T7噬菌体展示文库在鉴定单克隆抗体所识别的抗原及表位中的应用

Identification of protein antigen for antibody DBD02 by biopanning of T7 phage display library

目的:用T7噬菌体展示文库鉴定1株单克隆抗体(mAb)DBD02的特异性。方法:用Protein G Sepharose结合mAb后对T7噬菌体展示文库进行2轮生物淘洗,用洗脱的通过特异mAb富集的噬菌体铺板后,以DBD02为一抗进行Dotblot筛选,阳性克隆进一步通过Western blot检测,PCR扩增阳性噬菌体插入的肝脏cDNA片段,测序后,通过BLAST比对确定mAb DBD02所识别的抗原。结果:在2轮淘洗后得到了>50个阳性克隆,取其中2个点扩增后进行了Western blot检测,并对这2个阳性克隆进行了PCR,序列测定后鉴定了此mAb所识别的抗原为乙醇脱氢酶,并将此mAb识别的表位限定于22个多肽内。结论:T7噬菌体展示文库可应用于mAb特异性的鉴定,与cDNA表达文库的免疫筛选相比,具有省时、省力和经济的特点,是mAb特异性鉴定的有力工具。

AIM： To identify the specificity of mAb DBD02, we developed and optimized a new method by biopanning of T7 Select human liver cDNA phage display library. METHODS： After two rounds of biopanning, eluted phages were subjected to Dot blot using mAb DBD02 as primary antibody. The positive PFUs （plaque forming unit） which recognized by DBD02 were further confirmed by Western blot, PCR and sequencing. RESULTS： The antigen recognized by DBD02 was identified as alcohol dehydrogenase. And the epitope for mAb DBD02 was further mapped within a peptide of 22 amino acids （QDYKKPIQEVLKEMTDGGVDFS）. CONCLUSION： Our data indicate that biopanning of T7 phage display library by rnAbs is time, cost, and laborsaving and specific tool for protein antigen identification.