纤溶酶抑制剂textilinin-1在毕氏酵母的表达及应用

目的研究真核表达的textilinin-1蛋白纯化品对纤溶酶抑制作用。方法根据textilinin-1的天然氨基酸序列,按照毕氏酵母偏好密码子进行优化,合成textilinin-1基因,重组到pPICZα载体上,并转化至Pichia p.X-33菌种中,实现高效分泌表达,并进行重组textilinin-1活力单位的定义及小鼠断尾试验。结果通过高密度发酵及两步柱层析纯化,最终从10L发酵液中得到6g纯度达97.0%以上的重组textilinin-1。活性研究表明,重组textilinin-1对纤溶酶的活性具有抑制作用,并对tPA所致的小鼠出血倾向有一定的抑制作用。结论重组textilinin-1可抑制纤溶酶活性,并对纤溶性出血小鼠模型有止血作用,具有开发为止血药的潜力。

Expression and application of textilinin-1,a plasmin inhibitor,in Pichia p astoris

Objective To investigate the inhibition of textilinin-1 ,purified protein of eukaryotic expression ,to the plasmin . Methods On basis of the nature amino acid sequence of textilinin-1 and preference codon in Pichia pas-toris ,the synthetic textilinin-1 gene ,restructuring to the pPICZαcarrier ,and into Pichia p .X-33 strains ,was highly secretory expressed .The definition of recombinant textilinin-1 active unit and tail cutting test in mice were also studied . Results 6 g purity of more than 97.0% of recombinant textilinin-1 was received from 10 L fermentation broth by high density fermentation and two-step column chromatography purification .Activity research indicated that recombinant textilinin-1 can inhibit plasmin ,and can also inhibit bleeding caused by tPA in mice . Conclusion Recombinant textilinin-1 inhibits plasmin ,and has hemostatic effect on fibrinolytic hemorrhages mouse models , so it has potential to be used to prevent or reduce bleeding in surgical .