毕赤酵母表达重组人凝血酶原2及活性分析

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GenBank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pPICZαA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。

Expression and analysis of recombinant human prothrombin-2 in Pichia pastoris

Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.