CLONED AND EXPRESSION OF THE INTEGRATED PROTEIN P DOMAIN EPITOPE HBSAG AND EPITOPE HBCAG (NOV GII. 4) USING THE MALTOSE BINDING PROTEIN (MBP) FUSION SYSTEM ON ESCHERICHIA COLI BL21 (DE3)

• Main Author: Mellyani, Karlina
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48717
• Institution: Institut Teknologi Bandung

There have been 3000 cases of Hepatitis B in the world every year and up to 2019 there are 2.9 million cases in Indonesia. Vaccines are one of the precautions that can be done to deal with Hepatitis B (HBV). The prevention of vaccine use can be an alternative and does not cause viral resistance as posed by anti-viral. Vaccine therapy may also help to increase immunity from liver cirrhosis patients. The effectiveness of vaccines can be increased using intranasal pathways and mucosal immune systems. In addition, it is necessary to improve the immune response from the antigen on the vaccine using the P particle of Norovirus as the vaccine platform. In this study, used NoV GII. 4 which is the P Domain of Norovirus Genegorup II genotype 4 and integrated epitope of HBV namely HBsAg and HBcAg. The recombinant protein of NoV GII. 4 enhanced its solubility with the addition of the protein maltose binding protein (MBP) fusion tag on the pMAL-C5X plasmids. An insertion of the NoV GII. 4 gene in the pMAL-C5X plasmids is done with the NcoI and EcoRI restriction enzymes. Then, NoV GII. 4 insertion result with pMAL-C5X plasmids will be cloned on Escherichia coli TOP10 (E.coli TOP10) and confirmed using polymerase chain reactions (PCR) colony and PCR confirmation. Furthermore, pMAL-C5X_NoVGII. 4 was transformed into E.coli BL21 (DE3) to be performed expression optimization and analysis of recombinant protein solubility. Optimization of expression is done with the induction of IPTG and confirmed through the results of sodium dodecyl Sulphate-polyacrylamide gel electrophoresis (SDS PAGE). Also conducted analysis of the solubility of recombinant proteins with dissolved fraction and insoluble fraction results in the induced IPTG protein expression and confirmed using the SDS PAGE. Based on the results of the study, it was obtained that the insertions of NoV GII. 4 on the pMAL-C5X plasmids and cloning on E.coli TOP10 as well as the transformation of E.coli BL21 (DE3) was successfully completed. This is evidenced by the results of the transformation confirmation using PCR colonies and PCR confirmations. Besides, optimization of protein expression and solubility analysis has not been done successfully.