本文關鍵詞：人NDRG1的原核表達、純化及相互作用蛋白分析　出處：《西北農林科技大學》2005年碩士論文　論文類型：學位論文

  更多相關文章： 人 ndrg 家族 原核表達 HHCC western 雙向電泳

【摘要】：人ndrg2 (N-myc downstream regulated gene 2) 基因是第四軍醫(yī)大學生化教研室【11,12】于1999 年從正常成人全腦cDNA 文庫中最先發(fā)現并克隆得到的。其染色體定位于14q11.2,含有16 個外顯子,15 個內含子;mRNA 全長為2,024 nt,編碼含有357 個氨基酸殘基,分子量約4.0×104的蛋白質。Ndrg 基因家族中最早被發(fā)現的是Ndrg1,它是在研究N-myc 下游調節(jié)基因時從小鼠胚胎中發(fā)現的一種新基因。目前已報道的該家族成員有4 個:ndrg1、ndrg2、ndrg3 和ndrg4,它們之間具有較高的同源性,但表達水平在組織發(fā)育階段和分布上差異明顯,具體功能還不清楚。NDRG 家族的表達受很多內外界因素的調節(jié),使NDRG1 表達量提高的因素有homocystein、tunicamycin、細胞氧化應激(缺氧、Ni2+、各種還原試劑)、視黃酸、P53 等;使NDRG1 表達量降低的因素有:Androgen、c-myc、n-myc 等。綜合已有文獻,推測NDRG 家族可能參與細胞生長分化、維持細胞氧化還原電勢平衡(氧化應激或生物轉化)、阻止腫瘤細胞惡化等。因此,檢測與其相互作用的蛋白,并由此研究其在細胞中的具體功能以及可能的信號通路將具有非常重要的意義。 鑒于上述事實,本實驗開展了以下幾方面的研究: 1. ndrg 家族成員的表達載體構建:從已成功構建并測序的T 載體pMD18-T-ndrg1、pMD18-T-ndrg2、pMD18-T-ndrg4b 中酶切下ndrg1、ndrg2、ndrg4b 片段,克隆至pGEX-4T-1空載體中,構建成pGEX-4T-1-ndrg1、pGEX-4T-1-ndrg2、pGEX-4T-1-ndrg4b 表達載體。 2. ndrg 家族成員的原核表達:分別用IPTG 誘導表達pGEX-4T-1-ndrg1、pGEX-4T -ndrg2、pGEX4T-1-ndrg4b,發(fā)現pGEX-4T-1-ndrg1 部分可溶,而pGEX-4T-ndrg2、pGEX4T-1-ndrg4b 均不溶。 3. NDRG1 蛋白的純化及western 驗證:用谷胱甘肽瓊脂糖珠-4B 珠親和層析pGEX-4T-1-ndrg1 表達的融合蛋白GST-NDRG1,結果在Mr 6.6×104 處出現兩條帶,經western 驗證發(fā)現它們均是GST-NDRG1。 4. NDRG1 高表達細胞系的選育:選取三種腫瘤細胞系7721、A549、HHCC,培育后裂解細胞,western 驗證,發(fā)現HHCC 表達的NDRG1 含量最高。 5.NDRG1 相互作用蛋白的檢測:將HHCC 細胞裂解液和純化的GST-NDRG1 孵育,同時將GST-NDRG1 與Tris.CL 裂解液孵育作為對照,在同樣條件下雙向電泳,發(fā)現樣品至少在Mr 4.3×104,pH6.0 左右處比對照多出一個點,此點可能就是與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.
【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：Q78

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本文編號：1358283