Accepted Manuscript Cloning and characterization of goose interleukin-17A cDNA Shuangshi Wei, Xiaomei Liu, Mingchun Gao, Wenlong Zhang, Yunhui Zhu, Bo Ma, Junwei Wang PII: DOI: Reference:
S0034-5288(13)00325-1 http://dx.doi.org/10.1016/j.rvsc.2013.10.008 YRVSC 2555
To appear in:
Research in Veterinary Science
Received Date: Accepted Date:
6 March 2013 20 October 2013
Please cite this article as: Wei, S., Liu, X., Gao, M., Zhang, W., Zhu, Y., Ma, B., Wang, J., Cloning and characterization of goose interleukin-17A cDNA, Research in Veterinary Science (2013), doi: http://dx.doi.org/ 10.1016/j.rvsc.2013.10.008
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1
Cloning and characterization of goose interleukin-17A cDNA
2
Shuangshi Wei1, 2, Xiaomei Liu1, 2, Mingchun Gao2, Wenlong Zhang2, Yunhui Zhu3, Bo Ma2*,
3
Junwei Wang2*
4
(2 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China
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3 School of Life Science, Sun Yat-Sen University, Guangdong 510006, PR China)
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1 Shuangshi Wei, Xiaomei Liu contributes equally as first authors.
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Correspondence and phone calls about the paper should be directed to Bo Ma and Junwei Wang at
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the following address, phone number and e-mail address：
9
Bo Ma
10
College of Veterinary Medicine
11
North-east Agricultural University
12
No. 59 Mucai Street, Harbin, Heilongjiang 150030, China
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Tel：+86-0451-55191244
14
Fax：+86-0451-55191672
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E-mail：[email protected]
16 17
Junwei Wang
18
College of Veterinary Medicine
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North-east Agricultural University
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No. 59 Mucai Street, Harbin, Heilongjiang 150030, China
21
Tel：+86-0451-55191244
22
E-mail：[email protected] 1
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Abstract: Interleukin-17 (IL-17 or IL-17A) is a proinflammatory cytokine produced by activated
24
T cells. IL-17A plays important roles in inflammation and host defense. In this study, the cDNA of
25
the goose IL-17A (GoIL-17A) gene was cloned from thymocytes. Recombinant GoIL-17A
26
(rGoIL-17A) was expressed using a baculovirus expression system and then biologically
27
characterized. The complete open reading frame (ORF) of GoIL-17A contains 510 base pairs that
28
encode 169 amino acid residues, including a 29-amino acid signal peptide and a single potential
29
N-linked glycosylation site. This protein has a molecular weight of 18.9 kDa. The amino acid
30
sequence showed 95.9%, 84.6%, 45.0% and 38.4% similarity with the corresponding duck,
31
chicken, rat, and human IL-17A sequences, respectively. The six conserved cysteine residues were
32
also observed in GoIL-17A. A recombinant, mature form of GoIL-17A was produced and its
33
biological activities in goose embryonic fibroblasts were investigated. RT-PCR analysis revealed a
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marked up-regulation of IL-6 and IL-8 mRNA expression in goose embryonic fibroblasts treated
35
with 1-50 μg of rGoIL-17A for 12 h. The GoIL-17A gene sequence and the biologically active
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recombinant protein may be useful for understanding the role of IL-17A in immune regulation.
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Keywords: goose; IL-17A; characterization; biological activity
38 39
1. Introduction
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It has been almost two decades since the identification of interleukin (IL)-17 by Rouvier et al.
41
(1993). IL-17 was cloned from a murine cytotoxic T lymphocyte hybridoma cDNA library as
42
CTLA-8 (CTL antigen-8). Subsequently, CTLA-8 was confirmed to be a novel cytokine that binds
43
to a novel cytokine receptor; the cytokine and receptor are now referred to as IL-17 and IL-17R,
44
respectively (Yao et al., 1995a). 2
45
Classically, effector T helper cells have been classified as type 1 (Th1) or type 2 (Th2) based
46
on their cytokine expression profiles and immune regulatory functions. A third subset of
47
IL-17-producing effector T helper cells, Th17 cells, was discovered and characterized in 2005
48
(Harrington et al., 2005, Park et al., 2005). IL-17 has six family members (IL-17A to IL-17F).
49
Although IL-17A and IL-17F share the highest amino acid sequence homology, they perform
50
distinct functions; IL-17A is involved in the development of autoimmunity, inflammation, and
51
tumours, and also plays important roles in the host defenses against bacterial and fungal infections,
52
whereas IL-17F is mainly involved in mucosal host defense mechanisms.
53
IL-17 acts as a proinflammatory cytokine that can induce the release of certain chemokines,
54
cytokines, matrix metalloproteinases (MMPs) and antimicrobial peptides. The release of these
55
molecules leads to the expansion and accumulation of neutrophils during innate immune responses
56
and links innate and adaptive immunity in vivo. Furthermore, increasing evidence indicates that
57
the IL-17 and IL-17-producing cells are involved in the pathogenesis of various diseases, such as
58
allergies, autoimmune diseases, allograft rejection and even malignancy (Xu and Cao, 2010).
59
Moreover, it is becoming apparent that IL-17 plays protective roles against infectious diseases,
60
especially in the mucosa (Dubin and Kolls, 2008). A critical characteristic of IL-17 in mucosal
61
immunology is its ability to increase the production of granulocyte colony-stimulating factor
62
(G-CSF) and CXC chemokines, resulting in the recruitment of neutrophils and contributing to
63
bacterial and fungal clearance at mucosal sites. IL-17 also increases the expression of
64
antimicrobial peptides and enhances epithelial repair functions that are important for controlling
65
extracellular fungal pathogens. In the setting of vaccine-induced immunity, Th17 cells can induce
66
the production of ligands for CXCR3 and enhance the recruitment of interferon-γ-producing Th1 3
67
cells to control the replication of intracellular pathogens (Khader et al., 2009).
68
The IL-17 genes of other species, including chickens IL-17A (Min and Lillehoj, 2002) and
69
IL-17F (Kim et al., 2012), pigs (Katoh et al., 2004), cows (Riollet et al., 2006), ducks (Yoo et al.,
70
2009) and horses (Tompkins et al., 2010), have been cloned previously. This is the first report of
71
the cloning of the goose IL-17A gene, its expression using a baculovirus system and the
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determination of its biological activities in primary cultures of goose embryonic fibroblasts
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(GEFs).The results of our experiments allow for a better understanding of the proinflammatory
74
effect of goose IL-17A and provides the basis for further studies on its potential use as a mucosal
75
vaccine adjuvant.
