EFFECT OF HBX T118N/K130M/V131I MUTANT PROTEIN ON COLONY FORMATION OF HEPG2 CELLS

• Main Author: Riskha Nurmalasari, Dewi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78936
• Institution: Institut Teknologi Bandung

Hepatitis B virus (HBV) infected more than two billions people in the world. HBV infection can occur as acute or chronic infection. The development of chronic HBV infection towards hepatocellular carcinoma (HCC) is affected by viral particle load, genotype and the HBV X protein (HBx). The existence of HBx mutant protein has been studied to be correlated with HCC incidence, e.g. HBx K130M/V1311. Presence of HBx K130M/V1311 and Tl 18N mutant are the most common mutants reported in Indonesia. Preliminary study has reported the presence 'Of HBx T118N/K130M/V1311 mutant combination, meanwhile the effect this mutant combination on HCC development was never been studied before. The aim of this research was to determine the effect of HBx Tl 18N/K130M/V1311 on the colony formation of 4epG2 cells. The gene encoding for HBx Tl 18N and K130M/VI 311 has been obtained as synthetic gene, while the control plasmid pcDNA, plasmid with HBx wild-type gene (wtHBx) and plasmid with HBx Tl 18N/K130M/V1311 gene were generated in this study. The construction of pcDNA plasmid without DNA insert was performed by removing the HBx gene using EcoRI. The construction of pcDNA wtHBx was performed by inserting wtHBx gene from pD609 wtHBx plasmid into pcDNA. The construction of pcDNA Tl 18N/K130M/V13 li plasmid was performed using site directed mutagenesis. The generated plasmids were characterized using migration and restriction analyses as well as nucleotide sequence analysis. The plasmids containing gene for wild-type and mutant HBx were then transfected into HepG2 cells. In the colony formation assay, the transfected cells were selected using Zeocin and the optimum concentration was determined at 200 pg/ml. The result of colony formation assay showed less number of colonies formed upon expression ofHBx Tl 18N/K130M/Vf3 as compared to wtHBx.