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目的通过毕赤酵母(Pichia pastoris)表达制备重组人凝血酶原2(prothrombin-2),并利用锯鳞蝰(Echis carinatus)凝血酶原激活剂ecarin将其活化为凝血酶,分析凝血酶的活性。方法根据Gen Bank公布的人凝血酶原2和ecarin的cDNA序列,设计并优化合成凝血酶原2和ecarin基因。将凝血酶原2基因构建至表达载体p PICZαA中,转化并筛选重组凝血酶原2的糖基工程酵母菌,诱导工程酵母分泌表达凝血酶原2,培养上清中的目的蛋白经两步阳离子层析纯化制备,并利用酶切N-糖链和肽指纹图谱分析鉴定目的蛋白。将ecarin基因构建到表达载体pcDNA3.1(+),瞬转HEK 293T细胞,收集培养上清。将HEK 293T工程细胞培养上清与纯化的凝血酶原2反应,利用凝血酶发色底物S-2238,测反应产物的酶促活性;利用血浆纤维蛋白原测反应产物的血凝时间、分析血凝活性。结果酵母表达凝血酶原2的培养上清经纯化获得37×103的目的蛋白,去除N-糖链的蛋白相对分子质量降为35×103,与凝血酶原2理论分子量一致。经肽指纹图谱鉴定,纯化得到的蛋白为凝血酶原2。制备的凝血酶原2经HEK 293T细胞表达ecarin的培养上清处理后,可催化S-2238解离产生黄色的对硝基苯胺(pNA),且pNA产生的光密度值随着处理的凝血酶原2的增加而升高;经ecarin处理的凝血酶原2能促进血浆凝结,与凝血酶试剂的血凝时间接近,且血凝时间随着处理的凝血酶原2的量减少而延长。结论利用毕赤酵母制备了重组人凝血酶原2,被ecarin活化为具有活性的凝血酶,有望替代从血浆中提取的凝血酶原,用于战伤救治和临床研究。
OBJECTIVE: To prepare recombinant human prothrombin-2 by expression of Pichia pastoris and activate it with thrombin using ecarin, an Echis carinatus prothrombin activator, to analyze the activity of thrombin . Methods Based on the cDNA sequences of human prothrombin 2 and ecarin released by Gen Bank, the prothrombin 2 and ecarin genes were designed and optimized. The prothrombin 2 gene was constructed into expression vector p PICZαA, transformed and screened recombinant Saccharomyces cerevisiae 2 Saccharomyces cerevisiae, induction of engineering yeast secretion expression prothrombin 2, the culture supernatant of the target protein by two-step cation Chromatography purification, and the use of enzyme-N-glycan and peptide fingerprinting analysis identified the target protein. The ecarin gene was constructed into the expression vector pcDNA3.1 (+) and transiently transfected into HEK 293T cells, and the culture supernatant was collected. The HEK 293T engineering cell culture supernatant was reacted with purified prothrombin 2, and the enzymatic activity of the reaction product was measured by using thrombin chromogenic substrate S-2238. The hemagglutination time of the reaction product was measured by using plasma fibrinogen Hemagglutination activity. Results The recombinant protein of prothrombin 2 was purified to obtain 37 × 103 protein. The relative molecular weight of the protein with N-glycan removal was reduced to 35 × 103, which was consistent with the theoretical molecular weight of prothrombin 2. Identified by peptide fingerprinting, the purified protein was prothrombin 2. The prepared prothrombin 2 treated with the culture supernatant of ecarin expressed in HEK 293T cells could catalyze the dissociation of S-2238 to produce yellow pNA (pNA), and the optical density value of pNA produced as the treated thrombin Prothrombin 2 treated with ecarin promoted plasma coagulation, approaching the clotting time of the thrombin agent, and the clotting time was prolonged as the amount of prothrombin 2 treated decreased. Conclusion Recombinant human prothrombin 2 was prepared by using Pichia pastoris and activated by ecarin to be an active thrombin. It is expected to replace prothrombin extracted from plasma for the treatment and clinical research of war injuries.