摘要目的 探讨应用生物信息学技术对人中性粒细胞明胶酶相关脂质运载蛋白(NGAL)分子进行B细胞表位的预测的可行性,筛选出具有诊断价值的线性表位分子,构建并表达重组线性多表位抗原肽并进行免疫原性鉴定.方法 2015年7月南京医科大学第二附属医院检验科查询GenBank中NGAL氨基酸序列,利用Predicted、ABCpred、BepiPred、BcePred及Lasergene软件进行B细胞线性表位预测,构建、原核表达预测出的可能表位,通过免疫印迹实验筛选出可与现有NGAL多克隆抗体反应的单表位抗原,最后将获得的单表位抗原构建、表达出多表位抗原肽,并对其进行免疫反应性鉴定.结果 综合预测得出了8个可能的表位,通过构建pET32a-N1～N8原核表达载体,经表达、纯化、免疫印迹筛选,获得了3条具有较强抗原性的表位,随后构建出的多表位原核表达载体pET22b-Ngal_MEP1,获得了可溶性表达的融合蛋白,通过Ni2+亲和柱纯化,成功纯化出了融合蛋白.免疫印迹试验表明,该重组融合蛋白具有较强抗原性.结论构建的NGAL多表位线性抗原肽在原核表达系统中能获得高效可溶性表达,并具有较强免疫反应性,可用于后续抗体制备.

abstractsObjective 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.