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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 : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/11

In order Lo create an antisense construct of the gene encoding Ornithine Decarboxylase (ODC) from tobacco (Nicotiana 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 i olated 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 . Â

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 : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

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.

Pengaruh Ekstrak Etanol Daun Murbei (Morus Alba L.) dengan Glibenklamid Terhadap Ekspresi Gen CYP3A4 pada Kultur Sel HepG2 Nuralih, Nuralih; Churiyah, Churiyah; Pambudi, Sabar; Tamat, Swasono R.; Meila, Okpri
Pharmacon: Jurnal Farmasi Indonesia Vol 15, No 1 (2018)
Publisher : Universitas Muhammadiyah Surakarta

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.23917/pharmacon.v15i1.5766

Mulberry leaf is a traditional herb and predicted has ecdysteronecompound which act as antihyperglicemid. Glybenclamide is a synthetic medicine used to cure diabetes mellitus type 2. The leaf reported as competitive inhibitor of CYP3A4 enzyme, which metabolizingglibenclamide. However, in many cases, combination of herbs with synthesis drugs causes interaction if used at the same time. This research aimed to see interaction of ethanol mulberry leaf extract with glibenclamide through CYP3A4 gene expression in HepG2 cell culture.Sample of mulberry extract, glibenclamide, and combination both sample were tested into cell HepG2 culture. Then RNA were isolated and purification using real time PCR to see the gene CYP3A4 expression. As a result, mulberry extract acts as inhibitor enzyme CYP3A4, while glibenclamide is enzyme substrate.The combination of mulberry and glibenclamide showed increased of expression of CYP3A4 gene, means greater enzyme produced, and lower medicine on blood plasma.

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)

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-GuÃƒÂ©rin (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, expressionÃ‚Â 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-GuÃƒÂ©rin (BCG). Namun demikian, vaksin BCG ini memiliki kelemahan berupa terjadi efek preventif yang bervariasi dari satu individu terhadap individu lainnya.Ã‚Â 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.Ã‚Â 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.

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

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-GuÃ©rin (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, expressionÂ 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-GuÃ©rin (BCG). Namun demikian, vaksin BCG ini memiliki kelemahan berupa terjadi efek preventif yang bervariasi dari satu individu terhadap individu lainnya.Â 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.Â 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.

Pengaruh Ekstrak Etanol Daun Murbei (Morus Alba L.) dengan Glibenklamid Terhadap Ekspresi Gen CYP3A4 pada Kultur Sel HepG2 Nuralih Nuralih; Churiyah Churiyah; Sabar Pambudi; Swasono R. Tamat; Okpri Meila
Pharmacon: Jurnal Farmasi Indonesia Vol 15, No 1 (2018)
Publisher : Universitas Muhammadiyah Surakarta

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.23917/pharmacon.v15i1.5766

Mulberry leaf is a traditional herb and predicted has ecdysteronecompound which act as antihyperglicemid. Glybenclamide is a synthetic medicine used to cure diabetes mellitus type 2. The leaf reported as competitive inhibitor of CYP3A4 enzyme, which metabolizingglibenclamide. However, in many cases, combination of herbs with synthesis drugs causes interaction if used at the same time. This research aimed to see interaction of ethanol mulberry leaf extract with glibenclamide through CYP3A4 gene expression in HepG2 cell culture.Sample of mulberry extract, glibenclamide, and combination both sample were tested into cell HepG2 culture. Then RNA were isolated and purification using real time PCR to see the gene CYP3A4 expression. As a result, mulberry extract acts as inhibitor enzyme CYP3A4, while glibenclamide is enzyme substrate.The combination of mulberry and glibenclamide showed increased of expression of CYP3A4 gene, means greater enzyme produced, and lower medicine on blood plasma.

VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE Ira Djajanegara; Wayan Artama; Retno Lestari; Sabar Pambudi
JURNAL PENELITIAN BIOLOGI BERKALA PENELITIAN HAYATI Vol 11 No 1 (2005): December 2005
Publisher : The East Java Biological Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.23869/460

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

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

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 Ira Djajanegara; Sabar Pambudi; Retno Lestari; Nina Artanti
ANNALES BOGORIENSES Vol 9, No 2 (2004): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

In order Lo create an antisense construct of the gene encoding Ornithine Decarboxylase (ODC) from tobacco (Nicotiana 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 i olated 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 Titta Novianti; alfero Putra Iryanto; feby feby; callista marsya; putri mega utami; febriana dwi wahyuni; henny saraswati; seprianto; adri nora; roaslein putri; nie nie; sabar pambudi
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 10 No. 1 (2023): Juni 2023
Publisher : Balai Bioteknologi, Badan Pengkajian dan Penerapan Teknologi (BPPT)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.29122/jbbi.v10i1.5653

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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