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目的设计和构建新基因内皮高表达脂多糖相关因子1(EOLA1)诱导表达载体,建立EOLA1强制表达模型,观察其对细胞增殖的影响。方法逆转录聚合酶链反应扩增EOLA1开放阅读框片段,引入NoⅠt和XhoⅠ酶切位点,定向亚克隆入可调控真核表达的载体pOPRSVⅠ,测序验证后共转染pOPRSVⅠ-EOLA1和pCMVLacⅠ质粒于ECV304细胞,经潮霉素和G418筛选,获得稳定转染细胞株。对异丙硫半乳糖苷(IPTG)诱导和非诱导的稳定转染细胞株进行计数,绘制细胞生长曲线。结果成功构建了EOLA1可调控真核表达载体pOPRSVⅠ-EOLA1。诱导后第4天,EOLA1稳定表达的ECV304细胞数量为(44±17)×104个,显著多于未诱导细胞的数量(27±11)×104个(P<0.01)。结论强制高表达EOLA1蛋白具有促进ECV304细胞增殖的作用。
Objective To design and construct a new gene expression vector induced by lipopolysaccharide related factor 1 (EOLA1) in endothelial cells and to establish EOLA1 forced expression model to observe its effect on cell proliferation. Methods The open reading frame of EOLA1 was amplified by reverse transcription polymerase chain reaction (RT-PCR). The restriction endonucleases NoⅠt and XhoⅠ were digested with restriction endonucleases. The pOPRSVⅠ vector was subcloned into plasmid pOPRSVⅠ. After sequencing, pOPRSVⅠ-EOLA1 and pCMVLacⅠwere co- ECV304 cells, screened by hygromycin and G418 to obtain stable transfected cell lines. The stable transfected cell lines induced and not induced by isopropylthiogalactoside (IPTG) were counted, and the cell growth curve was drawn. Results The eukaryotic expression vector pOPRSVⅠ-EOLA1 with EOLA1 was successfully constructed. On the 4th day after induction, the number of ECV304 cells stably expressing EOLA1 was (44 ± 17) × 104, significantly higher than that of non-induced cells (27 ± 11) × 104 (P <0.01). Conclusion Forced overexpression of EOLA1 protein can promote the proliferation of ECV304 cells.