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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 a monoclonal antibody (mAb) that is a non-coordinating molecule 5 homologous protein B (UNC5B) and analyze its function. Methods The UNC5B gene fragment was ligated with the prokaryotic expression vector pET-32a. The recombinant product was transformed into E. coli BL21 (DE3) and induced by prokaryotic expression. BALB / c mice were immunized with UNC5B recombinant protein. The hybridoma cell lines secreting anti - UNC5B were screened by hybridoma technique and identified by Western blot, ELISA and flow cytometry. Effect of UNC5B mAb on Migration of Melanoma Cells by Cell Scratch Test. Results The recombinant protein of UNC5B was expressed and purified. One high titer 2C9 mAb was screened out. UNC5B was identified by ELISA, Western blot and flow cytometry. Scratch experiment proved that under the condition of netrin-1 UNC5B mAb promotes melanoma cell migration. Conclusion The specific m Ab of UNC5B was successfully prepared, which can promote the migration of melanoma cells in the presence of netrin-1.