摘要
目的构建基于细胞穿透肽靶向细胞核运输的蛋白表达载体,并研究其蛋白转导功能。方法在带His标记的增强型绿色荧光蛋白(EGFP)表达载体pET14b-HE(pET14b-His-EGFP)基础上,利用点突变的方法构建依次含细胞穿透肽(CPP)、连接子、核定位信号(NLS)序列和EGFP的融合蛋白表达载体pET14b-HC(L)NE(pET14b-His-CPP-Linker-NLS-EGFP);经酶切、测序鉴定载体构建正确后,将重组质粒转化BL21(DE3)宿主菌,经异丙基硫代-β-D-半乳糖苷诱导表达后、用Ni2+亲和层析纯化得到融合蛋白;将融合蛋白透析、过滤除菌后加入培养的真核细胞中,荧光显微镜下观察并进行Westernblot检测分析其在活细胞的蛋白转导功能。结果酶切、测序证实载体构建成功,融合蛋白在大肠杆菌中可有效表达;蛋白转导实验可见融合蛋白可快速穿透细胞膜并进入细胞核,并且这种内化转导功能存在时间和剂量依赖效应。结论成功构建了基于细胞穿透肽的蛋白表达运输载体,建立了可携带外源蛋白进入细胞浆及细胞核的运输系统,为研究蛋白或多肽的细胞内功能以及运输药物提供了一种经济有效的工具。
Objective To construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction. Methods The fusion protein expression vector pET14b-HC （L）NE （pET 14-b-His-CPP-Linker-NLS-EGFP） incorporating cell penetrating peptide （CPP）, nuclear localization signal（NLS）, linker and enhanced green fluorescent protein （EGFP） was constructed based on His-tagged pET14b-HE （pET14b-His-EGFP） by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21 （DE3） strain. The HC（L）NE fusion protein was expressed following IPTG induction and purified with Ni^2＋-NTA affinity chromatography. After dialysis and filtration, the HC （L）NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting. Results Enzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC（L）NE vector, and the fusion protein efficiently expressed in E.coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent. Conclusion The cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2006年第10期1394-1399,1407,共7页
Journal of Southern Medical University
基金
国家高技术研究发展(863)计划课题(2001AA234061)
广州市科技计划项目(2001-Z-035-01-1)
广东省自然科学基金重点项目(13058)
广东省科技计划项目(A1090202)~~
关键词
细胞穿透肽
增强型绿色荧光蛋白
核定位信号
蛋白转导
内化
cell penetrating peptide
enhanced green fluorescent protein
nuclear localization signal
protein transduction
internalization