76 77
2. Materials and methods
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2.1. RNA extraction and cDNA synthesis
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Total RNA was isolated from 50 mg of goose thymus tissue using the E.Z.N.A. ® HP Total
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RNA Isolation Kit (OMEGA Bio-Tek, Doraville, Georgia, USA). Its concentration and purity
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were determined using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Hudson, NH,
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USA). RNA was stored at −80◦C until required for cDNA synthesis. The cDNA was synthesized
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from the total RNA isolated from goose thymus tissue using SMART (Switching Mechanism at 5’
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End of RNA Transcript) Reverse Transcriptase (Clontech, Palo Alto, CA, USA).
85 86
2.1.1. First-strand cDNA synthesis
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One microliter of the First-dT20 primer (Table 1) was added to 3.5 μl (59 ng/μl) of total RNA.
88
The tube was then mixed, spun briefly and then placed at 72°C for 3 min, followed by incubation 4
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at 42°C for 2 min. The cDNA synthesis was performed with a prepared mix of 1 μl of
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SMARTScribe Reverse Transcriptase (Clontech, Palo Alto, CA, USA) in the presence of 2 μl of
91
5× First-Strand Buffer, 0.25 μl of DTT (100 mM), 1 μl of dNTP Mix (10 mM), 0.25 μl of RNase
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Inhibitor (TaKaRa Bio, Otsu, Japan), 3G primer (Table 1) and deionized water to a final volume of
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10 μl. This reaction mixture was incubated at 42°C for 1.5 h.
94 95
2.1.2. cDNA PCR amplification reaction
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Two microliters of cDNA from the previous reaction was amplified with 1 μl of
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PrimeSTARTM HS DNA Polymerase (TaKaRa Bio, Otsu, Japan) in the presence of 20 μl of 5×
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PrimeSTAR Buffer, 2 μl of dNTP Mix (2.5 mM), 1 μl each of the 5’ PCR primer and the 3’ PCR
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primer (Table 1) and deionized water to a final volume of 100 μl. The reaction contents were then
100
mixed. The PCR was completed using a Mastercycler ep Gradient thermocycler (Eppendorf,
101
Hamburg, Germany) with the following program: 95°C for 3 min followed by 30 cycles of 98°C
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for 30 sec, 65°C for 30 sec, and 72°C for 6 min. The dsDNA product was stored at -20°C until
103
use.
104 105
2.2 Cloning of Go-IL17A
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GoIL-17A-specific primers (Table 1) were designed based on the sequence of chicken
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IL-17A (GenBank ID: NM_204460.1) and were used to acquire the actual sequence of goose
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IL-17A. Touchdown PCR was performed as follows: an initial step at 94°C for 5 min, followed by
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30 cycles each of denaturation at 94°C for 1 min, annealing at a variable temperature (65°C to
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50°C) for 30 sec, and extension at 72°C for 1 min. For the first cycle, the annealing temperature 5
111
was set to 65°C. For each of the 29 subsequent cycles, the annealing temperature was decreased
112
by 0.5°C. These 30 cycles were followed by 10 cycles of 94°C for 1 min, 50°C for 30 sec, and
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72°C for 1 min. Amplified fragments were inserted into the pEASY-Blunt vector (TransGen
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Biotech, Beijing, China). DNA sequencing was performed using the dideoxy chain termination
115
method. Sequences were initially analyzed using a BLAST search to confirm that the correct gene
116
had been cloned. The CLUSTALW (Larkin et al., 2007) program was used to align the sequences,
117
and ESPript (Gouet et al., 1999) was used to format the multiple sequence alignments in a single
118
postscript file.
119 120
2.3 Expression of GoIL-17A in E. coli and baculovirus-infected insect cells
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To subclone the GoIL-17A cDNA without the signal peptide region, sense and antisense
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primers were designed that included BamHI and HindIII restriction sites at the 5’-ends of the
123
primers. After digestion with BamHI and HindIII, the PCR fragment was ligated into both the
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pET32a (Novagen) expression vector and the pFastBac HTB donor vector, which is part of the
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baculovirus expression system (Invitrogen).
126
The pET32a expression vector containing the GoIL-17A gene was transformed into the E.
127
coli Rosetta (DE3) pLysS strain (Promega). Transformants were selected for on LB-ampicillin
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agar plates. Log phase cultures (approximate OD600=0.6) were induced at 37°C for 4 h by adding
129
IPTG (Sigma) to a final concentration of 1 mM. The cells were harvested by centrifugation (5000
130
g for 15 min) and suspended in PBS buffer. The cells were disrupted by sonication, and the
131
insoluble material was collected by centrifugation (5000 g for 20 min).
132
The baculovirus donor vector pFastBac HTB (Invitrogen) was sequenced to confirm the 6
133
insertion of the GoIL-17A gene. The recombinant vector was then transformed into DH10BAC
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bacterial cells (Invitrogen) for recombination of the GoIL-17A cDNA with the genetically
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modified baculovirus genome (bacmid). Positive recombinant bacmids were transfected into Sf9
136
(Invitrogen) insect cells. All procedures were performed according to the manufacturer’s protocols
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(Bac-to-Bac, Invitrogen). The recombinant baculovirus was submitted to four rounds of
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amplification (72 h each) by infecting Sf9 monolayers to generate a high titer of recombinant virus.
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The virus stocks were protected from light at +4°C or -80°C. Protein expression was analyzed by
140
12% SDS-PAGE.
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2.4 Purification and renaturation
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The prokaryotic rGoIL-17A protein was dissolved in 2 ml of denaturing buffer (100 mM
144
NaH2PO4, 10 mM Tris-Cl, 8 M urea, 10 mM imidazole, pH 8.0), sonicated for 15 min in an ice
145
bath, and then centrifuged at 5000 g for 10 min. The supernatant was loaded onto a Ni-NTA
146
agarose (QIAGEN) column that had been equilibrated with denaturing buffer, and the column
147
contents were mixed gently by shaking for 60 min at room temperature. The column was washed
148
twice with 4 ml of wash buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, 20 mM imidazole
149
pH 8.0), and the protein was eluted using a 4 ml gradient of 50-250 mM imidazole in the same
150
buffer. The collected fractions were analyzed by SDS-PAGE. The concentration of prokaryotic
151
rGoIL-17A was measured using a BCA Protein Assay Kit (Beyotime, Jiangsu, China).
152
Recombinant baculovirus-expressed GoIL-17A was purified as prokaryotic rGoIL-17A.
