative efficacy
of recombinant IL-2’s to induce armadillo blasts on a per pmol basis. (C) rep-
resents the recombinant human IL-2 (rHuIL-2) and (:) represents recombi-
nant D. novemcinctus IL-2 (rDnIL-2). Each point represents the mean  SD
of triplicate wells.
We left the his-tag intact on the N-terminus of rDnIL-2 and
this should not have had any significant untoward effect. It has
been found that the presence of the six histidine residues from
the his-tag of glutathione S-transferase fusion proteins on the
amino terminus of the ovine IL-2 fusion protein did not affect
the proliferative stimulus of recombinant IL-2 [12]. The lower
activity of rDnIL-2 when compared to that of rHuIL-2 may be
due to a portion of the rDnIL-2 having been irreversibly dena-
tured and biologically inactive after purification from inclu-
sion bodies leading to some loss of activity; however,
rDnIL-2 was clearly biologically active and sustained prolifer-
ation in CTLL-2 cells and armadillo lymphoblasts.
This is the first published report of a protein from
D. novemcinctus to be overexpressed and bioassayed. rDnIL-
2 shows properties similar to other mammalian IL-2’s. This
protein plays an essential role in the immune response and
can be used effectively in model studies of the pathogenesis
of leprosy. With the D. novemcinctus genome nearly complete,
a vast array of armadillo cell markers, receptors, and cytokines
will likely evolve in the near future and these animals will ad-
vance as important translational models for leprosy research.
 J.E. Adams et al. / Cytokine 32 (2005) 219e225
Acknowledgements
The authors would like to thank Kyle Andrews, Tana Pitt-
man, Naoko Robbins, and Heidi Zhang for their technical
assistance and express our appreciation to Dr. Jean Chang at
the Broad Institute for information regarding the Dasypus
novemcinctus sequencing project.
Financial support: This study was supported in part by the
National Hansen’s Disease Program and funds from the Na-
tional Institute for Allergy and Infectious Disease, contract
number YI-AI-2646-01.
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