人NDRG1的原核表达、纯化及相互作用蛋白分析

发布时间：2017-12-31 05:11

本文关键词：人NDRG1的原核表达、纯化及相互作用蛋白分析　出处：《西北农林科技大学》2005年硕士论文　论文类型：学位论文

【摘要】：人ndrg2 (N-myc downstream regulated gene 2) 基因是第四军医大学生化教研室【11,12】于1999 年从正常成人全脑cDNA 文库中最先发现并克隆得到的。其染色体定位于14q11.2,含有16 个外显子,15 个内含子;mRNA 全长为2,024 nt,编码含有357 个氨基酸残基,分子量约4.0×104的蛋白质。Ndrg 基因家族中最早被发现的是Ndrg1,它是在研究N-myc 下游调节基因时从小鼠胚胎中发现的一种新基因。目前已报道的该家族成员有4 个:ndrg1、ndrg2、ndrg3 和ndrg4,它们之间具有较高的同源性,但表达水平在组织发育阶段和分布上差异明显,具体功能还不清楚。NDRG 家族的表达受很多内外界因素的调节,使NDRG1 表达量提高的因素有homocystein、tunicamycin、细胞氧化应激(缺氧、Ni2+、各种还原试剂)、视黄酸、P53 等;使NDRG1 表达量降低的因素有:Androgen、c-myc、n-myc 等。综合已有文献,推测NDRG 家族可能参与细胞生长分化、维持细胞氧化还原电势平衡(氧化应激或生物转化)、阻止肿瘤细胞恶化等。因此,检测与其相互作用的蛋白,并由此研究其在细胞中的具体功能以及可能的信号通路将具有非常重要的意义。鉴于上述事实,本实验开展了以下几方面的研究: 1. ndrg 家族成员的表达载体构建:从已成功构建并测序的T 载体pMD18-T-ndrg1、pMD18-T-ndrg2、pMD18-T-ndrg4b 中酶切下ndrg1、ndrg2、ndrg4b 片段,克隆至pGEX-4T-1空载体中,构建成pGEX-4T-1-ndrg1、pGEX-4T-1-ndrg2、pGEX-4T-1-ndrg4b 表达载体。 2. ndrg 家族成员的原核表达:分别用IPTG 诱导表达pGEX-4T-1-ndrg1、pGEX-4T -ndrg2、pGEX4T-1-ndrg4b,发现pGEX-4T-1-ndrg1 部分可溶,而pGEX-4T-ndrg2、pGEX4T-1-ndrg4b 均不溶。 3. NDRG1 蛋白的纯化及western 验证:用谷胱甘肽琼脂糖珠-4B 珠亲和层析pGEX-4T-1-ndrg1 表达的融合蛋白GST-NDRG1,结果在Mr 6.6×104 处出现两条带,经western 验证发现它们均是GST-NDRG1。 4. NDRG1 高表达细胞系的选育:选取三种肿瘤细胞系7721、A549、HHCC,培育后裂解细胞,western 验证,发现HHCC 表达的NDRG1 含量最高。 5.NDRG1 相互作用蛋白的检测:将HHCC 细胞裂解液和纯化的GST-NDRG1 孵育,同时将GST-NDRG1 与Tris.CL 裂解液孵育作为对照,在同样条件下双向电泳,发现样品至少在Mr 4.3×104,pH6.0 左右处比对照多出一个点,此点可能就是与NDRG1 相互作用的蛋白。
[Abstract]:Human ndrg2 N-myc downstream regulated gene 2 gene is a biochemistry teaching and research department of 4th military Medical University [11]. In 1999, it was first found and cloned from the whole brain cDNA library of normal adults. Its chromosome was located at 14q11.2 and contained 15 introns in 16 exons. The total length of mRNA is 2,024 NT, encoding 357 amino acid residues. Ndrg1 is the earliest protein. Ndrg gene family with a molecular weight of 4. 0 脳 10 ~ 4. It is a novel gene found in mouse embryos during the study of N-myc downstream regulatory genes. Four members of the family have been reported to have 4: ndrg1ndrg2. Ndrg3 and ndrg4 had high homology, but the expression level was different in the development stage and distribution of tissue. The specific function is not clear. NDRG family expression is regulated by a lot of internal and external factors, the increase of NDRG1 expression factor is homocystein. Tunicamycin, cell oxidative stress (anoxic acid Ni2, various reductive reagents, retinoic acid, p53, etc.); The factors contributing to the decrease of NDRG1 expression were c-mycn- myc and c-mycn- myc. It was suggested that the NDRG family might be involved in cell growth and differentiation. Maintain cell redox potential balance (oxidative stress or biotransformation, prevent tumor cells from deteriorating, etc.) therefore, detect proteins interacting with them. Therefore, it will be of great significance to study its specific function and possible signal pathway in cells. In view of the above facts, this experiment carried out the following aspects of research: 1. Construction of expression vector for members of ndrg family: pMD18-T-ndrg1 pMD18-T-ndrg2 was successfully constructed and sequenced from T vector pMD18-T-ndrg1. The ndrg1ndrg2ndrg4b fragment was digested from pMD18-T-ndrg4b and cloned into pGEX-4T-1 empty vector. The expression vector pGEX-4T-1-ndrg1 pGEX-4T-1-ndrg4b was constructed. 2. Prokaryotic expression of ndrg family members: the expression of pGEX-4T-1-ndrg1pGEX-4T -ndrg2 was induced by IPTG, respectively. PGEX4T-1-ndrg4b, pGEX-4T-1-ndrg1 partially soluble, while pGEX-4T-ndrg2. PGEX4T-1-ndrg4b is insoluble. 3. Purification and western validation of NDRG1 protein: glutathione agarose bead 4B bead affinity chromatography pGEX-4T-1-ndrg1. Expressed fusion protein GST-NDRG1. Results two bands were found at Mr 6.6 脳 10 ~ 4, which were all GST-NDRG1 by western. 4. Selection of high expression NDRG1 cell lines: three tumor cell lines 7721A549HHCC were selected, and then the cells were identified by western assay. It was found that HHCC expressed the highest NDRG1 content. 5. Detection of NDRG1 interaction protein: HHCC cell lysate was incubated with purified GST-NDRG1. At the same time, GST-NDRG1 was incubated with Tris.CL lytic solution as control. Under the same conditions, the sample was found to be at least 4. 3 脳 10 ~ 4. There is one more point on the left and right side of pH6.0 than the control, which may be the protein interacting with NDRG1.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78

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