Cloning, expression and identification of CgFUT5 gene associated with Lewis antigen synthesis of Oyster norovirus receptors

Lewis antigen is regarded as a specific binding receptor for norovirus, and Lewis-like antigen is also present in oysters as a vehicle for norovirus transmission, but the pathway for synthesis of this carbohydrate in oysters has not been elucidated. To clarify the pathway of norovirus receptor-like Lewis antigen synthesis in oysters, we cloned the CgFUT5 gene from Pacific oyster (Crassostrea gigas) genome and analyzed the expression in five tissues. The full sequence of CgFUT5 gene was obtained by rapid amplification of cDNA ends (RACE) and bioinformatically analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). A prokaryotic expression plasmid was constructed to transform Escherichia coli for heterologous expression, and immunogenicity was identified by immunoblotting (Western blot). The cDNA sequence of CgFUT5 gene with 1 173 bp open reading region was obtained by cloning, and phylogenetic tree shows that CgFUT5 gene was genetically related to the rockweed glycosyltransferase gene that had the function of synthesizing Lewis antigen in several species. The recombinant CgFUT5 protein could be overexpressed in E. coli, and the expressed recombinant CgFUT5 protein specifically bound to both anti-human FUT5 antibody and anti-6×His tag antibody. To sum up, CgFUT5 gene was successfully cloned and found to be abundantly expressed in oyster gill tissue, and CgFUT5 protein has similar immunogenicity to human FUT5 protein. It is hypothesized that a Lewis-like antigen synthesis pathway exists in oysters, and the genes regulating Lewis-like antigen synthesis in oysters also have differential tissue expression.