CLONING AND EXPRESSION OF HEPATITIS B SURFACE ANTIGEN (HBsAg) GENE IN E. coli, Pichia AND Coleus forskohlii
| dc.contributor.advisor | RAMANJINI GOWDA, P. H | |
| dc.contributor.author | SATISH KUMAR, K. | |
| dc.date.accessioned | 2017-08-05T05:24:00Z | |
| dc.date.available | 2017-08-05T05:24:00Z | |
| dc.date.issued | 2015-07-19 | |
| dc.description.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). | en_US |
| dc.identifier.other | Th-11130 | |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810027523 | |
| dc.language.iso | en | en_US |
| dc.pages | 158 | en_US |
| dc.publisher | UNIVERSITY OF AGRICULTURAL SCIENCES GKVK, BENGALURU | en_US |
| dc.sub | Plant Biotechnology | en_US |
| dc.subject | null | en_US |
| dc.theme | CLONING AND EXPRESSION OF HEPATITIS B SURFACE ANTIGEN | en_US |
| dc.these.type | M.Sc | en_US |
| dc.title | CLONING AND EXPRESSION OF HEPATITIS B SURFACE ANTIGEN (HBsAg) GENE IN E. coli, Pichia AND Coleus forskohlii | en_US |
| dc.type | Thesis | en_US |