STANDARDIZATION OF TRANSFORMATION PROTOCOL AND EXPRESSION OF HEPATITIS B SURFACE ANTIGEN (HBsAg ) IN Coleus forskholii
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Date
2013-08-27
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Hepatitis B Virus (HBV) infection remains a major worldwide
infectious disease with serious long-term morbidity and mortality. It is
estimated that two billion people have been infected with the virus, and
more than 360 million have chronic HBV infections. The effective way to
control the virus is to take HBV vaccine. Hepatitis B surface antigen
(HBsAg) is an effective protective antigen suitable for vaccine
development for prevention of this disease. Hence, plant-based
production of vaccine for hepatitis B may be an economically feasible
alternative. The present study lays emphasis on production of a
recombinant protein/vaccine (HBsAg, Hepatitis B surface antigen) in
Coleus forskohlii against hepatitis-B disease through Agrobacterium
mediated gene transfer. MS basal media with 6mg/L BAP and 0.4mg/L
IAA was effective to induce a good callus as well as multiple shoot
production from leaf explant. The shoots were also rooted on same MS
basal media. The control of Agrobacterium overgrowth was effective in OD
dilution of 1:40 and 1:50with OD value of 0.6, immersion time of 2
minutes and co-cultivation period of one day. The transformed plants
were confirmed by Polymerase Chain Reaction (PCR) analysis which has
shown a band size of 681bp. Expression of HBsAg was confirmed by
Sodium Dodecyl Sulphate (SDS) and DOT-BLOT. The expected protein
band size of 24 KDa on gel confirmed expression of HBsAg S-protein in
transgenic plants. Since Coleus forskohlii is a medicinal plant and nature
of propagation is vegetative, it is advantageous to produce recombinant
HBsAg S-protein in this plant.
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Keywords
planting, proteins, biological phenomena, genes, genetic processes, dna, diseases, transgenics, concentrates, bacteria