酵母双杂交研究小鼠肝脏再生TGF-β调控通路中相关转录因子的相互作用
网络出版日期: 2011-05-27
基金资助
国家高技术研究发展计划(“八六三”计划)(2007AA02Z149)
Transcription factor interactions of TGF-beta signaling pathway identified by yeast two-hybrid technique during mice liver regeneration
Online published: 2011-05-27
Supported by
National High-tech R&D Program of China, 2007AA02Z149
目的 采用酵母双杂交系统研究肝脏再生调控途径转化生长因子β(TGF-β)信号通路中4种转录因子的相互作用。方法 经CCl4腹腔注射诱导建立小鼠肝损伤再生模型,提取模型小鼠肝脏组织总RNA作为模板,经RT-PCR获得cDNA。经肝脏再生基因芯片检测显示差异表达且与TGF-β信号通路相关的激活转录因子3(ATF3)、DNA结合抑制因子3(ID3)、DNA损伤诱导的转录因子3(DDIT3)和LIM蛋白家族成员FHL2(FHL2)作为候选基因,进行基因克隆和载体构建,PCR及DNA测序鉴定。采用酵母双杂交系统进行自激活检测实验和酵母双杂交实验,观察ATF3、FHL2、DDIT3和ID3之间的相互作用。结果 PCR及DNA测序鉴定表明,ATF3、FHL2、DDIT3、ID3基因克隆与载体构建成功。自激活检测显示ATF3、FHL2、DDIT3和ID3不存在自激活现象;酵母双杂交实验发现7对蛋白互作,分别为ID3/ATF3、FHL2/ATF3、ATF3/ATF3、FHL2/FHL2、ID3/FHL2、ID3/ID3和ID3/DDIT3。结论 ATF3、FHL2和ID3间的蛋白互作可能对TGF-β信号通路具有调控作用;蛋白互作网络可作为肝损伤再生机制研究的途径之一。
谢 超, 王方圆, 袁运生, 等 . 酵母双杂交研究小鼠肝脏再生TGF-β调控通路中相关转录因子的相互作用[J]. 上海交通大学学报(医学版), 2011 , 31(5) : 551 . DOI: 10.3969/j.issn.1674-8115.2011.05.006
Objective To investigate the interactions of four transcription factors of transforming growth factor-β (TGF-β) signaling pathways using yeast two-hybrid technique. Methods CCl4-induced liver injury mouse models were established, and the total RNA of liver tissues were extracted as the template for cDNA by RT-PCR. Activating transcription factor 3 (ATF3), inhibitor of DNA binding 3 (ID3), DNA-damage-inducible transcript 3 (DDIT3) and LIM domains 2 (FHL2), which were differentially expressed detected by liver regeneration gene chip and associated with TGF-β signaling pathway, were served as candidate genes. After gene clone, vector construction and PCR and DNA sequencing, yeast two-hybrid system was used to indentify the interactions among ATF3, ID3, DDIT3 and FHL2 with auto-activation test. Results PCR and DNA sequencing indicated that ATF3, FHL2, DDIT3 and ID3 were successfully cloned, and there was no auto-activation phenomenon after auto-activation test. Seven protein interactions (ID3/ATF3, FHL2/ATF3, ATF3/ATF3, FHL2/FHL2, ID3/FHL2, ID3/ID3 and ID3/DDIT3) were identified by yeast two-hybrid technique. Conclusion Protein interactions among ATF3, FHL2 and ID3 may be one of the possible ways to regulate the TGF-β signaling pathway, and protein-protein interactions may be a key gateway to liver regeneration.
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