论文部分内容阅读
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。
Objective: To improve the antithrombin activity of hirudin by using the DNA family shuffling method. METHODS: The hirudin HV1, HV2, and HV3 genes were mixed in equal amounts, digested with DNase I, and 50 bp fragments were recovered. PCR was performed with and without primers and primers to obtain the same shuffling molecule of the same size as the original gene. The reprogrammed library was screened by thrombin affinity and ELISA was used to screen highly active hirudin variants. RESULTS: After DNA shuffling and phage affinity screening, hirudin variant HV2-N47K with improved antithrombin activity was successfully obtained. Conclusion: The high-activity hirudin variant protein can be screened by DNA family shuffling and affinity panning, which provides a reference for the reorganization of other molecules.