THE POTENCY OF RECOMBINANT STAPHYLOCOCCAL ENTEROTOXIN B (SEB) AS ANTICANCER AGAINST MCF-7 BREAST CANCER CELL LINE

• Main Author: Rodiansyah, Achmad
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/71392
• Institution: Institut Teknologi Bandung

Staphylococcal enterotoxin B (SEB), a toxin produced by Staphylococcus aureus, may be used as a therapeutic protein to eradicate cancer cells. The immune system is activated by SEB, or SEB directly attacks target cells with the potential to trigger apoptosis. This study investigated the cytotoxic effects of recombinant SEB (rSEB) in MCF-7 cells. This research was conducted by plasmid construction that carried optimized SEB encoding gene for Escherichia coli BL21(DE3) expression host. The recombinant plasmid was transformed with the heat shock method. After that, colony PCR and DNA sequencing were used to confirm the transformants. Gradient IPTG concentrations and incubation after inductions were performed to obtain the optimal condition for protein production. The protein target was then purified with nickel-NTA resin and dialyzed with the dialysis tubing method. The purified protein was concentrated, and its concentration was measured using the Bradford method. The purified rSEB protein was then tested for cytotoxicity assay on MCF-7 cancer cells using the MTT method, and the RT-qPCR was used to assess the expression profile of the gene that regulates apoptosis. The recombinant protein production results showed that the rSEB could be detected in both the soluble and insoluble fractions. Induction of 0.75 mM IPTG followed by 16 hours of incubation was the optimal condition for protein production. The result of purification, dialysis, and verification using Western blotting showed that rSEB protein could be purified. The inhibitory concentration (IC)-50 for the MCF-7 cell line was 223.75 ?g/mL. Treatment with different dosages of IC25, IC50, and IC75 showed that rSEB could enhance the expression of the TNF-?, NF-?B, and Bax genes better than the apoptosis control (paclitaxel). In MCF-7 cells, rSEB at an IC75 concentration increased the relative expression of NF-?B and Bax about 12.05±0.92 and 12.34±0.86 folds, respectively. However, the highest expression of TNF-? was found in the IC50 group, around 12.31±1.78. These findings suggest that expressed rSEB is toxic to the MCF-7 cell line and leads it to apoptosis.