title: Superexpression of tuberculosis antigens in plant leaves.
abstract: Recent developments in genetic engineering allow the employment of plants as factories for 1/foreign protein production. Thus, tuberculosis (TB) ESAT6 antigen was expressed in different plant systems, but the level of vaccine protein accumulation was extremely low. We describe the technology for superexpression of TB vaccine proteins (Ag85B, ESAT6, and ESAT6:Ag85B fusion) in plant leaves which involves: (i) construction of tobacco mosaic virus-based vectors with the coat protein genes substituted by those for TB antigens; (ii) Agrobacterium-mediated delivery to plant leaf tissues of binary vectors containing the cDNA copy of the vector virus genome; and (iii) replication of virus vectors in plant cells under conditions suppressing the virus-induced gene silencing. This technology enables efficient production of the TB vaccine proteins in plants; in particular, the level of Ag85B antigen accumulation was not less than 800 mg/kg of fresh leaves. Expression of TB antigens in plant cells as His(6)-tagged proteins promoted their isolation and purification by Ni-NTA affinity chromatography. Deletion of transmembrane domains from Ag85B caused a dramatic increase in its intracellular stability. We propose that the strategy of TB antigens superproduction in a plant might be used as a basis for the creation of prophylactic and therapeutic vaccine against TB.
authors.affiliation: Department of Virology and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia. dorokhov@genebee.msu.su <dorokhov@genebee.msu.su>
authors.forename: Yuri L
authors.initials: YL
authors.lastname: Dorokhov
authors.forename: Anna A
authors.initials: AA
authors.lastname: Sheveleva
authors.forename: Olga Y
authors.initials: OY
authors.lastname: Frolova
authors.forename: Tatjana V
authors.initials: TV
authors.lastname: Komarova
authors.forename: Anna S
authors.initials: AS
authors.lastname: Zvereva
authors.forename: Peter A
authors.initials: PA
authors.lastname: Ivanov
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Atabekov
mesh_terms: D016960:Agrobacterium tumefaciens; D000942:Antigens, Bacterial / Q000032:analysis / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000276:immunology; D005822:Genetic Vectors; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D018515:Plant Leaves / Q000276:immunology*; D030821:Plants, Genetically Modified / Q000276:immunology*; D010957:Plasmids; D014026:Nicotiana; D014027:Tobacco Mosaic Virus / Q000276:immunology; D032581:Tuberculosis Vaccines / Q000096:biosynthesis
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D032581:Tuberculosis Vaccines