[Preparation of a uncoordinated-5 homolog B monoclonal antibody and its effect on melanoma cell migration in vitro].

OBJECTIVE:To prepare and characterize the monoclonal antibody (mAb) against uncoordinated-5 homolog B (UNC5B) and analyze its effect on the migration of melanoma cells. METHODS:UNC5B gene fragment was cloned into the prokaryotic expression vector pET-32a. The recombinant UNC5B protein was expressed in E.coli BL21 (DE3) and purified by affinity chromatography. BALB/c mice were immunized with the recombinant protein and the hybridoma cell clones stably secreting UNC5B antibody were screened by traditional hybridoma technique. ELISA, Western blotting and flow cytometry were used to characterize the specificity of the antibodies. In addition, the effect of the mAb on melanoma cell migration was analyzed by wound healing assay. RESULTS:The recombinant UNC5B protein was expressed and purified. One high-titer antibody 2C9 was obtained. ELISA, Western blotting and flow cytometry all demonstrated that 2C9 antibody specifically recognized the UNC5B protein. Wound healing assay indicated that the UNC5B mAb could promote melanoma cell migration at the presence of netrin-1. CONCLUSION:A UNC5B-specific monoclonal antibody was prepared and proved to have the ability of promoting melanoma cell migration at the presence of netrin-1.