pEGFP-N3-t-PA真核表达质粒的构建及其在成纤维细胞中的表达

目的利用基因工程技术克隆人组织纤溶酶原激活物（t—PA）基因并构建一种能高效、安全表达t—PA的pEGFP—N3-t—PA真核表达重组质粒，为进一步研制转基因动物奠定基础．方法采用高效Trizol试剂快速从黑色素瘤细胞中提取总RNA，RT—PCR获得t—PAcDNA，并将真核表达质粒pEGFP—N3和t—PA基因片段分别双酶切，将回收的pEGFP—N3大片段（4．7kb）与t—PA基因片段（1．1kb）重组．对pEGFP—N3-t—PA质粒进行酶切鉴定和基因测序鉴定．脂质体介导pEGFP—N3-t—PA转染成纤维细胞（NIn313），并用RT—PCR法从mRNA水平检测t—PA的表达情况，用倒置荧光显微镜检测、分析其在NH 3T3细胞中的表达．结果成功地从黑色素瘤细胞中克隆了t—PA基因，并构建了以pEGFP—N3为载体的真核表达质粒载体，并能在真核细胞中表达、分泌t—PA．结论含t—PA基因真核表达质粒构建成功．

CLONING OF THE t- PA GENE AND CONSTRUCTION OF pEGFP- N3 - t- PA EXPRESSION VECTOR

Objective To construct a recombinant eukaryotic expressing vector pEGFP-N3-t-PA including functional region of human tissue-type plasminogen activator(t-PA) gene to provide a basis for further study on the research of transgenic animals.Methods The t-PA cDNA was obtained from melanoma cell with RT-PCR and inserted into the Hind III and BamH I site of the plasmid pEGFP-N3,then,pEGFP-N3-t-PA was transfected into NIH 3T3 mediated by Lipfectin 2000TM.The recombinant plasmid was proved successful by restriction ...