文档介绍：HSP70诱饵载体的构建及其在酵母双杂交系统中自激活作用的检测
作者:李静,杨守京,刘彦仿,周鹏,韩骅
【关键词】热休克蛋白70
【Abstract】 AIM: To construct the bait vector (pGBKT7Hsp70) of MATHMAKER GAL4 TwoHybrid System 3, and test it for its autonomous activation. METHODS: Hsp70 gene was amplified by PCR from pBBS212Hsp70 plasmid, and the EcoRI and BamHI sites were introduced into it. The Hsp70 gene, after sequenced, was cloned into pGBKT7. Selfactivation of the bination vector (pGBKT7Hsp70) was tested. RESULTS: The pGBKT7Hsp70 vector was essfully constructed and had no autonomous activation. CONCLUSION: The pGBKT7Hsp70 was proved to be suitable for further research on the genes in tumors, which interact with the Hsp70.
【Keywords】 Hsp70; yeast two hybrid system; autonomous activation
【摘要】目的:构建用于酵母双杂交系统的“诱饵”载体pGBKT7Hsp70,:用PCR的方法从质粒pBBS212Hsp70中扩增出Hsp70基因片段,引入酶切位点EcoRⅠ,BamH
Ⅰ.测序正确后,将Hsp70基因克隆至载体pGBKT7中,并检测pGBKT7Hsp70在MATHMAKER GAL4 TwoHybrid System 3中的自激活作用. 结果: 成功构建了“诱饵”载体pGBKT7Hsp70,并证明其在酵母双杂交系统中无自激活作用. 结论: pGBKT7Hsp70可以用于酵母双杂交系统中,寻找与Hsp70相互作用的分子.
【关键词】热休克蛋白70;酵母双杂交系统;自激活作用
0引言
热休克蛋白(heat shockproteins, HSPs)是广泛存在于生物体内的一类高度保守的蛋白质,它参与蛋白质的合成、折叠、装配及转运,是细胞生存所必需的,且也是肿瘤细胞生存所必需的. 反义hsps转染,可导致肿瘤细胞生长停滞或死亡[1-3]. 热休克蛋白70(HSP70)是目前研究得较为深入的一个家族,与肿瘤的发生发展及预后关系密切. 为寻找与HSP70相互作用的蛋白,并查明他们的相互作用位点及其体内外结合特性,我们拟采用酵母双杂交系统进行研究,构建酵母双杂交系统的诱饵载体pGBKT7HSP70,并转化酵母菌AH109,观察其是否具有自激活作用.
1材料和方法
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