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目的构建一种无形成病毒及激活原癌基因可能的真核表达载体ｐｃＤＮＡ３．１（＋）ｔＰＡ，以探讨其对纤溶活性影响，方法用分子克隆方法，将人ｔＰＡｃＤＮＡ与真核表达载体ｐｃＤＮＡ３．１（＋）重组，并对重组质粒进行ＤＮＡ测序。结果重组质粒ｐｃＤＮＡ３．１（＋）ｔＰＡ经ＫｐｎⅠ和ＸｂａⅠ酶切后，出现了５．４ｋｂ与２．０ｋｂ两个片段；经ＰｓｔⅠ酶切后出现了７８ｂｐ，４１４ｂｐ，６２２ｂｐ，２．０ｋｂ及４．２ｋｂ五个片段；经ＥｃｏＲＩ酶切后，出现了４７２ｂｐ及６．９ｋｂ两个片段；测序结果证明为人全长ｔＰＡｃＤＮＡ。结论该新型表达质粒的构建为探讨基因防治脑梗死等血栓性疾病奠定了基础。
Objective To construct a possible eukaryotic expression vector pcDNA3.1 (+) tPA without virus and activating proto-oncogene to investigate its effect on fibrinolytic activity.Methods Cloning human tPA cDNA and eukaryotic expression vector pcDNA3 .1 (+) recombinant, and the recombinant plasmid DNA sequencing. Results The recombinant plasmid pcDNA3.1 (+) tPA was digested with Kpn Ⅰ and Xba Ⅰ, and two fragments of 5.4kb and 2.0kb appeared. After digested with Pst Ⅰ, 78bp, 414bp, 622bp, 2.0kb and 4 appeared. 2kb five fragments; after EcoRI digestion, appeared 472bp and 6.9kb two fragments; sequencing results show that the human full-length tPAcDNA. Conclusion The construction of this new expression plasmid lays a foundation for exploring gene therapy for thrombotic diseases such as cerebral infarction.