CAJ | 학술논문

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.
목적 구건저양유도인자1 α(hypoxia inducible factor-1α,HIF-1α)기인(야생、점돌변)적량개만병독진핵표체재체pEGFP-N1-HIF-1α화pEGFP-N1-muHIF-1 α,이기위후속간세포적기인전염제공괄의재체.방법 근거야생형인원HIF-1α기인서렬신식화학정매절위점적점돌변형서렬신식,설계인물,진행취합매련반응(polymerase chain reaction,PCR)확증、연후대목적기인PCR산물급목적재체진행매절、최후용중조PCR방법장목적편단여목적재체련접적산물전화지DH-5α감수태세포,도선양성극륭진행측서급서렬비대,험증중조극륭중삽입편단서렬여설계적과핵감산(oligo)서렬시부일치.결과 중조극륭적균락PCR、중조극륭적매절결과화질립극륭측서결과균표명,극륭중삽입편단서렬여설계적oligo서렬일치,HIF-1α기인(야생、점돌변)적량개진핵표체재체pEGFP-N1-HIF-1α화pEGFP-N1-muHIF-1α구건성공.결론 본항연구성공구건료HIF-1α기인pEGFP-N1-HIF-1α화pEGFP-N1-muHIF-1 α량개만병독진핵표체재체,위전염골수간충질간세포등후속실험전정료기출.
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.