摘要
目的:运用DNA家族改组方法提高水蛭素的抗凝血酶活性。方法:将水蛭素HV1、HV2、HV3基因等量混合,用DNaseI消化,回收50bp左右的片段,再经无引物和有引物PCR扩增获得与原基因大小相同的改组分子,构建噬菌体展示的水蛭素改组文库,经凝血酶亲和淘选,ELISA筛选高活性的水蛭素变异体。结果:经过DNA改组和噬菌体亲和筛选,成功获得抗凝血酶活性提高的水蛭素变异体HV2-N47K。结论:用DNA家族改组及亲和淘选技术能够筛选出高活性的水蛭素变异体蛋白,这为其他分子的改组提供了借鉴。
AIM： To directed promote the antithrombotic activity of hirudin in vitro with DNA family shuffling method. METHODS： PCR products of HV1, HV2 and HV3 genes of hirudin were combined and digested by DNase I. Then random fragments about 50 bp were purified and reassembled to the same size of hirudin gene by two amplifications of PCR with or without primers. The shuffled hirudin genes were inserted into phagemid pCANTAB5E vector and the hirudin mutants were displayed on the surface of bacteriophage M13. Mutants with increased specific activity of antithrombin were screened by affinity panning with decreased amounts of thrombin, RESULTS, After shuffling and selection, a clone （HV2-N47K） with high antithrombotic activity was obtained, CONCLUSION： Hirudin variants with improved properties could be hopefully aquired by DNA family shuffling and affinity panning.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期167-170,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30200359)
