细胞穿透肽YARA介导增强型绿色荧光蛋白转导入人结直肠癌细胞的实验研究

目的 构建原核表达载体pET15b-YARA EGFP,表达并纯化出融合蛋白YARA-EGFP,观察其穿透人结直肠癌细胞的情况.方法 用分子克隆技术构建出表达型载体pET15b-YARA-EGFP,在E.coli BL21(DE3)中表达融合蛋白YARA-EGFP,将鉴定和纯化后的融合蛋白加入体外培养的人结直肠癌细胞.结果 得到纯化的融合蛋白YARA-EGFP,能高效地转导入体外培养的人结直肠癌细胞,并表现出剂量和时间依赖性,MTT法检测在其高浓度时仍对细胞无毒性.结论 YARA-EGFP融合蛋白能高效地转导入体外培养的人结直肠癌细胞.

Transduction of enhanced green fluorescent protein mediated by a cell penetrating peptide-YARA into human colorectal cancer SW480 cells

Objective To construct the expression vector pET15b-YARA-EGFP and purify the fusion protein YARA-EGFP.And evaluate the penetrating ability of YARA in the transduction of enhanced green fluorescent protein(EGFP) into human colorectal cancer line SW480.Methods The recombinant plasmid pET15b-YARA-EGFP was constructed by inserting YARA-encoding DNA into pET15b-EGFP.The fusion protein YARA-EGFP expressed in E.coli BL21(DE3),which were purified and added in cultured human colorectal cancer line SW480.Results The highly purified YARA EGFP fusion protein was obtained,and it could be transduced into SW480 cells in a time-and dosedependent manner when added exogenously in a culture media.The cytotoxicity of YARA-EGFP detected by MTT method demonstrated there were no statistical significantly differences between different concentrations.Conclusion The fusion protein YARA-EGFP could efficiently transduce into human colorectal cancer line SW480 which provides a basis for YARA-mediated biomacromolecule transduction in protein therapy.