用噬菌体表面显示肽库技术筛选乙酰胆碱酯酶有效抗原表位的研究

目的：筛选和鉴定乙酰胆碱酯酶的有效抗原表位。方法：收集乙酰胆碱受体抗体阴性而乙酰胆碱酯酶抗体阳性的重症肌无力病人血清，采用溴化氰活化的琼脂糖柱（CNBr-actived sepharose^TM4B）纯化抗乙酰胆碱酯酶的多克隆抗体并定量；用纯化的抗乙酰胆碱酯酶的抗体对随机的12肽噬菌体表面显示肽库进行5轮免疫学筛选，随机挑取克隆；采用Western blot免疫识别，识别为阳性的克隆进行核苷酸序列的测定，并与乙酰胆碱酯酶的氨基酸序列进行同源性比较。将获得的不同表位的阳性克隆分别免疫小鼠，采用Western blot筛选能刺激小鼠产生抗乙酰胆碱酯酶抗体的阳性克隆，进行生物学活性的鉴定。结果：经5轮免疫学筛选后挑取的49个克隆中，经Westem blot识别15个克隆能被抗乙酰胆碱酯酶抗体识别。核苷酸序列分析发现共有7种不同的表位，其中1种表位与乙酰胆碱酯酶有较高的同源性，其余6种表位与其无一级结构的同源性。经动物免疫初步实验筛选，共有5个免疫原性较强的阳性克隆，其免疫的鼠血清均可识别人乙酰胆碱酯酶。结论：获得了5种乙酰胆碱酯酶的有效抗原表位，1种可能为结构表位，4种为模拟表位。

Identification of potent epitopes of acetylcholinesterase with phage-displayed random peptide library

Objective:To identify the epitopes of acetylcholinesterase(AChE).Methods:The sera of patients with seronegative myasthenia gravis were collected. The antiserum was furtherly purified with CNBr-actived sepharose~ TM4B and quantified to obtain polyclonal antibody against acetylcholinesterase. A 12-mer phage display peptide library was then used to screen the epitopes of acetylcholinesterase with purified polyclonal antibody against acetylcholinesterase. Five rounds of biopanning were carried out and forty-nine phage clones from the fifth round of biopanning were randomly selected and identified by Western blot analysis. The positive clones were sequenced and their deduced amino acid sequences were compared with those of acetylcholinesterase. The BALB/c mice were immunized with the obtained positive clones and the sera of the mice were identified with human acetylcholinesterase by Western blot to screen the clones which could stimulate the mice to produce anti-AChE antibody.Results:Fifteen of the forty-nine selected clones could be recognized by anti-AChE antibody with Western blot. The fifteen positive clones included seven different epitopes. The amino acid sequence of one epitope showed high homology with that of acetylcholinesterase, the other six epitopes showed no homology with acetylcholinesterase. Human acetylcholinesterase was recognized by the sera of the mice immunized with the seven different positive phages and five epitopes stimulated the mice to produce anti-AChE antibodies as analysed by Western blot.Conclusion:Five potent epitopes of acetylcholinesterase were obtained. One may be structure epitope and the other four are mimotopes.