OPTIMASI OVERPRODUKSI DAN PEMURNIAN ESAT-6 REKOMBINAN (RESAT-6) SERTA UJI IMUNOREAKTIVITASNYA TERHADAP SERUM PENDERITA TUBERKULOSIS

• Main Author: Putri, Anil
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/62204
• Institution: Institut Teknologi Bandung

Tuberculosis is caused by Mycobacterium tuberculosis and have infected 9.4 million world population. Accurate diagnosis is essential for prevention, treatment and control of tuberculosis. Early Secreted Antigenic Target 6 kDa (ESAT-6) is a secreted protein of M. tuberculosis which is a potential diagnostic component for tuberculosis. Open reading frame (orf) of ESAT-6 of clinical isolate M. tuberculosis from Indonesia was cloned into expression vector pET32b. In this research pET32b esat-6 has been transformed into Escherichia coli BL21(DE3) and characterized by migration analysis, digested with BamH1 and PCR. rESAT-6 was overproduced using various IPTG (isopropyl ?-D-1- thiogalactopyranoside) concentration, incubation time and temperature induction (0.1, 0.25, 0.5, 1mM IPTG; 2, 3, 4 hours; 4 ºC, 20 ºC, 37 ºC temperature). Purification of rESAT-6 was optimized with various concentration of imidazole by Nickel affinity column chromatography and characterized by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Immunoreactivity was analyzed by Western Blot using sera of tuberculosis patient. Characterization of pET32b esat-6 using three methods gave positive results i.e. slower migration of plasmid than pET32b without inserted DNA, was digested by BamH1 and resulted 6047 bp linier DNA fragment in size. An 275 bp PCR product was obtained by using specific primer for esat-6. rESAT-6 soluble protein 32 kDa in size was overproduced at optimum condition 0.1 mM IPTG, at 37 ºC for 3 hours incubation time. Purified rESAT-6 was obtained by using 100 mM imidazole in elution buffer. Immunoreactivity assay showed 1 of 6 patient sera (BTA (+) and BCG (-)) positively recognized rESAT-6.