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All Journal HAYATI Journal of Biosciences Microbiology Indonesia Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Eksakta : Berkala Ilmiah Bidang MIPA Health Information : Jurnal Penelitian Narra J Makara Journal of Health Research Makara Journal of Science Jurnal Penyakit Dalam Indonesia Berita Biologi
. Andriansjah
Departemen Mikrobiologi, Fakultas Kedokteran Universitas Indonesia/RSUPN Cipto Mangunkusumo, Jakarta
Agriculture, Biological Sciences & Forestry Astronomy Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Civil Engineering, Building, Construction & Architecture Computer Science & IT Dentistry Earth & Planetary Sciences Energy Engineering Environmental Science Health Professions Immunology & microbiology Materials Science & Nanotechnology Mathematics Mechanical Engineering Medicine & Pharmacology Neuroscience Nursing Public Health

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Journal : Makara Journal of Science

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Optimization of pGEX System to Express and Isolate Mycobacterium tuberculosis Inclusion Body Protein in Combining with Modified Refolding Method Rukmana, Andriansjah; Burhanuddin, Burhanuddin; Yasmon, Andi
Makara Journal of Science Vol. 22, No. 4
Publisher : UI Scholars Hub

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Antigen sub units for vaccine studies are typically isolated from recombinant proteins in an expression system. However, not all protein expression systems are used to express the specific protein. In this study,we optimized the pGEX system combined with the modified protein refolding to express and isolate M. tuberculosis proteins, especially proteins that are expressed as an inclusion body. Resuscitation promoting factor B (RpfB) protein is one of the Resuscitation promoting factor (Rpf) family of proteins that has been studied for its ability to induce cellular immunity in animal tests. Silico analyses demonstrate how RpfB is included in cell wall and cell processes. The Rpf family proteins are promising antigens that can be used as a TB vaccine candidate. The polymerase chain reaction was briefly performed using specific primers to amplify the full length of the rpfB. PCR amplification products were then purified, cut by restriction endonucleases, and cloned into pGEX 6-P1. Protein expression was done in the Escherichia coli BL21 strain, and expressed protein was isolated using themodified protein refolding and solubilization method. The complex protein expression that appeared as inclusion bodies were successfully isolated and can be detected as complex GST-RpfB through the western blotting process. Our study results indicate that this system and our modified method are suitable for M. tuberculosis inclusion body protein expression and isolation.
Construction of pcDNA3.1 Vector Encoding RpfD Gene of Mycobacterium tuberculosis Rakhmawati, Aprilia; Rukmana, Andriansjah; Karuniawati, Anis
Makara Journal of Science Vol. 22, No. 3
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Tuberculosis (TB) is an infectious diseasecaused by Mycobacterium tuberculosis (M. tuberculosis). TB is still a major health problem. The Bacillus Calmette-Guérin (BCG) vaccineis the only one available for TB and is known to confer variable levels of protection. Because of thisvariability, a new vaccine is needed to control TB. Proteins secreted by M.tuberculosisare known to induce protective immunity. Within the genome of M. tuberculosis, there is a family of proteins called resuscitation promoting factor (Rpf), which playsa role in the reactivation of M. tuberculosis. RpfD is amember of the Rpf family that has been shown to be immunogenic, makingitsuitable for use as a TB vaccine. The rpfD gene of the M. tuberculosis Beijing strain from the bacterial stock of the Department of Microbiologyat the Medical Facultyof theUniversitas Indonesia was amplified using polymerase chain reaction (PCR) and then insertedintothemammalian expression vector pcDNA3.1(+). Then, the pcDNA3.1(+)-rpfD vector was transformed to Escherichia coli DH5α. A 465-bp target fragment was obtained, and the accuracy ofthecloning was confirmed using colony PCR, restriction enzyme digestion, and sequencing. We expect that this recombinant plasmid will induce immunity in future animal models and thus will prove itself to be a candidate for an M. tuberculosis vaccine.
Cloning of pe11 (LipX, Rv1169c) gene of Mycobacterium tuberculosis Beijing strain to pcDNA3.1 plasmid vector Supardi, Lulut Azmi; Rukmana, Andriansjah; Sjatha, Fithriyah
Makara Journal of Science Vol. 25, No. 1
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Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. It is a persistent global health problem with a high mortality rate. Currently, TB is controlled by administering the Bacillus Calmette-Guerin (BCG) vaccine, but the effectiveness of its protection varies among individuals in a population. The pe/ppe gene family comprises a typical group of genes that play a role in avoiding the host immune response and inducing persistent TB infection. Based on in silico analysis, the pe11 gene has estimated immunogenicity and potential as a TB seed vaccine candidate. The pe11 gene from an Indonesian isolate of an M. tuberculosis Beijing strain was amplified by polymerase chain reaction (PCR) and inserted into the mammalian expression vector pcDNA3.1. The recombinant vector pcDNA3.1-pe11 was used to transform Top10 competent Escherichia coli. Clones from the transformation were subjected to colony PCR to confirm the direction of the insert. Sequencing was performed to confirm the correctness of the insert sequence. In this study, the pe11 gene was successfully cloned into the pcDNA3.1 vector in the correct direction to assure PE11 expression. No mutations were found in the pe11 gene insert, compared with the M. tuberculosis H37Rv sequence as the standard. A pcDNA3.1 vector containing the pe11 gene derived from an M. tuberculosis Beijing strain was successfully constructed.
Efficacy of Tuberculosis Vaccine Candidate pcDNA3.1-rpfB in Inhibiting the Growth of Mycobacterium tuberculosis In Vitro with Mycobacterial Growth Inhibition Assay Pujilestari, Ratih; Rukmana, Andriansjah; Karuniawati, Anis
Makara Journal of Science Vol. 26, No. 1
Publisher : UI Scholars Hub

