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All Journal Jurnal Bioteknologi & Biosains Indonesia (JBBI) Makara Journal of Science Annales Bogorienses Jurnal Bioteknologi & Biosains Indonesia
Pambudi, Sabar
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Immunology & microbiology

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EKPRESI ANTIGEN Ag85B DARI Mycobacterium tuberculosis PADA GALUR SEL MAMALIA Pambudi, Sabar; Widayanti, Tika; Stephanie, Nadya
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 7 No. 1 (2020): June 2020
Publisher : Balai Bioteknologi, Badan Pengkajian dan Penerapan Teknologi (BPPT)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (354.863 KB) | DOI: 10.29122/jbbi.v7i1.3840

Abstract

Expression of Mycobacterium tuberculosis Ag85B Antigen in Mammalian Cell CultureTuberculosis (TB) continues to be a major health problem worldwide, affecting millions of people each year. The only vaccine approved for the prevention of TB is Bacillus Calmette-Guérin (BCG). However, one of the limitations of BCG is that its preventive effect against pulmonary TB varies from person to person. Therefore, there arises a need for a new TB vaccine to replace BCG. This study aims to obtain the Ag85B recombinant protein which has characteristics similar to the native Ag85B antigen from Mycobacterium tuberculosis. In this study, we cloned and expressed recombinant Ag85B in mammalian cell culture. In the initial step, we cloned synthetic Ag85B into mammalian expression vector pFLAG-CMV4 and expressed the gene in CHO-K1 cells. Interestingly, a specific band around 30 kDa was observed in the culture media of transfected cells by Western blot analysis. The results from our research showed the potency of mammalian expression system to produce recombinant protein Ag85B for new TB vaccine candidate.Keywords: Ag85B, mammalian cells, tuberculosis, vaccine, expression ABSTRAKTuberkulosis (TB) terus menjadi salah satu masalah kesehatan dunia yang mempengaruhi jutaan manusia setiap tahun. Satu-satunya vaksin untuk TB yang ada adalah Bacillus Calmette-Guérin (BCG). Namun demikian, vaksin BCG ini memiliki kelemahan berupa terjadi efek preventif yang bervariasi dari satu individu terhadap individu lainnya.  Oleh sebab itu diperlukan pengembangan vaksin TB yang dapat menggantikan vaksin BCG yang sudah ada. Penelitian ini bertujuan memperoleh protein rekombinan Ag85B yang memiliki karakteristik mirip dengan antigen Ag85B native dari Mycobacterium tuberculosis. Pada penelitian ini, telah dilakukan kegiatan pengklonaan dan ekspresi gen Ag85B pada galur sel mamalia.  Pada tahap awal dilakukan pengklonaan gen sintesis Ag85B ke dalam plasmid pada sel mamalia pFLAG-CMV4 dan diekspresikan gennya pada sel CHO-K1. Hasil analisis Western blot menunjukan tersekresinya gen target berukuran 30 kDa pada media kultur dari sel mamalia yang ditransfeksi. Hasil dari penelitian ini menunjukkan potensi dari sistem ekpresi untuk protein rekombinan Ag85B pada galur sel mamalia sebagai kandidat vaksin TB yang baru.
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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Abstract

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.
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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Abstract

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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Abstract

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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Abstract

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

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 ANOM BOWOLAKSONO Arbianto, Alfan Danny Dwiranti, Astari HENI RACHMAWATI Ira Djajanegara Nina Artanti Rahma Micho Widyanto Rahmana E. Kartasasmita Rajagukguk, Selly Setiati Restiani, Dewi Esti Retno Lestari Stephanie, Nadya Sudiro, Tjahjani Tjahjani Sukmadjaja Asyarie Tarwadi, Tarwadi Tika Widayanti Utomo, Doddy Irawan Setyo Vanny Narita