t-PA基因新型真核表达载体的构建及鉴定

目的构建人组织型纤溶酶原激活因子(tPA)基因的新型真核表达载体,为血栓性疾病的基因治疗奠定基础。方法将tPA基因克隆到真核表达载体pcDNA3.1MycHisB()中,进行酶切鉴定及DNA测序分析,并将携带有tPA基因的该真核表达载体,通过脂质体介导,转染鼠血管平滑肌细胞,底物发色法测定转基因血管平滑肌细胞tPA活性。结果经双酶切鉴定和测序证实已将tPA基因DNA片段正确插入到真核表达载体中,并能在真核细胞中表达、分泌有活性的tPA。结论成功构建pcDNA3.1MycHisB()/tPA基因新型真核表达载体。

Construction and Indentification of Human tissue-type Plasminogen Activator Gene Recombinant Eukaryotic Expressing Vector

Objective To construct a recombinant eukaryotic expressing vector pcDNA3.1-Myc-His B(-)/t-PA including functional region of human tissue-type plasminogen activator(t-PA) gene to provide a basis for further study on the therapy of thrombotic disease. Methods The t-PA gene fragment was obtained from PBS/t-PA cloning vector by restricting enzyme digesting and cloning into pcDNA3.1-Myc-His B(-) eukaryotic expression vector. Analysis by restricting enzyme digesting and DNA sequencing was carried out to demonstrate the sequence of the plasmid, and pcDNA3.1-Myc-His B(-)/t-PA was then transfected into VSMC mediated by Lipfectin2000TM. Results It was comfirmed that t-PAcDNA was inserted into the eukaryotic expression vector correctly by using digestion indification and sequencing. The biological activity of t-PA in the media could be detected in the group with transfected t-PA gene. Conclusion The recombinant eukaryotic expression vector pcDNA3.1-Myc-His B(-)/t-PA is successfully constructed.