Construction and identification of recombinant human neutrophil gelatinase-associated lipocalin linear multi-epitope peptide

Objective The feasibility of predicting the B-cell epitopes of human Neutrophil Gelatinase-Associated Lipocalin (NGAL) was discussed by applicating bioinformatics technology. Linear epitope molecules that have diagnostic value were screened and these recombinant linear multi-epitope peptides were constructed, and expressed. The immunogenicity of the recombinant linear multi-epitope peptides were also identified. Methods NGAL amino acid sequence was got from GenBank in the Department of Clinical Laboratory of the Second Affiliated Hospital of Nanjing Medical University in July 2015, the Predicted, ABCpred, BepiPred, BcePred, and Lasergene softwares were used to predict the linear B cell epitope prediction. The predict epitopes were constructed and prokaryotic expressed, and then the single epitope antigens which could reacted with commercially available polyclonal NGAL antibody were screened out by Western blot. Finally, the multi-epitope peptide was constructed, expressed, and identified through immunoreactions. Results Eight possible epitopes were obtained after prediction. pET32a-N1-N8 prokaryotic expression vector were used to express the predict epitopes.After purification and Western blot analysis, three of the epitopes have strong antigenicity, and then a soluble fusion protein was expressed and obtained from the multi-epitope prokaryotic expression vector pET22b-Ngal_MEP1. The fusion protein was successfully purified by Ni2+ affinity column. Western blot analysis showed that the fusion protein had a strong antigenicity. Conclusions The constructed multi-epitope linear NGAL antigen peptides can obtain high soluble expression in prokaryotic expression system, and have a strong immunoreactivity, which can be used in subsequent antibody preparation.(Chin J Lab Med, 2016, 39: 380-385) Key words: Lipocalins; Amino acid sequence; B-cell maturation antigen; Epitopes; Immunoblotting