用抗HCV多抗从随机15肽库中筛选抗原表位

Identification of HCV antigen epitopes from a random 15-peptide library by anti-HCV polycloral antibody

目的:分析丙肝患者血清中抗HCV抗体的抗原表位。方法: 制备Protein A亲和层析柱,从丙肝患者血清中纯化、制备抗HCV多克隆抗体;并以此为筛选配基,对噬菌体表面展示的随机15肽库进行亲和筛选。结果:三轮筛选的投入产出比逐轮升高至3.3×10-3,假阳性率逐轮降低至0.2％,提示具有良好的富集效果。对从第3轮挑选出的16个克隆进行结合试验,发现9个克隆只与HCV抗体有较强的结合力而不与正常人血清起反应。测序表明,8个克隆的外源肽含有核心序列WPWS。用阳性噬菌体克隆检测20例丙肝患者血清,有程度不等的阳性反应。结论:用多克隆抗体从噬菌体随机肽库中,筛选到有一定功能的模拟表位。

AIM: To analyze epitope recognized by anti-HCV antibodies; patients suffered from hepatitis C. METHODS; Anti-HCV Abs were purified from the patients' serum throngh an affinity chromatography column which was prepared with sepharose 4B coupled with protein A. These Abs were used for biopanning of a phage-displayed random 15-peptide library. RESULTS: After 3 rounds of bioparning, the ratio of output to input increased to 3.3 × 10-3 and the false positive rate reduced to 0.2% , suggesting that the enrichment was effective. After the third round of bioparning, sixteen clones were selected to conduct binding test to Abs from the patients' and normal person' s sera. Nine of them were proved to react specifically to the sera from the patients. From the deduced insert sequence in the coat protein VIII, the core sequence of WPWS was found in 8 clones. The positive phage clones could react to different patients' and not react to normal person's sera. CONCLUSION: These findings indicate that WPWS motif in the short peptide may mimic the HCV epitope recognized by anti-HCV Abs.