摘要
目的将兔出血症病毒(RHDV)VP60全长基因在昆虫细胞-杆状病毒系统中表达,验证重组蛋白形成病毒样颗粒(VLPs)的能力及其生物学特性,探讨VLPs作为检测抗原及亚单位疫苗的潜力。方法用Bac-to-Bac系统体外表达RHDVVP60全长基因。以免疫荧光及Western blotting检测蛋白表达情况及确定蛋白最佳表达条件;免疫电镜观察VLPs形态,并对VLPs的血凝性、免疫原性进行检测。结果SDS-PAGE电泳分析表明,表达的重组蛋白分子量大小约为68KDa,在免疫荧光、琼脂扩散、ELISA试验中均与RHD多克隆抗血清特异性反应;接种重组病毒的Sf9细胞裂解液在电镜下可观察到与RHDV形态相似的VLPs;该VLPs可凝集人“O”、“B”型红细胞,凝集可被RHD多克隆抗血清所抑制;含VLPs的Sf9细胞裂解液可不经纯化用作间接ELISA抗原,所建立的ELISA方法与进口商品化试剂盒相比,特异性良好,敏感性、检出率稍低;将含VLPs的细胞裂解液加氟氏佐剂免疫兔,HI效价可达1∶40,可经受致死量病毒攻击。结论RHDV-VLPs的获得及其良好的免疫原性,为RHD血清学检测试剂的标准化、亚单位疫苗研制应用奠定基础,同时在转移载体及RHDV受体方面研究亦有潜在应用价值。
Objective To express full length VP60 gene of rabbit hemorrhagic disease virus （RHDV） in insectbaculovirus system, and to characterize the formation of RHDV-Like particles （RHDV-VLPs） and its biological properties, and verify the potential of RHDV-VLPs as detection antigen and subunit vaccine to RHD. Methods Immunofiuorescence and Western blotting assay were used to detect the VP60 protein expression and determine its optimal expression condition in Bac-to-Bac system. The morphology of VLPs was viewed by immunoelectron microscopy. Their hemagglutination and immunogenicity were also assayed. Results SDS-PAGE analysis showed that the expression protein is about 68 KDa in size. Its immunogenicity was identified by immunofluorescence, Western blotting, agar gel immunodiffusion and ELISA tests. Immunoelectron microscopical view demonstrated that the recombinant VP60 protein in Sf9 cytoplasm could serf-assemble into virus-like particles （VLPs）, which were similar in size and morphology to native RHDV. In hemagglutination assays, the VLPs could bound to human ＂0＂ and ＂B＂ erythrocytes and this reaction could be inhibited by RHD polyclonal antiserum. Sf9 cells lysate containing RHDV-VLPs could be used as coating antigen in indirect ELISA without purification. The sensitivity of indirect ELISA was proper,and the specificity and positive ratio were lower than those obtained by commercial kits of Spain. Sf9 cells lysate containing RHDV-VLPs were emulsified with Freund＇ s adjuvant and inoculated to rabbits. The rabbits survived challenge with lethal dose of RHDV and dipicted high haemagglutination inhibiting （HI） titer （1：40） in sera. Conclusion RHDV-VLPs from recombinant baculovirus-infected Sf9 cells has been obtained and their satisfactory antigenicity demonstrated. The RHDV-VLPs can be used as detection antigen and potential subunit vaccine instead of native RHDV, as RHDV has no propagation system in vitro so far. Otherwise, it makes possible to study RHDV-VLPs in gene transfer vector and RHDV receptor in future.
出处
《中国比较医学杂志》
CAS
2007年第8期448-454,F0003,共8页
Chinese Journal of Comparative Medicine
基金
中央级科研院所科技基础性工作专项计划(No.2001DEB20055)
黑龙江省博士后资金项目(LRB05-358)。
关键词
兔出血症病毒
基因
VP60
昆虫细胞
病毒样颗粒
Rabbit hemorrhagic disease virus （RHDV）
VP60 gene
Insect cell
Virus-like particles, VLPs
