通过基因突变提高噬菌体蛋白Ⅷ介导的抗体Fab段的展示

Improvment the pⅧ mediated phage display of Fab by mutation of geneⅧ

目的 构建基因Ⅷ变种文库 ,筛选表面展示效率高的突变体 ,改进抗体分子在噬菌体表面的呈现效果。方法 通过重叠PCR ,在基因Ⅷ引入随机突变 ,克隆到抗乙肝表面抗原 (HBsAg)的噬菌体抗体表达载体中 ,使抗HBsAg的Fab段通过蛋白Ⅷ展示于噬菌体表面 ,构建蛋白变种Ⅷ文库 ,筛选能使HBsAg表面呈现效果得到提高的蛋白Ⅷ突变体。结果 构建了一个库容为 6× 10 8的基因Ⅷ变种文库 ,使用游离抗原竞争、硫氰酸盐洗涤等方法进行淘筛 ,获得多个展示活性高于野生型蛋白Ⅷ的突变体 ,其中 2个最好的变种可使表面呈现效果提高 5 0～ 70倍。结论 通过对基因Ⅷ的改造可提高其对抗体Fab段的展示性能 ,获得 2株可使展示活性提高 5 0～

Objective To improve pⅧ mediated phage display of Fab by constructing and screening of gene Ⅷ variants library. Methods Random mutations were introduced into gene Ⅷ by overlap PCR. A phage antibody library was constructed by inserting the gⅧ variants into an anti HBsAg phage antibody expression vector p8HB. The library was used to select better binder by bio panning. Results A gene Ⅷ variants library containing 6×10 8 clones was constructed. Bio panning through free antigen competition or thiocyanate elution resulted in several gⅧ variants with higher Fab display activity. The best two of the selected clones exhibited 70 and 50 folds increase than wild type gⅧ. Conclusion The display of antibody Fab fragments via protein Ⅷ can be improved by mutagenesis of gene Ⅷ. Two variants with 50 70 folds higher capacity in displaying Fab have been obtained.