Synthesis and assembly of a cholera toxin B subunit SHIV 89.6p Tat fusion protein in transgenic potato.
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 G(M1)-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 4.6mg per 100g 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.
Kim
Tae-Geum
TG
Department of Biochemistry and Microbiology, Center for Molecular Biology and Gene Therapy, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
Ruprecht
Ruth
R
Langridge
William H R
WH
D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015696:Gene Products, tat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D015302:Simian Immunodeficiency Virus / Q000235:genetics*; D011198:Solanum tuberosum / Q000235:genetics*; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D002772:Cholera Toxin