GSThDaxx蛋白的构建及其原核表达产物的鉴定.docVIP

GSThDaxx蛋白的构建及其原核表达产物的鉴定 【摘要】目的构建GSThDaxx蛋白并在原核细胞中诱导表达GSThDaxx融合蛋白，并研究其与p53的结合能力。方法构建GSThDaxx融合蛋白原核细胞表达载体pGEX4T/hDaxx，在大肠杆菌（E.coli）中用异丙基βD硫代半乳糖苷（IPTG）诱导表达。表达产物用亲和层析柱加以纯化后，Western blot鉴定表达产物与纯化物。通过共免疫沉淀反应与Western blot观察hDaxx与p53的结合反应。结果在原核细胞中成功表达了GSThDaxx融合蛋白，hDaxx与p53可发生共免疫沉淀反应。结论1.GSThDaxx融合蛋白成功表达; 2.hDaxx和p53的相互结合提示它们可能与肿瘤的发生有关。 【关键词】 GST hDaxx 融合蛋白 p53 Identification of GSThDaxx Protein Construction and Its Prokaryotic Expression Products TANG Xuhong, ZHU Cuiming, WAN Yanping Department of Diagnosis of Western Medicine, Hunan Chinese Medical College, Changsha 410007, China；Institute of Pathogenic biology, Nanhua University Abstract：Objective To construct a recombinant protein of GSThDaxx and to induce the expression of its fusion protein. Methods By constructing pGEX4T/hDaxx recombinant, GSThDaxx fusion protein was induced to express by IPTG in E.coli BL21, and purified by glutathione resin. hDaxx fusion protein was analyzed by Western blot. The direct binding of hDaxx and p53 was studied by coimmunoprecipitation reaction. Results hDaxx fusion protein was induced to express by IPTG in E.coli successfully. Soluble GSThDaxx fusion protein was purified and identified by Western blot. The binding reaction of hDaxx and p53 was observed by coimmunoprecipitation reaction and Western blot. Conclusion GSThDaxx fusion protein was expressed and purified. The binding of hDaxx and p53 suggested that they may play important role in tumorigenesis. Key words：GST; hDaxx; fusion protein; p53 0 引言 hDaxx是细胞核内的一种新型转录调控蛋白， 它与Fas死亡结构域（death domain, DD）相互作用诱导细胞凋亡［1］。hDaxx蛋白定位6p21.3， 即位于主要组织相容性复合体(the major histocompatibility complex, MHC), 可能与自身免疫性疾病和癌症的发病机理存在着一定的关系［2］。p53是细胞DNA损伤应答过程中的主要成分， 它在细胞周期控制、DNA修复、细胞凋亡、细胞分化及血管生成中起关键调控作用［3］。hDaxx可能与p53在病毒感染及细胞凋亡和细胞转化中有重要生物学作用。本文用谷胱甘肽S转移酶（GST）与hDaxx融合蛋白表达载体， 在E.coli BL21中成功地用IPTG诱导表达了GSThDaxx融合蛋白， 并用亲和层析柱加以纯化， 利用Western blot分析纯化产物及其与p53发生共免疫沉淀反应后的反应产物， 以便为在体内外研究hDaxx与p53相互作用及作用效果提供前期实验依据。 1 材料与方法 1.1 主要仪器与试剂 电穿孔机及低温超速离心机分别系