高原鼠兔HIF-1α基因真核表达载体的构建及细胞表达和定位

Construction of Eukaryotic Expression Vector of HIF-1α Gene in Plateau Pika and Its Expression and Localization in Cell

利用PCR从克隆质粒PMD18-T-pHIF-1α中扩增鼠兔HIF-1α;构建重组质粒pcDNA3.0/6Xmyc-p HIF-1α,用重组质粒转染HEK-293T,HeLa和COS-7细胞,通过免疫印迹分析,检测HIF-1α表达蛋白在细胞中的分布。EcoRI和XhoⅠ酶切鉴定和序列分析表明,构建的pcDNA3.0/6Xmyc-pHIF-1α真核表达载体序列正确;HIF-1α在真核细胞中能够正确表达,且其表达产物主要存在于细胞核中。

HIF-1α in cloning plasmid PMD18-T-pHIF-1α was amplified by PCR,and the recombinant plasmid pcDNA3.0/6Xmyc-pHIF-1α was constructed and was transfected into HEK-293T,Hela and COS-7 cells.The distribution of the protein expressed by HIF-1α in cells was detected by western blot analysis.The results of enzyme digestion and sequence analysis showed that the sequence of the constructed eukaryotic expression vector was correct.The HIF-1α gene could correctly expressed in eukaryotic cells and its expression products mainly exist in cell nuclear.