title: Molecular cloning, expression and functional analysis of interleukin-8 (IL-8) in South African clawed frog (Xenopus laevis).
abstract: In this study, an IL-8 homologue has been cloned and identified from South African clawed frog Xenopus laevis (designated XlIL-8). The open reading frame (ORF) of XlIL-8 consists of 312 bases encoding a protein of 103 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in South African clawed frog IL-8. By quantitative real-time PCR, mRNA transcript of XlIL-8 was detectable in all the examined tissues with higher level in spleen and kidney. The temporal expression of XlIL-8 mRNA in the monocytes was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at about 6h post-stimulation. Recombinant soluble XlIL-8 (XlsIL-8) was fused with a small ubiquitin-related modifier gene (SUMO) to enhance the soluble expression level in Escherichia coli BL21 (DE3). The fusing protein SUMO-XlsIL-8 was purified using metal chellate affinity chromatography (Ni-NTA) and cleaved by a SUMO-specific protease, then confirmed by SDS-PAGE and Western blotting analysis. Chemotaxis assays showed that lymphocytes but not monocytes could be recruited toward SUMO-XlsIL-8 or XlsIL-8 protein in a dose-dependent manner in vitro. The present study may be useful for understanding the anti-bacteria immunity in amphibian and gives the potential to use the recombinant proteins to manipulate the immune response.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Xianwei
authors.initials: X
authors.lastname: Cui
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Han
authors.forename: Yangbin
authors.initials: Y
authors.lastname: Pan
authors.forename: Xingzhou
authors.initials: X
authors.lastname: Xu
authors.forename: Wenhua
authors.initials: W
authors.lastname: Ren
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016209:Interleukin-8 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics*; D013154:Spleen / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D016209:Interleukin-8; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins