BRD-7基因溴区结构域的真核表达及其特征研究(英文)

Analysis of Bromodomain of BRD-7 Gene and Its Prokaryotic Expression

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 & Objective: BRD 7 is a novel gene (AF:152604), containing a bromodomain, was cloned in our lab. Previous studies showed that BRD 7 plays an obviously suppressive role on NPC cell growth. In order to clarify the function mechanism of this gene,we investigate an important motif of BRD 7, the bromodomain. Methods: The bromodomain of BRD 7 was analyzed by homology based amino sequence and secondary structure analysis. In addition, we constructed a prokaryotic expression vector of bromodomain.Western blot analysis was used to confirm the expression of the bromodomain protein in Escherichia coli. Results: Homology based sequence analysis revealed that the bromodomain of BRD 7 possibly contains four α helices (Z, A, B and C), and a hydrophobic pocket which is an important structure to recognize acetylated histone peptide. This bromodomain encoding a 12.8kD protein, was introduced into Escherichia coli using the pGEX 4T 2 expression vector. After isopropyl β D thiogalactopyranoside(IPTG) induction, a new anticipated protein of 38.8 kD appeared on SDS PAGE and the result was confirmed by Western blot analysis. Conclusion: BRD 7 of bromodomain protein is similar to three proteins containing known structural bromodomain motif by bio informatics analysis, suggesting the bromodomain of BRD 7 should belong to co activator subgroup and may have similar function that can selectively interact with acetylated histone peptide.