Wang Yong-shan, Fan Hong-jie, Zhang Xiao-min, Li Yin, Xiao Huan-juan
Military Medical Institute of Nanjing Command, Center for Disease Control and Prevention of Nanjing Command, Nanjing 210002, China.
Wei Sheng Wu Xue Bao. 2007 Feb;47(1):121-5.
Five mimic epitopes of Infectious bursal disease virus (IBDV) have been identified from a 12-mer phage-displayed peptide library by 5 monoclonal antibodies. Based on the sequences of the five epitopes, multiple epitope gene 5epis was constructed by the five epitopes being tandemly arranged and linked with 4-peptide GGGS. The expression plasmid pET-5epis was constructed and successfully expressed in E. coli. The resultant protein of 5epis was called r5EPIS. The results from SDS-PAGE analysis showed that the proportion of r5EPIS was 15% of the total bacterial proteins and the molecular weight of r5EPIS was 10kDa. By use of parallel immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of r5EPIS against IBDV have been verified. Rabbits were subcutaneously injected with r5EPIS (400 micro g per injection), twice with 7 days interval. The titers of the IBDV-specific antibody measured by indirect ELISA were up to 1:4000 at the 7th day after first immunization and 1:256000 at the 14th day after the second immunization. To determine the protective ability of r5EPIS to the challenge of IBDV, chickens were injected intramuscularly with r5EPIS in adjuvant twice with 7 days interval (501g per injection) and the resultant antibody titer was up to 1:12800 at the 7'h day after the second immunonization. After challenge with 200ELD50 of virulent IBDV GX8/99 strain, all the chicken in r5EPIS-immunized group were survived in contrast to the mortality of 86.7%(13/15) in adjuvant control group, suggesting that r5EPIS had a potent ability to generate protective immune response and it implied that the constructed gene 5epis is a prospective candidate for the development of epitope-based IBD vaccine.
通过5种单克隆抗体从一个12肽噬菌体展示肽库中鉴定出传染性法氏囊病病毒（IBDV）的5个模拟表位。基于这5个表位的序列，通过将5个表位串联排列并与4肽GGGS连接，构建了多表位基因5epis。构建了表达质粒pET-5epis并在大肠杆菌中成功表达。5epis的表达产物称为r5EPIS。SDS-PAGE分析结果表明，r5EPIS占细菌总蛋白的比例为15%，分子量为10kDa。通过使用相应的单克隆抗体和多克隆抗体进行平行免疫印迹试验，验证了r5EPIS对IBDV的免疫特异性和反应性。给兔子皮下注射r5EPIS（每次注射400μg），间隔7天注射2次。首次免疫后第7天，通过间接ELISA测定的IBDV特异性抗体效价高达1:4000，第二次免疫后第14天高达1:256000。为了确定r5EPIS对IBDV攻击的保护能力，给鸡肌肉注射含佐剂的r5EPIS，间隔7天注射2次（每次注射50μg），第二次免疫后第7天产生的抗体效价高达1:12800。用200ELD50的强毒IBDV GX8/99株攻击后，r5EPIS免疫组的所有鸡均存活，而佐剂对照组的死亡率为86.7%（13/15），这表明r5EPIS具有产生保护性免疫反应的强大能力，这意味着构建的基因5epis是基于表位的IBD疫苗开发的一个有前景的候选物。
