摘要
目的:为了研制新型特异性抗血栓制剂。方法:利用噬菌体展示文库技术筛选与vWF- A1区有高亲合力单链抗体(ScFv) ;基因重组的方法构建高效表达载体pET 2 0b(+) ScFv ,在大肠杆菌中诱导表达;夹心ELISA方法鉴定此单抗的抗原结合活性;瑞斯脱霉素诱导的血小板聚集试验(RIPA)测定ScFv对血小板聚集的抑制作用。结果:噬菌体展示技术筛选的ScFv在大肠杆菌中成功地诱导表达,表达的ScFv占菌体总蛋白的4 1% ,以包涵体形式存在;经纯化复性的ScFv可以与vWF、rvWF- A1、rvWF- A1 A3结合,但不与rvWF A3、BSA反应;ScFv具有抑制RIPA功能,抑制率为73. 7%。结论:在大肠杆菌中高效表达的噬菌体展示技术筛选的抗vWF A1区ScFv可特异性与vWF- A1区结合,而抑制瑞斯脱霉素诱导的血小板聚集,显示出很好的应用前景,为研制新型抗栓药物奠定基础。
Objective:For development of drugs for the treatment or preention of arterial thrombosis.Methods:The single chain antibody with high affinity to vWF-A1 was screened with phage display technology;and high-level expression vector(pET20b-ScFv) was constructed,then was transformed into E.coli (DE3),and induced by IPTG.Sandwich ELISA was applied to identification of the ScFv binding to antigens.Ristocetin-induced platelet aggregation assay was adopted to detect the inhibitory effect of the ScFv on RIPA.Results:High level ScFv was expressed in DE3,the target protein amounted to 41% of total protein,the yield of ScFv was 850 mg/L.The resulting protein was refolded by dialysis.The ScFv specially reacted with vWF,rvWF-A1,rvWF-A1/A3,not with rvWF-A3,BSA,meanwhile,it inhibited with an IC 50 of 0.83 μmol/L,its maximal inhibiting rate was 73.7%.Conclusion:Our data suggest that the single chain antinody with high affinity to vWF-A1 possess characteristic of inhibition of RIPA,and it could be a new agent for the treatment or prevention of arterial thrombosis.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2005年第4期308-311,314,共5页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目 (3 0 0 70 3 2 2 )
关键词
VWF
血栓
抗体/ScFv
表达
vWF
Thrombosis
Antibody/signle chain Fv
Expression
