Eukaryotic expression of von Willebrand factor A1A2A3 triplet and its biological activity / 中国实验血液学杂志

ABSTRACT

The aim of this study was to construct the eukaryotic expression vectors harboring human vWF-A1A2A3 gene and to investigate its expression in CHO cells and biologic function so as to provide a basis for further exploring the biologic activity of vWF-A1A2A3. The primers were designed according to published sequences; the human vWF-A1A2A3 was amplified by PCR from vWF cDNA; the fragment of interest was inserted into eukaryotic expression vector pSectag2b by using restriction enzyme and ligase after vWF-A1A2A3 was confirmed by sequencing. The recombinant expression plasmid was transfected into CHO cells and the stable expression product (rvWF-A1A2A3) was detected by using Western blot. The results showed that the eukaryotic expression vector pSectag2b-A1A2A3 was successfully constructed and expressed its corresponding protein efficiently. The recombinant protein was identified to be able to bind collagen and ristocetin-induced platelets. It is concluded that the pSectag2b-A1A2A3 is successfully constructed and can express in CHO cells. The rvWF-A1A2A3 protein established in this study provides a basis for the further study on its biological structure, function and clinical application.