人von Willebrand因子A1区基因的克隆和表达

为了深入研究血栓形成的机制以及开发新的抗血栓药物 ,将人vonWillebrand(vWF)因子A1区cDNA在大肠杆菌中表达 ,以获得高效表达的具有生物学活性的重组A1区蛋白 ,应用PCR方法从含有人全长vWFcDNA质粒中扩增A1区基因片段 ,并将其克隆至pUCm T载体中 ,经DNA序列分析后 ,将其重组于有 6×HisTag的融合蛋白表达载体pQE 31中 ,并在大肠杆菌M1 5中诱导表达。采用Ni NTA柱进行纯化 ,用Western印迹鉴定。结果表明 ,PCR扩增得到 854bp的A1区基因片段 ,序列测定结果与文献报道一致。IPTG诱导 5小时后表达的vWF因子A1蛋白占菌体总蛋白的 30 %左右 ,经Ni NTA纯化后其纯度在 95 %以上。Western印迹检测显示A1蛋白具有很好的抗原性和特异性。结论 :成功地在大肠杆菌中高效表达了人vWFA1区蛋白 ,为进一步研究vWF在血栓形成与止血中的作用奠定了基础

Molecular Cloning of Human vWF/A1 Gene and Its Expression

To study the machanism of thrombogenesis and search new anti thrombotic agent, the cDNA for human vWF A1 domain was high level expressed in E.coli and recombinant protein of vWF A1 with biologic activity was obtained. The gene encoding A1 domain was amplified by PCR from plasmid containing full length cDNA of human vWF. After confirming by DNA sequencing analysis, the recombinant expression plasmid pQE31 vWF/A1 was constructed and introduced into E.coli M15 strain, then induced by IPTG; the expressed protein was purified with Ni NTA agarose, identified by Western blotting. The results showed that the 854 bp DNA fragment was obtained by PCR from the plasmid containing full length cDNA for human vWF and its sequence was identical to the published sequence. High level expression of A1 protein was yielded after 5 hour induction, which amounted to 30% of total bacteria protein in inclusion body. Western blot demonstrated it possessed good antigenicity and high specificity. It is concluded that cDNA for vWF/A1 had been cloned successfully, high level expression of A1 protein was achieved in E.coli. This study will provide a basis for the further clinical and basic research on the role of vWF in thrombosis and hemostasis.