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背景与目的:BRD-7基因是我室自己克隆的一个新基因,它包含一个溴区结构域。研究表明BRD-7基因能明显抑制鼻咽癌细胞的生长,是一个良好的鼻咽癌候选抑瘤基因。为了阐明BRD-7基因的作用机制,我们首先对其溴区结构域进行了初步的研究。方法:通过定向克隆,我们构建了BRD-7基因溴区结构域的真核表达载体,并进行了诱导表达,Western-EP迹检验结果的真实性。另外,我们运用生物信息学对BRD-7溴区结构域进行了分析。结果:我们构建重组表达质粒PGEX-4T-2/bromodomain,经酶切鉴定和测序验证表达载体构建正确。经IPTG诱导,SDS-PAGE分析在大肠杆菌中成功诱导表达了分子量为38.8KD的融合蛋白,用Western-EP迹证实了融合蛋白的表达。通过氨基酸序列的一级结构,二级结构的分析及其同源匹配,我们发现BRD-7溴区蛋白与三个结构已知的溴区蛋白具有高度相似性:可能含有四个α螺旋(Z,A,B,C),螺旋与螺旋之间通过环伴连接(ZAloop,BCloop),并且含有一个由疏水氨基酸形成的“口袋”,通过这个结构,能特异性识别乙酰化的组蛋白。结论:通过同源比较,BRD-7溴区蛋白应属与溴区蛋白家族联合作用因子亚群,并且可能具有特异性识别乙酰化组蛋白的功能。
BACKGROUND & AIM: The BRD-7 gene is a novel gene cloned by our laboratory and contains a bromodomain. Studies have shown that BRD-7 gene can significantly inhibit the growth of nasopharyngeal carcinoma cells, is a good nasopharyngeal cancer candidate tumor suppressor gene. To elucidate the mechanism of action of BRD-7, we first conducted a preliminary study of its bromodomain. Methods: By directional cloning, we constructed the eukaryotic expression vector of BRD-7 gene bromodeoxyribonucleic acid domain and induced it by Western-blot analysis. In addition, we used bioinformatics to analyze the BRD-7 bromodomain. Results: We constructed the recombinant expression plasmid PGEX-4T-2 / bromodomain, which was confirmed by restriction enzyme digestion and sequencing. After induced by IPTG, the fusion protein was successfully induced in E.coli by SDS-PAGE and the molecular weight was 38.8KD. Western-blot analysis confirmed the expression of the fusion protein. Through the primary structure, secondary structure analysis and homologous matching of amino acid sequences, we found that the BRD-7 bromodomain protein has high similarity with three known bromodomain proteins: it may contain four α-helices (Z , A, B, C). The helix and helix are linked by a loop (ZAloop, BCloop) and contain a “pocket” of hydrophobic amino acids. By this structure, acetylated histones are specifically recognized. Conclusion: By homologous comparison, the BRD-7 bromodomain protein should belong to the co-acting factor subfamily of the bromodomain family and may have the function of specifically recognizing acetylated histone.