[通过DNA改组构建噬菌体展示文库及筛选高效水蛭素变体]

[Construction of phage-displayed library by DNA shuffling and the screening of potent hirudin variants].

作者信息

Long Yin, Liu Jia-yun, Liu Li, Wang Zong-ren

机构信息

Department of Traditional Chinese Medicine, Fourth Military Medical University, Xi'an 710032, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Mar;22(2):167-70.

PMID:16507250

Abstract

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.

摘要

目的

采用DNA家族改组方法在体外定向提高水蛭素的抗血栓活性。

方法

将水蛭素HV1、HV2和HV3基因的PCR产物进行合并，并用DNase I进行消化。然后纯化约50 bp的随机片段，并通过有引物或无引物的两轮PCR扩增将其重新组装成与水蛭素基因大小相同的片段。将改组后的水蛭素基因插入噬菌粒pCANTAB5E载体中，使水蛭素突变体展示在噬菌体M13表面。通过用减少量的凝血酶进行亲和淘选，筛选出抗凝血酶比活性增加的突变体。