穿膜肽介导的新型基因转运载体的功能研究  

Function of a Novel DNA Delivery System Mediated by Cell-Penetrating Peptides

作　　者：张宏达[1] 绳纪坡[1] 于芳[1] 黄君健[1] 胡宝成[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2013年第6期782-786,共5页Letters in Biotechnology

基　　金：国家自然科学基金(31070760;30770651;30670616)

摘　　要：目的:借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体(LacI HPM)高亲和力结合DNA的特性,建立一种安全高效、无基因插入片段大小限制的基因转导系统。方法:在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a(+)-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果:表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论:初步证实TAT-LacI HPM融合蛋白作为一种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。Objective： To establish a novel DNA delivery system which is safe, high efficient and no limitation of inserting DNA fragments, by using cell-penetrating peptides TAT with the ability of high efficient cellular penetration and LacI to specifically bind to its recognizing DNA sequence with high affinity. Methods： GST-tag was inserted into either C- or N-terminal of TAT-LacI HPM（LacI headpiece mutant） fragment to obtain the expression plasmids. TAT-LacI HPM fusion proteins expressed in E.coli were purified by glutathione Sepharose 4B beads, and were dialyzed against buffer to form the TAT-LacI HPM directs. Whether the transduced dimeric TAT-LacI HPM fusion proteins could penetrate in cells was tested by immunofluorescence, and the ability of TAT-LaeI HPM dimers to deliver DNA into the cells were examined by observing the expression of the reporter EGFP gene and by Western blotting. Results： The expression plasmids of pET-28a（＋）-TAT-LacI HPM-GST and pGEX-GST-TAT-LacI HPM were obtained, after expression and purification, the dimers were concentrated, the transduced dimerie TAT-LaeI HPM fusion proteins could penetrate in cells and mediate EGFP transfection and expression in human cells. Conclusions： Our data suggest that the TAT-LacI HPM fusion proteins may have the potential to serve as a novel safe, efficient DNA delivery system which may improve the application of DNA vaccines in the future.

关 键 词：穿膜肽 LacI前头肽突变体 二聚体 DNA转运 

分 类 号：Q78[生物学—分子生物学]