1607-6729/04/0304- © 2004 MAIK “Nauka / Interperiodica” 0104 Doklady Biochemistry and Biophysics, Vol. 395, 2004, pp. 104–107. Translated from Doklady Akademii Nauk, Vol. 395, No. 5, 2004, pp. 704–707. Original Russian Text Copyright © 2004 by Turchinovich, Deineko, M. Filipenko, Khrapov, Zagorskaya, E. Filipenko, Sennikov, Kozlov, Shumnyi. Studies conducted over the last decade have shown that genetically modified plants are potentially less expensive and safer sources of recombinant proteins than the commonly used expression systems based on bacteria, yeast, and cultured mammalian and insect cells. World’s leading biotech companies have already created transgenic plants producing hormones, cytok- ines, growth factors, and enzymes. The recombinant proteins from plants were found to be similar in biolog- ical activity to their counterparts derived from other expression systems [1]. Transgenic plants that produce epitopes of human and animal pathogens are promising for oral immunization [2]. Vaccines against hepatitis B, rabies, Norwalk virus, and diarrhea, obtained from plants, have now passed first clinical trials [3, 4]. How- ever, when subunit vaccines are used, immunological tolerance may develop to the pathogen [5] (for exam- ple, no immune response is observed to antigens con- sumed every day in food). To improve the immuniza- tion efficacy of “edible vaccines” and to prevent the development of immunological tolerance, researchers actively seek proteins that can act as adjuvants [6]. Interleukin-18 (IL-18) was found to be a promising adjuvant in laboratory animals subjected to intranasal vaccination [7]. It was possible to avoid the induction of immunological tolerance to ovalbumin in newborn mice by combining this antigen with oral administra- tion of IL-18; their combination elicited the systemic and mucosal immune responses to ovalbumin in the mice [8]. In this context, it is of interest to study the possibility of supplementing edible vaccines with IL- 18 as an adjuvant. We believe that transgenic plants may be convenient vehicles for delivery of IL-18 by the oral route to gastrointestinal mucosae. It is also of inter- est to study whether pure recombinant IL-18 can be obtained by affine chromatography. The goal of this study was to develop transgenic tobacco plants producing human IL-18. To this end, a genetic construct was prepared containing the human IL-18 nucleotide sequence under control of the double cauliflower mosaic virus 35 S promoter. Transgenic Nic- otiana tabacum SR1 plants containing single-copy inserts were obtained by Agrobacterium-mediated transformation. Their transgenic status was confirmed by a number of molecular biological methods. The pro- duction level of recombinant IL-18 in transgenic tobacco leaves was more than 0.01% of the total cell protein. Note that, in the Russian-language scientific litera- ture, there are no publications describing attempts at expressing human or animal interleukins, potential mucosal adjuvants, in plants. The target gene used in plant transformation was the nucleotide sequence encoding a chimeric protein, in which the thrombin cleavage site is inserted between the leaderless form of human IL-18 and the N-terminal hexahistidine region. Plasmid pET15bIL18 was con- structed earlier by researchers of the Group of Pharma- cogenomics (Institute of Chemical Biology and Funda- mental Medicine, Siberian Division, Russian Academy of Sciences, Novosibirsk). Transformation of Nicotiana tabacum SR1 plants was performed by coculturing leaf disks with Agrobacterium tumefaciens LBA4404 cells in suspension, as described previously [9]. Genomic DNA was isolated from the plants as described in [10]. Transgenic tobacco plants with one transgene insert per genome were selected based on a 3 : 1 ratio of kanamy- cin-resistant to susceptible phenotypes among progeny derived from self-pollination of original transformants under selective conditions (Murashige and Skoog medium [11] containing 100 mg/ml of kanamycin). To prove that transgenic plants harbor contain single-copy inserts, Southern blot analysis was used. Genomic Transgenic Tobacco Plants Producing Human Interleukin-18 A. A. Turchinovich*, E. V. Deineko*, M. L. Filipenko**, E. A. Khrapov**, A. A. Zagorskaya*, E. A. Filipenko*, S. V. Sennikov***, Academician of the RAMS V. A. Kozlov***, and Academician V. K. Shumnyi* Received October 15, 2003 BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY * Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 10, Novosibirsk, 630090 Russia ** Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia *** State Research Institute of Clinical Immunology, Siberian Division, Russian Academy of Medical Sciences, ul. Yadrintsevskaya 14, Novosibirsk, 630099 Russia