153
Renaturation of the denatured eukaryotic rGoIL-17A was performed using urea gradient
154
size-exclusion chromatography according to Gu et al., (2001) with some modifications. A 7
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pre-packed Superdex G25 column connected to an N3000 chromatographic workstation (Zhejiang
156
University, Zhejiang, China) was used for the chromatography process. Refolding buffer
157
contained PBS (pH 6.8), 1 mM EDTA, 3 mM GSH, and 0.3 mM GSSG/. Equilibration buffer was
158
refolding buffer to which 8 mol/L urea had been added. Packed Superdex 25 columns were first
159
equilibrated with mixed buffers consisting of refolding buffer and equilibration buffer in various
160
ratios. The columns were then treated with gradients of various concentrations up to a final urea
161
concentration of 8 mol/L (100% equilibration buffer). After the equilibration, 2 mg of the
162
denatured rGoIL-17A was applied to the column and eluted with PBS.
163 164
2.5 Western blot analysis
165
Individual New Zealand rabbits were immunized with 1 mg of prokaryotic rGoIL-17A
166
protein mixed with an equal volume of complete Freund's adjuvant (Sigma) and were boosted
167
with the same amount of prokaryotic rGoIL-17A in 50% incomplete Freund’s adjuvant (IFA)
168
every two weeks for six weeks. Blood samples were collected one week after the last
169
immunization, and the sera were prepared by centrifugation. The titres of the antibodies against
170
the purified His-tagged rGoIL-17A were determined by ELISA.
171
Renaturation of the denatured eukaryotic rGoIL-17A was examined using non-reducing
172
SDS-PAGE followed by western blotting. The denatured and the renatured eukaryotic expressed
173
protein samples were mixed with equal volumes of sample buffer (0.125 M Tris–HCl, pH 6.8, 4%
174
SDS, 20% glycerol, and 0.004% bromophenol blue), resolved on 10-12% SDS-polyacrylamide
175
gels and transferred to nitrocellulose. The membranes were blocked in PBST containing 5%
176
nonfat dry milk for 16 h at 4°C. The membranes were then shaken for 2 h at room temperature 8
177
with rabbit polyclonal antibodies against GoIL-17A (1:500), followed by three washes with PBST.
178
The bound antibody was allowed to react with HRP-conjugated goat anti-rabbit IgG (G+L)
179
(1:5000, ZSGB-BIO, Beijing, China) in PBST for 40 min at room temperature. The membrane
180
was then washed three times with PBST and one time with PBS. Visualization was performed
181
with the AEC Staining Kit (Sigma).
182 183
2.6 Biological effect of eukaryotic rGoIL-17A on GEFs
184
The biological activity of eukaryotic rGoIL-17A was assessed by RT-PCR. RT-PCR was
185
performed to detect the mRNA levels of cytokines IL-6 and IL-8 (Wang et al., 2012, Wu et al.,
186
2008) in GEFs after exposure to eukaryotic rGoIL-17A. GEFs were prepared from 10-day-old
187
goose embryos (Brown et al., 1995). Cells (1×107) were stimulated with 1-50 μg of eukaryotic
188
rGoIL-17A for 12 h in DMEM (Gibco). PBS was used as a control. After extraction of the total
189
RNA, reverse transcription was performed with an oligo d(T)20 primer. The primers used in the
190
PCR are listed in Table 2. The PCR conditions were as follows: one cycle for 3 min at 94°C and
191
25-30 cycles of 30 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C. The PCR products were
192
analyzed by 1% agarose-gel electrophoresis followed by ethidium bromide staining, and the gels
193
were photographed with an AlphaImager 2200 (Alpha Innotech Corporation).
194 195
3. Results
196
3.1 Molecular cloning and structure of the GoIL-17A cDNA
197
A DNA fragment of the expected size (574 bp) was obtained by PCR amplification of the
198
SMART ds cDNA using the designed primers, and a BLAST search revealed that the sequence of 9
199
this DNA fragment was similar to the sequences of known IL-17A genes (GenBank accession No.
200
JN887437). The ORF of this cDNA encoded a putative protein of 169 amino acids, including a
201
29-amino acid signal peptide and a single potential N-linked glycosylation site (Fig. 1). This
202
protein has a predicted molecular mass of 18.9 kDa (non-glycosylated) and a calculated isoelectric
203
point of 9.11. Subsequent analysis of the translated cDNA sequence indicated that GoIL-17A
204
shares 95.9%, 84.6%, 45.0% and 38.4% identity with the duck, chicken, rat and human IL-17A,
205
respectively. The six cysteine residues conserved in bird and mammalian IL-17s were observed in
206
GoIL-17A at positions 33, 94, 99, 129, 144 and 146 (stars, Fig. 1). These cysteine residues form
207
an unusual pattern of intrachain disulfide bonds, demonstrating that IL-17A is a structural
208
homolog of members of the cysteine knot family (Weaver et al., 2007).
209 210
3.2 Expression and purification of mature rGoIL-17A
211
rGoIL-17A was produced both in E. coli and baculovirus-infected insect cells. Purification
212
was performed using affinity chromatography under denaturing conditions because the proteins
213
were expressed in insoluble form. The purified prokaryotic protein was used to produce polyclonal
214
antibodies in rabbits. The polyclonal antibodies were tested by Western blotting and were found to
215
detect the monomeric and dimeric forms of IL-17A when the antibodies were diluted 1:500. These
216
antibodies were used to detect the expression of recombinant goose IL-17A by Western blot
217
analysis.
218
The refolded eukaryotic protein was considered to be a homo-dimeric protein because under
219
non-reducing conditions, a band at 40 kDa reacted with the polyclonal antibodies, and under
220
reducing conditions, the 40 kDa band disappeared, and a 20 kDa band was observed (Fig. 2). The 10
221
biological activity of the secreted protein was then tested.
222
3.3 Biological activity of eukaryotic rGoIL-17A
223
To evaluate the biological activity of eukaryotic rGoIL-17A, the induction of cytokine
224
production in primary cultures of goose embryonic fibroblasts by eukaryotic rGoIL-17F was
225
investigated. As shown in Fig. 3, refolded eukaryotic rGoIL-17A induced higher levels of IL-6 and
226
IL-8 mRNA expression than did the control. These results indicate that the eukaryotic rGoIL-17A
227
produced in this study was biologically active.
228 229
4. Discussion
230
In this study, the SMART reverse transcriptase approach was used to clone and characterize
231
the cDNA encoding IL-17A in the goose thymus. The GoIL-17A sequence is predicted to encode a
232
protein which has 95.9%, 84.6%, 45.0% and 38.4% sequence homology with the duck, chicken,
233
rat and human protein sequences, respectively. The levels of sequence homology between the
234
goose IL-17A and the duck, chicken, and human IL-17s are similar to those observed for other
235
goose cytokines and their homologs in these species (Zhou et al., 2005, Li et al., 2006).
