EXPRESSION AND MOLECULAR CHARACTERIZATION OF HEPATITIS B PROTEIN PRODUCED IN Escherichia coli AND Coleus forskohlii
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Date
2016-07-13
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UNIVERSITY OF AGRICULTURAL SCIENCES GKVK, BENGALURU
Abstract
Hepatitis B virus (HBV) infection is a worldwide health problem, which can lead
to severe liver disease mainly hepatocellular carcinoma and cirrhosis. The present
investigation lays emphasis on expression of HBsAg gene in E. coli and C. forskohlii. The
confirmed recombinant pET28A+HBsAG clone was transformed into E. coli. The
positive clones were used for protein expression studies and induction parameters viz.,
IPTG concentration, temperature, induction time and pH of the medium were
standardized to produce optimum HBsAg protein yield. The clone 2 was selected for
optimization of protein expression. The highest protein expression was recorded when
cells were subjected to 1.5 μM IPTG (384 μg mL-1), pH at 8 (378 μg mL-1) and induction
time of 8 h (360 μg mL-1) with temperature of 35 °C. The purified protein was subjected
to SDS-PAGE analysis. The presence of ~25 kDa protein band confirmed the expression
of HBsAg protein in E. coli which was further confirmed through dot blot. The
Agrobacterium strain LBA4404 carrying confirmed recombinant pHB118 vector was
used for agroinfiltration of C. forskohlii leaves and the protein was extracted after 24, 48,
72, and 96 h. The extracted HBsAg protein was subjected to SDS-PAGE analysis which
showed the presence of ~25 kDa protein. Further this protein was confirmed by dot blot
and ELISA for its specificity. The highest expression was observed in leaves harvested
after 72 h (1.5 mg g-1 fresh weight of C. forskohlii leaf).
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