DEVELOPMENT OF COLORIMETRIC-BASED REPORTER SYSTEM FOR SCREENING OF CANDIDATE INHIBITOR OF RNA POLYMERASE INFLUENZA VIRUS

• Main Author: Ainun Qolbi Wasdili, Fini
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44437
• Institution: Institut Teknologi Bandung

Influenza virus is an enveloped virus with segmented negative-sense RNA genome. Infection of influenza virus can cause seasonal flu epidemic and pandemic. Development of novel anti-influenza candidate require quantification method based on virus replication and reporter system. Reporter system for quantification of influenza virus uses Green Fluorescent Protein (GFP) and luciferase. This system requires a special instrument to measure fluorescent and luminescent. The aim of this research is to develop of colorimetric-based reporter system using Secreted Alkaline Phosphate (SEAP) which can be used in screening of anti-influenza candidate. SEAP reporter system was designed as synthetic gene which contain gene encoding for SEAP with negative orientation, RNA Polymerase I Promoter, RNA Polymerase I Terminator, 5’and 3’UTR from segment 6 of influenza virus. SEAP reporter system was ordered in pcDNA3.1(+) plasmid and named as pSEAP-Flu. To detect SEAP expression from reporter system, Vero cell were transfected with pSEAP-Flu, plasmid carrying the gene encoding for influenza nucleoprotein (NP) and RNA polymerase subunit (PA, PB1, PB2). SEAP activity was measured by addition of p-nitrophenyl phosphate (pNPP) substrate to growth medium, and measurement of color intensity at 405 nm. pSV-?-Galactosidase (pSV-?-Gal) plasmid was also transfected into Vero cells as transfection control. ?-Galactosidase activity was performed by addition of Chlorophenol Red-?-D-galactopyranoside (CPRG) to Vero cell lysate and then measured at 570 nm. Variation in transfection time, the incubation time of SEAP activity and amount of pSEAP-Flu was performed to optimize SEAP reporter system. The selected reporter system condition is 24 hours transfection time, 24 hours incubation time of SEAP activity and 200 ng pSEAP-Flu. The addition of ribavirin concentration 0-300 µM showed decrease of SEAP expression, detected from its activity.