一种优化的DNA家族改组方法

An optimized DNA family shuffling method

目的 :优化DNA家族改组方法 ,增加改组分子库的多样性。方法 :以人、猴、兔的BPI2 3基因为模板 ,首先将3种基因的PCR产物等量混合后用DNaseⅠ消化 ,回收 5 0bp左右的片段 ,再经过无引物和有引物两步PCR反应获得与原基因大小相同的改组分子 ,将其与载体连接获得改组分子库。随机挑取 4个克隆测序观察改组效果。结果 :4个克隆均为 3种模板基因片段的杂合体 ,其中 3个克隆还包含氨基酸点突变 ,表明改组分子库多样性较大 ,改组方法较为成功。结论 :该方法的建立为进一步筛选高活性的BPI2 3分子打下基础 ,并为改组其他基因家族提供了借鉴。

Objective:To optimize DNA family shuffling method for increasing the diversity of shuffled libraries.Methods:First, equal PCR products of BPI 23 genes derived from human, monkey and rabbit were combined and digested by DNaseⅠ.Then random fragments about 50?bp were purified and reassembled to the same size of BPI 23 genes by two steps of PCR:first without primer and then with primers. Pools of mutant genes were ligated with a vector and four randomly picked clones were sequenced to observe the efficiency of shuffling.Results:It was found that all the four clones were hybrids of three original genes and three clones carried several additional amino acid substitutions not found in the templates. Conclusions:These results show that a highly shuffled library of BPI 23 genes has been obtained which can be used to screen chimerical BPI 23 molecules with high bioactivity, and the method can also be used to evolve other gene families.