236
E. coli-expressed rGoIL-17 does not have biological activity. However, the monomeric
237
protein was used to produce rabbit polyclonal antibodies that recognize both the monomeric and
238
dimeric forms of the protein in Western blot analyses. An active protein was produced by Sf9 cells
239
infected with recombinant baculovirus, and this protein stimulated cytokine synthesis in GEFs. In
240
previous studies, it has been shown that IL-17A stimulates the production of IL-6 and IL-8 by
241
fibroblasts (Yao et al., 1995b, Kehlen et al., 2003). In our IL-17A-mediated proinflammatory
242
model, the levels of the IL-6 and IL-8 mRNAs were up-regulated in GEFs; in contrast these 11
243
mRNAs were undetectable in the control group (Fig. 3).
244
IL-17 has been shown to play important roles in inflammation and host defense (Curtis and
245
Way, 2009). The role of IL-17A in host defense, specifically in protection against bacterial, fungal
246
and viral infections, especially at mucosal sites, was investigated. The stimulation of lung and gut
247
epithelial cells with IL-17 has been shown to induce the expression of CXCL-1, CXCL-5,
248
CXCL-2 and CCL20, which are neutrophil chemoattractants (Awane et al., 1999, Kao et al., 2005)
249
and induce the migration of neutrophils into the mucosa. The treatment of bronchial epithelial
250
cells with IL-17 stimulates the production of CXC chemokines such as IL-8 and G-CSF and the
251
expression of antimicrobial proteins such as human β-defensin 2 (Kao et al., 2004). IL-17
252
treatment also stimulates the production of IL-19 (Huang et al., 2008), which may have an
253
important role in regulating Th2 responses in the mucosa. Additionally, IL-17 induces the
254
expression of the polymeric immunoglobulin receptor and Th17 cytokines that have been shown
255
to be critical for generating mucosal IgA responses (Jaffar et al., 2009).
256
Further studies will be valuable to elucidate the role of IL-17A in mucosal immune system.
257
Additionally, it is necessary to determine if this protective effect can be exploited in the
258
development of T-cell lineage-specific adjuvants.
259 260 261
Acknowledgments
262
This research was financially supported by a major technology project from the Education
263
Department of Heilongjiang Province (10541Z004) and by the Scientific and Technological
264
Project of Heilongjiang Province (GB01B503-02 and GB04B504).
265
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the expanding diversity of effector T cell lineages. Annual Review of Immunology 25,
326
821-852.
327
Wu, Y. F., Shien, J. H., Yin, H. H., Chiow, S. H. Lee, L. H., 2008. Structural and functional
328
homology among chicken, duck, goose, turkey and pigeon interleukin-8 proteins. Veterinary
329
immunology and immunopathology. 125, 205-215.
330 331
Xu, S. Cao, X. T., 2010. Interleukin-17 and its expanding biological functions. Cellular and Molecular Immunology 7, 164-174. 15
332
Yao, Z. B., Fanslow, W. C., Seldin, M. F., Rousseau, A. M., Painter, S. L., Comeau, M. R., Cohen,
333
J. I. Spriggs, M. K., 1995a. Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds
334
to a novel cytokine receptor. Immunity 3, 811-821.
335
Yao, Z., Painter, S. L., Fanslow, W. C., Ulrich, D., Macduff, B. M., Spriggs, M. K. Armitage, R. J.,
336
1995b. Human IL-17: a novel cytokine derived from T cells. Journal of Immunology 155,
337
5483-5486.
338
Yoo, J., Jang, S. I., Kim, S., Cho, J. H., Lee, H. J., Rhee, M. H., Lillehoj, H. S. Min, W., 2009.
339
Molecular
340
Immunopathology 132, 318-322.
341 342 343 344
characterization
of
duck
interleukin-17.
Veterinary
Immunology
and
Gu, Z. Y., Su, Z. G., Janson , J. C., 2001. Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding. Journal of Chromatography A 918, 311-318. Zhou, J. Y., Chen, J. G., Wang, J. Y., Wu, J. X. Gong, H., 2005. cDNA cloning and functional analysis of goose interleukin-2. Cytokine 30, 328-338.
345 346 347 348 349 350 351 352 353 16
354
Figure legends
355 356
Figure 1. Alignment of goose IL-17A with avian and mammalian IL-17A proteins. The sequences
357
were aligned using the CLUSTAL W (1.81) program (www.ebi.ac.uk/clustalw/). Identical residues
358
in the different sequences are highlighted by black boxes. The six conserved cysteine residues are
359
marked by stars and one N-glycosylation site is marked by triangles Sequences were retrieved
360
from public databases — — monkey: XP_001106391.1; human: NP_002181.1; manatee:
361
XP_004388776.1; rabbit: XP_002714544.1; bat: EPQ19560.1; fox: ELK00212.1; ferret:
362
XP_004771940.1; dog: NP_001159350.1; walrus: XP_004415698.1; horse: NP_001137264.1;
363
goat: ADB25062.1; sheep: XP_004018936.1; pig: NP_001005729.1; mouse: NP_034682.1; rat:
364
NP_001100367.1; duck: XP_005014110.1; chicken: CAD38489.1；
365 366
Figure 2. Western blot analysis of eukaryotic rGoIL-17A. Eukaryotic rGoIL-17A proteins were
367
run on a 12% gel under non-reducing conditions. Immunodetection of the Western blot was with a
368
rabbit polyclonal antibody against rGoIL-17A (1:500) and then revealed with HRP-conjugated
369
goat anti-rabbit IgG. Lane 1: denatured eukaryotic rGoIL-17A; lane 2: refolded eukaryotic
370
rGoIL-17A.
371 372 373
Figure 3. RT-PCR analysis of GEFs stimulated with 1-50 μg of rGoIL-17 or the control (PBS).
