摘要
目的:构建人低氧诱导因子1α(HIF-1α)真核表达质粒及其在人肝癌细胞(HepG2)中的表达。方法:从HepG2细胞中提取总RNA,通过RT-PCR逆转录,获得HIF-1α的cDNA,双酶切后构建真核表达载体pcDNA3.1(+),测序验证碱基序列。用脂质体法将质粒pcDNA3.1(+)-HIF-1α转染至HepG2,以免疫荧光和Western Blot法分别对转染细胞进行HIF-1α表达分析。结果:扩增出HIF-1α全长cDNA,构建真核表达质粒,测序结果正确。经检测的质粒转染至HepG2细胞后,HIF-1α蛋白水平高于转染空载细胞。结论:成功构建人HIF-1α基因真核表达质粒pcDNA3.1(+)-HIF-1α,并证明其能在HepG2细胞内表达。
Objective：To construct the eukaryotic expression plasmid of human hypoxia-inducible factor-1α and study its expression in HepG2 cells. Methods：Total RNA was isolated from HepG2 cells and cDNA library was constructed by reverse transcriptional PCR method. The cDNA prepared was inserted into pcDNA3.1（＋） vector. All sequences amplified by PCR were confirmed by complete sequencing. After lipofectamine-mediated transient transfection of HepG2 with pcDNA3.1 （＋）-HIF-1α plasmid, the expression levels of HIF-1α protein were determined by immunofluorescent staining and Western bloting. Results：DNA sequence analysis demonstrated the pcDNA3.1（＋）-HIF-1α plasmid was obtained, which Could express HIF-1α protein in HepG2 in normixa. Conclusion： The pcDNA3./（＋）-H/F-1α plasmid has been successfully constructed with efficient expressions in HepG2.
出处
《交通医学》
2009年第2期119-121,124,共4页
Medical Journal of Communications
基金
国家自然科学基金(30770806)
江苏省普通高校研究生科研创新计划项目
关键词
低氧诱导因子1Α
真核表达
质粒构建
hypoxia-inducible factor-lot
eukaryotic expression
plasmid construction
