人巨细胞病毒单克隆抗体识别的抗原表位的筛选

Screening phage displayed random hexapeptide library against HCMV McAb

目的找出人巨细胞病毒（ＨＣＭＶ）单克隆抗体识别的抗原表位以进行ＨＣＭＶ多肽疫苗的研制。方法通过链亲和素－生物素的作用将ＨＣＭＶ单抗固定在酶联板上，加入噬菌体随机六肽文库，经过三轮捕捉、洗脱、扩大培养的筛选过程，并逐轮增加冲洗强度，与ＨＣＭＶＭｃＡｂ具有亲合力的噬菌体得到了富集；从最后得到的三级库中，随机挑选１６个单克隆，测定其所携带的外源ＤＮＡ的序列，找出一致序列并进行同源性查询。结果１６个克隆中有１５个序列相同，５＇－ＡＧＴＧＣＴＧＧＴＴＧＧＧＣＴＴＣＴ－３＇，对应的氨基酸序列为Ｓｅｒ－Ａｌａ－Ｇｌｙ－Ｔｒｐ－Ａｌａ－Ｓｅｒ，此序列同源性查询结果显示与ＨＣＭＶ病毒膜糖蛋白ＵＬ０１有７８．８％的同源性，其中有４个氨基酸残基（ＡＧＷＡ）是完全相符的。结论所筛选到的六肽序列可能为ＨＣＭＶ抗原表位。

Objectives To define the epitopes of HCMV with McAb aiming at preparing peptide vaccine in future. Methods (a) Affinity purification of monoclonal antibody; (b)Screening against a phage displayed random peptide library using the purified antibody ;(c)Plating the enriched phage on agar plates, randomly picking clones;(d)DNA sequencing and conducting a homology search against the protein sequence databank. Results A consensus DNA sequence has been found, 5' AGTGCTGGTTGGGCTTCT 3', the amino acid sequence was Ser Ala Gly Trp Ala Ser, which had the highest match (78.8%) with a fragment of HCMV UL01 protein. Conclusions Screening of bioactive peptides from random peptide libraries using monoclonal antibodies as ligands is an effective method to define the epitopes of protein antigens; the sequence that we found might be the epitope of HCMV.