374
The RT-PCR products were resolved on an agarose gel. Ne: negative control. PBS: GEFs treated
375
with PBS. 17
Table
Table 1 Oligonucleotide primers used to amplify cDNAs for Goose IL-17 Gene name cDNA
ds cDNA
Goose IL-17
Ex IL-17
◦
Primer sequence
Ann T ( C)
First-dT20
TCTAGAGTCGACCTGCACATTTTTTTTTTTTTTTTTTTTGC
65
3G primer
GAGCTCGAATTCACTTAGTATAGCGCGCGGG
65
5’PCR primer
TCTAGAGTCGACCTGCACAT
52.5
3’PCR primer
CTCGAATTCACTTAGTATAGCG
52.5
IL-17 S
GGGTCGCCCAGCACAAGCA
62.2
IL-17 A
ACTCCTGTGCTGTGGGCTCCCT
61.9
mIL-17 S
CGCGGATCCATGAAGGTGATACGGCCC
65
mIL-17 A
CCCAAGCTTTTAAGCCTGGTGCTGGATCAA
65
Primer name
Table 2 Oligonucleotide primers used to assess biologic activity of IL-17 Gene name IL-6
IL-8 β-actin
Primer name
Primer sequence
IL-6 S
GCGGTCTCCGACTCCTCC
IL-6 A
ATAGCGAACAGCCCTCACG
IL-8 S
GCTGTCCTGGCTCTTCTCCT
IL-8 A
GCACACCTCTCTGTTGTCCTTC
β-actin S
CCACACCGTGCCCATCTAT
β-actin A
GGTCGTATTCCTGCTTGCTG
Goose IL-6: JF437643.1; Goose IL-8: DQ393274; Goose β-actin: M26111
Size (bp) 546 bp
210 bp
610 bp
Figure 1
Figure 2
Figure 3
S0034-5288(13)00325-1 http://dx.doi.org/10.1016/j.rvsc.2013.10.008 YRVSC 2555
To appear in:
Research in Veterinary Science
Received Date: Accepted Date:
6 March 2013 20 October 2013
Please cite this article as: Wei, S., Liu, X., Gao, M., Zhang, W., Zhu, Y., Ma, B., Wang, J., Cloning and characterization of goose interleukin-17A cDNA, Research in Veterinary Science (2013), doi: http://dx.doi.org/ 10.1016/j.rvsc.2013.10.008
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1
Cloning and characterization of goose interleukin-17A cDNA
2
Shuangshi Wei1, 2, Xiaomei Liu1, 2, Mingchun Gao2, Wenlong Zhang2, Yunhui Zhu3, Bo Ma2*,
3
Junwei Wang2*
4
(2 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China
5
3 School of Life Science, Sun Yat-Sen University, Guangdong 510006, PR China)
6
1 Shuangshi Wei, Xiaomei Liu contributes equally as first authors.
7
Correspondence and phone calls about the paper should be directed to Bo Ma and Junwei Wang at
8
the following address, phone number and e-mail address：
9
Bo Ma
10
College of Veterinary Medicine
11
North-east Agricultural University
12
No. 59 Mucai Street, Harbin, Heilongjiang 150030, China
13
Tel：+86-0451-55191244
14
Fax：+86-0451-55191672
15
E-mail：[email protected]
16 17
Junwei Wang
18
College of Veterinary Medicine
19
North-east Agricultural University
20
No. 59 Mucai Street, Harbin, Heilongjiang 150030, China
21
Tel：+86-0451-55191244
22
E-mail：[email protected] 1
23
Abstract: Interleukin-17 (IL-17 or IL-17A) is a proinflammatory cytokine produced by activated
24
T cells. IL-17A plays important roles in inflammation and host defense. In this study, the cDNA of
25
the goose IL-17A (GoIL-17A) gene was cloned from thymocytes. Recombinant GoIL-17A
26
(rGoIL-17A) was expressed using a baculovirus expression system and then biologically
27
characterized. The complete open reading frame (ORF) of GoIL-17A contains 510 base pairs that
28
encode 169 amino acid residues, including a 29-amino acid signal peptide and a single potential
29
N-linked glycosylation site. This protein has a molecular weight of 18.9 kDa. The amino acid
30
sequence showed 95.9%, 84.6%, 45.0% and 38.4% similarity with the corresponding duck,
31
chicken, rat, and human IL-17A sequences, respectively. The six conserved cysteine residues were
32
also observed in GoIL-17A. A recombinant, mature form of GoIL-17A was produced and its
33
biological activities in goose embryonic fibroblasts were investigated. RT-PCR analysis revealed a
34
marked up-regulation of IL-6 and IL-8 mRNA expression in goose embryonic fibroblasts treated
35
with 1-50 μg of rGoIL-17A for 12 h. The GoIL-17A gene sequence and the biologically active
36
recombinant protein may be useful for understanding the role of IL-17A in immune regulation.
37
Keywords: goose; IL-17A; characterization; biological activity
38 39
1. Introduction
40
It has been almost two decades since the identification of interleukin (IL)-17 by Rouvier et al.
41
(1993). IL-17 was cloned from a murine cytotoxic T lymphocyte hybridoma cDNA library as
42
CTLA-8 (CTL antigen-8). Subsequently, CTLA-8 was confirmed to be a novel cytokine that binds
43
to a novel cytokine receptor; the cytokine and receptor are now referred to as IL-17 and IL-17R,
44
respectively (Yao et al., 1995a). 2
45
Classically, effector T helper cells have been classified as type 1 (Th1) or type 2 (Th2) based
46
on their cytokine expression profiles and immune regulatory functions. A third subset of
47
IL-17-producing effector T helper cells, Th17 cells, was discovered and characterized in 2005
48
(Harrington et al., 2005, Park et al., 2005). IL-17 has six family members (IL-17A to IL-17F).
49
Although IL-17A and IL-17F share the highest amino acid sequence homology, they perform
50
distinct functions; IL-17A is involved in the development of autoimmunity, inflammation, and
51
tumours, and also plays important roles in the host defenses against bacterial and fungal infections,
52
whereas IL-17F is mainly involved in mucosal host defense mechanisms.
53
IL-17 acts as a proinflammatory cytokine that can induce the release of certain chemokines,
54
cytokines, matrix metalloproteinases (MMPs) and antimicrobial peptides. The release of these
55
molecules leads to the expansion and accumulation of neutrophils during innate immune responses
56
and links innate and adaptive immunity in vivo. Furthermore, increasing evidence indicates that
57
the IL-17 and IL-17-producing cells are involved in the pathogenesis of various diseases, such as
58
allergies, autoimmune diseases, allograft rejection and even malignancy (Xu and Cao, 2010).
59
Moreover, it is becoming apparent that IL-17 plays protective roles against infectious diseases,
60
especially in the mucosa (Dubin and Kolls, 2008). A critical characteristic of IL-17 in mucosal
61
immunology is its ability to increase the production of granulocyte colony-stimulating factor
62
(G-CSF) and CXC chemokines, resulting in the recruitment of neutrophils and contributing to
63
bacterial and fungal clearance at mucosal sites. IL-17 also increases the expression of
64
antimicrobial peptides and enhances epithelial repair functions that are important for controlling
65
extracellular fungal pathogens. In the setting of vaccine-induced immunity, Th17 cells can induce
66
the production of ligands for CXCR3 and enhance the recruitment of interferon-γ-producing Th1 3
67
cells to control the replication of intracellular pathogens (Khader et al., 2009).
