PENGARUH PROTEIN X MUTAN T118N DAN K130M/V131I VIRUS HEPATITIS B TERHADAP EKSPRESI GEN HSPA6 PADA SEL HEPG2

• Main Author: Hans, Calvin
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49827
• Institution: Institut Teknologi Bandung

Hepatitis B virus (HBV) infection can cause hepatitis B disease and becomes the main cause of the development of hepatocellular carcinoma. One of the viral factors that induce tumor cell transformation is X protein or HBx. Mutations in the HBV x gene cause the formation of HBx mutants such as HBx T118N mutant and HBx K130M/V131I mutant which are found with the highest prevalence in Indonesia’s clinical samples. Previous next generation sequencing analysis has shown that HBx T118N and K130M/V131I upregulate hspa6 gene in HepG2 cells compared to HBx wild type. This study aims to confirm the effect of HBx T118N and HBx K130M/V131I on the expression of hspa6 gene in HepG2 cells using quantitative polymerase chain reactions (qPCR). Primers for the hspa6 gene were obtained from literature, commercial catalogs (OriGene ™), and designed using NCBI Primer-BLAST. Plasmids as expression vectors for HBx wild type and their mutants were confirmed by migration analysis, restriction analysis, and Sanger sequencing. Total RNAs from HepG2 cells that had been transfected by the plasmids were isolated and reverse transcribed into cDNA as a template for qPCR analysis. HSPA6 primer set 2 was chosen based on the results of qPCR optimization and the primer efficiency was 100.35%, calculated using LinRegPCR software. The expression of hspa6 gene was determined by Livak relative quantification method with hgapdh as a reference gene. The presence of HBx T118N and HBx K130M/V131I increased the expression of hspa6 gene in HepG2 cells as compared to HBx wild type which confirmed the NGS result.