GLIADIN QQPYPQPQPF-SOD CITRUS LIMON PERMEABILITY ACROSS RAT EPITHELIAL INTESTINE IN VITRO}

• Main Author: Andineke Nurhanifa, Aa
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78769
• Institution: Institut Teknologi Bandung

Superoxide dismutase (SOD) is antioxidant protein found in nature and is potent to be active ingredient for many pathophysiological condition caused by radical oxygen. One of the SOD which is potent as therapeutic protein candidate is SOD from lemon (Citrus limon L.). In Biotechnology Laboratory of Pharmacy ITB, SOD from C. limon has been produced and fused with gli@din peptide QQPYPQPQPF (GliSOD P61). The gliadin peptide is supposed to be SOD permeability enhancer to cross intestinal epithelial layer. In this research, permeability testing of GliSOD P61 to penetrate rat intestinal epithelial cells was conducted. Overproduction of GliSOD P61 was done in Escherichia coli BL21(DE3) and the purification was done using Nickel-nitrilotriacetic acid (NitNTA) column. Permeability testing was conducted by incubating the intestine with lysozim, bovine serum albumin, and GliSOD P61 (0.4 and 4 ng) inside the intestine. The existence of protein inside and outside the intestine was analyzed by protein electrophoresis and zymography. In this research, gene expression analysis for gene encoding zonulin and claudin-3 was also evaluated by quantitative Polymerase Chain Reaction (qPCR). The results showed that protein electrophoresis showed the existence of lysozim, albumin, and GliSOD P61 outside the intestine, but they were not detected in intestine control without GliSOD P61. Zymography result from the solution outside the intestine showed there was superoxide dismutase activity from GliSOD P61. Primer for zonulin could not amplify the gene target. Primer claudin-3 and ß-actin could amplify the gene target specifically at 58 cc, but the efficiency score was out of the acceptable range. The conclusion is GliSOD P6i could penetrate rat intestinal epithelial membrane and the gliadin peptide could help lysozim and albumin to penetrate the epithel. Gene expression analysis is still on progress.