68
The IL-17 genes of other species, including chickens IL-17A (Min and Lillehoj, 2002) and
69
IL-17F (Kim et al., 2012), pigs (Katoh et al., 2004), cows (Riollet et al., 2006), ducks (Yoo et al.,
70
2009) and horses (Tompkins et al., 2010), have been cloned previously. This is the first report of
71
the cloning of the goose IL-17A gene, its expression using a baculovirus system and the
72
determination of its biological activities in primary cultures of goose embryonic fibroblasts
73
(GEFs).The results of our experiments allow for a better understanding of the proinflammatory
74
effect of goose IL-17A and provides the basis for further studies on its potential use as a mucosal
75
vaccine adjuvant.
76 77
2. Materials and methods
78
2.1. RNA extraction and cDNA synthesis
79
Total RNA was isolated from 50 mg of goose thymus tissue using the E.Z.N.A. ® HP Total
80
RNA Isolation Kit (OMEGA Bio-Tek, Doraville, Georgia, USA). Its concentration and purity
81
were determined using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Hudson, NH,
82
USA). RNA was stored at −80◦C until required for cDNA synthesis. The cDNA was synthesized
83
from the total RNA isolated from goose thymus tissue using SMART (Switching Mechanism at 5’
84
End of RNA Transcript) Reverse Transcriptase (Clontech, Palo Alto, CA, USA).
85 86
2.1.1. First-strand cDNA synthesis
87
One microliter of the First-dT20 primer (Table 1) was added to 3.5 μl (59 ng/μl) of total RNA.
88
The tube was then mixed, spun briefly and then placed at 72°C for 3 min, followed by incubation 4
89
at 42°C for 2 min. The cDNA synthesis was performed with a prepared mix of 1 μl of
90
SMARTScribe Reverse Transcriptase (Clontech, Palo Alto, CA, USA) in the presence of 2 μl of
91
5× First-Strand Buffer, 0.25 μl of DTT (100 mM), 1 μl of dNTP Mix (10 mM), 0.25 μl of RNase
92
Inhibitor (TaKaRa Bio, Otsu, Japan), 3G primer (Table 1) and deionized water to a final volume of
93
10 μl. This reaction mixture was incubated at 42°C for 1.5 h.
94 95
2.1.2. cDNA PCR amplification reaction
96
Two microliters of cDNA from the previous reaction was amplified with 1 μl of
97
PrimeSTARTM HS DNA Polymerase (TaKaRa Bio, Otsu, Japan) in the presence of 20 μl of 5×
98
PrimeSTAR Buffer, 2 μl of dNTP Mix (2.5 mM), 1 μl each of the 5’ PCR primer and the 3’ PCR
99
primer (Table 1) and deionized water to a final volume of 100 μl. The reaction contents were then
100
mixed. The PCR was completed using a Mastercycler ep Gradient thermocycler (Eppendorf,
101
Hamburg, Germany) with the following program: 95°C for 3 min followed by 30 cycles of 98°C
102
for 30 sec, 65°C for 30 sec, and 72°C for 6 min. The dsDNA product was stored at -20°C until
103
use.
104 105
2.2 Cloning of Go-IL17A
106
GoIL-17A-specific primers (Table 1) were designed based on the sequence of chicken
107
IL-17A (GenBank ID: NM_204460.1) and were used to acquire the actual sequence of goose
108
IL-17A. Touchdown PCR was performed as follows: an initial step at 94°C for 5 min, followed by
109
30 cycles each of denaturation at 94°C for 1 min, annealing at a variable temperature (65°C to
110
50°C) for 30 sec, and extension at 72°C for 1 min. For the first cycle, the annealing temperature 5
111
was set to 65°C. For each of the 29 subsequent cycles, the annealing temperature was decreased
112
by 0.5°C. These 30 cycles were followed by 10 cycles of 94°C for 1 min, 50°C for 30 sec, and
113
72°C for 1 min. Amplified fragments were inserted into the pEASY-Blunt vector (TransGen
114
Biotech, Beijing, China). DNA sequencing was performed using the dideoxy chain termination
115
method. Sequences were initially analyzed using a BLAST search to confirm that the correct gene
116
had been cloned. The CLUSTALW (Larkin et al., 2007) program was used to align the sequences,
117
and ESPript (Gouet et al., 1999) was used to format the multiple sequence alignments in a single
118
postscript file.
119 120
2.3 Expression of GoIL-17A in E. coli and baculovirus-infected insect cells
121
To subclone the GoIL-17A cDNA without the signal peptide region, sense and antisense
122
primers were designed that included BamHI and HindIII restriction sites at the 5’-ends of the
123
primers. After digestion with BamHI and HindIII, the PCR fragment was ligated into both the
124
pET32a (Novagen) expression vector and the pFastBac HTB donor vector, which is part of the
125
baculovirus expression system (Invitrogen).
126
The pET32a expression vector containing the GoIL-17A gene was transformed into the E.
127
coli Rosetta (DE3) pLysS strain (Promega). Transformants were selected for on LB-ampicillin
128
agar plates. Log phase cultures (approximate OD600=0.6) were induced at 37°C for 4 h by adding
129
IPTG (Sigma) to a final concentration of 1 mM. The cells were harvested by centrifugation (5000
130
g for 15 min) and suspended in PBS buffer. The cells were disrupted by sonication, and the
131
insoluble material was collected by centrifugation (5000 g for 20 min).
132
The baculovirus donor vector pFastBac HTB (Invitrogen) was sequenced to confirm the 6
133
insertion of the GoIL-17A gene. The recombinant vector was then transformed into DH10BAC
134
bacterial cells (Invitrogen) for recombination of the GoIL-17A cDNA with the genetically
135
modified baculovirus genome (bacmid). Positive recombinant bacmids were transfected into Sf9
136
(Invitrogen) insect cells. All procedures were performed according to the manufacturer’s protocols
137
(Bac-to-Bac, Invitrogen). The recombinant baculovirus was submitted to four rounds of
138
amplification (72 h each) by infecting Sf9 monolayers to generate a high titer of recombinant virus.
139
The virus stocks were protected from light at +4°C or -80°C. Protein expression was analyzed by
140
12% SDS-PAGE.
