携带tPA基因转移的真核表达载体的构建研究

Construction of pcDNA3.1(+) tPA experession vector

目的 构建一种无形成病毒及激活原癌基因之虞的真核表达载体ｐｃＤＮＡ３．１（＋ ）ｔＰＡ，以探讨其对纤溶活性的影响。方法 用分子克隆方法，将人ｔＰＡ ｃＤＮＡ 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Ｋ ｐｎＩ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ｔＰＡ ｃＤＮＡ。结论 该新型表达质粒的构建为探讨基因防治脑梗塞等血栓性疾病提供了新工具。

Objective To construct a new kind of recombinant vector containing human tissue type plasminogen activator(t PA) cDNA for studing the feasibility of enhancing fibrinolytic activity by transplantation of genetic engineering cells.Methods We recombinated human tPA cDNA with expression vector pcDNA3.1(+) by using the method of molecular cloning,sequenced the plasmid DNA,and cut the plasmid by using enzymes.Results The plasmid pcDNA3.1(+)tPA was divided into 5.4Kb and 2.0Kb segments respectively by using Kpn I and Xba I,into 78bp,414bp,822bp,2.0Kb,and 4.2Kb segments respectively by using Pst I,into 472bp and 6.9Kb segments respectively by using EcoR I.Sequenoling result showed that it is the whole human tPA cDNA.Conclusions The new kind of recombinant expression vector could serve as new tools and methods for prevention of thrombogenic diseases. [