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目的采用噬菌体展示肽技术鉴定抗日本血吸虫14-3-3蛋白单克隆抗体5C6的抗原识别表位。方法将纯化的抗日本血吸虫14-3-3蛋白单克隆抗体5C6作为固相配基筛选噬菌体随机展示12肽库,按照吸附-洗脱-扩增的淘洗过程进行3轮生物淘洗,富集特异性结合的噬菌体颗粒。随机挑取第3轮洗脱的独立噬菌体克隆,分别依次进行噬菌体扩增、基因组DNA抽提和DNA序列分析。选择外源性插入DNA序列完全相同或高度相似的同源性噬菌体,采用ELISA及免疫印迹分析对其免疫反应性作进一步鉴定,以确定单克隆抗体5C6的抗原识别表位。结果第3轮淘洗物中33个独立噬菌体克隆的DNA测序结果显示,其中25个噬菌体编码外源性插入肽的核酸序列均为5′-CCACCTAGTAGCAGACCGATTCT-CAGTCGAAGGAAA-3′,其对应的演绎氨基酸序列为PPSSRPILSRRK,该肽与日本血吸虫信号蛋白14-3-3及自然界其他已知蛋白的氨基酸序列均无同源性。免疫印迹试验证实此短肽可被自然感染的日本血吸虫病人血清所识别,而不与健康人血清反应。结论本研究成功鉴定了抗日本血吸虫14-3-3蛋白单克隆抗体5C6所识别的模拟抗原表位,并证明该表位为空间构像表位。
OBJECTIVE: To identify epitopes of anti-Schistosoma japonicum 14-3-3 monoclonal antibody 5C6 by phage display peptide technique. Methods Purified anti-Schistosoma japonicum 14-3-3 monoclonal antibody 5C6 was used as a solid-phase ligand for phage display of 12-mer peptide library. Three rounds of bio-elutriation were performed according to the elution-adsorption- Set specific binding of phage particles. Randomly picked the third round of independent phage clones eluted, followed by phage amplification, genomic DNA extraction and DNA sequence analysis. The homologous phage with the same or highly similar exogenous insert DNA sequences were selected and their immunoreactivity was further identified by ELISA and Western blot analysis to determine the antigen recognition epitope of monoclonal antibody 5C6. Results The DNA sequencing results of 33 independent phage clones in the third round of panning showed that the nucleic acid sequences of the 25 phage encoding exogenous insertion peptides were all 5’-CCACCTAGTAGCAGACCGATTCT-CAGTCGAAGGAAA-3 ’, and the corresponding deduced amino acid sequence For PPSSRPILSRRK, the peptide has no homology with the amino acid sequence of Schistosoma japonicum signaling protein 14-3-3 and other known proteins in nature. Western blotting confirmed that this short peptide was recognized by the sera of patients with naturally infected Schistosoma japonicum, but not with healthy human serum. Conclusions This study successfully identified the mimic epitope recognized by the anti-Schistosoma japonicum 14-3-3 monoclonal antibody 5C6 and proved that the epitope was a spatial conformational epitope.