E. B. Rukavtsova

Expression and Characterization of Hepatitis B Surface Antigen in Transgenic Potato Plants

Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 μg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1

Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F0 and F1 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.

Tissue specific expression of hepatitis B virus surface antigen in transgenic plant cells and tissue culture

The tobacco plants (Nicotiana tabacum L.) carrying the HBsAg gene controlled by (Aocs)3AmasPmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh1, maize alcohol dehydrogenase gene intron were obtained. The presence of the adh1 gene intron did not significantly change the level of expression of the HBsAg gene in plants. The analysis of expression of hepatitis B virus surface antigen (HBs-antigen) in transformed plants expressing the HBsAg under the control of different promoters was made. The level of HBs-antigen in plants carrying the HBsAg gene controlled by (Aocs)3AmasPmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to 0.01% of total amount of soluble protein. The level of HBs-antigen in plants carrying the HBsAg gene controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant and made up to 0.06% of the total amount of soluble protein. Hairy root and callus cultures of plants carrying the HBsAg gene and expressing the HBs-antigen were obtained.

The use of RNA interference for the metabolic engineering of plants (Review)

The metabolic engineering of plants is aimed at the realization of new biochemical reactions by transgenic cells. These reactions are determined by enzymes encoded by foreign or self-modified genes. Plants are considered to be the most interesting objects for metabolic engineering. Although they are characterized by the same pathways for the synthesis of basic biological compounds, plants differ by the astonishing diversity of their products: sugars, aromatic compounds, fatty acids, steroid compounds, and other biologically active substances. RNA interference aimed at modifying metabolic pathways is a powerful tool that allows for the obtainment of plants with new valuable properties. The present review discusses the main tendencies for research development directed toward the obtainment of transgenic plants with altered metabolism.

Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen

The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01–0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.

Analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis B virus

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBs antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HBsAg gene under a single 35S promoter was 0.0001–0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002–0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants.

Plant Growth and Morphogenesis in vitroIs Promoted by Associative Methylotrophic Bacteria

The effects of aerobic methylotrophic bacteria Methylovorus mayson growth and morphogenesis were studied in in vitropropagated tobacco, potato, and flax. Colonization of plant explants with the methylo-trophic bacteria led to the stable association of bacteria and plants and enhanced the growth and the capacity of the latter for regeneration and root formation. When colonized by the methylotrophic bacteria, the rootless transgenic tobacco plants carrying the agrobacterial cytokinin gene iptrestored their ability to form roots. These data indicate the possibility to employ methylotrophic bacteria as a tool in experimental biology and plant biotechnology.

The ways to produce biologically safe marker-free transgenic plants

The review considers the basic strategies used to produce biologically safe marker-free transgenic plants and analyzes their advantages and disadvantages. The systems of positive and negative selection as safer approaches for transformant identification are briefly described. The application of co-transformation, transposition, and site-specific recombination for production of marker-free plants is described. Special attention is paid to novel approaches to create marker-free plants initially containing no selective genes in their genomes.

Immunogenicity of biologically safe potato tubers synthesizing hepatitis B surface antigen

110 Plants could be considered to be safe alternative sources of production of various proteins, vaccines and antibodies. To date, plants synthesizing dozens of valuable proteins, including antibodies, vaccines, immunomodulators, growth regulators, industrials enzymes, biopolymers, and reagents for biochemistry and molecular biology, have been obtained [1]. Post translational modifications of monomeric proteins and their intracellular translocations are similar in plant and animal cells, which give advantage for the plant cells producing multimeric proteins instead of recombinant microbial producers [2]. It has been demonstrated that valuable synthesis of the target anti gens resulting in active immune response could occur in plants. Viral and bacterial antigens synthesized in plants stimulated immunoglobin formation as a response to the corresponding pathogens in animals [3–7]. At present, transgenic plants producing vac cines against various infectious agents causing human diseases, including vaccines against hepatitis B virus, have been created and investigated [8–10]. Some vac cines based on transgenic plants are being clinically trialed [1]. The present study dealt with preclinical trials of the edible vaccine synthesized on the basis of transgenic potato with increased biological and ecological safety synthesizing hepatitis B surface antigen (HBsAg, HBs antigen) on mice. The marker free transgenic potato Solanum tuberosum L. cultivar Desiree constructed with the use of the recombinant pBM Ag plasmid was used in the study [11]. These plants contained no unfavorable (in some cases, even harmful) genes of selective resistance to antibiotics and herbicides in their genome. The HBs antigen synthesized in plants has no dif ferences in its physical, chemical or immune proper ties from the recombinant antigen synthesized by yeast cells and stimulates the formation of specific IgGs when injected into the body [9]. The HBs antigen from the transgenic plants is more efficient than purified recombinant yeast antigen in the case of peroral administration. Since the HBs antigen is collected in multimeric particles accumulated inside the mem brane vesicles in the plant cells, such natural “bioen capsulation” in the plant cell protects the antigen from the aggressive action in the digestive tract until it con tacts the effectors of intestine mucosal system [10]. The HBs antigen content in the obtained potato was up to 0.05% of the total soluble protein. Western blot analysis of HBs antigen was conducted after its immunoaffinity purification. The molecular weight of the purified protein was 24 kDa, which corresponded to the molecular weight of the major protein of hepa titis B virus envelope. Gel filtration demonstrated that the product of the HBsAg gene expression in trans genic plants was present in a high molecular weight multimeric form [12]. Thus, the assembly of mono meric HBs antigen forms in immunogenic multimeric aggregates that could be used as the substrate for the synthesis of hepatitis B virus vaccine occurs in the cells of both transgenic plants and recombinant strains pro ducing HBsAg. For obtaining potato tubers, transgenic potato con taining HBsAg gene was grown in green house condi tions in the Biotron artificial climate station. The amount of the HBs antigen in potato tubers detected using a test system for the immunoenzymatic detec tion of HBsAg (VectoHep B HBs antigen kit, Vec torBest, Russia) was up to 1 μg/g tuber weight. To study the immunogenicity of the antigen syn thesized in potato tubers, three groups consisting of 10 outbred NMRI mice 23–25 g in weight were used. The animals from the first and second groups were fed by transgenic potato tubers on days 1, 7, and 14 of the experiment (20 g of tubers or 20 μg of HBsAg per mouse), in addition, the mice from the first group were given 20 μg of glycopin (GMDP) as an adjuvant. The control group was fed with untransformed potato tubers. The use of recombinant vaccines characterized by a low immunogenicity suggests the necessity of adju GENERAL BIOLOGY

Obtaining marker-free transgenic plants

143 Selection genes conferring resistance to antibiotics and herbicides [1] or reporter genes [2] are conventionally used for selection of transgenic plants. Currently used commercial transgenic plants resistant to pests and herbicides pose a considerable potential biological and environmental hazard associated with their adverse effect on useful organisms of agrobiocenoses. In view of this, the development of methods for obtaining newgeneration transgenic plants without genetic garbage (such as selection genes conferring resistance to antibiotics and herbicides and other marker genes) is a topical problem.