酵母双杂交系统鉴定hTCF4相互作用

利用酵母双杂交试验技术，鉴定了Ｗｎｔ／Ｆｒｉｚｚｌｅｄ信号通路中的关键信号分子ＴＣＦ４自身的相互作用．通过 ＴＣＦ４突变体的同源相互作用的双杂交反应（ｔｗｏ ｈｙｂｒｉｄｉｚａｔｉｏｎ），确定了转录因子ＴＣＦ４在发挥作用过程中形成双聚体，推测以ＨＬＨ或ＬＺ的结构花式（ｍｏｔｉｆ）调控下游基因的表达．

Interaction of hTCF4 by yeast two-hybrid system

To investigate the interaction of hTCF4, the yeast two-hybrid system has been used for testing the interaction of mutants of hTCF4 with themselves. Mutants of hTCF4 (hTCF4 Ⅰ ) and hTCF4 Ⅱ) have been obtained by polymerase chain reaction (PCR). Bait (hTCF4 Ⅰ -pDBLeu and hTCF4Ⅱ-pDBLeu) and prey (hTCF4 Ⅰ -pPC86 and hTCF4Ⅱ-pPC86) have been constructed by DNA recombination for yeast two-hybrid. Interaction of hTCF4Ⅱ with itself is found in the reverse yeast two-hybrid system (GIBCOBRL Co.). However, no interactions are found in hTCF4Ⅱ with hTCF4 Ⅰ and in hTCF4 Ⅰ with hTCF4 Ⅰ . These results suggest that hTCF4 could interact with itself to form homodimer or homocopolymer and perform the transcriptional activating function through LZ or HLH motifs in nucleus of renal cell carcinoma.

hTCF_4酵母双杂交载体的构建

目的 研究Wnt Frizzled信号传导通路上的主要信号分子T细胞因子 (TCF4)在细胞核内转导的辅激活物和辅阻遏物。方法 通过PCR法以真核表达载体pCDNA TCF4为模板 ,扩增获得了15 0 0bp的TCF4基因的末端及DNA结合域片段 ,经过SalI ,NotI酶切后 ,基因重组的方法定向克隆于酵母双杂交载体pPC86和pDBleu中 ,构建形成pDBleu TCF4和pPC86 TCF4真核表达载体。结果 经转化 ,提取质粒酶切鉴定表明构建成功。结论 hTCF4酵母双杂交载体的构建成功为Wnt Frizzled信号通路的深入研究奠定了基础。