摘要
目的:构建重组人HIF-lα真核表达质粒,为进行人HIF-lα基因的克隆与表达做准备。方法:从人外周血细胞中提取总RNA,逆转录聚合酶链反应(RT-PCR)法反转录合成cDNA。分别以含有绿色荧光蛋白(EGFP)片段的质粒PIREGFP质粒和反转录合成的cDNA为模板,加入所设计的3对引物[EGFP-linker、HIF-1α)上游(up)和下游(down)引物]所扩增目的基因片段分别与T载体连接后转化大肠杆菌DH5α,LB/Amp平板培养基筛选菌落,提取质粒。测序正确后经酶切先后克隆进入pVAX1载体。结果:通过聚合酶链反应(PCR)分别获得约870、1199和672bp的特异性DNA片段,分别与T载体连接后测序,测序结果与GenBank所提供的基因序列相同。将以上3个片段依次连入pVAX1载体后,所得质粒经酶切鉴定后获得约1800bp片段,与预想结果一致。结论:成功构建了重组人HIF-1α真核表达质粒pVAX1-EGFP-linker-HIF-1α。
Objective To construct the eukaryotic expression plasmid of recombinant human hypoxia-inducible factor-1α（HIF-1α） and get ready for the coloning and expressing of HIF-1α gene. Methods The total RNA was isolated from blood cells, and cDNA library was constructed by reverse transcription PCR method. PIREGFP containing EGFP fragment and cDNA were used as templet, the three designed primers（EGFP-linker, HIF-1α upstream and HIF-α downstream） were put into, after amplification the target gene fragment was inserted into the shuttle vector T, and transfected with E.coli DH5α, the bacterial colonies containing recombinant plasmids were identified by LB/KANA-agar plate, and the recombinant plasmids were extracted and purified. All sequences amplified by PCR were confirmed by complete sequencing. The correct sequences were cloned into the pVAX1 vector. Results The amplified fragments were about 870, 1 I99, 672 bp by PCR, they were inserted into the shuttle vector T and sequenced. The result of sequencing was identical with that provided by GenBank. The final plasmid about 1 800 bp was obtained by the identification of enzyme digestion and it had same molecular size as expected. Conclusion The eukaryotic expression plasmid pVAX1-EGFP-linker-HIF-1α of recombinant human HIF-1α is successfully constructed.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2009年第2期318-321,共4页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅科研基金资助课题(20070105)
关键词
缺氧诱导因子1
载体
质粒
hypoxia inducible factor-1
vector
plasmids
