噬菌体随机肽库筛选抗恶性疟原虫乳酸脱氢酶单抗2A5的识别表位

目的利用噬菌体随机肽库技术筛选恶性疟原虫乳酸脱氢酶(pfLOH)抗体2A5的识别表位。方法以抗pfLDH的单抗2A5筛选噬菌体随机12肽库,挑取阳性克隆测定DNA序列,推导其氨基酸序列并进行同源性分析。通过ELISA、竞争抑制试验鉴定获得的噬菌体短肽与单抗2A5之间的结合特性。结果从噬菌体随机12肽库中筛选出21株可与2A5单抗特异结合的噬菌体克隆,其中多数克隆呈现核心序列SQK(R)L,该序列与pfLDH的一级序列具有一定同源性。竞争抑制实验显示,带有核心序列的噬菌体克隆可与pfLDH竞争结合单抗2A5。结论 SQK(R)L是pfLDH单抗2A5特异识别的表位。

Selection of Antigenic Determinants Recognized by Monoclonal Antibody 2A5 Against pfLDH from a 12-mer Random Peptide Library

Objective To identify the epitope of monoclonal antibody against lactate dehydrogenase of Plasmadium falciparum (pfLDH). Methods Monoclonal antibody 2A5 against pfLDH was used to screen a 12-mer random peptide library.Positive clones were obtained by three rounds of biopanning, and the reactivity of each clone binding to 2A5 antibody was examined by sandwich ELISA. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. Results 25 phage clones from the third round were randomly selected,and 21 clones of them could bind to the 2A5 antibody.Analysis of DNA and amino acid sequences indicated that most selected peptides show a sequence motif SQK (R)L which showed high homogeneity to the primary sequence of pfLDH amino acid (residues 165~ 168 ). The competitive ELISA tests proved that phageborne peptides contained SQK (R)L motif could competitively inhibit the binding of 2A5 antibody with pfLDH. Conclusion SQK(R)L was antigenic determinants of pfLDH.