一种高效人源性穿膜肽的生物活性研究

摘要

Objective To study the transduction activity of Cyt c77-101, a human-derived cell-penetrating peptide(CPP), in HeLa cells. Methods The oligonucleotide chains encoding Cyt c 77-101were synthesized artificially, and a recombinant vector of pET28a-EGFP-Cyt c77-101 was constructed. The fusion protein was expressed in E.coli BL21 and purified by Ni2+-NTA affinity chromatography. The penetration activity of the CPP in HeLa cells was tested by fluorescence microscope and laser scanning confocal microscope(LSCM). Results Sequencing analysis proved that the recombinant plasmid pET28a-EGFP-Cyt c77-101 was constructed successfully. And the fusion protein was expressed in E.coli BL21. After about 10 h incubation of HeLa cell with EGFP-Cyt c77-101, an intensive green fluorescence was observed. Conclusion The CPP of Cyt c 77-101shows a strong transduction activity.%　　目的研究人类细胞色素C中Cyt c77-101对HeLa细胞的穿膜活性。方法人工合成编码Cyt c77-101的寡核苷酸链，构建重组质粒pET28a-EGFP-Cyt c77-101，并转化大肠杆菌BL21进行原核表达，进一步通过Ni2+-NTA亲和层析纯化带有绿色荧光蛋白EGFP的Cyt c77-101融合蛋白，并经荧光显微镜和激光共聚焦检测Cyt c77-101的穿膜活性。结果测序分析表明重组质粒pET28a-EGFP-Cyt c77-101构建成功，且在大肠杆菌BL21中成功表达。融合蛋白EGFP-Cyt c77-101与HeLa细胞保温10 h后，可在细胞内观察到强绿色荧光。结论人源性穿膜肽Cyt c77-101具有较强的穿膜活性。