兔脑组织因子基因在大肠杆菌中的克隆与表达

Cloning and Expression of Rabbit Brain Tissue Factor Gene in Escherichia coli

重组组织因子(recombinant tissue factor,rTF)是新一代凝血酶原时间检测(PT)试剂的主要成分.从新鲜兔脑组织提取总RNA,通过RT-PCR得到TF全长编码基因,并克隆到pMD19-T载体.以测序验证正确的重组子为模板,扩增TF基因并构建成融合表达质粒pMAL-c2X-TF,并将其转化到DH5α中.SDS-PAGE和Western blotting证明,菌体诱导后形成可溶性的融合表达产物,该融合蛋白通过亲和层析纯化和凝血因子Ⅹa水解成为游离的TF蛋白,该蛋白通过脂化,制备成重组PT试剂.凝血试验表明,该试剂对血浆有明显的促凝活性.

The tissue factor gene was amplified by the method of RT-PCR from the template of total RNA extracted from fresh rabbit brain, then cloned into the pMD19-T vector and the DNA sequence was determined. The correct identified recombinant was used as a template for amplification of TF gene by PCR and the product was inserted into the fusion expression vector pMAL-c2X. SDS-PAGE and Western blotting showed that the recombinant pMAL-c2X-TF had expressed a soluble fusion protein in E. coli DH5α. The fusion protein was purified by affinity chromatography ,and the TF protein obtained after Factor X a cut had coagulant activity.