Abstract：von Willebrand factor (vWF) mediates platelet adhesion through binding to platelet glycoprotein Ⅰb(GPⅠb). vWF domain A1 (vWF\|A1) was involved in binding GPⅠb, ristocetin or botrocetin. To further investigate the mechanism of thrombosis and develop a new remedy of anti thrombosis, reverse transcription PCR was used to amplify cDNA of vWF domain A1 from endothelial cells. After sequence analysis, the amplified DNA fragment was inserted into expression vector with 6×his Tag pET20b(+). The recombinant expression vector was transformed into E.coli (strain DE3) and induced by IPTG. The recombinant fragment, comprising residues 449—728 of mature vWF subunit, was designated rvWF A1. It was purified by chromatography on Ni NTA agarose column and renatured by Tris HCl buffer containing GSH and GSSG. FACS was adopted to analyse the rvWF A1 function of binding to platelets and Chinese hamster ovary cells transfected with pcDNA3.1 GP Ⅰb, meanwhile, platelet aggregometer was applied to detect its effect on the inhibition of ristocetin induced platelet aggregation. The rvWF A1 was expressed in E.coli , accounted for 30% of total bacterial protein. Its purity was over 95% after chromatography on Ni NTA agarose. It was proved to be able to bind GPⅠb. Its rates of binding platelets and the transfected CHO K1 cells were 78 60% and 96 90%, respectively. The activity to inhibit ristocetin induced platelet aggregation was dose dependent, its maximal inhibiting rate was 84 70% at 1 4 μmol/L. The results indicated that high level expression of rvWF A1 was achieved in E.coli and rvWF A1 might be an effective antithromotic agent in preventing thrombosis.
祝怀平,王迎春,赵小娟,季顺东,白霞,阮长耿. 人vWF-A1多肽的融合表达及生物学活性鉴定[J]. 中国生物化学与分子生物学报, 2003, 19(05): 582-587.
ZHU Huai ping, WANG Ying chun , ZHAO Xiao juan, JI Shun dong, BAI Xia, RUAN Chang geng. Expression of von Willebrand Factor-A1 Fragment in E.coli and Identification of Its Biological Activities. Chinese Journal of Biochemistry and Molecular Biol, 2003, 19(05): 582-587.
