摘要
采用一种新的PCR定点诱变技术成功地对野生型水蛭素Ⅲ进行了定点诱变 ,这种方法快速、简便 ,有效。通过突变 ,将野生型水蛭素Ⅲ分子的活性功能非必需区的指状结构顶端第 33～36位的氨基酸残基替换为RGDS序列 ,并进行了高效分泌表达 ,表达产物分泌到细胞外发酵液中。改构的水蛭素突变体与野生型水蛭素Ⅲ相比 ,两者的抗凝血酶活性基本一致 ,但体外抗血小板凝集试验结果表明 ,前者除原有的抗凝活性外还具有显著的抗ADP诱导的血小板凝集活性 ,该研究为水蛭素Ⅲ分子的结构功能关系研究以及新型抗凝药物的研制打下了基础。
A hirudin Ⅲ Mutant with replacement of amino acid residues 33～36 with RGDS motif was constructed by a novel PCR directed-mutagenesis method.The mutein was efficiently expressed in E.coli using the L-Asparaginase II signal sequence and the product was secreted into the culture medium.The hirudin Ⅲ mutant shows the similar anticoagulant activity to the native Hirudin Ⅲ,suggesting that residues 33～36 in native Hirudin Ⅲ is not necessary for it to exert its thrombin inhibition activity.However,compared with native hirudin Ⅲ,the hirudin Ⅲ mutant shows the significant inhibition of ADP-induced rat platelet aggregation with an IC 50 of 6.05×10 3ATU/ml. ;
出处
《中国生物工程杂志》
CAS
CSCD
2003年第8期69-73,共5页
China Biotechnology
基金
国家自然科学基金 (No .3 0 0 0 2 14 )
国家教育部霍英东优秀青年教师奖励基金 (No .810 3 2 )资助课题
