Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato |
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Authors: | Tae-Geum Kim Andreas Gruber Ruth M. Ruprecht William H. R. Langridge |
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Affiliation: | (1) Department of Biochemistry and Microbiology and Center for Molecular Biology and Gene Therapy, School of Medicine, Loma Linda University, 92350 Loma Linda, CA;(2) Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, JFb-809, 02115-6084 Boston, MA |
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Abstract: | A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5′ to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016–0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques. |
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Keywords: | SIV Solanum tuberosum edible vaccine AIDS Gag |
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