Molecular Cloning And Characterization Of A Lectin Gene CfLec-2 From Zhikong Scallop Chlamys Farreri

Zhikong scallop Chlamys farreri is one of the most important aquaculture species in the northern Chinese provinces. Since the summer in 1997, a large scale mortality of C. farreri scallop has caused catastrophic losses to scallop aquaculture and threatened the sustainable development of this industry. Understanding the immune defense mechanisms of scallop may contribute to the development of management strategies for disease control and long-term sustainability of scallop farming. Based on EST sequencing and 3'RACE technology, a scallop lectin gene CfLec-2 was cloned and characterized in the present study.The full length cDNA of CfLec-2 was of 708 bp, containing a 5'-UTR of 59 bp and an unusual long 3'-UTR of 163 bp which contains seven polyadenylation signal sequences AATTAAA and a poly (A) tail. The CfLec-2 cDNA encoded a polypeptide of 162 amino acids with pI of 4.54 and a theoretical mass of 16.8 kDa. SignalP analysis indicated that a putative signal peptide was cut between VEA-QSL. SMART analysis revealed a typical long-form carbohydrate-recognition domain (CRD) of 130 residues. Clustal W alignment indicated that CfLec-2 possessed the conserved cysteine residues and carbohydrate recognition domain. The deduced amino acid sequence of CRD region in CfLec-2 shared high homologue with Brevican in human, C-type lectin-1 and C-type lectin-2 in Anguilla japonica, CD23 in Rattus norvegicus. The cysteine residues Cys49, Cys125 Cys141, Cys149 were predicated to form the CRD internal disulfide bridges, while the other two cysteine residues (Cys21, Cys32) at N-terminus formed another disulfide bridge. Glu115-Pro116-Asp117 determined the ligand-binding specificity.The cDNA fragment encoding the mature peptide was subcloned into pET32a(+) vector and expressed in E. coli Rosetta-gami(DE3). The recombinant protein CfLec-2 could agglutinate Gram-positive Staphylococcus haemolyticus in a calcium independent manner. Moreover, CfLec-2 could specifically inhibit the growth of E. coli TOP10F'. The results revealed that CfLec-2 may be involved in not only the immune recognition, but also the pathogen clearance. It was interesting that CfLec-2 displayed distinct characteristics to CFLec-1. The comprehensive study on lectins is crucial to better understand the immune recognization and defense mechanisms in scallops, and give new insights into health management and diseases control in mollusk aquaculture.