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Evaluation of Tuberculosis Vaccine Candidate, pcDNA3.1-rpfD using Mycobacterial Growth Inhibition Assay (MGIA) Mifa Nurfadilah; Andriansjah Rukmana; Fithriyah Sjatha
HAYATI Journal of Biosciences Vol. 29 No. 1 (2022): January 2022
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.29.1.1-8

Abstract

Resuscitation-promoting factor D (RpfD) is a protein involved in the resuscitation of dormant bacteria. A new tuberculosis vaccine carrying the rpfD gene has been successfully constructed, pcDNA3.1-rpfD. It was demonstrated that this vaccine exhibits cellular and humoral immune responses. Therefore, within this study, the efficacy of this new vaccine candidate was evaluated using mycobacterial growth inhibition assay (MGIA). MGIA is a functional assay that measures the complex host immune response, peripheral blood mononuclear cell (PBMC) and splenocyte from BALB/c mice against mycobacteria. With BACTECTM MGITTM 960 automated system, the effect of vaccination on bacterial growth was reported as a time to positivity (TTP) in hours. The mean of TTP from the vaccinated group (both pcDNA3.1-rpfD and BCG) was higher than the negative control group. These results suggest that pcDNA3.1-rpfD may be effective in controlling tuberculosis growth and may provide a clue for the development of the tuberculosis vaccine. In addition, despite previous evidence that IFNγ was essential for tuberculosis immunity, IFNγ (interferon gamma) production was found not to be correlated with mycobacterial inhibition. Therefore, these findings offer an alternative method to evaluate vaccine candidates than the assessment using IFNγ only.
Cloning and Expression of Nonstructural Protein NS1 of Dengue Virus Serotype 2 BETI ERNAWATI DEWI; FITHRIYAH FITHRIYAH; ANDRIANSJAH RUKMANA; PAISAL PAISAL; DEKA LARASATI; TJAHJANI MIRAWATI SUDIRO
Microbiology Indonesia Vol. 6 No. 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (614.911 KB) | DOI: 10.5454/mi.6.1.3

Abstract

Early diagnosis of dengue virus (DENV) infection is affirmative for patient management and control of the disease. Detection of nonstructural-1 (NS1) antigen has been proven to provide early detection of DENV infection. Commercial NS1 antigen assays are available in Indonesia with variable sensitivity. In an attempt to develop an NS1-based diagnostic test, we successfully cloned NS1 gene of DENV2 to a glutathione Stransferase- based vector pGEX6P-1 in Escherichia coli system. The recombinant protein (pG2NS12) was expressed in E. coli BL21. After induction with isopropyl-β-D-thiogalactoside 0.1 mM for 4 h at 25 °C a recombinant protein GST-NS1 with molecular size of approximately 75 kDa was  obtained. The fusion protein was insoluble and found in the pellet fraction of the cell lysate. Addition of lysozyme (10 mg mL-1) and DNase-I (7.2 mg mL-1) in the lysis buffer was necessary to collect proteins from the pellet fraction. The proteins in the cell pellet were fractionated through Sephadex-G100 column, and GST-NS1 was further purified with Glutathione-Sepharose 4B beads. To obtain pure recombinant NS1 protein to be used in the immunization of mice, the fusion protein was cut with PreScission Protease® by addition of 0.075% Triton-X 100 was necessary to cut the fusion protein. We found that antibodies that recognized the recombinant NS1 protein and DENV2 virus were produced in mice immunized with purified NS1 protein. Therefore, our recombinant NS1 could be used to produce antibody that is potentially useful for developing diagnostic assay to determine the presence of dengue virus NS1 antigen in patient sera.
Development of a Tuberculosis Vaccine Seed: Construction of Resuscitation-Promoting Factor B DNA Vaccine and its Expression in Vitro and in Vivo Saraswati, Ratih D; Rukmana, Andriansjah; Fithriyah, Fithriyah; Rakhmawati, Aprilia
Makara Journal of Health Research Vol. 22, No. 1
Publisher : UI Scholars Hub

