神经元蛋白3.1结合蛋白酵母双杂交筛选体系的建立

Construction a yeast two-hybrid system for screening P311 binding proteins

神经元蛋白3.1(P311)是肺泡发育的上游调节因子。以pEGFP-P311重组质粒为模板,利用PCR方法扩增P311基因编码序列。通过Nde I和BamH I位点插入诱饵载体pGBKT7,构建重组诱饵载体pGBKT7-P311。重组体转化酵母菌AH109进行自激活和毒性检测,结果 DNA-BD-P311融合蛋白无单独激活报告基因作用,对酵母菌亦无毒性。以出生11 d小鼠肺组织为材料,提取总RNA。逆转录产生单链cDNA,通过长距离PCR进行扩增。扩增产物ds cDNA电泳后可见大小为0.2～3.0 kb间的弥散状分布条带,说明文库cDNA可满足筛选要求。诱饵载体pGBKT7-P311的构建及相应小鼠肺组织cDNA文库的建立,为进一步利用酵母双杂交技术探讨P311功能奠定了基础。

P311 is an up regulator of lung alveolar development. Coding sequence of P311 was amplified by PCR using plasmid pEG- FP-P311 as template. It was then inserted between NdeI and BamHI sites of bait vector pGBKT7 to get recombinant bait vector pG- BKT7-P311. The recombinant was transformed into yeast AH101 to test transcriptional activation and toxicity of DNA-BD fusion. The results showed that DNA-BD-P311 fusion protein had no ability to autoactive reporter genes of yeast, and had no toxicity for the host. Total RNA was isolated from postnatal day 11 mouse lungs. After got first strand cDNA by reverse transcription, a LD-PCR was per- formed to produce ds eDNA. It represented a qualified library due to the ds eDNA appeared a moderately strong smear from 0. 2 to 3.0kb on agarose gel.