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主要探讨禽网状内皮组织增生症病毒(REV)亚单位疫苗联合CpG-ODN/Poly(I:C)缓释佐剂对雏鸡的免疫保护作用。根据已发表的REV囊膜蛋白Gp90的基因序列,设计引物,从感染REV的细胞中扩增出目的基因,构建原核重组表达载体,在Rosseta(E.coli)菌中诱导表达,对表达蛋白进行纯化和Western blot鉴定;制备的重组蛋白与CpG-ODN/Poly(I:C)缓释佐剂联合接种7日龄雏鸡,2次加强免疫后第2周进行REV攻毒,接种后每周检测血清抗体和攻毒后检测REV病毒血症,评价免疫保护效果。结果显示,成功构建重组原核表达质粒,并获得相对分子质量为51 000的重组表达产物,该产物能够与REV单克隆抗体特异性结合;与CpG-ODN/Poly(I:C)缓释佐剂联合接种后能够诱导雏鸡产生较单一免疫Gp90蛋白抗体效价显著增加的血清抗体,经二次加强免疫全部鸡只产生抗体;攻毒后接种雏鸡病毒血症为0%(0/13),而对照组和单一免疫Gp90蛋白组分别为69%(9/13)和15%(2/13)。结果表明,本研究制备的REV Gp90重组蛋白联合CpG-ODN/Poly(I:C)缓释佐剂对雏鸡具有完全的保护力,这为开展REV疫苗防控提供科学数据。
The main purpose of this study was to investigate the immunoprotective effect of poultry reticuloendotheliosis virus (REV) subunit vaccine and CpG-ODN / Poly (I: C) sustained-release adjuvant on chickens. According to the published sequence of GFP90 gene, primers were designed to amplify the target gene from REV-infected cells. The prokaryotic recombinant expression vector was constructed and induced in Rosseta (E.coli) Purified and identified by Western blot. The prepared recombinant protein was inoculated with CpG-ODN / Poly (I: C) sustained-release adjuvant in 7-day-old chicks and challenged with REV 2 weeks after 2 booster immunizations. Serum antibodies and challenge REV viremia after challenge, evaluation of immune protection. The results showed that the recombinant prokaryotic expression plasmid was successfully constructed and the recombinant product with a relative molecular mass of 51 000 was obtained and the product could specifically bind to the REV monoclonal antibody. Compared with CpG-ODN / Poly (I: C) After combined vaccination, the chickens could be induced to produce the serum antibody with significantly higher antibody titer than the single immunized Gp90 protein. All the chickens were immunized twice to produce antibody. After vaccination, the chick viremia was 0% (0/13) The control group and the single immunized Gp90 protein group were 69% (9/13) and 15% (2/13), respectively. The results showed that the REV Gp90 recombinant protein combined with the CpG-ODN / Poly (I: C) sustained-release adjuvant prepared in this study had complete protection for the chickens, which provided scientific data for the prevention and control of REV vaccine.