Bang-Geul Kim

Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system

To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM1-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.

Expression of a Synthetic Cholera Toxin B Subunit in Tobacco Using Ubiquitin Promoter and bar Gene as a Selectable Marker

A protocol has been developed to produce a cholera toxin B subunit (CTB) in tobacco tolerant to the herbicide phosphinothricin (PPT) by means of in vitro selection. The synthetic CTB subunit gene was altered to modify the codon usage to that of tobacco plant genes. The gene was then cloned into a plant expression vector and was under the control of the ubiquitin promoter and transformed into tobacco plants by Agrobacterium-mediated transformation. Transgenic plantlets were selected in a medium supplemented with 5 mg/L PPT. Polymerase chain reaction analysis confirmed stable integration of the synthetic CTB gene into a chromosomal DNA. A high level of CTB (1.8% of total soluble protein) was expressed in transgenic plants, which was 18-fold higher than that under the control of the expressed CaMV 35S promoter with native gene. The transgenic plants when transferred to a greenhouse proved to be resistant to 2% PPT.

Production of monoclonal antibody against FimA protein from Porphyromonas gingivalis in rice cell suspension culture

Major fimbriae (FimA) of Porphyromonas gingivalis play an important role in the adherence of the bacterium to oral surfaces, making them a target for the development of a vaccine against periodontal disease. In our previous study, monoclonal antibodies to FimA were expressed in tobacco plants using the agroinfiltration method. In this report, for easy purification, monoclonal antibodies to FimA were produced and accumulated in rice callus suspension culture. The accumulated antibodies were purified by protein G-affinity chromatography. The plant-produced monoclonal antibodies inhibited the binding of P. gingivalis to saliva-coated hydroxyapatite beads, as well as the invasion of oral epithelial KB cells due to the bacterium. The antibodies enhanced the intracellular killing of P. gingivalis by polymorphonuclear neutrophils. These results suggest that FimA-specific monoclonal antibodies produced in a rice suspension culture were easily purified and biologically active against P. gingivalis, and thus may be used for passive immunization to prevent P. gingivalis-induced periodontal disease.

Production of monoclonal antibodies against the FimA protein of Porphyromonas gingivalis in Nicotiana benthamiana

Porphyromonas gingivalis, a gram-negative anaerobic oral bacterium, causes periodontal disease by binding to saliva-coated oral surfaces. The FimA protein from P. gingivalis is a crucial pathogenic component of the bacterium and a target for vaccine development against periodontal disease. Complementary DNAs encoding the heavy and light chains of two monoclonal antibodies that bind specifically to the FimA protein were cloned into a plant expression vector under the control of the duplicated Cauliflower Mosaic Virus 35S promoter, and agroinfiltration was used to allow the vectors to infiltrate tobacco plants. The expressions of the heavy and light chains in the leaf tissue were detected using antibodies specific to each antibody chain. Western blot analysis showed the specific binding of the plant-derived monoclonal antibodies to the native FimA protein purified from P. gingivalis. Our finding that plant-derived monoclonal antibodies bound specifically to the native FimA protein indicates that plantderived monoclonal antibodies can protect against P. gingivalis invasion.