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利用寡核苷酸诱导的定位突变技术及DNA重组技术构建了组织型纤溶酶原激活剂(t-PA)的PAI-1结合位点Lys296～Gly302缺失的突变体de1(296～302),K1,K2区去糖基化突变体N117Q/N184Q,以及两者的组合突变体GGI,并在COS-7细胞中获得了暂时性表达,并进一步在CHO细胞中稳定表达了组合突变体GGI.在此基础上对表达产物的生物学特性进行了分析,结果显示de1(296～302)和组合突变体GGI获得了PAI-1抗性,组合突变体GGI在与纤维蛋白亲和力未受损害的同时,特异活性约提高46％,半衰期延长约1倍.此组合突变体很可能成为优于野生t-PA的新型溶栓剂候选株.
A de1 (296 ~ 302) mutant of Lys296 ~ Gly302 with a PAI-1 binding site of tissue-type plasminogen activator (t-PA) was constructed by oligonucleotide-directed mutagenesis and DNA recombination technology. The K1, K2 region deglycosylated mutants N117Q / N184Q, as well as the combined mutant GGI of both, obtained transient expression in COS-7 cells and further stably expressed the combined mutant GGI in CHO cells. Based on these results, the biological characteristics of the expressed product were analyzed. The results showed that PA1-1 was obtained from de1 (296-302) and the combined mutant GGI. The combination mutant GGI was not damaged at the same time as the affinity of fibrin , The specific activity increased by about 46% and the half-life prolonged by about 1. The combination mutant is likely to become a new candidate for thrombolytic agent over wild t-PA.