CONSTRUCTION AND EXPRESSION OF A GENE ENCODING E1 RECOMBINANT PROTEIN FROM CHIKUNGUNYA VIRUS IN Escherichia coli

• Main Author: MAMIZAR (NIM:20516046), IMANA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28005
• Institution: Institut Teknologi Bandung

Chikungunya is an infection disease caused by chikungunya virus (CHIKV). CHIKV is <br /> <br /> transmitted from one human to another human by Aedes aegypti and Aedes albopictus <br /> <br /> mosquitos. The symptoms start to appear during 3–12 days incubation period are fever, skin <br /> <br /> rash, joint and muscle pain. Chikungunya is not a deadly desease, however the joint and <br /> <br /> muscle pain can last up to several months without proper handling. There are clinical <br /> <br /> symptoms similarities between this disease and several other diseases such as dengue fever <br /> <br /> and zika fever. These causing the importance of early diagnostic to make sure the right <br /> <br /> handling. <br /> <br /> CHIKV genom is about 11,8 kb and has two open reading frames (ORF) which encode four <br /> <br /> non-struktural proteins (nsP1, nsP2, nsP3, nsP4), and five struktural proteins (capsid, E3, <br /> <br /> E2, 6K, E1). One of the structural protein, E1 protein, has important rule in virus infection to <br /> <br /> host cell process and can lead specific immune response from the patient infected by CHIKV. <br /> <br /> Hence, E1 protein is potentially becoming a basic to make diagnostic kit. The aim of this <br /> <br /> research are to construct and express gene encoding E1 CHIKV protein in pET-32b system <br /> <br /> using Escherichia coli (E. coli) as the host cell. <br /> <br /> In this research, E1 recombinant protein is produced as fusion protein together with <br /> <br /> thioredoxin (Trx) as fusion partner in order to enhance its solubility, and 6× histidin-tagged <br /> <br /> to facilitate the purification process. Trx-E1 fusion protein recombinant was produced <br /> <br /> udergo several steps including amplification of a gene encoding E1 CHIKV protein using <br /> <br /> Polymerase Chain Reaction (PCR), gene cloning, subcloning, and expression using two types <br /> <br /> of (E. coli) as hosts cell. Amplified E1 CHIKV gene was cloned to pGEM-T vector. Gene <br /> <br /> expression was performed in pET-32b system using E. coli BL21(DE3) and E. coli <br /> <br /> BL21(DE3)plysS as hosts cell within 3 hours production time with 0,5 mM IPTG as inducer. <br /> <br /> The results of this study showed that the expression level of E1 CHIKV gene are much higher <br /> <br /> in E. coli BL21(DE3)plysS The sodium dodecyl sulphate polyacrylamide gel electrophoresis <br /> <br /> (SDS-PAGE) analysis showed ~66,8 kDa band which was corresponding to protein target <br /> <br /> actual size. This protein appear more dominant at dissolved phase, so it can be concluded <br /> <br /> that Trx as protein fusion partner can enhance the solubility of protein target. The dissolved <br /> <br /> phase of Trx-E1 protein was then purified by using nickel nitrilotriacetic acid (Ni-NTA) <br /> <br /> chromatography affinity. The SDS-PAGE showed that target protein (~66,8 kDa protein) was <br /> <br /> dominant at eluted fraction using Tris-HCl pH 8,0 elution buffer that contain 200 mM <br /> <br /> imidazole.