RESUMEN
Expression of the protective F1 and V antigens of Yersinia pestis, as a fusion protein, in carrot was pursued in an effort to develop an alternative vaccine production system against the serious plague disease. Transgenic carrot plants carrying the F1-V encoding gene were developed via Agrobacterium-mediated transformation. Presence, integration, and expression of the F1-V encoding gene were confirmed by polymerase chain reaction (PCR), DNA gel blot analysis, and reverse-transcriptase (RT)-PCR analyses, respectively. An ELISA assay confirmed the antigenicity of the plant-derived F1-V fusion protein. Immunogenicity was evaluated subcutaneously in mice using a soluble protein extract of freeze-dried transgenic carrot. Significant antibody levels were detected following immunization. These results demonstrated that the F1-V protein could be expressed in carrot tap roots, and that the carrot F1-V recombinant protein retained its antigenicity and immunogenicity.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Daucus carota/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Yersinia pestis/metabolismo , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Daucus carota/genética , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Peste/inmunología , Vacuna contra la Peste/genética , Vacuna contra la Peste/inmunología , Vacuna contra la Peste/metabolismo , Raíces de Plantas/genética , Plantas Modificadas Genéticamente/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Yersinia pestis/genéticaRESUMEN
Yersinia pestis is a pathogenic agent that causes the bubonic and pneumonic plague. The development of an efficient and low-cost oral vaccine against these diseases is highly desirable. In this study, the immunogenic fusion protein F1-V from Y. pestis was introduced into lettuce via Agrobacterium-mediated transformation, and putative transgenic lines were developed. The presence of the transgene in these putative transgenic lines was determined using polymerase chain reaction (PCR), and transgene integration and transgene copy number were confirmed following Southern blot analysis. The presence of specific F1-V transcripts was confirmed by reverse-transcriptase (RT)-PCR. Using monoclonal antibodies, ELISA and western blot analysis revealed that the expected antigenic F1-V protein was successfully expressed in transgenic lines. Mice immunized subcutaneously with lettuce expressing the F1-V antigen developed systemic humoral responses as 'proof of concept' of using lettuce as a production platform for the F1-V immunogen that could be used as a candidate plant-based vaccine against plague.