Genomic organization, promoter characterization and expression analysis of the leukocyte cell-derived chemotaxin-2 gene in Epinephelus akaraa.
ABSTRACT Leucocyte cell-derived chemotaxin 2 (LECT2) was first identified as a chemotactic factor and has been subsequently proven to be a multifunctional protein that mediates the regulation of liver regeneration, carcinogenesis and Natural killer T (NKT) cell homeostasis in mammals. In fish, it has been recently found to be critical for the inflammatory response to stimuli. However, the in vivo function of LECT2 in fish remains obscure. Base on the full-length cDNA of the Epinephelus akaraa LECT2 (EaLECT2) gene we previously isolated, we sought to analyze its genomic structure and context. The genomic DNA of the EaLECT2 gene spans 2866bp from the transcription start site to the termination codon. As in most LECT2 genes in other vertebrates, the EaLECT2 genomic DNA contains four exons and three introns. An analysis of the promoter region revealed the presence of a TATA box and several putative transcription factor-binding sites. And transcriptional activity analysis suggested that most basal DNA regulatory elements required for EaLECT2 transcriptional activity might be contained within the 581bp region upstream of the transcription start codon. A real-time PCR analysis showed that the EaLECT2 expression levels were slightly increased in the head kidney, liver, gill and brain by bacterial challenge with Vibrio harveyi. Furthermore, the transcriptional level of the EaLECT2 gene in the liver was significantly up-regulated within 1h and reached its peak level at 12h post-stimulation. Higher levels of LECT2 expression were also observed in head kidney in challenged individuals.The expression pattern demonstrates the role of EaLECT2 in the immune response and its functions under other conditions. Additionally, we found that the recombinant EaLECT2 could be expressed as a soluble protein using a prokaryotic expression system with the expression vector pET32a(+) and the soluble protein was further proved to be the recombinant EaLECT2 with the rat antiserum against EaLECT2 we obtained. This work provides a unique basis for substantial work in future projects.