title: Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).
abstract: For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.
authors.affiliation: Department of Plant Molecular biology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Sharma
authors.forename: Nirmal Kumar
authors.initials: NK
authors.lastname: Singh
authors.forename: Dewal
authors.initials: D
authors.lastname: Jani
authors.forename: Rama
authors.initials: R
authors.lastname: Sisodia
authors.forename: M
authors.initials: M
authors.lastname: Thungapathra
authors.forename: J K
authors.initials: JK
authors.lastname: Gautam
authors.forename: L S
authors.initials: LS
authors.lastname: Meena
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Singh
authors.forename: Amit
authors.initials: A
authors.lastname: Ghosh
authors.forename: Akhilesh Kumar
authors.initials: AK
authors.lastname: Tyagi
authors.forename: Arun Kumar
authors.initials: AK
authors.lastname: Sharma
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D002772:Cholera Toxin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D022121:Cholera Vaccines / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D018551:Solanum lycopersicum / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics* / Q000276:immunology
chemical_list: D022121:Cholera Vaccines; D011993:Recombinant Fusion Proteins; C085807:TcpA protein, Vibrio cholerae; D036781:Fimbriae Proteins; D002772:Cholera Toxin