Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato.
A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P2 promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.
Choi
Nak-Won
NW
Center for Molecular Biology and Gene Therapy, Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, CA 92354, USA.
Estes
Mary K
MK
Langridge
William H R
WH
D000956:Antigens, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001709:Biotechnology; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D018515:Plant Leaves; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011198:Solanum tuberosum / Q000235:genetics* / Q000378:metabolism*; D014170:Transformation, Genetic
D000956:Antigens, Viral; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C050779:VP7 protein, Rotavirus; D002772:Cholera Toxin