Expression, Purification, And Refolding Of Leukocyte Cell-derived Chemotaxin2and Its Effect On Gene Expression Of Head Kidney-derived Macrophages Of A Teleost Fish, Ayu (Plecoglossus Altivelis)

Recently, studies on the immune response in some fish showed that LECT2transcripts increaseddramatically after bacterial infections, shedding new lights on the function of this gene in fish. Mostrecently, we found for the first time that ayu LECT2mature peptide (aLECT2m) could interact with thesingle extra-cellular C-type lectin domain of a C-type lectin receptor (CLR), which is one of patternrecognition receptors (PRRs) present on fish immune cells.Therefore, it would be interesting to make clearwhether aLECT2m could regulate ayu macrophages. Before that, sufficient amount of protein is required inits active form.In this study, the BL21plys E/pET28a-aLECT2strain, which was constructed and stored in our lab,was induced to express ayu LECT2. The raw products were inclusion bodies. Then a superdex75columnwith a urea gradient was used to refold the recombinated ayu LECT2. The result showed that the purity ofrefolding protein was about95%. In order to detect the activation of the refolded LECT2, the primary headkidney cell and primary head kidney-derived macrophages were isolated. In vitro, the refolded and purifiedayu LECT2was added into the medium, and the chemotactic activity of the head kidney cell or headkidney-derived macrophage had been observed.On the basis above, we used the suppression subtractive hybridization (SSH) to identify differentiallyexpressed genes in head kidney-derived macrophages between ayu LECT2treated and untreated control.500random clones were selected and sequenced from the forward subtractive libraries.473ESTs withgood qualities were obtained. After assembling and BLAST analyses,272(71.2%) unigenes from theforward library were annotated. These known unigenes in the forward subtractive libraries could fall into20categories of biological processes at Gene Onotology (GO) annotation level2. Genes involved incellular process, metabolic process, and biological process were the most abundant in the forward library,about19%,14%,12%， respectively.Subsequently,10immune-related genes from the forword subtractive libraries were selected to furtherevaluate by real-time quantitative PCR (RT-qPCR) analyses. According to the sequenced ESTs, the primerwas designed and used in the RT-qPCR. The results revealed that the immune-related genes in headkidney-derived macrophages was significant up-regulated after this macrophages treated with LECT2,suggesting LECT2’s role in fish immune response.In this study, active recombinant ayu LECT2was first obtained by in vitro refolding. In an invitro model of head kidney-derived macrophages, LECT2activated the macrophages confirmed by SSH. Itis the first report that LECT2has an effect on macrophages, which will provide a new insight for furtherstudy in the signal pathway of the immunorevelant protein LECT2in fish..