【作者】 周广举；

【导师】 梁自文；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 硕士

【摘要】 人类基因组计划完成后,绝大多数基因得到了定位。深入研究这些基因的功能,阐明其在生命活动中的意义成为后基因组时代的主要任务。迄今为止,大部分基因功能得到了较深的认识,但还有部分基因的功能和转录调控机制仍不清楚,这其中包括内皮高表达脂多糖相关因子1(EOLA1)基因。EOLA1基因是我们实验室在前期研究工作中应用抑制消减杂交(SSH)和cDNA末端扩增技术(RACE),从脂多糖(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,故命名为内皮高表达脂多糖相关因子1(endothelial-overexpressed lipopolysaccharide-associated factor 1, EOLA1)。对该基因的特性已完成的研究显示其转录序列全长为1404个bp,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、蛋白激酶C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,在正常成人心脏、骨胳肌、肾脏、肝脏和胎盘有较强的表达,在脾脏、结肠和小肠表达较弱,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、慢性白血病K-562细胞系都有表达,以在白血病细胞系MOLT-4和腺癌细胞系SW480表达为最高,但在肺癌细胞系A549和黑素瘤细胞系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,调控细胞增殖,参与炎症反应时细胞内保护机制。但确切的EOLA1基因功能和其调控机制仍不清楚。为帮助明确基因的功能,了解基因在不同组织和细胞中差异表达的原因,阐明其转录调控机制是必不可少的步骤。基因转录调控主要由启动子、增强子和沉默子、顺式作用元件和与其相互结合的反式作用因子构成。其中,启动子提供了基本的转录活性,多数基因启动子具有典型的TATA盒结构,而不具有TATA盒的启动子基因,常有多个转录起始位点。有研究显示EOLA1基因mRNA转录本存在两种不同的剪切形式,其原因可能与转录起始位点不同相关。另外,转录因子与顺式作用元件的结合既可能增强启动子的活性促使转录,也可能抑制启动子的活性阻滞转录,从而使目的基因在不同细胞或同一细胞不同状态时表现出转录的差异。为此,本研究首先克隆EOLA1基因5’-侧翼区约2.4 kb的基因组DNA序列,再缺失突变为两个不同长度的片段,分别插入双荧光素酶报告基因表达质粒,转染细胞后,瞬时表达,通过检测报告基因活性的变化来反映5’-侧翼区内启动子区域,利用生物信息学方法预测其启动子并与实验结果相对照,为全面阐明EOLA1表达调控机制提供基本数据。本研究的主要结果和结论如下:培养ECV304细胞,从该细胞中抽提基因组DNA,并以该基因组DNA作模板采用PCR方法扩增到EOLA1基因上游3个不同长度的基因片段,然后应用pGL3-Basic空质粒和PCR产物构建重组质粒,转化到大肠杆菌DH5a,筛选阳性克隆,构建得到不同长度片段启动子-荧光素酶载体:pGL3-Basic -1076bp、pGL3-Basic-1723bp、pGL3-Basic-2426bp。并经限制性内切酶酶切鉴定,测序证明克隆基因片段插入方向正确,插入的接头处核苷酸序列正常,未发现碱基突变。因此pGL3-Basic-EOLA1系列重组体构建成功。将上面构建所得的重组质粒与内参照质粒pRL-TK共转染ECV-304细胞,检测报告基因相对荧光活性结果显示,与pGL3-Basic空载体相比,3种构建的质粒(pGL3-Basic-1076bp、pGL3-Basic-1723bp、pGL3-Basic-2426bp)荧光活性均明显增强(p﹤0.01或p﹤0.05),提示相应的基因序列均具有启动报告基因转录的能力。其中pGL3-Basic -1723bp的转录活性最高。利用生物信息学软件预测启动子所在区域结果显示:在EOLA1基因5’侧翼区-671～-721区存在启动子的预测值为1.00分,在-74～-124区预测值为0.96,在-2091～-2141区预测值为0.83。而-671～-721和-74～-124均位于-1672～+51内,因此预测结果与本实验的结果是一致的。总之,本研究的结果表明EOLA1基因启动子存在于EOLA1基因5’侧翼区-1672～+51内,为进一步研究该基因的表达调控机制和功能奠定了基础。更多还原

