[Eukaryotic expression of von Willebrand factor A1A2A3 triplet and its biological activity].
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.
Key Laboratory of Thrombosis and Hematasis Subordinated to Ministry of Health, Jiangsu Provincial Institute of Hematology, The First Affiliated Hospital, Soochow University, Suzhou 215007, Jiangsu Province, China.
Ting-Ting
TT
Zhang
Yi-Ming
YM
Zhao
Miao
M
Jiang
Chang-Geng
CG
Ruan
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D018911:Trinucleotide Repeats; D014841:von Willebrand Factor / Q000235:genetics*
D011994:Recombinant Proteins; D014841:von Willebrand Factor