人HIF-1α真核表达载体质粒的建立和鉴定

CONSTRUCTION OF pcDNA3-HIF-1α EUKARYOTIC EXPRESSION PLASMID

目的:建立低氧诱导因子lα(HIF-1α)真核表达质粒。方法:从人胎肝细胞中提取总RNA,RT-PCR法反转录合成HIF-1α cDNA,与T载体连接后转化感受态细胞TOP10,测序正确后克隆入pcDNA3真核表达载体,酶切鉴定重组子。结果:获得人HIF-1α真核表达载体质粒,酶切及测序结果证明表达质粒的DNA序列完全正确。结论:成功克隆和建立人HIF-lα基因真核表达质粒,为进一步研究HIF-1α对风湿性疾病的靶向治疗奠定了基础。

Objective： To construct pcDNA3-HIF-1α eukaryotic expression p1αsmid. Methods. The total RNA was extracted from the liver of human fetus, and the intact eDNA of HIF-1α was applied to RTPCR. The product of RT-PCR was digested by Hind Ⅲ and BamH Ⅰ. HIF-1α eDNA was inserted into pGM-T after sequencing, and then cloned into the pcDNA3 vector. Results： The amplified products were confirmed to be the cDNA of HIF-1α by DNA sequencing, and the pcDNA3-HIF-1α obtained was verified by endonuclear enzyme digestion. The sequence of pcDNA3-HIF-1α is identical to HIF-1α sequence in the Genbank. Conclusion： The pcDNA3-HIF-1α eukaryotic expression p1αsmid is successfully constructed and will be useful for further research.