从随机9肽库中筛选HCV抗原表位

Biopanning of HCV antigen epitopes from a random peptide library

目的 :用患者血清中抗HCV抗体从随机 9肽库中筛选HCV抗原表位。方法 :将病人血清用硫酸铵粗提后 ,用ProteinA亲和层析柱纯化和制备抗HCV多克隆抗体 ;以此为筛选配基 ,对噬菌体表面展示的随机 9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 5 0× 10 - 3 、假阳性率逐轮降低至 0 2 % ,提示具有良好的富集效果。从第三轮挑选出的 15个克隆进行结合试验 ,发现 7个克隆只与HCV抗体有较强的结合力而不与正常人血清反应 ;测序表明 6个克隆的外源肽含有核心序列SPVAXVLXT。用阳性噬菌体克隆检测 2 0例病人血清 ,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得到了有一定功能的模拟表位。

Objective:To analyze the epitopes of HCV antigen by peptide library biopanning.Methods:Anti-HCV Abs were purified from patients sera using an affinity chromatography column which constructed with sepharose 4B and protein A. These Abs were used for biopanning of a phage-displayed random peptide library of 9 amino acid residues.Results:After 3 rounds of screening, the ratio of output to input increased to 5.0×10 -3 and the false positive rate reduced to 0.2%, which means the enrichment was effective. At the third round of screening, fifteen clones were selected for binding test with Abs from patient's and normal sera. Seven of the clones was proved to specifically react with the sera from patient. From the deduced insert sequence in the coat protein III, the core sequence of SPVAXVLXT was found in them. The positive clones could react with different patients and not react with normal sera.Conclusion:These findings indicate that SPVAXVLXT motif in the short peptide could be the mimic of HCV epitope that can be recognized by HCV Abs.