CONSTRUCTION AND CLONING OF ENCODED GENE OF STRUCTURAL PROTEIN OF HEPATITIS C VIRUS INTO pVAX1 EXPRESSION VECTOR AND ITS EXPRESSION IN CHINESE HAMSTER OVARY CELLS

• Main Author: Setiadji, Hidayat
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45359
• Institution: Institut Teknologi Bandung

Hepatitis C Virus (HCV) is a member of Hepacivirus genus in Flaviviridae family which genome consisting of positive single stranded RNA molecule. Polyprotein precursor encoded by this RNA would be was cleaved by co-translational and post-translational process to generate structural (core and envelope, C, E1, E2) and non-structural proteins. This study was aimed to obtain recombinant HCV structural protein as a vaccine candidate that has CD4 + T cells and CD8 + T cells immune response, and could be manufactured in production scale. In order to obtain HCV structural protein as a vaccine component, the nucleotide sequence of C, E1, E2 (CE1E2) protein was constructed based on konsensus of amino acid sequence of CE1E2 protein of genotype 1b which was derived from multiple sequence alignment of amino acid sequences that were retrieved from several web sites. The amino acid sequence was examined by homology analysis to determine the numbers of coresponding amino acid sequences. Furthermore, the sequence was analyzed to search for B and T cell epitopes, signal peptide and codon preference of its nucleotide sequence was determined to CHO cell. The synthetic DNA sequence was ligated into pVAX1 © expression vector. The recombinant DNA were transformed into Escherichia coli. After the DNA insert was confirmed by Polymerase Chain Reaction, migration and sequencing analyses, the recombinant DNA was transfected into CHO cell to express HCV structural protein. Expression of HCV structural protein was characterized by internal cellular staining using fluorescence-activated cell sorting (FACS). In this study, cloning and construction the nucleotide sequence of CE1E2 protein was sucessfully done. However, recombinant CE1E2 protein was produced in little quantity because the transfection of recombinant DNA into CHO cells was not optimal.