Estein Silvia M, Cheves Pablo C, Fiorentino María A, Cassataro Juliana, Paolicchi Fernando A, Bowden Raúl A
Laboratorio de Inmunoquímica y Biotecnología, Departamento de Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, 7000 Tandil, Argentina.
Vet Microbiol. 2004 Sep 8;102(3-4):203-13. doi: 10.1016/j.vetmic.2004.05.004.
Detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli was previously identified as a protective immunogen against B. ovis in mice. In this study, we evaluated the immunogenicity of rOmp31extract in rams. This immunogen was emulsified in an oil adjuvant and administered three times with 4 and 8 weeks intervals. Antibody response was measured in serum by whole B. ovis ELISA. Specific antibodies to purified rOmp31 (pET-Omp31) were detected by Western blotting and indirect ELISA. In addition, isotype specific antibodies were measured in tears. Serum bactericidal activity against B. ovis in the presence of complement was measured in vitro. Cellular immune response was explored by intradermal testing with purified rOmp31. Immunization with rOmp31 extract induced IgG specific antibodies in serum able to bind to whole B. ovis cells. Furthermore, strong inhibition in a competitive ELISA (with an Omp31-specific monoclonal antibody) suggested that a proportion of Omp31-specific antibodies were directed against a loop containing a protective epitope. Serum antibodies killed efficiently B. ovis in vitro in the presence of either guinea pig or ovine serum. Tears had both IgG and IgA antibodies to equivalent titers. Finally, immunized rams showed skin reactivity to Omp31. These data demonstrate that B. melitensis Omp31, a protective antigen identified in the mouse model, induces antibody and cellular immune mechanisms in sheep.
先前已确定，在大肠杆菌中产生的来自羊种布鲁氏菌的去污剂提取重组Omp31（rOmp31提取物）是小鼠抗绵羊布鲁氏菌的一种保护性免疫原。在本研究中，我们评估了rOmp31提取物在公羊中的免疫原性。将这种免疫原乳化于油佐剂中，并间隔4周和8周给药3次。通过全绵羊布鲁氏菌ELISA检测血清中的抗体反应。通过蛋白质印迹法和间接ELISA检测针对纯化的rOmp31（pET - Omp31）的特异性抗体。此外，还检测了泪液中的同种型特异性抗体。体外测定了在补体存在下血清对绵羊布鲁氏菌的杀菌活性。通过用纯化的rOmp31进行皮内试验来探索细胞免疫反应。用rOmp31提取物免疫诱导血清中能够结合全绵羊布鲁氏菌细胞的IgG特异性抗体。此外，在竞争性ELISA（使用Omp31特异性单克隆抗体）中强烈的抑制作用表明，一部分Omp31特异性抗体针对含有保护性表位的环。在豚鼠或绵羊血清存在下，血清抗体在体外能有效杀死绵羊布鲁氏菌。泪液中IgG和IgA抗体的滴度相当。最后，免疫的公羊对Omp31表现出皮肤反应性。这些数据表明，在小鼠模型中鉴定出的一种保护性抗原——羊种布鲁氏菌Omp31，可在绵羊中诱导抗体和细胞免疫机制。
