UNC5B单克隆抗体的制备及对黑素瘤细胞体外迁移能力的影响

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

目的制备非协调性分子5同源蛋白B(UNC5B)的单克隆抗体(mAb),分析其功能。方法将UNC5B基因片段与原核表达载体pET-32a连接,连接产物转化大肠杆菌BL21(DE3),进行原核诱导表达并纯化重组蛋白。用UNC5B重组蛋白免疫BALB/c小鼠,经杂交瘤技术筛选可稳定分泌抗UNC5B的杂交瘤细胞株,并用Western blot法、ELISA和流式细胞术鉴定。利用细胞划痕实验观察UNC5BmAb对黑素瘤细胞迁移能力的影响。结果表达并纯化了UNC5B重组蛋白;筛选出1株高效价的2C9mAb,通过ELISA、Western blot法和流式细胞术证实该m Ab特异性识别UNC5B;划痕实验证明在netrin-1存在的条件下,UNC5BmAb可促进黑素瘤细胞迁移。结论成功制备了UNC5B的特异性m Ab,在netrin-1存在时,该抗体可促进黑素瘤细胞迁移。

Objective To prepare and characterize the monoclonal antibody（m Ab） 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-32 a. The recombinant UNC5 B 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 themAb 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 UNC5 BmAb 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.