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目的扩增内皮细胞受内毒素刺激后上调表达的新序列标签ST55(GenBank No.BM121646)全长cDNA序列并研究其结构和生物学功能。方法应用快速扩增cDNA末端技术延伸ST55获得其全长cDNA序列,以Northern杂交检测其组织分布,通过酵母双杂交筛选其胞内相互作用蛋白,在ECV304细胞中稳定转染目的基因并强制表达后观察细胞生长变化。结果获得1个全长为1404碱基的cDNA序列,作为人类新mRNA被GenBank接受(AY074889),命名为内皮高表达脂多糖相关因子1(endothelial-overexpressedlipopolysaccharide-associatedfactor1,EOLA1)。生物信息学分析显示EOLA1基因含5个外显子,定位于染色体Xq27.4,编码蛋白质由158个氨基酸组成,分子量为1789。Northern印迹显示EOLA1在人各组织和癌细胞系有不同的表达;以EOLA1cDNA作为诱铒,进行酵母双杂交筛选人肝cDNA文库,鉴定了金属硫蛋白2A(metallothionein2A,MT2A)为其相互作用蛋白并采用免疫共沉淀验证了这一结果。高表达EOLA1显著促进了ECV304细胞增殖。结论EOLA1是人内皮活化相关新基因,EOLA1与MT2A的相互作用可能在炎症反应中细胞内保护方面发挥作用。
Objective To amplify the full-length cDNA sequence of ST55 (GenBank No. BM121646), which is up-regulated after endotoxin stimulation in endothelial cells, and to study its structure and biological function. Methods The full-length cDNA sequence of ST55 was amplified by rapid amplification of cDNA ends. The tissue distribution was detected by Northern blotting. The intracellular interaction proteins were screened by yeast two-hybrid system. The target gene was stably transfected in ECV304 cells Observation of cell growth changes. As a result, a cDNA sequence of 1404 bases in length was obtained, which was accepted as a new human mRNA by GenBank (AY074889) and named as endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1). Bioinformatics analysis showed that the EOLA1 gene contains five exons, located on chromosome Xq27.4. The encoded protein consists of 158 amino acids and has a molecular weight of 1789. Northern blotting showed that EOLA1 was expressed differently in human tissues and cancer cell lines. Yeast two-hybrid screening of human liver cDNA library using EOLA1 cDNA as bait and identification of metallothionein 2A (MT2A) as an interacting protein Co-immunoprecipitation demonstrated this result. High expression of EOLA1 significantly promoted the proliferation of ECV304 cells. Conclusion EOLA1 is a new gene related to human endothelial activation. The interaction between EOLA1 and MT2A may play an important role in the intracellular protection of inflammatory response.