组织型纤溶酶原激活剂真核表达载体的构建及其在乳癌细胞系MCF-7中的瞬时表达

Construction of tissue plasminogen activator eukaryotic expression vector and its expression in MCF-7 cell line

目的:构建组织型纤溶酶原激活剂(t- PA)真核表达载体,并检测其在乳癌细胞系MCF-7中的瞬时表达。方法:以含t PAcDNA的质粒PETPFR为模板,PCR扩增目的片段t PA,HindⅢ和SalⅠ双酶切t- PA,HindⅢ和XhoⅠ双酶切pcDNA3真核表达载体。SalⅠ和XhoⅠ的黏性末端互补,体外连接酶切产物,重组体转化感受态JM109细菌,筛选菌落和测序鉴定。以脂质转染胺试剂LipofectamineTM2000将pcDNA3t PA转染入乳癌细胞系MCF-7,G418筛选。应用原位杂交技术,以地高辛标记的t PAcDNA为探针,显示t PA在MCF7中的瞬时表达。结果:电泳获得目的片段t PA的条带2.1kb,初步鉴定为阳性重组体,测序证实为正确的t PA序列。MCF-7细胞原位杂交显示大部分细胞表达阳性。结论:组织型纤溶酶原激活剂真核表达载体构建成功,可在乳癌细胞系MCF7中表达。

Aim ： To construct eukaryotic expression vector of tissue plasminogen activator （t-PA） and detect its transient expression in MCF-7. Methods：The t-PA eDNA was amplified by PCR using PETPFR plasmid containing t-PA eDNA as the templete. The PCR fragment of t-PA was digested with Hind Ⅲ and Sal Ⅰ and PcDNA3 was digested with Hind Ⅲ and Xho Ⅰ. The fragment of t-PA and pcDNA3 were linked in vitro. The recombinant was transformed into the competent JM109 bacterials and was selected by enzymes digestion. The recombinant was indentified by sequencing . The pcDNA3-t-PA DNA was transfected into MCF-7 with LipofectamineTM2000. The t-PA transient expression was demonstrated by in situ hy- bridization using Dig-labeled t-PA eDNA as probe. Results： The fragment of 2 100 bp t-PA was obtained with electrophoresis. The positive clones identified primarily was confirmed by sequencing . The positive signals of in situ hybridization appeared in most cells of MCF-7 cells selected by G418. Conclusion： The eukaryotic expression vector of pcDNA3-t-PA is successfully constructed and can be expressed in MCF-7 cells.