t-PA cDNA基因在AGZY83-a细胞系的稳定表达

目的 建立稳定表达人组织型纤溶酶原激活剂(tissuc-type plasminogen activator,t-PA)的细胞系。方法 将t-PA的反义DNA(t-PA cDNA)连接在真核表达载体pcDNA3．1( )的Kpn I和Xba I酶切位点上,构建成重组质粒pcDNA3．1( )t PA,用FuGENE6介导法转入人类AGZY83-a细胞系中．经G418筛选形成阳性克隆,接种于培养基中培养,检测表达产物。结果 各种酶切图谱及筛选后单克隆形成,证明tPA cDNA已正确组装在载体上．培养液中rt-PA的含量高于对照组且可持续12周,表明pcDNA3．1( )tPA已可在AGZY83-a细胞中得到表达。结论 转染tPA cDNA的AGZY83-a细胞已可稳定表达人类tPA,为血栓性疾病的基因治疗奠定了一定的基础。

Stable Expression of t - PA cDNA in AGZY83-a Cells

Objective To establish a cell strain secreted recombinant tissue- type plasminogen activator (rt - PA). Methods Recombinant plasmid pcDNA3. 1tPA was constructed by insertion of t - PA cDNA o-riginatcd from pATA18tPA into cukaryotic expression vectors pcDNA3. 1 at Kpn I and Xba I sites, and trans-fccted into AGZY83 - a cell by FuGENE 6 mediated transfcction methods. After screened with G418, the positive clones were selected and further purified in conditional culture medium. Results Immunoreactive human t - PA of the medium was more than 32 ng/24 h. The expression level of t - PA cDNA clone did not change after 85days. Conclusions A genetic engineering cell strain secreted rt - PA is successfully constructed.