摘要
研究人源性穿膜肽HBD的N端融合蛋白的跨膜转导作用。通过DNA重组技术将HBD融合在EGFP蛋白的氨基端(N端),构建重组质粒pET28b-HBD-EGFP-Histag,经IPTG诱导表达和Ni 2+-NTA纯化,获得重组蛋白HBD-EGFP。通过激光共聚焦显微镜定性观察及流式细胞仪定量检测发现,融合在EGFP氨基端的HBD跨膜转导效率比融合在EGFP羧基端(C端)的HBD转导效率提高了约10倍。实验结果表明,N端融合的HBD-EGFP对于外源蛋白具有跨膜运输效果,且比C端融合的HBD具有更高的跨膜转导效率。该结果可为HBD在抗癌药物递送方面的有效应用提供理论依据。
To study the transmembrane activity of Heparin-Binding Domain（HBD）derived from human fused at N-terminal of heterologous protein,the expression plasmid pET28b-HBD-EGFP-Histag was constructed by genetically fusing HBD to the N-terminal of enhanced green fluorescent protein（EGFP）.The fusion protein HBD-EGFP was expressed in E.coli and purified by Ni 2＋-NTA affinity chromatography.The results show that green fluorescence can be observed in HeLa cells using laser scanning confocal microscope when HeLa cells were co-cultured with HBD-EGFP.Flow-cytometry results show that the transmembrane efficiency of HBD fused at the N-terminal of EGFP is improved about 10 times compared with the HBD fused at the C-terminal of EGFP.These results demonstrated that HBD fused to N-terminal of heteologous protein exhibited tansmembrane activity.The HBD fused at the Nterminal of EGFP had a higher efficiency than the HBD fused at the C-terminal of EGFP.These results will provide theoretical foundation for the efficient application of HBD in the field of the delivery of anticancer drugs.
出处
《华东理工大学学报（自然科学版）》
CAS
CSCD
北大核心
2014年第2期176-181,共6页
Journal of East China University of Science and Technology
基金
国家自然科学基金(81171449)