141 142
2.4 Purification and renaturation
143
The prokaryotic rGoIL-17A protein was dissolved in 2 ml of denaturing buffer (100 mM
144
NaH2PO4, 10 mM Tris-Cl, 8 M urea, 10 mM imidazole, pH 8.0), sonicated for 15 min in an ice
145
bath, and then centrifuged at 5000 g for 10 min. The supernatant was loaded onto a Ni-NTA
146
agarose (QIAGEN) column that had been equilibrated with denaturing buffer, and the column
147
contents were mixed gently by shaking for 60 min at room temperature. The column was washed
148
twice with 4 ml of wash buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, 20 mM imidazole
149
pH 8.0), and the protein was eluted using a 4 ml gradient of 50-250 mM imidazole in the same
150
buffer. The collected fractions were analyzed by SDS-PAGE. The concentration of prokaryotic
151
rGoIL-17A was measured using a BCA Protein Assay Kit (Beyotime, Jiangsu, China).
152
Recombinant baculovirus-expressed GoIL-17A was purified as prokaryotic rGoIL-17A.
153
Renaturation of the denatured eukaryotic rGoIL-17A was performed using urea gradient
154
size-exclusion chromatography according to Gu et al., (2001) with some modifications. A 7
155
pre-packed Superdex G25 column connected to an N3000 chromatographic workstation (Zhejiang
156
University, Zhejiang, China) was used for the chromatography process. Refolding buffer
157
contained PBS (pH 6.8), 1 mM EDTA, 3 mM GSH, and 0.3 mM GSSG/. Equilibration buffer was
158
refolding buffer to which 8 mol/L urea had been added. Packed Superdex 25 columns were first
159
equilibrated with mixed buffers consisting of refolding buffer and equilibration buffer in various
160
ratios. The columns were then treated with gradients of various concentrations up to a final urea
161
concentration of 8 mol/L (100% equilibration buffer). After the equilibration, 2 mg of the
162
denatured rGoIL-17A was applied to the column and eluted with PBS.
163 164
2.5 Western blot analysis
165
Individual New Zealand rabbits were immunized with 1 mg of prokaryotic rGoIL-17A
166
protein mixed with an equal volume of complete Freund's adjuvant (Sigma) and were boosted
167
with the same amount of prokaryotic rGoIL-17A in 50% incomplete Freund’s adjuvant (IFA)
168
every two weeks for six weeks. Blood samples were collected one week after the last
169
immunization, and the sera were prepared by centrifugation. The titres of the antibodies against
170
the purified His-tagged rGoIL-17A were determined by ELISA.
171
Renaturation of the denatured eukaryotic rGoIL-17A was examined using non-reducing
172
SDS-PAGE followed by western blotting. The denatured and the renatured eukaryotic expressed
173
protein samples were mixed with equal volumes of sample buffer (0.125 M Tris–HCl, pH 6.8, 4%
174
SDS, 20% glycerol, and 0.004% bromophenol blue), resolved on 10-12% SDS-polyacrylamide
175
gels and transferred to nitrocellulose. The membranes were blocked in PBST containing 5%
176
nonfat dry milk for 16 h at 4°C. The membranes were then shaken for 2 h at room temperature 8
177
with rabbit polyclonal antibodies against GoIL-17A (1:500), followed by three washes with PBST.
178
The bound antibody was allowed to react with HRP-conjugated goat anti-rabbit IgG (G+L)
179
(1:5000, ZSGB-BIO, Beijing, China) in PBST for 40 min at room temperature. The membrane
180
was then washed three times with PBST and one time with PBS. Visualization was performed
181
with the AEC Staining Kit (Sigma).
182 183
2.6 Biological effect of eukaryotic rGoIL-17A on GEFs
184
The biological activity of eukaryotic rGoIL-17A was assessed by RT-PCR. RT-PCR was
185
performed to detect the mRNA levels of cytokines IL-6 and IL-8 (Wang et al., 2012, Wu et al.,
186
2008) in GEFs after exposure to eukaryotic rGoIL-17A. GEFs were prepared from 10-day-old
187
goose embryos (Brown et al., 1995). Cells (1×107) were stimulated with 1-50 μg of eukaryotic
188
rGoIL-17A for 12 h in DMEM (Gibco). PBS was used as a control. After extraction of the total
189
RNA, reverse transcription was performed with an oligo d(T)20 primer. The primers used in the
190
PCR are listed in Table 2. The PCR conditions were as follows: one cycle for 3 min at 94°C and
191
25-30 cycles of 30 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C. The PCR products were
192
analyzed by 1% agarose-gel electrophoresis followed by ethidium bromide staining, and the gels
193
were photographed with an AlphaImager 2200 (Alpha Innotech Corporation).
194 195
3. Results
196
3.1 Molecular cloning and structure of the GoIL-17A cDNA
197
A DNA fragment of the expected size (574 bp) was obtained by PCR amplification of the
198
SMART ds cDNA using the designed primers, and a BLAST search revealed that the sequence of 9
199
this DNA fragment was similar to the sequences of known IL-17A genes (GenBank accession No.
200
JN887437). The ORF of this cDNA encoded a putative protein of 169 amino acids, including a
201
29-amino acid signal peptide and a single potential N-linked glycosylation site (Fig. 1). This
202
protein has a predicted molecular mass of 18.9 kDa (non-glycosylated) and a calculated isoelectric
203
point of 9.11. Subsequent analysis of the translated cDNA sequence indicated that GoIL-17A
204
shares 95.9%, 84.6%, 45.0% and 38.4% identity with the duck, chicken, rat and human IL-17A,
205
respectively. The six cysteine residues conserved in bird and mammalian IL-17s were observed in
206
GoIL-17A at positions 33, 94, 99, 129, 144 and 146 (stars, Fig. 1). These cysteine residues form
207
an unusual pattern of intrachain disulfide bonds, demonstrating that IL-17A is a structural
208
homolog of members of the cysteine knot family (Weaver et al., 2007).
209 210
3.2 Expression and purification of mature rGoIL-17A
211
rGoIL-17A was produced both in E. coli and baculovirus-infected insect cells. Purification
212
was performed using affinity chromatography under denaturing conditions because the proteins
213
were expressed in insoluble form. The purified prokaryotic protein was used to produce polyclonal
214
antibodies in rabbits. The polyclonal antibodies were tested by Western blotting and were found to
215
detect the monomeric and dimeric forms of IL-17A when the antibodies were diluted 1:500. These
216
antibodies were used to detect the expression of recombinant goose IL-17A by Western blot
217
analysis.
218
The refolded eukaryotic protein was considered to be a homo-dimeric protein because under
219
non-reducing conditions, a band at 40 kDa reacted with the polyclonal antibodies, and under
220
reducing conditions, the 40 kDa band disappeared, and a 20 kDa band was observed (Fig. 2). The 10
221
biological activity of the secreted protein was then tested.
