摘要
研究含外显子8的prosaposin蛋白亚型(PsapE8)与同源异型框蛋白Rhox5蛋白间的相互作用.重叠延伸PCR法扩增PsapE8的cDNA序列,将其按照通读框方式分别克隆至pGBKT7和pGADT7酵母双杂交载体中构建pGBKT7-PsapE8和pGADT7-PsapE8重组质粒.酵母双杂交实验检测PsapE8与Rhox5蛋白在酵母体内的结合;体外转录翻译S35标记的PsapE8蛋白,GSTpull-down实验检测PsapE8与Rhox5蛋白在体外的结合情况.成功地构建了pGBKT7-PsapE8和pGADT7-PsapE8重组质粒,酵母双杂交实验表明PsapE8蛋白在酵母体内可以结合Rhox5蛋白;GST pull-down实验再次验证了两者在体外的结合;表明含有外显子8的prosaposin蛋白亚型PsapE8可以与Rhox5蛋白相结合.
To study the interaction between prosaposin with exon 8 isform and Rhox5 protein,the prosaposin cDNA including exon 8 spliced forms was amplified using overlap extension PCR and then was subcloned into pGBKT7 and pGADT7 vectors to construct pGBKT7-Psap^E8 and pGADT7-Psap^E8 plasmids.Yeast two-hybrid assay was used to detect whether Psap^E8 could bind Rhox5 protein in yeast.Psap^E8 proteins was translated in vitro and labeled with 35S-Met using a TNT T7 Quick Coupled transcription/translation reaction kit.GST pull-down was used to confirm the interaction between Psap^E8 and Rhox5 protein in vitro.The recombinant plasmids pGBKT7-Psap^E8 and pGADT7-Psap^E8 were constructed successfully.Yeast two-hybrid assay indicated Psap^E8 could bind RHOX5 in yeast.GST pull-down assay further confirmed this interaction in vitro.The results suggested that Prosaposin with exon 8 isform（Psap^E8） could interact with Rhox5 protein.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2010年第3期307-311,共5页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
广东省自然科学基金项目(8151063201000013)
高等学校博士学科点专项科研基金项目(20060559006)
