摘要
筛选日本乙型脑炎病毒(JEV)的E抗原表位,为开展用JEV模拟表位探索JEV的防治研究创造了条件,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的线索和依据。以抗JEV E蛋白的单克隆抗体作为固相筛选分子,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7肽库,挑取噬菌体单克隆培养并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(GGADSMSMAGMAVSY)cDNA序列,与pGEX-KG构建重组表达载体,诱导表达重组多肽并West-ern blot验证。经过4轮筛选后,噬菌体得到高度富集,挑取单克隆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多抗。应用上述方法成功筛选出JEV结构蛋白E的特异性噬菌体模拟表位,为下一步研究奠定了基础。
Screening E-epitope of Japanese encephalitis virus （JEV）, creating the conditions for JEV prevention and treatment research by mimotope,providing important clues and basis for establishing specific serological diagnostic methods and exploring peptide vaccine of JEV. With the monoclonal antibody molecules of JEV E protein as solid phase screening, screening the phage heptapeptide library by a rule of reduction, combined, elution, amplification. Picked monoclonal phages and detect them by ELISA, the positive clones were sequenced. Designed and synthesised a cDNA sequence with 15 peptides （GGADSMSMAGMAVSY） and conducted a recombinant expression vector with pGEX-KG,induced to express the recombinant polypeptide and dectected it by Western blot. Phages were highly en- riched after four rounds of screening, it is proved that there were 22 positive clones by ELISA. The Western blot show that the recombinant polypeptide can bind to rabbit polyelonal anti-JEV antibody specifically. We identified the structure of JEV E protein-specific phage mimotope successfully,it laid a good foundation for luther study.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2012年第6期866-870,共5页
Chinese Journal of Veterinary Science
基金
国家高技术研究发展计划(863计划)资助项目(SQ2009AA10Z4483730)
