title: Prokaryotic expression, purification, and refolding of leukocyte cell-derived chemotaxin 2 and its effect on gene expression of head kidney-derived macrophages of a teleost fish, ayu (Plecoglossus altivelis).
abstract: Leukocyte cell-derived chemotaxin 2 (LECT2) is reported to be an immunorelevant protein in ayu (Plecoglossus altivelis). In this study, ayu LECT2 mature peptide (aLECT2m) was expressed as insoluble inclusion bodies in Escherichia coli. The denatured recombinant aLECT2m (raLECT2m) was refolded by a size-exclusion chromatography refolding process achieved by using arginine-containing mobile phase and a decreasing urea gradient. The in vitro chemotactic activity assay showed that the refolded raLECT2m had the bioactivity. By using suppression subtractive hybridization (SSH) method, we further identified up-regulated genes in ayu macrophages treated with refolded raLECT2m. These genes were tightly involved in endocytosis, hydrolysis, transcriptional regulation, signal transduction, and so on. Moreover, real-time quantitative PCR (RT-qPCR) results confirmed that selected 10 genes expression was significantly up-regulated in refolded raLECT2m-treated ayu macrophages. This study provides a basis for further studies of the mechanism of cytokine LECT2 in fish immune responses.
authors.affiliation: Faculty of Life Science and Biotechnology, Ningbo University, Ningbo city 315211, Zhejiang Province, People's Republic of China.
authors.forename: R C
authors.initials: RC
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: C H
authors.initials: CH
authors.lastname: Li
authors.forename: X J
authors.initials: XJ
authors.lastname: Lu
authors.forename: Y H
authors.initials: YH
authors.lastname: Shi
mesh_terms: D000818:Animals; D002630:Chemotactic Factors / Q000378:metabolism*; D002633:Chemotaxis / Q000235:genetics; D002850:Chromatography, Gel / Q000662:veterinary; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005786:Gene Expression Regulation / Q000276:immunology*; D060226:Head Kidney / Q000166:cytology; D002479:Inclusion Bodies / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D036441:Osmeriformes / Q000276:immunology* / Q000378:metabolism; D058849:Protein Refolding; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary
chemical_list: D002630:Chemotactic Factors; D017931:DNA Primers