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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 methods and increase the diversity of shuffled molecular libraries. Methods: The BPI2 3 gene of human, monkey and rabbit was used as a template. The PCR products of the three genes were first mixed in the same amount and then digested with DNase Ⅰ, and the fragment of about 50 bp was recovered, and then obtained by two-step PCR without primer and primer The same size as the original gene shuffled molecules, and its connection with the vector to obtain shuffled molecular library. Four clones were randomly selected and sequenced to observe the shuffling effect. Results: All four clones were heterozygotes of three kinds of template gene fragments. Three of them also contained amino acid point mutations, indicating that the molecular diversity of the reorganization library was relatively large and the shuffling method was successful. Conclusion: The establishment of this method lays a foundation for further screening of highly active BPI2 3 molecules and provides a reference for the reorganization of other gene families.