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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

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

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.

CLONING OF DENGUE VIRUS TYPE 3 (INDONESIAN STRAIN D3-1703) NON STRUCTURAL-1 GENE INTO pYES2/CT VECTOR Narita, Vanny; Widyanto, Rahma Micho; Pambudi, Sabar; Sudiro, Tjahjani Tjahjani
Makara Journal of Science Vol. 15, No. 2
Publisher : UI Scholars Hub

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Dengue is an infectious disease caused by dengue virus. Dengue endemic region includes America, Western Pacific, Africa, East Mediterranian, and South East Asia including Indonesia. An early diagnostic system specific for Indonesia is needed to control dengue in Indonesia. In this research, cloning of Non Structural 1 (NS1) gene from dengue virus type 3 (Indonesian strain D3-1703) into pYES2/CT vector was performed. In the long run, NS1 recombinant protein will be expressed in Saccharomyces cerevisiae for diagnostic materials. Polymerase Chain Reaction (PCR) amplification of NS1 gene fragments were done with optimal annealing temperature at 55 ºC. NS1 gene fragment and pYES2/CT were cut by BamH I and Not I enzymes. The digested pYES2/CT was dephosphosrylated using Calf Intestine Alkaline Phospatase enzyme. Ligation with the vector:insert ratio of 1:12 and 1:20 resulted in 6 and 5 recombinant colony candidates respectively. Restriction enzyme and PCR verifications showed that 5 recombinant plasmids contained NS1 gene. Sequencing of the first 600 bp of one recombinant plasmid was performed. The blastn analysis showed that it had a 99% identity with dengue virus type 3 strain FW06. Finally, it was shown that NS1 clone within pYES2/CT was in the correct Open Reading Frame and ready to be expressed in S. cerevisiae.

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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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.

Cloning and Expression of SCAMP3 in Escherichia coli BL21(DE3) with In Silico Sequence-Based Cancer Epitopes Prediction Rajagukguk, Selly Setiati; Pambudi, Sabar; Dwiranti, Astari; Utomo, Doddy Irawan Setyo; Bowolaksono, Anom
Makara Journal of Science Vol. 29, No. 1
Publisher : UI Scholars Hub

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Secretory carrier membrane protein 3 (SCAMP3) is a crucial membrane protein involved in intracellular vesicle traffick-ing and exocytosis. The SCAMP3 expression has been observed in diverse cancer types, such as melanoma, glioma, hepatocellular and breast cancer. Increased SCAMP3 expression has been reported in certain cancer cells relative to that in normal cells, suggesting the potential role of SCAMP3 in cancer development or progression. In this study, we successfully cloned and expressed SCAMP3 in Escherichia coli strain BL21(DE3). SCAMP3 was amplified and insert-ed directionally into the prokaryotic expression vector pET21d(+). The transformation of recombinant plasmid into E. coli BL21(DE3) cells were performed for the protein expression. SDS–PAGE and Western blotting were performed to detect the expression product induced by IPTG, which confirmed the presence of a recombinant pET21d(+)-SCAMP3 at 38-kDa protein weight. Bioinformatics analyses helped discover several possible epitopes distributed throughout the SCAMP3 protein sequence. These findings together serve as a basis for future biochemical and functional studies on this important membrane protein alongside immunotherapy research related to SCAMP3 as a cancer biomarker.

DNA Condensation Study of Fully Synthesized Lipopeptide-Based Transfection Agent for Gene Delivery Vehicle Tarwadi, Tarwadi; Rachmawati, Heni; Kartasasmita, Rahmana E.; Pambudi, Sabar; Arbianto, Alfan Danny; Restiani, Dewi Esti; Asyarie, Sukmadjaja
Annales Bogorienses Vol. 22 No. 2 (2018): Annales Bogorienses
Publisher : BRIN

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The main requirement of transfection agent has to condense DNA in nanoparticle size, protect the DNA from nucleases and other degrading enzymes during its transport in cell cytoplasm and nucleus and should not toxic to target cells. In this research, lipopeptide composed of palmitoyl (C-16) and short peptide sequence have been designed fully synthesized and tested to DNA condensation capability and toxicity. The DNA condensation study was performed using EtBr exclusion assay and cytotoxicity determination was carried out by colorimetric MTT assay. It was revealed that lipopeptide-based transfection agent of Pal-CKKHH and Pal-CKKHH-YGRKKRRQRRR-PKKKRKV condensed DNA molecules efficiently. The lipopeptide was less toxic compared to Lipofectamine and Poly-L-Lysine, that shown by 90% of CHO-K1 cells remained viable when they were treated with 4.36 µM Pal-CKKHHYGRKKRRQRRR-PKKKRKV. Meanwhile, there were only ~75% and 80% of CHO-K1 viable cells when it was treated with PLL and Lipofectamine®2000, respectively. Moreover, cell viability of HepG2 was ~ 75% after treated with 2.18 µM of Pal-CKKHH-YGRKKRRQRRR-PKKKRKV and decreased to ~65% when the lipopeptide concentration increased to 8.72 M. In summary, the synthesized lipopeptide condenses DNA molecules efficiently, less toxic than Lipofectamine®2000 and PLL and has possibility to be explored as a non-viral gene delivery vehicle.

PCR Amplification of Ornithine Decarboxylase (ODC) Gene Fragment from Tobacco (Nicotiana tabacum L.) cv. Temanggung Djajanegara, Ira; Pambudi, Sabar; Lestari, Retno; Artanti, Nina
Annales Bogorienses Vol. 9 No. 2 (2004): Annales Bogorienses
Publisher : BRIN

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In order to create an antisense construct of the gene encoding Ornithine Decarboxylase (ODC) from tobacco (Nicoticum tabacum L.) cv. Temanggung, the target gene must be isolated. In this paper. we present the PCR amplification of a fragment from putative gene encoding ODC from tobacco cv. Temanggung. Leaf genomic DNA was isolated and used as the template for PCR. PCR optimization was done by adjusting the annealing temperature and the cycle number. Verification of the fragment obtained was also done using the second primer pairs.

MUTATION DETECTION OF MULTIDRUG-RESISTANT TUBERCULOSIS BY RT-PCR METHOD AS THE DIAGNOSTIC TOOL OF MDR-TB Pambudi, Sabar
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 10 No. 1 (2023)
Publisher : BRIN - Badan Riset dan Inovasi Nasional

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.55981/jbbi.2023.1741

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.

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