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Abstract

Background: Tuberculosis (TB) is a chronic infection disease caused by Mycobacterium tuberculosis (Mtb) and has a high death-rate worldwide. Bacillus Calmette-Guerin is the only TB vaccine which is currently available with several drawbacks, such as its different efficacy for different individuals, lack of protection for lung TB in adults and subsequent reactivation which lead the research for novel TB vaccine approach. Resuscitation-promoting factor (rpf) protein in Mtb is a protein cluster which play a big role in TB dormancy during latent infection. Member from this cluster protein is rpfB which shows the greatest biological and immunological characteristics among other proteins in the rpf family, now is widely explored as novel TB vaccine candidate. Methods: In this study, the rpfB gene of the Mtb Beijing strain was amplified using PCR and then cloned into pcDNA3.1 plasmids. The ability of recombinant pcDNA-rpfB to induce humoral immune response was tested through Balb/C mice immunization. Results: A positive recombinant rpfB protein ~66 kDa was detected through western blot analysis using immunized mice sera. Meanwhile, recombinant pcDNA-rpfB was transfected in to CHO-K1 mammalian cell line and recombinant rpfB antigen expression was confirmed through immunostaining. Conclusions: Therefore, we have succesfully express the recombinant rpfB proten of M.tb strain Beijing in mammalian expression system which proven to be antigenically induced humoral immune response in mice model.
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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Abstract

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
Publisher : UI Scholars Hub

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Abstract

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
Publisher : UI Scholars Hub

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Abstract

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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Abstract

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.
A comparison study of GeneXpert and In-House N1N2 CDC Real-Time RT-PCR for detection of SARS-CoV-2 infection Andi Yasmon; Lola Febriana Dewi; Fithriyah Fithriyah; Ariyani Kiranasari; Andriansjah Rukmana; Yulia Rosa Saharman; Fera Ibrahim; Pratiwi Sudarmono
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 54, No 3 (2022)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.19106/JMedSci005403202203

Abstract

COVID-19 is a disease caused by SARS-CoV-2, a new virus from genus β-coronaviruses. This disease has been declared a pandemic by WHO on 11 March 2020 until now. The nucleic acid tests are the most frequently used assays because of their high sensitivity and specificity. One of the tests is the GeneXpert, a real-time reverse transcription polymerase chain reaction (rRT-PCR)-based assay platform. The use of the GeneXpert shows great public health interest because of the rapid (50 min), the minimum number of trained staff, and less infrastructure and equipment. However, there are limited data on the application of the GeneXpert for the detection of SARS-CoV-2. Therefore, we conducted a comparative study between the GeneXpert and in-house N1N2 CDC rRT-PCR assay. Of 86 samples, 17 were rRT-PCR positive while 13 were GeneXpert positive. Of rRT-PCR positive 17 samples, 7 were GeneXpert negative [58.82% (10/17] sensitivity]. We also found that 3 GeneXpert positive samples showed rRT-PCR negative (95.65% [66/69] specificity). It is concluded that negative results by the GeneXpert can not rule out the possibility of SARS-CoV-2 infection, particularly in close-contact individuals and the interpretation of the positive result should be analyzed carefully, particularly amplification with Ct>40.
Potential of Colicin as an Antibacterial Agent in Escherichia coli Cynthia Gozali; Andriansjah Rukmana
EKSAKTA: Berkala Ilmiah Bidang MIPA Vol. 24 No. 02 (2023): Eksakta : Berkala Ilmiah Bidang MIPA (E-ISSN : 2549-7464)
Publisher : Faculty of Mathematics and Natural Sciences (FMIPA), Universitas Negeri Padang, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24036/eksakta/vol24-iss02/409

Abstract

The development of antibiotics calls for the critical consideration of instances of resistance. Infectious disorders brought on by resistant bacterial infections could affect the entire world. It is believed that the protein that the bacteria generate may one day replace antibiotics as an alternative antibacterial agent. Both Gram-positive and Gram-negative bacteria have the ability to manufacture bacteriocin. The bacteriocin type produced by Escherichia coli, notably colicin, has been demonstrated to inhibit the same bacteria through various essential methods. Colicin, a substance made by an E. coli cell, is also capable of protecting itself from attack; however, this defense mechanism has not yet been identified. The traits of colicin and the method by which it functions as a different antimicrobial agent to inhibit other bacteria will be covered in this article. We analyze the potential of colicin as an antibacterial agent in E. coli using PRISMA methods from diverse academic sources. Here, we found that the structure of the colicin, namely its central receptor domain, aids in the recognition of target cells. Promising results were found in recent studies on the antibacterial effects of the E. coli and colicin combination
Performance of Combination of Symptoms, Chest x-rays and MGIT 960 Culture for Diagnosis of Pulmonary Tuberculosis in HIV Patients Salwani, Desi; Nasir, Ujainah Zaini; Yunihastuti, Evy; Harimurti, Kuntjro; Andriansjah, Andriansjah
Jurnal Penyakit Dalam Indonesia Vol. 5, No. 2
Publisher : UI Scholars Hub

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Abstract