人新基因EOLA1的原核表达、蛋白纯化及多克隆抗体制备

发布时间：2018-03-10 14:49

本文选题：内皮高表达脂多糖相关因子1　切入点：内毒素　出处：《第三军医大学》2005年硕士论文　论文类型：学位论文

【摘要】：脓毒症(sepsis)是多器官功能障碍(MODS)的前兆,是当前危重病医学面临的难题之一,它也是导致严重烧伤、创伤患者死亡的主要原因之一;其发病机制与内毒素(LPS)激活血管内皮细胞诱发并加重全身炎症反应密切相关。脓毒症的病理生理机制复杂,现行的治疗措施有限,因此我们深入研究其发病机制就显得尤为重要。近年来,由于血管内皮细胞是内毒素作用的直接靶细胞之一,国内外对LPS激活内皮细胞的作用途径、特异受体、胞内信号转导等方面研究较多。本研究室前期应用抑制消减杂交(SSH)和cDNA末端扩增技术(RACE),从LPS刺激后的人脐静脉内皮细胞中克隆到一个人类新基因全长cDNA序列,经Northern blot验证后作为人类新基因被GenBank全长序列库所接受(No.AY074889)。因其在受LPS刺激的内皮细胞中上调表达,故暂时命名为内皮高表达脂多糖相关因子1(endothelial-overexpressed lipopolysaccharide-associated factor 1,EOLA1)。目前,国外尚无关于该基因的相关研究,本实验室对EOLA1基因做了一些前期研究工作,并经生物信息学分析显示:该基因全长1404个bp,其定位于染色体Xq27.4,编码蛋白由158个氨基酸组成,分子量17.89kDa,等电点6.43,亲水性好,为可溶性蛋白。其二级结构含α螺旋、β片层结构和β转角,并形成一个螺旋—转角—螺旋(HTH)基序。EOLA1氨基酸序列包含N-糖基化位点、PKC磷酸化位点和酪氨酸激酶2磷酸化位点。多组织Northern blot显示EOLA1基因除在LPS刺激的内皮细胞上调表达外,在骨胳肌、心脏、肾脏、肝脏和胎盘有较强的表达,在结肠、小肠、脾脏有较弱的表达,而在脑、胸腺、肺和外周白细胞则未见表达。在7种癌细胞株上,显示在早幼粒白血病系HL-60、海拉细胞系S3、慢性骨髓性白血病系K-562和肺癌细胞系A549都有表达,而在成淋巴细胞瘤性白血病MOLT-4和结直肠腺癌SW480表达水平最高,但在黑素瘤G-361无表达。在经过G418压力筛选后的稳定表达EGFP-EOLA1(enhanced green fluorescent protein-EOLA1)融合蛋白的ECV304细胞株中发现EOLA1蛋白为全细胞分布。酵母双杂交显示EOLA1蛋白与细胞内金属硫蛋白相互作用,可能参与细
[Abstract]:Sepsis (sepsis) is a precursor of multiple organ dysfunction (MODS) and one of the most difficult problems in critically ill medicine. It is also one of the main causes of death in patients with severe burns and trauma. Its pathogenesis is closely related to the activation of vascular endothelial cells (VEC) to induce and aggravate systemic inflammatory response. The pathophysiological mechanism of sepsis is complex and the current treatment measures are limited. Therefore, it is particularly important for us to study its pathogenesis in depth. In recent years, vascular endothelial cells (VECs) are one of the direct target cells of endotoxin, and the specific receptors for the activation of endothelial cells by LPS at home and abroad. We used suppression subtractive hybridization (SSH) and cDNA terminal amplification technique to clone a novel human cDNA sequence from human umbilical vein endothelial cells stimulated by LPS. As a novel human gene, Northern blot confirmed that it was accepted by the GenBank full-length sequence library no. AY074889. Because it was up-regulated in endothelial cells stimulated by LPS, it was temporarily named endothelial-overestimated lipopolysaccharide-associated factor 1EOLA1. At present, there is no related research on this gene in foreign countries. Our laboratory has done some preliminary research work on the EOLA1 gene. Bioinformatics analysis showed that the gene was 1404 BP, located on chromosome Xq27.4. the encoded protein was composed of 158 amino acids with molecular weight of 17.89 kDa, isoelectric point of 6.43 and good hydrophilicity. The secondary structure of the protein contains 伪 helix, 尾 lamellar structure and 尾 rotation angle, and forms a helico-turn-helix HTH) motif. EOLA1 amino acid sequence contains N-glycosylation site, PKC phosphorylation site and tyrosine kinase 2 phosphorylation site. Northern blot showed that the expression of EOLA1 gene was higher in skeletal muscle, heart, kidney, liver and placenta than in endothelial cells stimulated by LPS, and weakly expressed in colon, small intestine and spleen, but in brain and thymus. There was no expression in lung and peripheral leukocytes. In 7 kinds of cancer cell lines, HL-60, Haila cell line S3, K-562 and A549 were all expressed in promyelocytic leukemia cell line HL-60, Haila cell line S3, chronic myeloid leukemia cell line K-562 and lung cancer cell line A549. The expression of SW480 was the highest in lymphoblastic leukemia (LML) and colorectal adenocarcinoma (Colorectal adenocarcinoma). However, there was no expression of EOLA1 protein in melanoma G-361. The expression of EOLA1 protein was found to be a whole cell distribution in the ECV304 cell line with stable expression of EGFP-EOLA1(enhanced green fluorescent protein-EOLA1 fusion protein after G418 pressure screening. Yeast two-hybrid analysis showed that EOLA1 protein interacted with intracellular metallothionein. Possible participation
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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