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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IgG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-RhD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IgG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-RhD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IgG型单克隆抗-D与RhD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RhD结构与功能研究及研制针对RhD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 and validate mimic peptides of hemorrhagic D antigen using random phage 12 peptide library. Methods Monoclonal IgG anti-D was used to screen the phage random 12-peptide library to determine the binding of anti-RhD after each round of screening. The specific phage clones were screened by DNA sequencing. The deduced amino acid sequences of 12 peptides were compared by homology. The agglutination inhibition test was used to verify the reaction of the positive phage and IgG monoclonal anti-D. Results As the number of rounds of screening increased, the binding of phage eluate to anti-Rhd increased gradually. After 4 rounds of washing, 12 conserved peptide sequences with high affinity and high coincidence rate were obtained. Agglutination inhibition experiments showed that positive phage with the same amino acid sequence had an inhibitory effect on the agglutination reaction of IgG monoclonal anti-D and RhD-positive erythrocytes. Conclusion The mimic peptides of blood type D antigen screened by random phage 12 peptide library provide the experimental basis for studying the structure and function of RhD, developing new diagnostic antigens against RhD newborn hemolytic disease, preparing specific vaccines and exploring new therapeutic methods.