日本乙型脑炎病毒E抗原表位的筛选

为筛选日本乙型脑炎病毒(JEV)的E抗原表位,本实验以抗JEV E蛋白的单克隆抗体(MAb)作为固相筛选分子,应用噬菌体表面展示技术,按消减、结合、洗脱、扩增的顺序筛选噬菌体七肽库,挑取噬菌体单克隆培养并采用MAb包被的ELISA鉴定,对阳性克隆测序分析,确定JEV E抗原模拟表位的氨基酸序列.设计合成包含该表位的E抗原15肽(E-365GGADSMSMAGMAVSYE-379)cDNA序列,与pGEX-KG构建重组表达质粒,诱导表达重组多肽并进行western blot验证.经过4轮筛选后,噬菌体得到高度富集,挑取单克隆采用MAb包被进行ELISA鉴定,有22个克隆呈阳性.对重组多肽进行western blot验证,结果表明该重组多肽能够特异结合兔抗JEV多克隆抗体.本实验成功筛选出JEV结构蛋白E的特异性噬菌体模拟表位,为开展用JEV抗原表位探索JEV的防制研究创造了条件,为多肽疫苗研制、药物筛选以及特异血清学诊断方法的建立提供重要依据.

Screening and identification of epitope in glycoprotein E of Japanese encephalitis virus

To screen mimotope of Japanese encephalitis virus(JEV) E protein in phage peptide library,we used monoclonal antibody(MAb) against JEV E protein for the biopanning.A 7 mer phage peptide library was biopanned and positive clones were selected by ELISA and DNA sequencing.Phages were highly enriched after 4 rounds of screening and 20 positive clones were selected for DNA sequencing.According to the positive clone sequence result,an oligonucleotide encoding 15 peptides(GGADSMSMAGMAVSY) was synthesized,cloned and expressed in E.coli.The expressed polypeptide was specifically recognized by rabbit polyclonal anti-JEV identified by western blot.The identification of the epitope in JEV E protein provides a basis for further study on JEV E protein.