pcDNA3.1(+)/t-PA真核表达质粒的构建及其在血管内皮细胞中的表达

Cloning t-PA gene and constructing pcDNA3.1 (+)/t-PA expression vector

目的 利用基因工程技术克隆人组织纤溶酶原激活物 (t- PA)基因并构建一种能在血管内皮细胞 (VEC)中高效、安全表达 t- PA的 pc DNA3.1(+) / t- PA真核表达重组质粒。方法 采用高效 Trizol试剂快速从胎儿肺组织中提取总 RNA,RT- PCR获得 t- PA c DNA,并将真核表达质粒 pc DNA3.1(+)和 t- PA基因片段分别双酶切 ,将回收的 pc DNA3.1(+)大片段 (5 .0 kb)与 t- PA基因片段 (1.9kb)重组。对 pc DNA 3.1(+) / t- PA质粒进行酶切鉴定和测序。脂质体介导 pc DNA3.1(+) / t- PA转染血管内皮细胞 ,并用 RT- PCR法和 EL ISA法分别从 m RNA水平和蛋白质水平检测 t- PA的表达情况。结果 成功地从胎儿肺组织中克隆了 t- PA基因 ,并构建了以 pc DNA 3.1(+)为载体的真核表达质粒载体。pc DNA3.1(+) / t- PA转染血管内皮细胞后 ,t- PA表达增加 ,(n=6 ,P<0 .0 1)。结论 含t-

Objective Clone tissue-type plasminogen activator (t-PA) gene and construct a recombinant eukaryotic expression plasmid containing t-PA cNDA that could be highly expression in vascular endothelium cell(VEC). Methods The t-PA cDNA was obtained from fetal lung with RT-PCR and inserted into HindIII and BamhI site of the plasmid pcDNA3.1(+), then,pcDNA3.1(+)/t-PA was transfected into VEC by DEAE-Dextrana method,and the t-PA expression level was tested by RT-PCR and t-PA ELISA kit. Results The t-PA gene was cloned and pcDNA3.1(+)/t-PA was constrcted, and the t-PA expression level after pcDNA3.1(+)/t-PA transfection VEC was highly (n=6, P<0.01). Conclusion The recombinant eukaryon expression plasmid is successfully constructed.