【Abstract】 After finishing the human genomic plan,most genes have been located.Identification and characteristicing the human genes has been the primary work in back genomic era. Now, the function and transcription mechanism of most genes have been found, but some novel genes also maintain unkown,including EOLA1(endothelial-overexpressed lipopolysa -ccharide-associated factor 1, EOLA1).Endothelial cell (EC) is one of the main target cells that are directly activated by LPS, and many researches have been done in signal transmitting, specific receptor and the route of action in EC by LPS stimulation. The EOLA1 gene (GenBank Accession No. AY074889) was cloned by our laboratory by the effective method of mRNA differential display (differential gene expression between normal ECV304 and thosed treated with LPS). Using the SMART RACE technique, the full-length cDNA of the EOLA1 gene was cloned. This gene is 1404 base pair (bp) long and the open reading frame is 474bp, encoding a 158aa product. This gene locates at the human chromosome Xq27.4, containing 5 exons spans about 6294bp.α-helix,β-Lamellosa,β-turn and a helix-turn-helix (HTH) motif were found by bioinformatics analysis. This information indicated that EOLA1 may be as a transcription factor, which plays an important role in the process of activating Human EC.Northern blot analysis between EOLA1 and some human tissues and carcinoma cells revealed an extended expression profile of EOLA1. It was highly expressed in skeletal muscle, heart ,liver, kidney, placenta and expression in the spleen, colon and small intestine, and expression in brain, thymus, lung and peripheral blood leukocyte was not observed. EOLA1 was found to be expressed in different human cancer cells, such as promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4 and colorectal adenocarcinoma SW-480. Expression in lymphoblastic leukemia MOLT-4 and colorectal adenocarcinoma SW-480 was especially highly. But it was not found in lung carcinoma A-549 and melanoma G-361.The enhanced green fluorescent protein-EOLA1 was found in the whole ECV-304 after the pressure bolting of G418.The yeast two-hybrid experiment has showed the interaction of EOLA1 and MT2A, which may play some roles in the apoptosis, growth and anti-inflammation course in human EC. But the biological function of EOAL1 and its regulation is unknown.It is important to make the transcriptional control known before understanding the differential expression of identical gene. The transcriptional control system consists of promoter, enhancer, silencer, cis-acting element and the related trans-acting element, but it is the promoter that determines the basic transcriptional control. Most of the gene promoters have the TATA box, or there are many transcriptional start sites. There are two types of mRNA in EOLA1 due to the different transcriptional start site. Moreover, the transcriptional factor combining with the cis-acting element can boost the transcriptional activity of the promoter, or hold up its transcription. This is the reason why genes display different transcriptional activity in different cells or at the different stage in the same cell.The aim of this research was to seek the relatively accurate position of the promoter region of EOAL1 by Polymerase chain reaction (PCR), nested deletion, recombination , dual-luciferase reporter assay and Bioinformatics analysis, and this would be the foundation of subsequent research.First the genomic DNA was extracted from the ECV-304 cells, and this genomic DNA was used to clone the upstream regions of EOAL1 with different length. Then these DNA fragments were cloned into the luciferase reporter vector, pGL3-Basic, and three recombinant vectors with different length of upstream fragment of EOAL1 were obtained, naming pGL3-Basic-1076bp、pGL3-Basic-1723bp、pGL3-Basic-2426bp.These recombinant vectors were identified by specific endonuclease digestion and gene sequencing analysis to warrant the correctness. Then these vectors were contransefected into the ECV-304, with a interior control pRL-TK. Compared with pGL3-Basic,the relative luciferase activities of pGL3-Basic-1076bp, pGL3-Basic-1723bp and pGL3-Basic-2426bp are remarkably enhanced, and the improvement of pGL3-Basic-1723bp was especially significant. Bioinformatics analysis by promoter prediction program shows the region between -671 bp and -721 bp was the most probable promoter location region, with the score of 1.00. This result was consistent with that obtained in the reporter gene system.In conclusion, this research specified the promoter of EOAL1, which located between the region of -1672bp～+51bp. This work is of important means for the following research on the regulation and expression of EOAL1.更多还原