IDENTIFICATION OF REGION B AND C DNA FRAGMENT MUTATION ON REVERSE TRANSCRIPTASE DOMAIN AND CLONING OF OPEN READING FRAME ENCODING POLYMERASE OF HEPATITIS B VIRUS IN ESCHERICHIA COLI JM109

• Main Author: MARTOGI LORENSI HUTAPEA (NIM 20706012), HOTMA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/10618
• Institution: Institut Teknologi Bandung

Antiviral treatment effectively suppressing the viral replication of hepatitis B virus (HBV). Yet, drug resistant mutants arise with increased length of treatment. Some mutations have been investigated at reverse transcriptase (RT) domain, more specific in region B and C which were suggested to be related to antiviral resistance. The objective of this study is to investigate the mutation occur in HBV and to clone the open reading frame encoding polymerase protein of HBV (PolHBV). The amplification of DNA fragment was conducted by using PCR and Long PCR (LPCR) method, cloning was conducted by using ligation and transformation method, and the analysis of nucleotide sequence was conducted by using sequencing method. The DNA fragment of region B and C of reverse transcriptase (RT) domain was successfully amplified and the mutation were identified at nucleotides in the position 544 and 613 resulted mutational pattern A529V and M550I, respectively. The open reading frame encoding polHBV was amplified by using LPCR method. Subsequently, the amplicon was also successfully ligated into pGEM-Teasy vector and cloned into E. coli JM109. The restriction analysis using HindIII and XhoI revealed that recombinant plasmid contain of pGEM-T easy and DNA insert as shown in theoretical size. The mutations that are suggested to be important in antiviral resistance were identified. Recombinant plasmid pGEM-TPolHBV2 has been gained and characterized.