刺激ECV304细胞增殖的新基因EOLA1的克隆和功能研究

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．BMl21646）全长eDNA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Noithem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-overexpressed lipopolysaccharide-associated factor 1，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EOLA1 cDNA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（metallothionein 2A，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。

Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation

Objective To amplify the full-length cDNA and characterize the structure and biological function of a novel expression sequence tag ST55 (GenBank Accession No. BM121646). Methods Rapid amplification of cDNA ends was used to clone the full-length of cDNA of ST55 in this study. Then, its tissue distribution was checked by Northern blots, and the associated protein was screened by GAL 4-based yeast two-hybrid. The effect of stable transfection of the cDNA on cell proliferation was evaluated in ECV304 cells. Results A full-length 1404 bp cDNA was cloned, and it was accepted as a novel human mRNA by GenBank (No. AY074889), named endothelial-overexpressed lipopolysaccharide-associated factor 1 (?EOLA1). Bioinformatic analysis found that the?EOLA1 encoded 158 amino acids, 17.89 kDa protein, and mapped to chromosome Xq27.4 with 5 exons.?EOLA1 expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. The interaction between EOLA1 and MT2A was confirmed by co-immunoprecipitation experiments. Stable transfection of EOLA1 was noted to stimulate ECV304 cell proliferation (P<0.05). Conclusion The findings suggest that?EOLA1 is a novel gene and the interaction of EOLA1 and MT2A may play an important role in cell protection in inflammation reaction.