中文摘要：

目的表达并纯化TAT-N24穿膜融合多肽,初步探讨其生物学活性。方法构建pTAT-HA-N24原核表达载体,在BL21大肠埃希菌中表达并纯化TAT-N24融合多肽,利用SDS凝胶电泳检测纯化蛋白的纯度,利用免疫细胞化学和流式细胞术检测TAT-N24融合蛋白的穿膜能力和生物学活性。结果在原核表达系统中成功构建并表达纯化了TAT-N24穿膜融合多肽,其在6 mol/L尿素和DMSO中均具有较好的溶解性,纯化的融合多肽经SDS凝胶电泳分析未见明显杂蛋白混入,利用免疫细胞化学染色可见TAT-N24处理结肠HT29细胞12 h后90%以上的细胞中可检测到TAT-N24,流式细胞术检测细胞周期可见TAT-N24能够有效地诱导细胞周期阻滞,抑制结肠癌细胞生长。结论TAT-N24具有高效的穿膜能力,并能有效抑制结肠癌细胞增殖,可望开发成为有效的肿瘤分子治疗药物。

英文摘要：

Objective To express and identify cell-permeable TAT-N24 fusion peptide,and to investigate the biological activity of TAT-N24.Methods pTAT-HA-N24 plasmid was constructed,and TAT-N24 fusion peptide was expressed and identified in BL21 E.coli.The purity of the peptide was tested by SDS gel electrophoresis.The cell-permeable ability and biological activity of TAT-N24 fusion peptide were assayed by immunocytochemistry and flow cytometry.Results pTAT-HA-N24 plasmid was successfully constructed in the system of prokaryotic expression which can be dissolved in 6 mol/L urea and DMSO.No hybridprotein was found in the TAT-N24 fusion peptide after it was purified from E.coli by SDS gel electrophoresis.After HT29 cells were incubated with TAT-N24 for 12 h,TAT-N24 was detected in more than 90% cells.Flow cytometry revealed that TAT-N24 could effectively induce the cell cycle arrest of HT29 cells.Conclusion TAT-N24 fusion can penetrate cell member efficiently and induce the cell cycle arrest of colon cancer cells,and it may become an effective cancer molecular therapy agent.