摘要
利用ＰＣＲ扩增和合成突变引物的方法，将ＰＡＬ－１的Ｇｌｕ３５０和Ｇｌｕ３５１分别突变为Ｇｌｙ和Ｌｙｓ，在大肠杆菌中表达并分离纯化突变体ＰＡＬ－１（Ｅ３５０Ｇ，Ｅ３５１Ｋ），用盐酸胍激活并以ＥＬＩＳＡ法确定它与野生型ｒＰＡＩ－１的相对含量。通过对ｕ－ＰＡ，ｔ－ＰＡ抑制的动力学研究表明，突变体与野生型ｒＰＡＩ－１相比，对ｕ－ＰＡ和ｔ－ＰＡ的抑制活性都有明显下降，由活性态向潜伏态转变的半寿期也由０．８３ｈ缩短为０．５７ｈ。
Plasminogen activator inhibitor-1 (PAI-1 ) is a-physiological inhibitor of urine-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA).Using polymerase chain reaction (PCR) amplification and a synthesized mutation primer,a PAI-1 mutant, PAI-1 (E350G, E351K), was generated. The PAI-1 mutant expressed in E. coli was separated, followed by the activation with Guanidine HCI,and then the relative amounts of the mutant and wild type PAI-1 were determined by double-antibody ELISA. The kinetic studies showed that the second order rate constants , K_1, of wild type PAI-1 ,was 3. 38± 0. 20 towards u-PA and 3. 24± 0. 26 towards t-PA, while the K1 values of the mutant was 2. 84 ± 0. 12 towards u-PA and 2. 24± 0. 45 towards t-PA. It was also found that the half life of the mutant,for conversion from active form to latent form ,changed from 0. 83± 0. 11 h of wild type PAI-1 to 0. 57 ± 0. 04 h.The results suggested that the simultaneous mutations of Glu350 and Glu351might influence the inhibitory activities of PAI-1 towards u-PA and t-PA,as well as shorten the half life of its active form.
