Tet-on Advanced调控人血管生素-1基因真核表达载体的构建、表达与鉴定

Construction and identification of human Angiopoietin-1 eukaryotic expression vector regulated by Tet-on Advanced system

目的构建pTRE-Tight-Ang-1可调控性真核表达载体,并检测其在心脏细胞中表达的可调控性。方法巢式多聚酶链反应(nested PCR)从肺组织扩增Ang-l,测序正确后将其亚克隆入pTRE-Tight载体中,采用脂质体将pTet-on-Advanced质粒和pTRE-Tight-Ang-1共转染入新生SD大鼠心肌细胞,以不同浓度强力霉素(Dox)诱导,Western blot检测Ang-1蛋白表达水平。结果通过巢式PCR获得了Ang-1全长cDNA,与Genebank比对,序列完全一致。Western blot检测表明,所构建的pTRE-Tight-Ang-1可调控性表达载体能在心肌细胞内表达,表达量呈浓度依赖性。结论该实验成功构建pTRE-Tight-Ang-1真核表达载体,在心肌细胞中的表达受Dox的调控。

[Objective] To construct tetracychne （Tet） --controlled inducible vector pTRE-Tight-Ang-1, and then determinate expression character of Ang-1 under the regulation of Dox in cadioeyte. [Method] Ang-1 gene was amplified by nested PCR from the fresh lung tissue eDNA and the sequence was compared with Genebank data, and then sub-cloned into the pTRE-Tight plasmid after sequence analysis. PTRE- Tight-Ang-1 and pTet-on-Advanced were cotransfected with Lipofectamine 2000 TM to neonatal rat cadiocyte, expression of Ang-1 was assayed by Western blot after giving different concentration of Dox. [Results] The full-length of Ang-1 was obtained by nested PCR and identified by sequencing. Western blot showed that expression of Ang-1 could be induced by Dox in cadiocyte and shown in dose-dependent manner.[Conclusion] Tet-on inducible recombinant vector of pTRE- Tight-Ang-1 was successfully constructed, and its expression can be induced by DOX. This work has laid founda tions for further study on IHD （ischemic heart disease）.