筛选及鉴定IgG型单克隆抗-D噬菌体克隆的实验研究  

Screening and identification of IgG monoclonal anti-RhD phage clones

作　　者：吴凡[1] 庄乃保[1] 张印则[1] 苏宇清[1] 梁延连[1] 

机构地区：[1]深圳市血液中心,广东深圳518035

出　　处：《中国输血杂志》2017年第10期1121-1125,共5页Chinese Journal of Blood Transfusion

基　　金：深圳市卫生计生系统科研项目(201502008)

摘　　要：目的利用随机噬菌体12肽库筛选出血型D抗原的模拟多肽并验证。方法采用Ig G型单克隆抗-D筛选噬菌体随机12肽库,测定每轮筛选后洗脱物与抗-Rh D的结合情况。对经特异性筛选得到的阳性噬菌体克隆进行DNA序列测定,将推导出的12肽氨基酸序列进行同源性比较,通过凝集抑制试验验证阳性噬菌体与Ig G型单克隆抗-D的反应。结果随着筛选轮次增加,噬菌体洗脱物与抗-Rh D的结合力逐渐增强。经过4轮淘洗后,得到8个亲和力较强且具有较高重合率的保守区域的12肽序列。凝集抑制实验表明具有相同氨基酸序列的阳性噬菌体对Ig G型单克隆抗-D与Rh D阳性红细胞的凝集反应具有抑制作用。结论随机噬菌体12肽库筛选得到的血型D抗原模拟多肽,为Rh D结构与功能研究及研制针对Rh D新生儿溶血病的新型诊断抗原、制备特异疫苗以及探索新的治疗方法提供实验基础。Objective To screen peptide mimotopes of blood group D antigen and identify them with phage display peptide library. Methods A twelve mer phage peptide library was bio-panned with anti-RhD monoelonal antibody immobilized on plastic surface. The binding of the eluate to the anti-RhD antibody was determined for each round. Phage DNA of the positive phage clones obtained by specific screening was sequenced then deduced and compared the 12 peptide amino acid sequences. The resulting peptide mimotopes of blood group D antigen was verified by agglutination test. Results After the four rounds screening,phages were enriched. 8 12-peptide sequences with strong affinity and conserved regions with higher coincidence rates were obtained. Agglutination inhibition experiments showed that positive phages with the same amino acid sequence had an inhibitory effect on the agglutination of Ig G monoclonal anti-D and RhD-positive erythrocytes. Conclusion The results lay the foundation for further exploration of RhD structure and function,new diagnostic antigen development,specific vaccines preparation and explore the new treatment of RhD hemolytic diseases of newborn.

关 键 词：肽库 RHD抗原 细菌噬菌体 表位 

分 类 号：R457.11[医药卫生—治疗学]