双作用靶点水蛭素Ⅲ突变体库(R_(33)G_(34)D_(35)X_(36-r)HV3)的构建及最适突变体筛选  

Construction of R_(33)G_(34)D_(35)X_(36-r)HV3 Mutant Library and Screening for Optimal Dual-Site Targeting Hirudin Ⅲ

作　　者：许静[1] 王航[1] 谭树华[1] 

机构地区：[1]中国药科大学生命科学与技术学院分子生物学教研室,江苏南京210009

出　　处：《药物生物技术》2011年第3期195-200,共6页Pharmaceutical Biotechnology

基　　金：国家自然科学基金(No.30873191)资助项目

摘　　要：构建水蛭素Ⅲ(HVⅢ)突变体库R33G34D35X36-rHVⅢ,从中筛选出具有与重组水蛭素Ⅲ(rHV3)抗凝血酶活性比活力相当,同时又具有很强的抗ADP诱导的血小板聚集抑制率的双作用靶点水蛭素Ⅲ突变体。通过基因定点突变技术构建水蛭素Ⅲ突变库R33G34D35X36-rHVⅢ,表达纯化后通过凝血酶滴定方法测定其抗凝血酶比活性,同时测定其抗ADP诱导的血小板聚集比活性。结果显示所有突变体的抗凝血酶比活性未发生变化,但各突变体抗ADP诱导的血小板聚集的比活性各异,当第36位为Met和Ser时所表现的抗ADP诱导的血小板聚集的比活性最强。由此可知水蛭素Ⅲ(HVⅢ)突变体库R33G34D35X36-rHVⅢ中第36位氨基酸种类对其抗凝血酶活性影响不大,但对其抗ADP诱导的血小板聚集的比活性影响较大,当该位点为Met和Ser时突变体具有很好的抗凝血酶和抗ADP诱导的血小板聚集的双重药理活性。To construct recombinant hirudinⅢ（rHVⅢ） mutant library R33G34D35X36-rHVⅢ and to screen out the mutant which was well-matched in the anti-thrombin specific activity aganist recombinant hirudin Ⅲ（rHV3）,and also had bifunctional target with a strong activity of ADP induced platelet aggregation inhibition.Site-directed mutagenesis was used to build up hirudin Ⅲ mutant library R33G34D35X36-rHV Ⅲ.After expression and purification,the anti-thrombin specific activity was determined by titration of thrombin and the anti-platelet aggregation induced by ADP inhibition was also measured.The results showed that there was no difference in the anti-thrombin specific activity between all mutants,but there was difference in the ADP-induced platelet aggregation inhibition activity,which was the strongest when the site 36 was Met or Ser.These results indicated that the site 36 amino acid has little effect on the anti-thrombin specific activity in the recombinant hirudin Ⅲ（rHV Ⅲ） mutant R33G34D35X36-rHV Ⅲ,but it had a great influence on the activity of ADP induced platelet aggregation inhibition,especially when the site was Met or Ser.The mutant showed dual pharmacological activity-anti-thrombin activity and activity of ADP induced platelet aggregation inhibition.

关 键 词：水蛭素Ⅲ 定点突变 抗血小板聚集 RGD序列 

分 类 号：Q516[生物学—生物化学] Q81