CONSTRUCTION AND OVEREXPRESSION OPTIMIZATION OF GENE ENCODING HBCAG IN ESCHERICHIA COLI AS A THERAPEUTIC VACCINE COMPONENT
Hepatitis B is the most common liver infection worldwide. It is caused by Hepatitis B Virus (HBV) and may leads to cirrhosis and hepatocellular carcinoma. A therapeutic vaccine design is required to eliminate HBV from patient. Hepatitis B core Antigen (HBcAg) coded by HBcAg gene could induce Cytotox...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/78937 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Hepatitis B is the most common liver infection worldwide. It is caused by Hepatitis B Virus (HBV) and may leads to cirrhosis and hepatocellular carcinoma. A therapeutic vaccine design is required to eliminate HBV from patient. Hepatitis B core Antigen (HBcAg) coded by HBcAg gene could induce Cytotoxic T lymphocytes which plays a role in virus elimination. In this study, open reading frame (ORF) encoding for HBcAg from Indonesian isolate was codon optimized. The ORF encoding HBcAg was synthesized and inserted into an expression vector pET-28a(+) and the pET-28a HBcAg was transformed in Escherichia coli BL21(DE3). Optimization of growth condition of E. PET 28a HBcAg was conducted in two temperatures And agitation speeds, while optimization of gene overexpression was conducted in several IPTG concentrations, temperatures, and induction times using multifactorial design. The expression level were determined by SDS-PAGE analysis. The total HBcAg percentage (both soluble and insoluble) to soluble intracellular proteins was quantified using ImageJ software. Both significant and non-significant parameters affecting growth of E. coli and expression level were determined by Minitab software. A transforman of E. coli BL21(DE3) pET-28a HBcAg was used in optimization in a 10 mL culture. An SDS-PAGE analysis of the total intracellular proteins showed the presence of an intense protein band of 21.1 kDa, which was close to the theoretical size of HBcAg, 21 10m The optimization using multifactorial design showed that E. coli BL21(DE3) pET-28a HBcAg biomass was significantly affected by growth temperature, while the total HBcAg protein production was significantly affected by temperature and induction time (p<0,05). Using condition obtained from optimization in-culture volume of 200 mL, the percentage of soluble HBcAg at 25 0C (22.35%) was higher than percentage at 370C (15.96%). In conclusion, E. coli BL21(DE3) pET-28a HBcAg could produce HBcAg used as a component of therapeutic vaccine.
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