细胞穿膜肽pep-1与vMIP-Ⅱ的融合表达与纯化

The Fusion Expression and Purification on Cell Permeable Peptide pep-1 and Viral Macrophage Inflammatory Protein-Ⅱ

细胞穿膜肽是一类能携带大分子物质进入细胞的短肽,其穿膜能力不依赖经典的胞吞作用。本研究构建了含有细胞穿膜肽pep-1和病毒巨噬细胞炎症蛋白-Ⅱ(viral macrophage inflammatory protein-Ⅱ,vMIP-Ⅱ)的融合表达质粒pET15b-pep-1-vMIP-Ⅱ,并将其转化大肠杆菌BL21(DE3)plysS中经IPTG诱导表达,经SDS-PAGE和Western-blot鉴定出可溶性的融合蛋白pep-1-vMIP-Ⅱ。通过对IPTG浓度、温度等诱导表达条件进行优化,确定在IPTG浓度为0.2mmol/L、28℃下诱导7h可溶性蛋白的表达量相对较高,经Ni-NTA亲和层析,超滤除盐纯化获得高纯度的融合蛋白pep-1-vMIP-Ⅱ,将该蛋白作用于Hela细胞,激光共聚焦显微镜观察结果显示该融合蛋白能够携带目的基因穿透细胞膜。本文结果将为进一步研究vMIP-Ⅱ的功能和细胞穿膜肽技术的应用奠定基础。

Cell permeable peptide is a kind of small peptides that are capable of bringing micromolecules into cell memebrane,the cell permeation ability of which is independent of classic endocytosis.In this article,the plasmid pET15b-pep-1-vMIP-Ⅱ,synthesis from cell permeable peptide pep-1 and viral macrophage inflammatory protein-Ⅱ,was transducted into Escherichia coli BL21（DE3） plysS.The expression product induced by IPTG was identified and analysed by SDS-PAGE and Western-blot.The optimization of expression condition such as the concentration of IPTG and temperature led to the relatively highest expression level of soluble protein was at the circumstance of 7 hours of inducement under the condition of 0.2 mmol/L concentration of IPTG and 28℃.Fusion protein of high purity was cleaned by hyperfiltration after Ni-NTA affinity chromatography.When the fusion protein acted on the Hela cell,the function of carry target gene and penetrating cell membrane was abserved through LSCM.The results of this would lay the foundation for further research of the function of vMIP-Ⅱ and the application of cell cermeable technique.