人tPA基因真核表达质粒载体构建及其生物学功能研究

构建携带组织纤溶酶原激活物(tPA)基因真核表达质粒pcDNA3.1( )/tPA,并研究其有无生物学活性.用RT-PCR法从人心脏组织中克隆tPA基因并将其克隆至真核表达质粒pcDNA3.1( )中,对pcDNA3.1( )/tPA进行酶切鉴定和测序.脂质体介导pcDNA3.1( )/tPA转染血管平滑肌细胞,分别用Nothern Blot和斑点印迹法从mRNA和蛋白质水平检测tPA的表达,并用纤维蛋白板法测定表达产物的生物学活性.成功克隆了人tPA基因并构建了pcDNA3.1( )/tPA真核表达质粒,pcDNA3.1( )/tPA转染VSMC后,tPA mRNA和蛋白质表达增加,所表达的tPA蛋白质具有纤溶活性.

Construction of eukaryotic expression plasmid containing tPA genes and its expression in transfected VSMCs

In order to clone tissue-type plasminogen activator(tPA)gene,to construct the eukaryotic expres- sion plasmid pcDNA3.1(+)/tPA and to determine its biological activities,the tPA gene was obtained from human heart tissue by RT-PCR.tPA gene was cloned for eukaryotic expression of plasmid pcDNA3.1(+) by recombination strategy.The pcDNA3.1(+)/tPA was identified by restriction enzyme digestion and was sequenced.The pcDNA3.1(+)/tPA was transfected into vascular smooth muscle cell(VSMC)by using lipo- fection.The tPA expression level was detected by Nothern Blot and dot blot and the protein biological activities of tPA were detected by fibrin plate technique.The tPA gene was cloned and pcDNA3.1(+)/tPA was constructed.The tPA expression levels of mRNA and protein highly increased after pcDNA3.1(+)/tPA transfection VSMC and the protein of tPA expression has evident biological activities.The present study will lay a foundation for further animal testing of thrombus treatment resulting from transplanted organ by tPA gene transfecting.