【摘要】 目的构建重组人HIF-1α真核表达载体质粒,为进行治疗性血管再生研究做准备。方法从Hela细胞中提取总RNA,RT-PCR法反转录合成cDNA。设计两对引物分别调取目的基因片段A和B,与T载体连接后转化大肠杆菌JM109,LB平板培养基筛选菌落,抽提质粒。测序正确后双酶切先后克隆进入pcDNA4/HisMaxA载体。结果获得重组的人HIF-1α真核表达载体质粒,经PCR扩增获得的目的基因分子量与预计的相同,插入pcDNA4/HisMaxA载体部位正确。结论重组的人HIF-1α,所选用的pcDNA4/HisMax可高表达目的基因,为利用其进行治疗性血管再生研究奠定了基础。更多还原

【Abstract】 Objective To construct the eukaryotic expression plasmid pcDNA4-rhHIF-1α of recombinant human hypoxia-inducible factor-1α.Methods Total RNA was isolated from Hela cells,and cDNA library was constructed by reverse transcriptional PCR method.The two oligomers of primers were synthesized based on the reported sequences of HIF-1α,the cDNA prepared as above was used as the template,and the HIF-1α fragment A(amino acids 1～390) and fragment B(amino acids 786～826) were constructed and inserted into the shuttle vector T.Transformation of E.Coli JM109 with recombinant plasmids and identification of bacterial colonies containing recombinant plasmids by LB-agar plate containing 100 μg/ml of amplicillin were conducted,and recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.Correct sequences were cloned into the pcDNA4 vector.Results Recombinant eukaryotic expression plasmid pcDNA-rhHIF-1α was gained.The target gene obtained PCR amplification had the same molecular size as predicted.It was indicated that recombined pcDNA4 plasmid contained correct recombinant human HIF-1α sequences. Conclusion The pcDNA4 is high expressing eukaryotic vector,and can express the target gene in high level.The pcDNA4-rhHIF-1α plasmid is constructed successfully and will be useful for further research.更多还原