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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。
In order to screen the E antigen epitope of Japanese encephalitis virus (JEV), monoclonal antibody (MAb) against JEV E protein was used as the solid phase screening molecule in this experiment. The phage display technique was used to eliminate, bind and elute Phage display library was screened in the order of amplification. The phage clones were selected and cloned into MAb-coated ELISA. The positive clones were sequenced to determine the amino acid sequence of the mimotope of JEV E antigen. The cDNA sequence of E antigen 15 peptide (E-365GGADSMSMAGMAVSYE-379) containing this epitope was designed and synthesized. Recombinant plasmids were constructed with pGEX-KG to induce the expression of the recombinant polypeptide and verified by western blot. After 4 rounds of screening, the phages were highly enriched. Monoclonal antibodies were picked by MAb coating for ELISA, and 22 clones were positive. Western blot analysis of the recombinant polypeptide showed that the recombinant polypeptide could specifically bind to rabbit anti-JEV polyclonal antibody. This experiment successfully screened JEV structural protein E specific phage mimotopes for JEV antigen epitopes to explore JEV prevention and control to create the conditions for the development of peptide vaccine, drug screening and the establishment of specific serological diagnostic methods Important reference.