Molecular Cloning and mRNA Expression Analysis of Interleukin-1β, Cathepsin B and Cathepsin D from Siberian Sturgeon (Acipenser baerii)

Abstract

The innate immunity provides the first line and fundamental defense against infection by pathogen in fish. Sturgeons, as an extant primitive fish group might possess immune functions largely different from more advanced bony fishes and regarded as a useful model for evolutionary genomics of immunity in vertebrates. Siberian sturgeon (Acipenser baerii) is a threaten species and one of the most important species for sturgeon farming. The aim of this study was isolation, cloning, sequencing and analyzing of interleukin-1β (IL-1β), Cathepsin B (CTSB) and Cathepsin D (CTSD) gene from Siberian sturgeon and evaluation of the expression level in the embryonic developmental stages and in different tissues of juvenile specimen. The cDNA of the genes, obtained by RT-PCR were cloned, sequenced and analyzed. The Siberian sturgeon IL-1β transcript consisted of 849 bp open reading frame (ORF) which translated into a 282 amino acids (aa) peptide. The amino acid sequence lacked a signal peptide but contained interleukin-1 propeptide site and a clearly identifiable IL-1 family signature. The sequence showed low level of similarity with other known species IL-1β and the highest identity was 45% to Atlantic salmon IL-1β. The CTSB sequence consisted of 999 bp open reading frame (ORF) which encoded 332 aa. The amino acid sequence possessed a signal peptide, propeptide, mature peptide regions; active sites of cysteine, histidine and asparagine residues and also contain the papain family cysteine protease domain. The sequence showed the highest level of identity to orange-spotted grouper CTSB (81%). The CTSD sequence consisted of 1191 bp open reading frame (ORF) which encoded 396 aa. The amino acid sequence contained a signal peptide, propeptide domain, aspartic peptidase domain and two aspartic peptidase active sites. The sequence showed the highest level of identity to zebrafish CTSD (85%). The expression level of IL-1β gene mRNA was at a low level in early embryonic development stages and was up regulated from formation of large yolk plug stage up to first occurrence of advanced hatch stage but CTSB and CTSD gene mRNA showed up regulated expression for all the development stages. In the tissue-specific expression, CTSB and CTSD transcripts were relatively high in all examined tissue but IL-1β gene mRNA transcript was expressed relatively high in brain, fin, liver, and spleen than other tested tissue. The ubiquitously expression of IL-1β, CTSB and CTSD in every embryonic developmental stage and in all tested tissues revealed that these three genes may play a vital role in the innate immune functions of Siberian sturgeon. The information from this study could be a fundamental baseline data for detailed studying of innate immune functions and regulations in the development of this primitive fish species.