重组人纤维蛋白原基因在毕赤酵母中的高效表达

High level expression of recombinant human fibrinogen in Pichia pastoris

目的构建含有人纤维蛋白原基因的毕赤酵母表达系统,实现胞外高效分泌表达。方法全基因合成人纤维蛋白原3个基因FGA、FGB、FGG,构建表达载体pGAPZαA-FGB-FGG-FGA-AOX1,线性化后电转化导入毕赤酵母菌株SMD1168H,抗性筛选获得阳性克隆。发酵液经SDS-PAGE确定蛋白表达部位,ELISA检测目的蛋白表达量。表达产物超滤浓缩后利用AKTA蛋白纯化系统进行分离纯化,Western blot检测蛋白表达情况并对纯化产物进行生物学活性测定。结果基因工程菌株摇瓶培养上清液表达量约15mg/L,生物学活性分析重组蛋白具有凝集活性。结论成功获得了高效分泌表达重组人纤维蛋白原的毕赤酵母菌株,且分离纯化的蛋白具有生物凝集活性。

Objective To construct a eukaryotic expression vector in Pichia pastoris containing human fibrinogen gene,in order to achieve high level secretory expression in extracellular. Methods Expression plasmid,pGAPZαA-FGB-FGG-FGA-AOX1,was constructed by inserting the synthesized sequence encoding human fibrinogen（ FGA,FGB,FGG） and then introduced into Pichia pastoris SMD1168 H by electroporation. Transformants were availably screened by Zeocin resistance,the expression of recombinant protein was identified by SDS-PAGE and Western blot analysis,the protein yield was tested by ELISA assay. After ultrafiltration and purification,the biological activity of protein was detected. Results The crude yield of human fibrinogen in Pichia pastoris supernatant reached 15 mg/L in flask and the biological aggregation activity was determined. Conclusion The human fibrinogen gene was obtained and successfully expressed in Pichia pastoris and the active products were secreted into the medium.