CLONING AND EXPRESSION OF HEPATITIS B SURFACE ANTIGEN (HBsAg) GENE IN E. coli, Pichia AND Coleus forskohlii
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Date
2015-07-19
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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. Hepatitis B surface
antigen (HBsAg) is a glycoprotein found on the surface of the virus. The existing third
generation vaccine is a recombinant Hepatitis B surface antigen (rHBsAg) produced in
Yeast system (Saccharomyces cerevisiae or Pichia pastoris). Available vaccine is
costlier, hence low cost production of vaccine is necessary to reduce the incidence rate
especially in developing countries. Enhanced expression levels in yeast system or
alternative search for host system for low cost production of vaccine is necessary to meet
the demanding need of low cost vaccine. With these constrains in view, the present
investigation lays emphasis on expression of codon optimized HBsAg gene (coHBsAg) in
E. coli, Pichia pastoris (Intracellularly and Secretory) and Coleus forskohlii. HBsAg gene
was codon optimized and sub-cloned into expression vector E. coli (pET28a), Pichia
(pPICZA and pPICZ A) and transferred into E. coli BL21 and Pichia pastoris X-33
respectively. The positive clones were confirmed by DNA sequencing and restriction
enzyme analysis. E. coli clone (pET28a_HOP-C1) showed yield of 1.56 mg/L, Pichia
non-secretory clone (pPICZA_HOP-C10) recorded yield of 8.67 mg/L, Pichia Secretory
clone (pPICZ A_HOP-C5) yielded 11.85 mg/L. Recombinant protein isolated from
different host system was characterized by SDS-PAGE and Western blotting. Fused
recombinant protein isolated from E. coli and Pichia non-secretory showed size of 25.4
kDa, Pichia secretory and plant system showed band of size 34.7 and 24.0 kDa
respectively. Purified protein isolated from different host systems were used for
immunization studies in albino mice. Results showed that E. coli expressed HBsAg
showed highest titer value of 2.43 and plant produced HBsAg recorded second highest
with value of 1.70 on par with positive control (1.22).
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