Abstract:
Interleukin-17 (IL-17) is an important inflammatory factor, which is able to promote the production of various inflammation-related factors, such as
IL-1
β,
IFN-γ,
IL-6,
CCL20 and so forth. It plays an important role in autoimmune disease, host defense, inflammation and so on. In order to study the pro-inflammatory function of IL-17B in the
Cyprinus carpio, the
IL-17
B gene was cloned, the NusA-17B protein was recombinantly expressed by
Escherichia coli system and the pro-inflammatory effect of the IL-17B was then investigated by
in vivo and
in vitro experiments. In this study, by homology search and gene cloning method, we found two
IL-17
Bs in the genome of the
C. carpio,
CcIL-17
B1 and
CcIL-17
B2 respectively. Both of the two genes have open reading frames (ORF) of 597 bp, which are composed of three exons and two introns. The length of their exons are the same, 24, 320 and 253 bp respectively. While the introns are different, the
CcIL-17
B1 intron are 84 , 420 bp respectively and the
CcIL-17
B2 intron are 81, 851 bp respectively. The two
CcIL-17
Bs encode 198 amino acids, containing 2 disulfide bonds formed by 4 cysteines, which were unique to the IL-17 family. Sequence blast analysis showed that the consistency of the two CcIL-17Bs proteins reached 91.92%. The collinearity analysis results are as follows. During the doubling of the chromosomes of teleost,
IL-17
B and its nearby genes were lost. Most teleost have one
IL-17
B and
Danio rerio have both
IL-17
Bs that were lost. The common carp-specific chromosomes doubled resulting in the presence of two
CcIL-17
Bs. Real time quantitative PCR (qPCR) was used to measure the expression of
CcIL-17
B1 and
CcIL-17
B2 in fertilized eggs, larvae and different tissues of adult
C. carpio. The results showed that the expression levels of both
CcIL-17
B1 and
CcIL-17
B2 were significantly higher at 0-12 hours post fertilization than that at 25-90 hours post fertilization (
P<0.05). While the expression levels have no significant difference at 25-90 hours after fertilization and 1-14 days after hatching (
P>0.05). In adult fish, the expression level of two genes in testis and gonad are significantly higher than that in other tissues, such as spleen, heart, kidney, head kidney, intestine, gill, liver, skin, muscle, brain (
P<0.05). Further, using the
E. coli expression system, the soluble recombinant protein NusA-17B was obtained. Different concentrations of NusA-17B were injected into the anus to evaluate its pro-inflammatory effect. Histopathological results showed that intestinal villi defect, plenty of goblet cells and inflammatory cells appeared in intestines treated with high concentration NusA-17B (500 μg/kg) after 1 and 3 days. Meanwhile, qPCR showed that
IL-1
β,
IFN-γ,
IL-6,
CCL20 and
NF-κB were significantly up-regulated (
P<0.05). However, the abnormal changes in intestinal structure and expression of inflammation-related genes were restored to normal level after 7 days. In addition,
in vitro, the
C. carpio kidney cells were incubated into different concentrations (0.1, 1.0, 10.0 and 100.0 ng/mL) NusA-17B for 8 hours. The results showed that
IL-1β was significantly up-regulated under NusA-17B stimulation at 1.0, 10. and 100.0 ng/mL (
P<0.05);
IFN-γ and
NF-κB were significantly up-regulated under the stimulation of NusA-17B at 10.0 ng/mL and 0.1 ng/mL respectively (
P<0.05);
IL-6 and
CCL20 were significantly expressed under NusA-17B stimulation at 10.0 and 100.0 ng/mL (
P<0.05), and
TRAF6 was significantly expressed under NusA-17B stimulation at 0.1, 1.0 and 10.0 ng/mL (
P<0.05). In summary, the results of
in vivo and
in vitro experiments show that CcIL-17B participates in the inflammatory response.