利用反向双杂合系统分离干扰Ras-Raf相互作用的寡肽  

Isolation of Oligopeptides Targeting Ras- Raf Interactions via Reverse Two Hybrid Assays

作　　者：王淑丽[1] 陈丽[1] 唐曼[1] 付敏[1] 杜悦[1] 黄艳[1] 潘利超 刘秋云[1] 

机构地区：[1]广州中山大学生命科学学院,水产品安全教育部重点实验室,生物工程研究中心,广东广州510275

出　　处：《生物技术》2014年第5期58-63,共6页Biotechnology

基　　金：广东省自然科学基金项目("利用具有随机两臂核酸进行全基因组导向和高效同源重组";S201101000426);广东省科技计划项目("新型固氮酶基因的表达与转基因植物研究";2008B020100001);中山大学实验室开放基项目("动物感知地震的候选基因的合成与检测";KF201328);国家自然科学基金项目("科研训练及科研能力提高项目";J1310025)资助

摘　　要：目的:Ras/Raf代谢途径与癌症发生发展密切相关,但针对该途径的药物还不多。方法:结合反向双杂合系统和随机肽库对干扰Ras-Raf相互作用的寡肽进行了分离。首先构建了载体和嵌合基因库,然后对酵母进行了电激。对阳性克隆进行测序。结果:其中一个六肽EGPSGN对酵母在负选择培养基上的生长有一定促进作用。两个八肽TIARGPFE、FEGPSGNS在0.4 mmol/L时的抗癌作用比天蚕素和前人报道的干扰Ras-Raf相互作用的肽略高,均大于天蚕素2%和后者10%。结论:甘油(3%)/半乳糖(4%)选择培养基效果很好并分离到了阳性克隆,而前人建立的棉子糖/半乳糖选择培养基在该文的使用无效。该策略可增加基于Gal7突变株的经济的反向双杂合系统的应用范围。Objective： Targeted cancer therapies are gaining popularity in the clinic as these regimens have fewer side effects. The activation of Ras / Raf pathway has been implicated in human cancers. However, there are not many marketed drugs aimed at these proteins. Method： Here the isolation of oligopeptides inhibiting Ras- Raf interactions is presented,in a system that combines reverse two hybrid assays with cost effective random peptide libraries. Chimeric genes were initially constructed,and inserted to expression vectors,and electroporated to yeast. Putative positive clones were sequenced subsequently. Result： One of the hexapeptides EGPSGN identified showed modest activities in stimulating yeast growth under negative selection. Two octapeptides,TIARGPFE and FEGPSGNS demonstrated higher potency than a previously reported septapeptide（ 10%） and the antimicrobial peptide cecropin A（ 2%） in anticancer assays against human lung carcinoma A549 cells at 0. 40 mmol / L. Conclusion： The galactose（ 4%） / glycerol（ 3 %） solid media rendered the selection much tighter than previously reported galactose / raffinose selection,which may substantially expand the utility of the Gal7 mutant based and inexpensive reverse two hybrid assays.

关 键 词：反向双杂合系统 寡肽 Ras-Raf相互作用 随机肽库 甘油/半乳糖选择培养基 

分 类 号：Q75[生物学—分子生物学] R73-362[医药卫生—肿瘤]