RESUMEN
The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.
Asunto(s)
Cloroplastos/metabolismo , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Nicotiana/citología , Nicotiana/metabolismo , Plantas Tóxicas , Transgenes/genética , Southern Blotting , Western Blotting , Cloroplastos/genética , Toxina del Cólera/biosíntesis , Toxina del Cólera/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Gangliósido G(M1)/metabolismo , Expresión Génica , Vectores Genéticos/genética , Genoma de Planta , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Recombinación Genética/genética , Nicotiana/genética , Vacunas Comestibles/genética , Vibrio cholerae/genéticaRESUMEN
Chloroplast genetic engineering offers several advantages over nuclear genetic engineering, including gene containment and hyperexpression. However, introducing thousands of copies of transgenes into the chloroplast genome amplifies the antibiotic resistance genes. Two recent articles report different and novel strategies to either remove antibiotic resistance genes or select chloroplast transformants without using these genes. This should eliminate their potential transfer to microorganisms or plants and ease public concerns about genetically modified crops.