目的 构建低氧诱导因子1 α(hypoxia inducible factor-1α,HIF-1α)基因(野生、点突变)的两个慢病毒真核表达载体pEGFP-N1-HIF-1α和pEGFP-N1-muHIF-1 α,以期为后续干细胞的基因转染提供适宜载体.方法 根据野生型人源HIF-1α基因序列信息和确定酶切位点的点突变型序列信息,设计引物,进行聚合酶链反应(polymerase chain reaction,PCR)扩增、然后对目的基因PCR产物及目的载体进行酶切、最后用重组PCR方法将目的片段与目的载体连接的产物转化至DH-5α感受态细胞,挑选阳性克隆进行测序及序列比对,验证重组克隆中插入片段序列与设计的寡核苷酸(oligo)序列是否一致.结果 重组克隆的菌落PCR、重组克隆的酶切结果和质粒克隆测序结果均表明,克隆中插入片段序列与设计的oligo序列一致,HIF-1α基因(野生、点突变)的两个真核表达载体pEGFP-N1-HIF-1α和pEGFP-N1-muHIF-1α构建成功.结论 本项研究成功构建了HIF-1α基因pEGFP-N1-HIF-1α和pEGFP-N1-muHIF-1 α两个慢病毒真核表达载体,为转染骨髓间充质干细胞等后续实验奠定了基础.
Objective To construct two lentiviral eukaryotic expression vectors of pEGFP-N1-HIF-1α and pEGFP-N1-muHIF-1α and identificate the gene sequences.Methods Primers were designed according to wild-type human HIF-1 α gene sequence information and determined restriction sites of human HIF-1α point mutant sequence,taking human HIF-1α and constructed HIF-1α gene mutant clone as a template for polymerase chain reaction(PCR) amplification.Then,the target gene PCR product and purpose vector were digested.Adapter-ligated fragments of the target fragment with purpose vector were transformed into DH-5α competent cells using the recombinant PCR methods.Finally,the positive clones were selected and sequenced to verify whether the sequence of insert fragments in recombinant clones was consistent with oligo sequences designed or not.Results Through recombinant clone colony PCR,enzyme digestion of the recombinant clones and plasmid cloning and sequencing,the fragment sequences inserted in recombinant clones were consistent with the designed oligo sequences.Conclusions The two lentiviral eukaryotic expression vectors of pEGFP-N1-HIF-1α and pEGFP-NI-muHIF-1α were successfully constructed and applicable for follow-up experiments on transfection of bone marrow mesenchymal stem cells.