MLP增强生肌素对nAChRγ亚基基因启动子的结合活性

Muscle LIM Protein Enhances the Binding of Myogenin to the Nicotinic AChR γ-subunit Gene Promoter

构建表达GAL4 MLP融合蛋白的真核表达质粒pBind MLP .哺乳动物细胞双杂交试验表明 ,MLP与生肌素E12复合物存在细胞内的相互作用 .构建表达GST融合蛋白GST MLP和GST E12的原核表达质粒pGEX 2TK MLP以及pGEX 4T 2 E12 .在E .coliBL2 1中诱导表达GST MLP、GST 生肌素和GST E12 ,并用亲和层析法纯化了这 3种GST融合蛋白 .这 3种GST融合蛋白进行的凝胶阻滞试验表明 ,MLP可以增强生肌素 E12复合物对AChRγ亚基基因启动子的结合活性 .研究初步阐明了MLP增强nAChRγ亚基基因启动子在分化C2C12细胞中的转录活性的分子机制 .

The eukaryotic expression vector pBind MLP was constructed by subcloning MLP cDNA from pUC19 MLP in frame into pBind. Mammalian two hybrid assays found that MLP interacted with myogenin E12 complex in vivo . The prokaryotic expression vector pGEX 2TK MLP was constructed by subcloning MLP cDNA from pUC19 MLP in frame into pGEX 2TK. The prokaryotic expression vector pGEX 4T 2 E12 was constructed by subcloning E12 gene from pYN3218 E12 first into pUC18 and then in frame into pGEX 4T 2. The GST fusion proteins GST MLP,GST myogenin and GST E12 were expressed in E.coli BL21 and purified by affinity chromatography. Gel mobility shift assay indicated that MLP enhanced the binding of myogenin E12 complex to the E box sites in the promoter region of the AChR γ subunit gene. This study illustrated elementarily the molecular mechanisms of MLP enhancing the transcription activity of the AChR γ subunit gene promoter in differentiating C2C12 cells.