组织型纤溶酶原激活因子基因真核表达质粒的构建及其体外表达研究

Construction and in vitro Expression of Human Tissue-type Plasminogen Activator Gene Recombinant Eukaryotic Expression Vector

目的构建人组织型纤溶酶原激活因子(t-PA)基因的新型真核表达载体pcDNA3.1-Myc-His B(-)/t-PA,并观察其在人脐静脉内皮细胞株(hUVEC)中的表达情况。方法将t-PA基因克隆到真核表达载体pcDNA3.1-Myc-His B(-)中,经脂质体介导将pcDNA3.1-Myc-His B(-)/t-PA导入hUVEC,半定量逆转录聚合酶链反应(RT-PCR)检测t-PA基因在转染细胞内的转录水平,免疫印迹法(Western blot)检测转染细胞t-PA蛋白表达,底物发色法检测转染细胞t-PA活性。结果经双酶切鉴定和测序证实已将t-PA基因DNA片段正确插入到真核表达载体中,转基因组、空载体组、转绿色荧光蛋白组和空白对照组hUVEC t-PA mRNA相对含量分别(0.92±0.22)(、0.32±0.17)(、0.35±0.17)和(0.27±0.17),转t-PA基因组明显高于对照各组,其细胞培养上清t-PA活性分别为(48.90±8.06)、(5.44±1.09)(、5.17±0.95)和(5.01±1.03)U/106细胞.24h,转t-PA基因组t-PA活性明显高于各对照组(均P<0.01)。用抗Myc标签抗体可检测到转t-PA基因组外源性蛋白质表达,对照组则为阴性。结论成功构建pcDNA3.1-Myc-His B(-)/t-PA基因新型真核表达载体,并能在hUVEC中表达,从而为血栓性疾病的基因治疗研究奠定了基础。

Objective To construct a recombinant eukaryotic expression vector pcDNA3.1-Myc-His B（-）/t-PA including functional region of human tissue-type plasminogen activator（t-PA） gene, and investigate the expression of t-PA gene in transfected human umbilical vein endothelial cell lines （hUVEC）. Methods Recombinant plasmid pcDNA3.1-Myc-His B（ -）/t-PA was constructed by insertion of t-PA cDNA originated from pBS/t-PA into eukaryotic expression vector pcDNA3.1-Myc-His B （-） and transfected into hUVEC line cells mediated by lipofectamine. The transcription and expression of t-PA gene were detected by RT-PCR and Western blot respectively. The t-PA activity was measured by chromogenic substrate assay. Results t- PA cDNA was inserted into the eukaryotic expression vector correctly by using digestion identification and sequencing. In the transfected t-PA gene group, vector group, pEGFP group and blank group, the levels of t-PA mRNA were （0.92±0.22）, （0. 32±0.17）, （0.35±0.17） and （0.27±0.17） respectively. The content of t-PA mRNA in the transfected t-PA gene group was significantly higher than that in the control groups. The t-PA activity in the media was （48.90±8.06）, （5.44±1.09）, （5.17 ±0.95） and （5.01 ±1.03） U/10^6 cells/24 h respectively. The t-PA activity was increased significantly in the transfected t-PA gene group as compared with that in the control groups. The t-PA protein could be detected with anti-myc antibodies in transfected t-PA gene group. Conclusion The recombinant eukaryotic expression vector pcDNA3. 1-Myc-His B（-）/t-PA is successfully constructed and can be expressed in transfected hUVEC cell strains.