CONSTRUCTION AND EXPRESSION OF GENE ENCODING CHIKUNGUNYA VIRUS E1 ENVELOPE PROTEIN USING PMAL-C5X VECTOR IN 4 STRAINS OF ESCHERICHIA COLI

• Main Author: HAFIZH IQBAL NIM: 10514072, MUHAMMAD
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29217
• Institution: Institut Teknologi Bandung

Chikungunya disease is caused by chikungunya virus (CHIKV) infection against the patient <br /> <br /> through the bite of Aedes aegepty and Aedes albopictus mosquitoes. Symptoms include fever <br /> <br /> and joint pain, which could be mistaken for other mosquito-borne diseases, such as dengue <br /> <br /> fever and zika fever, so errors may occur while handling the patient. These errors can be <br /> <br /> avoided by making a proper diagnosis, but the existing chikungunya diagnosis techniques <br /> <br /> still have some drawbacks. Therefore, a rapid, precise, and economical diagnostic technique <br /> <br /> of chikungunya is needed. One of CHIKV structural proteins, which is E1 envelope <br /> <br /> (CHIKVE1) protein, plays a role in viral infection to host cell and in stimulating the specific <br /> <br /> immune response of the patient, so that CHIKVE1 protein can be used as the basis for <br /> <br /> making diagnostic kit for chikungunya disease. In this study, recombinant CHIKVE1 protein <br /> <br /> was produced and fused with Maltose Binding Protein and His tag, in Escherichia coli <br /> <br /> BL21(DE3), E. coli BL21(DE3) pLysS, E. coli ArcticExpress(DE3), dan E. coli Rosetta <br /> <br /> Gami B(DE3) cells. CHIKVE1 protein fusion with MBP was done to increase protein <br /> <br /> solubility, while fusion with His tag was done for purification process. Fusion proteins were <br /> <br /> obtained by expressing gene encoding CHIKVE1 protein which was amplified and modified <br /> <br /> by Polymerase Chain Reaction (PCR) technique. The amplified gene was subcloned <br /> <br /> downstream malE gene of pMAL-c5X expression vector. Fusion protein of ~90 kDa was <br /> <br /> well produced in the form of inclusion bodies by Escherichia coli Rosetta Gami B (DE3) <br /> <br /> cells based on Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) <br /> <br /> analysis. Therefore, protein refolding was carried out along with protein purification process <br /> <br /> using urea gradient in nickel affinity resin. The results of the SDS-PAGE analysis showed <br /> <br /> that the fusion protein solution was successfully eluted at a 50 mM imidazole concentration. <br /> <br /> Furthermore, the resulting MBP-CHIKVE1 protein was used to design CHIKV infection <br /> <br /> diagnostic kit prototype