Article

Analysis of Transgenic Tobacco Plants Carrying the Gene for the Surface Antigen of the Hepatitis B Virus

Russian Academy of Sciences; Sechenov Moscow Medical Academy; Combiotech AOZT
Russian Journal of Genetics (Impact Factor: 0.43). 12/2002; 39(1):41-45. DOI: 10.1023/A:1022066710010

ABSTRACT 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.

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    ABSTRACT: 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.
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