Production and application of monoclonal antibodies against C-type lectins in scallop Chlamys farreri

Abstract

C-type lectins, as types of pattern recognition receptors (PRRs), have crucial roles in innate immunity, and their conserved carbohydrate recognition domain (CRD) is recognized as the binding dominant to pathogen-associated molecular patterns (PAMPs). In this study, a C-type lectin in the scallop Chlamys farreri (lectin B, GenBank No. DQ209290) was heterologously expressed and the recombinant protein (rlectin B) with a molecular mass of approximately 42 kDa was purified. The rlectin B protein could induce the aggregation of Escherichia coli, Vibrio anguillarum and Staphylococcus aureus. Six monoclonal antibodies (mAbs) against rlectin B was developed, among which two mAbs (1A7 and 2B6) could react with the gills, hepatopancreas, adductor muscle, and haemocytes of C. farreri by indirect immunofluorescence assay and bound to proteins with molecular masses of 21, 28, 37, 45, 58, and 70 kDa in western blot assays. Using the two mAbs, C-type lectins protein levels were detected in C. farreri haemocytes after V. anguillarum infection, showing that that lectin levels rapidly increased, peaking at 12 h before gradually decreasing to the control group levels. Furthermore, the FACS results also confirmed that V. anguillarum infection significantly increasing C-type lectins expression in haemocytes. These data suggest that the mAbs can recognize various C-type lectins in C. farreri and are a useful immunological tool for the detection C-type lectins in scallops.