【摘要】 根据t PAcDNA的全长序列 ,设计了扩增t PA信号肽cDNA的引物 ,并进而获得了信号肽cDNA片段。将其回收纯化后 ,作为上游引物 ,结合原有的t PA下游引物 ,分别以t PA的缺失性原核表达载体pErA ,以及后者的点突变体pErA(K)为模板 ,进行PCR反应 ,从而使二者各自增加了信号肽部分。进一步将其分别克隆至pcDNA3 0 ,构建了相应的真核表达载体pCSRA与pCSRK。酶切及测序结果均证明了构建的正确性。经LIPOFECTAMINE[TM] 2 0 0 0Reagent将其分别转染至COS 7细胞 ,并同时设以pCDNA3 0为阴性对照。取转染后不同时间培养上清 ,以FAPA法进行检测。结果表明 ,上述构建载体的表达产物具有良好的溶圈活性。本实验为t PA突变体在真核细胞的稳定表达奠定了基础更多还原

【Abstract】 In according to the full sequence of t-PA cDNA, the cDNA of SP was acquired and purified.Then,pCSRA and pCSRK,eukaryotic expression vectors,were constructed.After both enzyme indigesting and sequencing proved its correction,they were transfected into COS-7 with liposome.The supernatant collected at different time was detected by FAPA.The result indicated that they have good biological activity.This laid a good foundation for the variants of t-PA stable expression in eukaryotic cell expressing system and its large scale production.;更多还原