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Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb). Bacille Calmette-Guérin (BCG) is the only licensed vaccine against TB, and it is effective in children but not in adults. The Vaccine Research Team, Department of Microbiology FKUI has developed a DNA-based TB vaccine candidate pcDNA3.1-rpfB. This candidate induces immune responses in mice, but its potency is unknown. The gold standard for potency testing of TB vaccine is the challenge method. The BSL3 animal laboratory for the challenge method is currently unavailable at FKUI. Therefore, mycobacterial growth inhibition assay (MGIA) was used as a preliminary test before the in vivo challenge test was conducted. The principle of MGIA is to reculture Mtb in a Mycobacteria Growth Indicator Tube (MGITTM) from co-cultured Mtb with mammalian cells that have been previously treated with pcDNA3.1-rpfB, pcDNA3.1 (negative control), and BCG (positive control). MGITTM shows the time to positivity, which is the time that has lapsed until a positive growth of Mtb is detected. In addition, measurements of interferon (IFN)γ levels by enzyme-linked immunosorbent assay were carried out. This study concluded that pcDNA3.1-rpfB can inhibit the growth of Mtb in vitro and showed no statistical difference from BCG. The IFNγ levels from co-culturing did not correlate with the level of inhibition of the growth of Mtb in vitro.
Co-Authors Abdul Munim Adiyaksa, Jongga Andi Yasmon Anis Karuniawati Aprilia Rakhmawati Aprilia Rakhmawati Arif Nurkanto Ariyani Kiranasari Beti Ernawati Dewi Burhanuddin Burhanuddin Burhanuddin Burhanuddin Cynthia Gozali DEKA LARASATI Desi Salwani, Desi Diah Erwati, Wa Ode Erlina, Linda Evy Yunihastuti Ewaldo, Muhammad Farrel Fera Ibrahim Fithriyah Fithriyah Fithriyah Fithriyah Harimurti, Kuntjro Heni Pujiastuti Kuntjoro Harimurti Lola Febriana Dewi Maria Tuntun Mifa Nurfadilah Nasir, Ujainah Zaini Paramitadevi, Yudith V. Pratiwi Pujilestari Sudarmono Priadi, Cindy R. Pujilestari, Ratih Puspitasari, Melya Rahmatika, Iftita Rakhmawati, Aprilia Ratih D Saraswati Saraswati, Ratih D SJATHA, FITHRIYAH Supardi, Lulut Azmi Syamsiar, Syamsiar Tjahjani Mirawati Sudiro Ujainah Zaini Nasir Yulia Rosa Saharman