OPTIMASI OVERPRODUKSI DAN PEMURNIAN ESAT-6 REKOMBINAN (RESAT-6) SERTA UJI IMUNOREAKTIVITASNYA TERHADAP SERUM PENDERITA TUBERKULOSIS
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...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/62204 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | 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.
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