DNA改组技术在水蛭素实验进化中的应用

蛋白质的改造是生物工程的重大研究课题.由于结构和功能预测的不精确性,而使按照三维结构信息进行定位诱变往往达不到预期的目的.近年来,另一条改造蛋白质的途径有较大的发展,即在实验室条件下模拟生物分子的自然进化,通过变异和靶功能的选择来获得改进性能的蛋白质[1],此过程称为生物分子实验定向进化.DNA改组(DNAshuffling)是一种改造基因和蛋白质的有效实验进化技术[2].它是在体外进行基因随机片段的重组,从而增加基因的多样性,促使有利变异与不利变异分离,通过选择使有利变异得到优化组合[3].DNA改组包含3个步骤:基因的随机片段化,自身引发PCR和重组合PCR.经过DNA改组的突变体库有可能选择到性能更优的突变体.为进行亲和淘选,需将突变体展示在噬菌体的表面[4].

Directed Evolution of Hirudin by DNA shuffling

Hirudin gene was mutated by error|prone PCR and DNA shuffling.The shuffled genes were inserted into phagemid pCANTAB|5G8.After the introduction of the recombinant DNA into E.coli TG1,the hirudin mutants were displayed on the surface of bacteriophage M13.A mutant with increased specific activity of antithrombin was selected by two rounds of panning with decreased amounts of thrombin,and was identified as N47K.Activity analysis of HV2 and its mutants N47K,N26S,D5A,P46L/S50G suggested that K47,P46 were important to the interaction of hirudin and thrombin.Hirudin mutants with improved properties could be hopefully aquired through directed evolution by DNA shuffling.