人宫颈癌Hela细胞穿透肽的噬菌体展示技术筛选  

Screening of display technology for phages targeting human cervical carcinoma Hela cells-penetrating peptides

作　　者：刘芸[1] 吴向玲[1] 江力玮[1] 姚琦[1] 邓鹏[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室,广东省功能蛋白质组学重点实验室,广州510515

出　　处：《解放军医学杂志》2009年第6期755-758,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30572151);广东省自然科学基金资助项目(05004730);广州市科技计划项目(2007J1-C0301);教育部长江学者和创新团队发展计划项目(IRT0731)

摘　　要：目的细胞穿透肽是近年来发现的具有穿透哺乳动物生物膜功能,并能介导大分子物质跨膜转导的一类小分子肽段。该肽段以其转导效率高,速度快,生物活性好,对细胞损害小等特点,成为药物导向治疗方法研究领域的热点。本研究目的在于利用噬菌体展示技术高通量、系统地筛选人宫颈癌Hela细胞的穿透多肽。方法以Hela细胞作为对象,利用噬菌体随机肽库,采用细胞筛选的方法,筛选具有细胞膜穿透作用的多肽。将筛选得到的穿透多肽与增强型绿色荧光(EGFP)融合表达,通过荧光显微观察技术快速对多肽的穿透功能进行验证。结果通过4轮的噬菌体文库筛选,目的多肽得到了有效而快速地富集。初步的测序和验证得到了3条具有明显介导融合蛋白内化的短肽序列。结论成功建立起基于噬菌体随机肽库的细胞穿透肽筛选技术,为肿瘤导向药物载体的研究开发奠定了基础。Objective Cell-penetrating peptides （CPP）, a class of small peptides, could translocate into eukaryotie cells, also mediate macromolecules transduction in recent reports. Such peptides were characterized with high efficiency, fast speed, full bio-activity and less harm to many cell lines, and became the focus of drug targeting therapy. The present study was designed to utilize the phage-display technology to screen CPP, absorbed by human cervical carcinoma Hela cells in high-throughput and systematic manners. Methods Hela cells were screened for CPP in whole cell screening mode through the Ph.D. 7TM phage display peptide library. The candidate peptides were fusion expressed with enhanced green fluorescent protein （EGFP）, and their abilities of internalization were monitored with fluorescent microscopy. Results After 4 cycles of screening, the positive clones were enriched fast and efficiently. Three peptides, which obviously could induce the internalization of fusion proteins, were identified by preliminary DNA sequencing and functional verification. Conclusion Screening method for CPPs through random phage pepfide library was established successfully, which may lay a foundation for the study and development of tumor targeting drug vector.

关 键 词：细胞穿透肽 噬菌体展示 筛选 功能验证 

分 类 号：Q932[生物学—微生物学]