CONSTRUCTION AND EXPRESSION OF AG85B FROM MYCOBACTERIUM TUBERCULOSIS FOR TB VACCINE DEVELOPMENT

• Main Author: DEMISYAREP (NIM : 10510018) ;Pembimbing Dr. Ihsanawati, Dr. Dessy Natalia, DEDANA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/18263
• Institution: Institut Teknologi Bandung

Tuberculosis (TB) is a deadly infectious disease caused by Mycobacterium tuberculosis. Until now, the only available vaccine against TB has had little impact on the TB disease. Therefore, researches on TB vaccines need to be developed to produce new vaccines for better protection against TB. Antigen 85 (Ag85) is one of the TB vaccine candidates, consisting of Ag85A, Ag85B, and Ag85C. Ag85B plays an important role in maintaining the integrity of the cell wall by transfering mycolic acid from cytosol to the cell wall. Moreover, Ag85B of TB patients shows immunogenic properties by induction of T-cell poliferation and secretion of IFN-γ. The purpose of this study is to construct and express Ag85B in Escherichia coli BL21(DE3). A gene encoding Ag85B was constructed in pMAL-c5x harbouring a gene for maltose binding protein. A gene encoding Ag85B was obtained successfully using Polymerase Chain Reaction (PCR) with a result of a 900 bp-band on agarose gel electrophoresis. The PCR-result had also been cloned using a pGEM-T cloning vector, producing a 3.9 kb-band on agarose gel electrophoresis. Subcloning of the gene encoding Ag85B into the pMAL-c5x vector generated a recombinant plasmid Ag85B-pMAL-c5x. The expression of Ag85B-pMAL-c5x showed bands at approximately 70 kDa on Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE). The SDS-PAGE band is correlated with a fusion protein between Ag85B (30 kDa) and the maltose binding protein (42.5 kDa).