MOLECULAR CHARACTERIZATION OF T4 TRANSGENIC TOBACCO WITH HEPATITIS – B GENE
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Date
40865
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Hepatitis B is a potentially life-threatening liver infection caused by the HBV and is a major global health problem. The worldwide problem of HBV infection eventually led to the development of a vaccine. Immunization with Hepatitis B vaccine is the most effective means of preventing Hepatitis B virus infection and its consequences. As the available recombinant vaccine is expensive, cheaper and improved vaccines are urgently needed to address the global scourge of infectious diseases. Plants are a potential source of HbsAg, in addition, a plant based HBsAg expression system makes possible as an oral immunization strategy by simply feeding the plant products. The primary means of transformation is the Agrobacterium mediated gene transfer which has provided a reliable means of creating transformants in a wide variety of species and also can express a wide variety of pharmaceutically important products including recombinant vaccines. The present investigation lays emphasis on study of integration and stability of the recombinant protein expressed in Tobacco plants. Restriction digestion analysis of the gene construct pHB118 with restriction enzymes EcoRI and BamHI yielded two separate bands of 9.7kb and 3.6kb size. The leaf explants of tobacco were transformed with Hepatitis B surface antigen gene along with npt-II as an antibiotic selection marker gene. The presences of HBsAg gene in putative transformants were confirmed by PCR analysis. All the putatively transformed tobacco plants showed the presence of 900 bp band in the PCR analysis. The crude protein obtained from the transformed tobacco plants were tested by SDS-PAGE for presence of 24 kDa protein, western-blot and ELISA confirmed the antigen specificity and immunogenic nature of the Hepatitis B surface antigen. The T4 generation seeds obtained from the transgenic tobacco plants were tested for the germination in the presence of kanamycin. It was observed that the segregation ratio was 3:1 indicating Mendelian inheritance. The growth parameters of T4 generation transgenic and control tobacco plants showed there is no major variation in growth between transgenic and control plants.