Hu L, Esposito J J, Scott F W
Cornell Feline Health Center,College of Veterinary Medicine, Cornell University, Ithaca, New York, 14853, USA.
Virology. 1996 Apr 1;218(1):248-52. doi: 10.1006/viro.1996.0186.
An infectious raccoon poxvirus (RCNV) was used to express the feline panleukopenia virus (FPV) open reading frame VP2. The recombinant, RCNV/FPV, was constructed by homologous recombination with a chimeric plasmid for inserting the expression cassette into the thymidine kinase (TK) locus of RCNV. Expression of the VP2 DNA was regulated by the vaccinia virus late promoter P11. Southern blot and polymerase chain reaction (PCR) analyses confirmed the cassette was in the TK gene of the RCNV genome. An immunofluorescent antibody assay using feline anti-FPV polyclonal serum showed the expressed viral antigen in the cytoplasm of infected cells. Radioimmunoprecipitation with the same antiserum detected a 67-kDa VP2 protein which exactly matched the migration of the authentic FPV VP2 protein by SDS-polyacrylamide gel electrophoresis. Nine five-month-old cats were vaccinated and 21 days later were boosted with the recombinant virus. Peroral FPV challenge 2 weeks after the booster showed that the cats were fully protected as measured by examining clinical signs and total white blood cell counts in peripheral blood. Cats not immunized developed low to very low leukocyte counts following peroral FPV challenge. The nine vaccinated cats showed high FPV neutralization antibody prior to challenge, whereas nonvaccinated cats formed anti-FPV antibodies only after challenge.
一种传染性浣熊痘病毒(RCNV)被用于表达猫泛白细胞减少症病毒(FPV)的开放阅读框VP2。重组病毒RCNV/FPV是通过与一个嵌合质粒进行同源重组构建而成的,该嵌合质粒用于将表达盒插入RCNV的胸苷激酶(TK)基因座。VP2 DNA的表达由痘苗病毒晚期启动子P11调控。Southern印迹和聚合酶链反应(PCR)分析证实该表达盒位于RCNV基因组的TK基因中。使用猫抗FPV多克隆血清进行的免疫荧光抗体检测显示,在感染细胞的细胞质中存在表达的病毒抗原。用相同抗血清进行的放射免疫沉淀检测到一种67 kDa的VP2蛋白,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),其迁移情况与天然FPV VP2蛋白完全一致。9只5月龄的猫接种了疫苗,21天后用重组病毒进行加强免疫。加强免疫2周后经口给予FPV攻击,通过检查临床症状和外周血白细胞总数来衡量,结果显示这些猫得到了完全保护。未经免疫的猫在经口给予FPV攻击后白细胞计数降低至极低水平。9只接种疫苗的猫在攻击前显示出高FPV中和抗体,而未接种疫苗的猫仅在攻击后形成抗FPV抗体。