222
3.3 Biological activity of eukaryotic rGoIL-17A
223
To evaluate the biological activity of eukaryotic rGoIL-17A, the induction of cytokine
224
production in primary cultures of goose embryonic fibroblasts by eukaryotic rGoIL-17F was
225
investigated. As shown in Fig. 3, refolded eukaryotic rGoIL-17A induced higher levels of IL-6 and
226
IL-8 mRNA expression than did the control. These results indicate that the eukaryotic rGoIL-17A
227
produced in this study was biologically active.
228 229
4. Discussion
230
In this study, the SMART reverse transcriptase approach was used to clone and characterize
231
the cDNA encoding IL-17A in the goose thymus. The GoIL-17A sequence is predicted to encode a
232
protein which has 95.9%, 84.6%, 45.0% and 38.4% sequence homology with the duck, chicken,
233
rat and human protein sequences, respectively. The levels of sequence homology between the
234
goose IL-17A and the duck, chicken, and human IL-17s are similar to those observed for other
235
goose cytokines and their homologs in these species (Zhou et al., 2005, Li et al., 2006).
236
E. coli-expressed rGoIL-17 does not have biological activity. However, the monomeric
237
protein was used to produce rabbit polyclonal antibodies that recognize both the monomeric and
238
dimeric forms of the protein in Western blot analyses. An active protein was produced by Sf9 cells
239
infected with recombinant baculovirus, and this protein stimulated cytokine synthesis in GEFs. In
240
previous studies, it has been shown that IL-17A stimulates the production of IL-6 and IL-8 by
241
fibroblasts (Yao et al., 1995b, Kehlen et al., 2003). In our IL-17A-mediated proinflammatory
242
model, the levels of the IL-6 and IL-8 mRNAs were up-regulated in GEFs; in contrast these 11
243
mRNAs were undetectable in the control group (Fig. 3).
244
IL-17 has been shown to play important roles in inflammation and host defense (Curtis and
245
Way, 2009). The role of IL-17A in host defense, specifically in protection against bacterial, fungal
246
and viral infections, especially at mucosal sites, was investigated. The stimulation of lung and gut
247
epithelial cells with IL-17 has been shown to induce the expression of CXCL-1, CXCL-5,
248
CXCL-2 and CCL20, which are neutrophil chemoattractants (Awane et al., 1999, Kao et al., 2005)
249
and induce the migration of neutrophils into the mucosa. The treatment of bronchial epithelial
250
cells with IL-17 stimulates the production of CXC chemokines such as IL-8 and G-CSF and the
251
expression of antimicrobial proteins such as human β-defensin 2 (Kao et al., 2004). IL-17
252
treatment also stimulates the production of IL-19 (Huang et al., 2008), which may have an
253
important role in regulating Th2 responses in the mucosa. Additionally, IL-17 induces the
254
expression of the polymeric immunoglobulin receptor and Th17 cytokines that have been shown
255
to be critical for generating mucosal IgA responses (Jaffar et al., 2009).
256
Further studies will be valuable to elucidate the role of IL-17A in mucosal immune system.
257
Additionally, it is necessary to determine if this protective effect can be exploited in the
258
development of T-cell lineage-specific adjuvants.
259 260 261
Acknowledgments
262
This research was financially supported by a major technology project from the Education
263
Department of Heilongjiang Province (10541Z004) and by the Scientific and Technological
264
Project of Heilongjiang Province (GB01B503-02 and GB04B504).
265
12
266
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Figure legends
355 356
Figure 1. Alignment of goose IL-17A with avian and mammalian IL-17A proteins. The sequences
357
were aligned using the CLUSTAL W (1.81) program (www.ebi.ac.uk/clustalw/). Identical residues
358
in the different sequences are highlighted by black boxes. The six conserved cysteine residues are
359
marked by stars and one N-glycosylation site is marked by triangles Sequences were retrieved
360
from public databases — — monkey: XP_001106391.1; human: NP_002181.1; manatee:
361
XP_004388776.1; rabbit: XP_002714544.1; bat: EPQ19560.1; fox: ELK00212.1; ferret:
362
XP_004771940.1; dog: NP_001159350.1; walrus: XP_004415698.1; horse: NP_001137264.1;
363
goat: ADB25062.1; sheep: XP_004018936.1; pig: NP_001005729.1; mouse: NP_034682.1; rat:
364
NP_001100367.1; duck: XP_005014110.1; chicken: CAD38489.1；
365 366
Figure 2. Western blot analysis of eukaryotic rGoIL-17A. Eukaryotic rGoIL-17A proteins were
367
run on a 12% gel under non-reducing conditions. Immunodetection of the Western blot was with a
368
rabbit polyclonal antibody against rGoIL-17A (1:500) and then revealed with HRP-conjugated
369
goat anti-rabbit IgG. Lane 1: denatured eukaryotic rGoIL-17A; lane 2: refolded eukaryotic
370
rGoIL-17A.
371 372 373
Figure 3. RT-PCR analysis of GEFs stimulated with 1-50 μg of rGoIL-17 or the control (PBS).
374
The RT-PCR products were resolved on an agarose gel. Ne: negative control. PBS: GEFs treated
375
with PBS. 17
Table
Table 1 Oligonucleotide primers used to amplify cDNAs for Goose IL-17 Gene name cDNA
ds cDNA
Goose IL-17
Ex IL-17
◦
Primer sequence
Ann T ( C)
First-dT20
TCTAGAGTCGACCTGCACATTTTTTTTTTTTTTTTTTTTGC
65
3G primer
GAGCTCGAATTCACTTAGTATAGCGCGCGGG
65
5’PCR primer
TCTAGAGTCGACCTGCACAT
52.5
3’PCR primer
CTCGAATTCACTTAGTATAGCG
52.5
IL-17 S
GGGTCGCCCAGCACAAGCA
62.2
IL-17 A
ACTCCTGTGCTGTGGGCTCCCT
61.9
mIL-17 S
CGCGGATCCATGAAGGTGATACGGCCC
65
mIL-17 A
CCCAAGCTTTTAAGCCTGGTGCTGGATCAA
65
Primer name
Table 2 Oligonucleotide primers used to assess biologic activity of IL-17 Gene name IL-6
IL-8 β-actin
Primer name
Primer sequence
IL-6 S
GCGGTCTCCGACTCCTCC
IL-6 A
ATAGCGAACAGCCCTCACG
IL-8 S
GCTGTCCTGGCTCTTCTCCT
IL-8 A
GCACACCTCTCTGTTGTCCTTC
β-actin S
CCACACCGTGCCCATCTAT
β-actin A
GGTCGTATTCCTGCTTGCTG
Goose IL-6: JF437643.1; Goose IL-8: DQ393274; Goose β-actin: M26111
Size (bp) 546 bp
210 bp
610 bp
Figure 1
Figure 2
Figure 3
