Expression of three Oreochromis niloticus Siglecs-like fusion proteins in COS-7 cells and characterization of their binding activities with GBS

Siglecs, sialic acid-binding immunoglobulin-like lectins is a class of membrane receptors which could specifically recognize sialic acid polysaccharide of self or some pathogens.In order to prepare Nile tilapia(Oreochromis niloticus)Siglecs-like fusion proteins and study their functions in the interaction of host-pathogen, we prepared O.niloticus Siglecs like fusion proteins using eukaryotic expression system.In this research, the extracellular domain of the three O.niloticus Siglecs like were respectively amplified from the cloned vectors containing the ORF of Siglec-1/-4b/-14 like and then inserted into the expression vector of pcDNA3.1(+)hIgG1 Fc.After double digestion and sequencing, they were transfected into the COS-7 cells in vitro.The expressions of target genes was detected by qPCR and Western-blot analysis.The fusion proteins were purified by affinity chromatography, and their purity was detected by SDS-PAGE electrophoresis.To study the biological activities of these Siglecs like fusion proteins, binding activities of the fusion proteins to Group B Streptococcus(GBS)were detected by ELISA.The results showed that these three recombinant plasmids containing extracellular domains of tilapia Siglecs like with human IgG1 Fc fragment were constructed successfully.These three genes were highly expressed in COS-7 cells both in mRNA and protein levels.Three Siglecs/Ex-Fc fusion proteins were proved to be of high purity.The binding activities between fusion proteins and control were significantly different(P < 0.05).This research showed we successfully obtained purified fusion proteins by COS-7 cells expression system.A preliminary study proved they could bind GBS.