#TITLE_ALTERNATIVE#

• Main Author: DWIMALIDA PUTRI (NIM : 20512045) ; Pembimbing Dr. Dessy Natalia, RISKI
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/19336
• Institution: Institut Teknologi Bandung

Dengue fever infection is caused by dengue virus from genus Flavivirus which was transmitted to human through Aedes aegypti mosquito. Dengue virus has four serotypes namely DENV - 1 , DENV - 2 , DENV - 3 and DENV - 4 with a high genetic homology. Infection by one serotype of dengue virus will result in lifelong immunity to that particular serotype, but there is no cross-protection mechanism against infections for other viral serotypes. Infection of dengue virus in human can give different clinical manifestations from mild dengue fever to severe clinical manifestations known as Dengue Hemorrhagic Fever and Dengue Shock Syndrome. Dengue virus genome has a 11 kb positive single-stranded RNA which encodes three structural proteins (M,C and E) and seven nonstructural protein (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). One of important nonstructural proteins is NS1 protein which plays a role in the maturation and replication of viral RNA, hence NS1 becomes an important element in immunopathogenesis of dengue fever. Most of currently available detection kits of dengue virus infection is based on the interaction between NS1 protein with anti NS1 antibody. The long term goal of this research is to produce a recombinant NS1 antigent for detection of human IgM /IgG in blood serum or for detection of the presence of NS1 in the early stage of dengue virus infection. <br /> <br /> <br /> <br /> <br /> The aims of this study were to express NS1 from DENV-2 in P. pastoris and to purify the recombinant DENV2- NS1. The DENV2-NS1 gene was synthetized commercially and inserted in pPICZ&#945;-A P. pastoris expression plasmid. The DENV2- NS1 expression was regulated by AOX1 promoter and the NS1 protein fused with pre-pro &#945; factor signal sequence for its secretion into culture media. The DENV2-NS1 expression cassette was integrated to the P. pastoris chromosome through single cross over homologous recombination in the AOX1 locus. P. pastoris transformants harboring DENV2-NS1 were subjected for multicopy selection in growth media containing zeocin in the range of 500-2000 &#956;g/mL. One of the P. pastoris transformants dengue designated as P. pastoris KM71-4 was capable to grow at 2000 &#956;g/mL zeocin. P. pastoris KM71-4 was <br /> <br /> <br /> <br /> <br /> iv <br /> <br /> <br /> <br /> <br /> then used for the NS1 expression study protein DENV2-NS1. Expression of DENV2- NS1 was induced by 2% (v/v) methanol for 72 hours at 30 °C. The resulted recombinant DENV2-NS1 was purified by Ni-NTA affinity chromatography. SDS-PAGE analysis showed that the recombinant DENV2-NS1 has a molecular weight of ~ 43 kDa which corresponds to its amino acid residues. Evaluation of recombinant DENV2-NS1 antigenicity using commercial diagnoctic kit showed that it has an ability to interact with monoclonal NS1 antibody. In conclusion the DENV2- NS1 recombinant obtained in this research is potential to be used as an antigen for development of dengue diagnostic kit.