重组人蛋白C在CHO/dhfr+细胞中的表达与分析

目的 :人蛋白C具有重要的抗凝功能 ,与复发性血栓性疾病、蛋白C缺陷症、创伤等有重要关系。血源蛋白C成本高、工艺复杂且存在纯度、稳定性、安全性等问题 ,研制重组人蛋白C具有必要性。目前尚无此类产品上市。本研究拟从人肝细胞中克隆蛋白C的cDNA ,构建人蛋白C的哺乳动物细胞表达载体 ,实现重组人蛋白C在CHO dhfr 中的表达。方法 :用RT_PCR从人胎肝细胞mRNA中扩增人蛋白C的全长cDNA片段 ,将之克隆入pGEM_T载体并测序。目的片段插入真核表达载体pIRESneo以构建重组表达质粒。磷酸钙共沉淀法转染CHO dhfr 细胞 ,G4 18筛选抗性克隆。Western印迹法检测细胞培养上清。HitrapQ进行色谱纯化。表达产物经蛙蛇蛇毒激活后以APTT法测定抗凝活性。结果 :RT_PCR扩增到长 1386bp的DNA片段 ,序列与已报道的人蛋白C一致。所构建的表达质粒pIREShPC转染CHO dhfr 细胞 ,经G4 18加压筛选得到 7株表达细胞。Western印迹法检测发现表达产物能与抗人蛋白C单克隆抗体结合 ,相对分子质量与人蛋白C一致。HitrapQ纯化重组蛋白回收率 >6 0 %。表达产物抗凝活性略低于天然人蛋白C。

Analysis and expression in CHO/dhfr+ of recombinant human protein C

Objective:Human protein C with major physiological importance in the control of hemostasis is a novel antithrombotic agent with a wider therapeutic indices than available anticoagulants. The present study is to develop a strategy for production of human protein C in mammalian cell by recombinant DNA technology.Methods: The cDNA of human protein C was amplified from human liver mRNA through RT_PCR. Then it was cloned into pGEM_T and sequenced. The expression vector pIREShPC for human protein C was constructed from the cDNA fragments coding human protein C and pIRESneo, and was transfected into CHO/dhfr by calcium phosphate DNA precipitates. The transformants were selected by growth on G418 and screened for expression products by SDS_PAGE and Western blot. Recombinant human protein C produced by CHO/dhfr was purified from serum_free media by Hitrap Q (AKTA_Explorer 100, Amersham Pharmacia Biotech). The anticoagulant activity was determined by measuring the prolongation of the clotting time in the activated partial thromboplastin time clotting assay.Results:The cDNA sequence amplified was identical to human protein C cDNA reported. Western blot analysis showed that the recombinant products as a secretion protein with molecular weight of 62*!000 reacted with an anti_human protein C monoclonal antibody. The level of expression for recombinant products was about 20-100*!ng/ml. The recombinant human protein C was purified from conditioned serum_free media on Hitrap Q with a recovery rate of at least 60%.Its anticoagulant activity approximated to 90% of human protein C from plasma. Conclusions:The cDNA fragments coding human protein C were isolated and the successful stable expression of recombinant human protein C in CHO/dhfr was achieved.