Synthesis and assembly of anthrax lethal factor-cholera toxin B-subunit fusion protein in transgenic potato.
A DNA encoding the 27-kDa domain I of anthrax lethal factor protein (LF), was linked to the carboxyl terminus of the cholera toxin B-subunit (CTB-LF). The CTB-LF fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated in vivo transformation methods and antibiotic-resistant plants were regenerated. The CTB-LF fusion gene was detected in transformed potato leaf genomic DNA by polymerase chain reaction (PCR)-mediated DNA amplification. Immunoblot analysis with anti-CTB and anti-LF primary antibodies verified the synthesis and assembly of biologically active CTB-LF fusion protein oligomers in transformed plant tuber tissues. Furthermore, the binding of CTB-LF fusion protein pentamers to intestinal epithelial cell membrane receptors measured by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) indicated that the CTB-LF fusion protein made up approx 0.002% of the total soluble tuber protein. Synthesis of CTB-LF monomers and their assembly into biologically active CTB-LF fusion protein pentamers in potato tuber tissues demonstrates the feasibility of using edible plants for production and delivery of adjuvanted LF protein for CTB-mediated immunostimulation of mucosal immune responses against anthrax toxin.
Kim
Tae-Geum
TG
Center for Molecular Biology and Gene Therapy, Departmentof Biochemistry and Microbiology, Loma Linda University, Loma Linda, CA 92350, USA.
Galloway
Darrell R
DR
Langridge
William H R
WH
D000942:Antigens, Bacterial / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; D002772:Cholera Toxin