CONSTRUCTION AND EXPRESSION OF RECOMBINANT PROTEIN EDIII OF DENGUE VIRUS SEROTYPE 1 IN PET-16B VECTOR ON ESCHERICHIA COLI BL21 CODONPLUS (DE3)-RIPL

• Main Author: Mai Versiska, Poppy
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/51976
• Institution: Institut Teknologi Bandung

Dengue Hemorrhagic Fever (DHF) is a disease caused by the Dengue virus, which is transmitted through the bite of the Aedes aegypti mosquito. Dengue virus infection is still one of the main health problems in the world, especially in tropical and subtropical areas. The World Health Organization (WHO) records Indonesia as a country with the highest DHF cases in Southeast Asia. The Ministry of Health of the Republic of Indonesia recorded cases of Dengue hemorrhagic fever in Indonesia from 2020 until July reaching 71.633 cases with the number of deaths reaching 459 people. The number of DHF sufferers each year shows that DHF is still one of the main public health problems in Indonesia that needs proper handling. To get the right treatment for a patient infected with the Dengue virus, an accurate and fast diagnosis is needed, so a diagnostic kit is needed. Diagnostic kit is a rapid test tool for diagnosing a disease that utilizes a specific interaction between an antibody and an antigen. One of the important components in the diagnostic kit is the EDIII protein. EDIII contains epitope that can induce antibody formation so that the blood of patients suspected of having Dengue will find specific EDIII antibodies that can interact with the EDIII antigen contained in the rapid test kit. The interactions that occur can cause a color change on the kit which indicates the presence of the Dengue virus in the patient. The final objective of this study was to obtain EDIII protein as the antigen required in the development of a Dengue fever rapid test kit. The EDIII (DENV1) protein coding gene fragment measuring 294 base pairs was amplified from a synthetic gene template of Dengue virus serotype 1 using the Polymerase Chain Reaction (PCR) technique. The EDIII (DENV1) gene fragment was ligated with the pGEM-T cloning vector for fragment propagation. The recombinant plasmid EDIII (DENV)-pGEM measuring 3320 base pairs was grown in E. coli TOP 10F’. Insertion success is confirmed by blue and white colony screening. Colony PCR analysis proved that the recombinant plasmid carried an insert in the form of the EDIII (DENV1) coding gene. Double digestion using restriction enzymes Nde1 and Xho1 resulted 2 fragments of about 294 and 3026 base pairs in size which indicating a linear fragment of the EDIII (DENV1) and pGEM protein coding genes. The alignment of the nucleotide sequences resulting from the recombinant EDIII (DENV)-pGEM plasmid sequence shows the nucleotide sequence that matches the reference template. The gene coding for the EDIII protein (DENV1) was then inserted into the pET16b expression vector on the Nde1 and XhoI restriction sites to obtain EDIII (DENV1)- pET16b recombinant plasmid measuring 6008 base pairs. Colony PCR analysis and restriction confirmed that the recombinant plasmid carried EDIII (DENV1) insert. The alignment of the nucleotide sequences resulting from the recombinant plasmid EDIII (DENV)-pET16b shows the nucleotide sequences that match the reference. This proves that there is no mutation during the recombination process. E. coli BL21 CodonPlus (DE3)-RIPL was transformed with recombinant plasmid EDIII (DENV1)-pET16b using the heat shock method and selected in LB medium containing ampicillin antibiotics. Transformants of E. coli BL21 CodonPlus (DE3)- RIPL/EDIII (DENV1)-pET16b were grown in ampicillin LB medium and induced with 0,3 mM IPTG for 4 hours at 37 oC. SDS-PAGE analysis showed that the recombinant protein EDIII (DENV1) has a molecular mass of ?11 kDa and forms inclusion bodies. The active EDIII protein (DENV1) was produced by refolding and purification using affinity chromatography for Ni-NTA resin. Enzyme-Linked Immunosorbent Assay (ELISA) analysis proved that the EDIII (DENV1) protein is able to form specific interactions with the universal IgM antibody of Dengue virus.