Identification and expression analysis of scavenger receptor SCARA4 in Nibea albiflora

Nibea albiflora is often infected by pathogens of Larimichthys crocea and other adjacent net-caught fish during the cage culture process, which brings great harm to its culture process. Scavenger receptors (SRs) are a class of membrane-anchored glycoproteins that play an essential role in innate immunity. In this study, the full length cDNA of SCARA4 in N.albiflora as the representative member in the scavenger receptor family (named as NaSCARA4, GenBank accession number: MN_871709) was identified based on its transcriptome data, which was 4050 bp including an open reading frame (ORF) of 2382 bp to encode 793 amino acid residues. Computational analysis via ExPASy revealed that the theoretical isoelectric point (pI) of NaSCARA4 was 5.58, and its molecular weight (Mw) was approximately 85.32 kDa. The carbohydrate recognition domain (CRD) was found in NaSCARA4 by SMART analysis, and ClustalW analysis showed that NaSCARA4 shared 78-94% homology with SCARA4 from other bony fish and branched into the same cluster, implying that this gene belonged to the scavenger receptor family. RT-qPCR analysis showed that NaSCARA4 distributed in the 7 tissues tested (spleen, liver, kidney, gill, stomach, intestine and heart), among which the highest expression was in the liver with 47.29-fold higher than those in the control group (heart). NaSCARA4 showed time-dependent up-regulation of pathogenic Vibrio (Vibrio alginolyticus and Vibrio parahaemolyticus) infection or Poly(I:C) stimulation, but the highest expression level and its appearing time was slightly different. The prokaryotic expression vectors pET32a-NaSCARA4_CRD was successfully constructed and expressed in the host bacteria of E. coli BL21 (DE3). Following IPTG induction, the target protein with the expected molecular weight of 34 kDa was obtained and purified to obtain a single high-purity recombinant protein for in vitro bacterial binding experiments after renaturation. It was observed that the recombinant protein exhibited binding affinity to all the assayed bacteria in the experiment (V.alginolyticus, V.parahaemolyticus and Vibrio harveyi) via Western blotting. The aforementioned findings suggested that NaSCARA4 was involved in the body's antimicrobial natural immune defense response as important membrane proteins.