Synthesis and assembly of a cholera toxin B subunit SHIV 89.6p Tat fusion protein in transgenic potato

Abstract

A cDNA encoding the simian-human immunodeficiency virus (SHIV 89.6p) Tat regulatory element protein was fused to the c-terminus of the cholera toxin B subunit gene (ctxB-tat) and introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The fusion gene was detected in the genomic DNA of transformed potato leaf cells by PCR DNA amplification. Synthesis and assembly of the CTB-Tat fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-Tat fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (G M1-ELISA). Based on the ELISA results, CTB-Tat fusion protein made up about 0.005-0.007% of total soluble tuber protein or approximately 4.6 mg per 100 g potato tuber tissue. The synthesis and assembly of CTB-Tat monomers into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using viral pathogen antigens synthesized in edible plants for mucosal immunization against HIV-1 infection. © 2004 Elsevier Inc. All rights reserved.