PRODUCTION AND PURIFICATION OF RECOMBINANT PROTEIN Ag85B AND FUSION ANTIGEN Ag85B-Rv2660c FROM Mycobacterium tuberculosis

• Main Author: PRADANA (NIM: 20514054), FENDI
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/22140
• Institution: Institut Teknologi Bandung

Tuberculosis (TB) is a deadly disease caused by Mycobacterium tuberculosis in humans. Until now, an effective vaccine to combat TB has not been found. Therefore, a lot of research are directed to find new TB vaccine. Two of the TB vaccines candidate are Ag85B and antigen Rv2660c. The Ag85B protein is involved in transfering mycolate acids from cytosol to the cell wall of M. tuberculosis colonies . The Ag85B also exhibits immunological function because it can induce the proliferation of T-cells and the secretion of IFN-&#947; in TB patients. The Rv2660c is a protein expressed constantly through the stages of M. tuberculosis infection which can be associated with latent infection. The Rv2660c can also induce immune response. The aim of this study is to produce and purify recombinant proteins Ag85B and fusion antigen Ag85B-Rv2660c. The two antigens are promising as TB vaccine molecules. Each gene encoding both proteins has been successfully constructed in pMAL-c5x expression vector containing the <br /> <br /> <br /> gene encoding Maltose Binding Protein (MBP), a marker of antibiotic, and the Tac promoter. Each protein was produced in LB medium containing 100 &#956;g/mL ampicillin, 0,3 mM IPTG (isopropyl &#946;-D-1-thiogalactopyranoside, the Tac promoter inducer) for 24 hours in 4oC. The gene expression analysis using Polyacrylamide Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE) showed a band at ~72 kDa and ~77 kDa, corresponding to the size of Ag85B and Ag85B-Rv2660c fusion proteins respectively. The proteins are soluble in buffer due to MBP. Purification of the proteins were carried out by the affinity <br /> <br /> <br /> chromatography method using the amylose resin are resulting 0,45 mg and 0,15 mg for Ag85B and Ag85B-Rv2660c fusion proteins respectively. Based on bands <br /> <br /> <br /> profile of the pure proteins in SDS-PAGE, the proteins have been successfully purified.