重组小纤溶酶的制备及其生物学活性

目的制备重组小纤溶酶,并检测其生物学活性。方法将重组质粒pET11a-miniPlg转化E.coli BL21(DE3),进行诱导表达。收获菌体,提取包涵体,经复性、层析纯化、尿激酶激活,制备小纤溶酶。进行初步理化鉴定后,采用生色底物法测定其体外活性,采用动物模型进行体内溶栓试验,并与重组组织型纤溶酶原激活剂(rt-PA)进行比较。结果小纤溶酶的表达量约占菌体总蛋白的30%～35%,纯化收率约为15%,得率为4.93mg/g菌体湿重,相对分子质量为38527,N-、C-端氨基酸序列与理论值一致。制备的小纤溶酶比活性为115IU/mg,动物模型溶栓试验中表现出良好的溶栓效果,对纤溶系统和凝血系统影响轻微。结论所制备的重组小纤溶酶具有良好的生物学活性,溶栓效果及安全性均优于rt-PA。

Preparation and Biological Activity of Recombinant Mini-plasmin

Objective To prepare recombinant mini-plasmin and determine its biological activity. Methods Transform recombinant plasmid pET11a-miniPlg to E. coli BL21(DE3) for expression under induction of IPTG. Inclusion body was extracted from the harvested bacteria, then re-naturalized, purified by chromatography, and activated by urokinase. The prepared mini-plasmin was preliminarily identified and determined for in vitro activity by chromophoric substrate method. Perform in vivo thrombolysis test on mini-plasmin in animal models, using recombinant tissue plasminogen activator(rt-PA) as control. Results The expressed mini-plasmin contained 30% ～ 35% of total somatic protein, and its recovery rate after purification, yield, relative molecular mass and specific activity were 15%, 4.93 mg / g wet bacteria, 38 527 and 115 IU / mg respectively. The amino acid sequences at N- and C- terminuses of the prepared mini-plasmin were consistent with the theoretical value. The mini-plasmin showed good thrombolytic effect in animal test but no significant influence on fibrinolysis and blood coagulation systems. Conclusion The prepared mini-plasmin showed good biological activity, and was superior to rt-PA in both thrombolytic effect and safety.