|Other Abstract||Interferon (IFN) is an important cytokine in antiviral immunity. The functions of IFN have been clearly elucidated in mammalian since IFN was first identified in 1957. To data, the IFN in mammalian was divided into three types: type I IFN (IFN-α/β), type II IFN (IFN-γ), and type III IFN (IFN-λ). In recent years, more and more research on the IFN in lower vertebrates such as teleost has been reported. The amphibian, as the transitional species that vertebrates evolve from hydrophilous to terricolous, and from ectotherm to homeotherm, has an important role in the biology evolution. However, little is known concerning the amphibian IFN and IFN related genes. Based on these, the type II IFN (IFN-γ), and type III IFN (IFN-λ) in the amphibian model animal, xenopus tropicalis (xe), were cloned, characterized, and their expression pattern were analyzed, aiming to reveal the function of amphibian IFN and the evolution story of the IFN system.
Using comparative bioinformatics approach, the xeIFN-γ gene locus was analyzed. In this locus, there are three cytokines: interlcukin (IL)-22, IL-26 and IFN-γ. The gene synteny analysis showed this locus is relatively conserved and the transcription orientation of the cytokine gens and the phase of the introns remain the same during evolution. The xeIFN-γ cDNA consisted of 793 bp encoding a 179 aa peptide, has a four-exon-three-intron genomic organization. The xeIL-22 cDNA is 1375 bp in length with a 579 bp open reading frame (ORF) encoding a 192-aa peptide. The xeIL-22 gene consisted of five exons and four introns. The xeIL-26 ORF is 525 bp in length, coding for a protein of 175 aa. Similar to xeIL-22, the xeIL-26 gene also possesses five exons and four introns. RT-PCR showed that xeIL-22 and xeIL-26 was not detected in all tissues tested, including heart, liver, spleen, lung, kidney, intestine, and stomach. Treatment of the LPS activated IL-22 expression in spleen and intestine 6 h after injection but had no effect on IL-26 expression. PolyI:C induced both IL-22 and IL-26 expression in spleen but not in other tissues. IFN-γ expression pattern was determined by real-time quantitative PCR. The result showed that constitutive expression of IFN-γ was detectable in spleen and intestine. Induction of the IFN-γ expression was apparent in liver, spleen, kidney, and intestine after PolyI:C treatment with increase of IFN-γ transcript level of 5-, 23-, 4-, 11-fold, respectively. In contrast, LPS significantly elevated IFN-γ transcript level by 14-folds in spleen but not in other tissues.
xeIFN-λs (xeIFN-λ1~ -λ5) are clustered on the same chromosome and have a multiple-exons with xeIFN-λ1, -λ2 and -λ5 possessing five exons and xeIFN-λ3 four exons. xeIFN-λ4 is a pseudogene which losts the exon 2 and exon 3. The xeIFN-λ1, -λ2 and -λ5 ORF is 540 bp in length, encoding 179 aa (xeIFN-λ1 and -λ5 shared 100% identity in nucleotide and amino acid level); xeIFN-λ3 ORF is 438 bp and encodes 145 aa. The xeIFN-λ receptors (xeIFNLR1 and xeIL-10R2) were also cloned. The xeIFNLR1 ORF is 1572 bp which encod 523 aa. xeIL-10R2 was located on Scaffold_1108 and ORF is 927 bp in length, encoding 308 aa. RT-PCR and realtime PCR showed that the xeIFN-λ could be detected in the tested tissues, including heart, liver, spleen, lung, kidney, intestine, and stomach. After PolyI:C treated, the xeIFN-λ expression increased in some tissue and had apparent increasing in spleen with elevating 33.13- ,9.34- and 11.86- folds. Further, the xeIFN-λs were analyzed in vitro. After treated the freshly isolated splenocytes for 4 h with 0.1, 1, 10 and 50μg/ml PolyI:C, the results showed that contrast to the control group, xeIFN-λs were upregulated when stimulated with 1 μg/ml PolyI:C. Then the xeIFN-λs expression level increased with the PolyI:C dose. xeIFNLR1 had a constitutive expression in all tested tissues, and had a significantly high expression in lung. Afer treated with PolyI:C, the xeIFNLR1 expression level increased in all tested tissues with the highest increasing in spleen and lung with 14.8 and 14.9-folds. In vitro, the xeIFNLR1 was upregulated when stimulated with 0.1 μg/ml PolyI:C. Similar to xeIFN-λ, the xeIFNLR1 expression level also increased with the PolyI:C dose. To further analysis the function of xeIFN-λ, the recombinant xeIFN-λ1 (RxeIFN-λ1) was expressed and purified. Then, the freshly isolated splenocytes were treated for 6 h with the RxeIFN-λ1 at doses of 0.001, 0.01, 0.1, and 1 mg/ml, respectively. Then the IFIT-5 gene expression level was tested with realtime-PCR. The result showed that IFIT-5 gene was increased with dose of RxeIFN-λ1 and increased 10.1-folds after stimulation with 1 mg/ml RxeIFN-λ1.
Peptidoglycan recognition proteins (PGRPs), as a family of pattern recognition receptors (PRRs), have an important role in the host innate immunity against bacteria pathogen. In this paper, a long type PGRP in xenopus tropicalis (xePGRP-L) was also cloned and characterized. The xePGRP-L cDNA is 1681 bp and encode 497 aa. At the N-terminal of xePGRP-L contained a conserved PGRP domain. X.ePGRP-L mRNA was clearly detected in the early development stages at 3 dpf. Realtime PCR and Western blotting results showed in the normal xenopus, the xePGRP-L was constitutive expressed with highest in liver, lung, intestine and stomach, lower in heart, spleen and kidney. After LPS stimulated, the x.ePGRP-L expression was apparently up-regulated in heart, liver, spleen and intestine after LPS treatment, and the transcript level increase 3-, 26-,7-, 33-fold, respectively. However, there is no significantly expression level change in lung, kidney and stomach.|