ANALISIS EKSPRESI GEN ZO-1, CLAUDIN-2 DAN OCCLUDIN SEL CACO-2 HASIL UJI PERMEABILITAS GliSOD_P51 DAN GliSOD_P61 CITRUS LIMON

• Main Author: Ghina Aqila Arham, Alya
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44428
• Institution: Institut Teknologi Bandung

Superoxide dismutase (SOD) is a natural antioxidant protein which catalyzes O2 - radical dismutation into O2 and H2O2. This enzyme is considered as a potential therapeutic compound which is important in dealing with oxidative stress. SOD has high molecular size, thus making it difficult to be absorbed in the intestinal tract. To overcome this problem, in previous research in Pharmaceutical Biotechnology Laboratory, Citrus limon SOD has been fused with gliadin peptide LGQQQPFPPQQPYPQPQPF (GliSOD_P51) and QQPYPQPQPF (GliSOD_P61). In this study, the mechanism of the gliadin peptides in increasing the permeability of the intestinal epithelium against SOD was evaluated. Research was initiated by overproduction, purification and concentration of unfused SOD (SOD Cl), GliSOD_P51 and GliSOD_P61. The purified protein was then characterized by SDS-PAGE and the activity was tested by zymography method. SODCl, GliSOD_P51 and GliSOD_P61 permeability was tested using Caco-2 cell culture method. SDS-PAGE analysis was performed to determine the presence of SOD at the apical and basolateral compartment after the permeability test had finished. Total RNA was isolated from permeability tested Caco-2 cell culture. RNA was reverse transcripted to obtain cDNA for qPCR. The primers specificity and efficiency were tested. Gene expression analysis of ZO-1, claudin-2 and occludin was conducted using qPCR and the result was analyzed by Livak relative quantification method. Gliadin peptides of GliSOD_P51 and GliSOD_P61 showed the ability to increase intestinal epithelial permeability against SOD. GliSOD_P51 and GliSOD_P61 were able to penetrate the Caco-2 cell monolayer, while the SODCl could not. Gliadin peptides opened the tight junction by lowering claudin-2 and occludin gene expression, without affecting ZO-1 gene expression.