摘要
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(NIH3T3),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NIH3T3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.
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 enzyme digestion and DNA sequencing. The expression of the chimeric protein was confirmed by fluorescent microscope and the t - PA expression level was tested by RT- PCR. The t- PA gene was cloned and the pEGFP- N3 - t- PA vector was constructed. The t - PA expression in the eukaryotic cell could be detected in the group with transfected t - PA gene. The recombinant eukaryotic expression plasmid is successfully constructed.
出处
《山东师范大学学报（自然科学版）》
CAS
2008年第4期109-112,共4页
Journal of Shandong Normal University(Natural Science)
基金
山东省优秀中青年科学家科研奖励基金资助项目(2004BS01012)
