[Molecular cloning of human vWF/A1 gene and its expression].
To study the mechanism 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. oli. This study will provide a basis for the further clinical and basic research on the role of vWF in thrombosis and hemostasis.
Zhu
Huai-Ping
HP
The First Affiliated Hospital of Suzhou University, Institute of Hematology of Province Jiangshu, Suzhou 215007, China.
Wang
Ying-Chun
YC
Ji
Shun-Dong
SD
Bai
Xia
X
Ruan
Chang-Geng
CG
D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis; D014841:von Willebrand Factor / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014841:von Willebrand Factor