Sequence analysis, expression patterns, acquisition of prokaryotic recombinant protein and pro-inflammatory effect of IL-17B in Cyprinus carpio

Interleukin-17 (IL-17) is an important inflammatory factor, which is able to promote the production of various inflammation-related factors, such as IL-1β, 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-17B 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-17Bs in the genome of the C. carpio, CcIL-17B1 and CcIL-17B2 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-17B1 intron are 84 , 420 bp respectively and the CcIL-17B2 intron are 81, 851 bp respectively. The two CcIL-17Bs 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-17B and its nearby genes were lost. Most teleost have one IL-17B and Danio rerio have both IL-17Bs that were lost. The common carp-specific chromosomes doubled resulting in the presence of two CcIL-17Bs. Real time quantitative PCR (qPCR) was used to measure the expression of CcIL-17B1 and CcIL-17B2 in fertilized eggs, larvae and different tissues of adult C. carpio. The results showed that the expression levels of both CcIL-17B1 and CcIL-17B2 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-1β, 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.