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All Journal Microbiology Indonesia Jurnal Bioteknologi & Biosains Indonesia (JBBI)
Sabar Pambudi
Center for Pharmaceutical and Medical Technology, Agency for the Assessment and Application of Technology, Jakarta 10340, Indonesia
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Environmental Science Health Professions Immunology & microbiology Medicine & Pharmacology

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Cloning and expression of NS2B/NS3 protein of DENV3 Indonesia strain in Saccharomyces cerevisiae expression system for the development of Dengue vaccine ASRI SULFIANTI; SABAR PAMBUDI; ISMA NUR AZIZAH; DODDY IRAWAN SETYO UTOMO; ABINAWANTO ABINAWANTO
Microbiology Indonesia Vol. 12 No. 2 (2018): June 2018
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (3936.725 KB) | DOI: 10.5454/mi.12.2.4

Abstract

NS3 protein is 618 amino acids (aa) in length containing serine protease and helicase domains required for DENV replication. Alignment of consensus amino acid sequences from all four DENV serotypes demonstrated that this protein is more conserved (78%) among the different dengue serotypes, which elicits a strong cellular immune response after viral infection in humans and animal models. Present study, a central hydrophilic region of NS3 cofactor, NS2B (NS2BH) with full length of NS3 genes DENV3 Indonesian strain were amplified from CDNA following PCR, and inserted to PYES2/CT shuttle vector. The recombinant plasmid was transformed and expressed in Saccharomyces cerevisiae expression system. As result, detection with Anti-His detector and Anti-NS3 shown NS2BH/NS3 was expressed as 83 KDA protein band. We found that addition of NS2BH on NS3 full length construction plasmid increase the yield and activity of protein expression in S. cerevisiae. In future study, our recombinant NS2B/NS3 protein can be used as recombinant protein in Dengue vaccine development.
The Potency of Aluminum Hydroxide Nanoparticles for Dengue Subunit Vaccine Adjuvant SABAR PAMBUDI; ETIK MARDLIYATI; SILMI RAHMANI; DAMAI RIA SETYAWATI; TIKA WIDAYANTI; ANGELINA GILL; ASRI SULFIANTI; WHINIE LESTARI
Microbiology Indonesia Vol. 12 No. 3 (2018): September 2018
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (984.8 KB) | DOI: 10.5454/mi.12.3.5

Abstract

The potency of aluminum hydroxide as an adjuvant in vaccine development is considered to depend on its particle size. In previous studies, we have successfully prepared two size particle, micro, and nano, aluminum hydroxide gel (alum) adjuvants. The potency of those particles as a candidate of adjuvant is needed to be characterized. In this study, we formulated our adjuvants with purified DENV3 pre Membrane Envelope (prM-E) recombinant protein and evaluated the induction of nitric oxide level in mouse macrophage RAW 264.7 cells. We prepared the alum adjuvant by precipitation-homogenization methods with an agitation rate at 11,000xg. Secreted prM-E recombinant protein was collected from Pichia pastoris X-33 fermentation which produced using bioreactor. Recombinant protein purification was carried out by anion exchange chromatography followed with size exclusion chromatography. The purified prM-E recombinant protein was observed as a single band around 70 -1k Da with a concentration of 105 mg mL . Complex nanoparticles alum with prM-E protein significantly (p<0.05) induced the nitric oxide level. Further analysis should be conducted in order to discover the detail molecular mechanism of nanoparticle alum adjuvant, recombinant protein, and cellular immune response.
MUTATION DETECTION OF MULTIDRUG-RESISTANT TUBERCULOSIS BY RT-PCR METHOD AS THE DIAGNOSTIC TOOL OF MDR-TB Sabar Pambudi
Jurnal Bioteknologi dan Biosains Indonesia Vol. 10 No. 1 (2023)
Publisher : BRIN - Badan Riset dan Inovasi Nasional

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Abstract

Eight percent of tuberculosis (TB) cases worldwide are resistant to rifampicin, with mutations occurring in the rpoB and katG genes. It is necessary to develop a specific multidrug-resistant (MDR) diagnostic technique using the RT-PCR method in Indonesia to aid in rapid and accurate diagnosis. In-silico testing using SnapGene software resulted in the design of DNA primers for the katG and rpoB genes, plasmids, and specific probes. This study employed a cross-sectional design using 30 non-MDR-TB and MDR-TB samples from RSUD Sitanala, Tangerang Banten, which were tested for amplification of the katG and rpoB genes using Sybr green RT-PCR. Validity testing was conducted using specific probes for the katG and rpoB genes. The amplification results showed that MDR-TB samples and MDR-TB plasmids required a longer time compared to non-MDR-TB samples and non-MDR-TB plasmids. The Quantification Cycle (Cq) value in non-MDR-TB samples was lower than the Cq value in MDR-TB samples. A t-test revealed a significant difference in Cq values of the rpoB and katG genes between MDR-TB and non-MDR-TB patients (p-value < 0.005). These differences in Cq values indicate that the findings of this study can serve as an initial reference for the development of an RT-PCR-based diagnostic kit for MDR-TB.
Co-Authors Abinawanto Abinawanto ANGELINA GILL ASRI SULFIANTI DAMAI RIA SETYAWATI DODDY IRAWAN SETYO UTOMO Etik Mardliyati Isma Nur Azzizah SILMI RAHMANI TIKA WIDAYANTI Whinie Lestari