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Tjahjani Mirawati Sudiro

Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta 10320, Indonesia

Author-ID : 404063

Biochemistry, Genetics & Molecular Biology Immunology & microbiology Medicine & Pharmacology

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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

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.

Expression of Recombinant Non Structural 1 Protein of Dengue Virus Serotype-2 in Mammalian Cell Line FITRIYAH SJATHA; OCTAVIA CHANDRA MUSTIKA; ANGKY BUDIANTI; TJAHJANI MIRAWATI SUDIRO
Microbiology Indonesia Vol. 13 No. 1 (2019): March 2019
Publisher : Indonesian Society for microbiology

Dengue infection is a global infectious disease with almost 100 million cases occur annually in over more than 100 endemic countries. Dengue virus (DENV), the causative agent of dengue infection, is an 11 kbp RNApositive strand virus which encode 3 structural and 7 non-structural protein within its genome. Non-structural 1 (NS1) protein of DENV is expressed in the earlier stage of infection and having pathogenic role in disease severity. NS1 gene of DENV serotype-2 Indonesian strain was amplified through PCR method using specific designated primers. NS1 amplicon were then cloned into pUMVC4.a and pcDNA3.1 mammalian expression vector which confirmed through colony PCR and sequencing method. Recombinant pUNS1 and pcNS1 plasmids were transfected into CHO-K1 mammalian cell line with lipid based method. Recombinant NS1 protein expression were analyzed through immunostaining using dengue patient sera and rapid NS1 detection kit. Recombinant pUNS1 and pcNS1 plasmids were successfully constructed and recombinant NS1 protein was expressed in CHOK1 mammalian cell line and shown to be reactive against dengue patient sera. Our recombinant NS1 protein also tend to be released outside the transfected CHO-K1 cells as detected in rapid NS1 detection kit. Recombinant dengue NS1 protein was expressed in mammalian cell line in both intra and extracellularly and shown to be immunogenic.

Levels of TNF-Î± in PBMC (Peripheral Blood Mononuclear Cells) Induced by Recombinant Non Structural 1 Protein of Dengue Virus Serotype-2 in vitro FITHRIYAH SJATHA; OKTIVIA CHANDRA MUSTIKA; BETI ERNAWATI DEWI; TJAHJANI MIRAWATI SUDIRO
Microbiology Indonesia Vol. 13 No. 2 (2019): June 2019
Publisher : Indonesian Society for microbiology

Dengue infection is a global health problem with an increasing incidence every year and now endemic in more than 100 WHO countries. Dengue infection is caused by dengue virus (DENV) which is an RNA virus with positive single strand, with Â±11kb genome size encoding 3 structural proteins, 7 non-structural proteins, and two Untranslated Region (UTR). NS1 protein is known to have a very important role in the development of severe DENV infection, by the direct effect causing host cells damage and indirect effect by activating immune response to induce the secretion of excess cytokines. This study aims to evaluate whether recombinant pcNS1 plasmids which have been proven expressing recombinant NS1 proteins in previous studies is able to induce cytokine secretion from Peripheral Blood Mononuclear Cells (PBMC). Transfected Chinese Hamster Ovary-K1 (CHO-K1) cells with recombinant pcNS1 plasmid was co-cultured with PBMC from healthy donor. After 48 h post co-cultured, cell supernatant was collected and TNF-Î± levels and NS1 recombinant were measured by ELISA. The results showed that recombinant NS1 protein was expressed in CHO-K1 mammalian cell line and able to induce TNF-Î± with higher levels compared to control.

Levels of CXCL10 Chemokine in Dengue Infected Hepatocyte Huh 7 it-1 Cell Line Co-cultured with Peripheral Blood Mononuclear Cells BETI ERNAWATI DEWI; EVA DAMAYANTI; TJAHJANI MIRAWATI SUDIRO; AGUS SYAHRURACHMAN
Microbiology Indonesia Vol. 13 No. 3 (2019): September 2019
Publisher : Indonesian Society for microbiology

Dengue is a mosquito borne virus that spreads rapidly in the world. At present, it is estimated that more than 3.9 billion people are at risk of being infected with dengue virus (DENV) and there are 96 million clinical cases that have been reported annually in 128 countries worldwide. In DENV infected patients often associated with liver dysfunction which hepatocyte and kuppfer cells as the main target of viral infections. DENV infection induced the expression of several chemokines, which might play an important role during the inflammatory response and pathogenesis of a disease. CXCL10 is known as a chemokine that activates lymphocytes for innate and adaptive immunity, induces tissue damage, and modulates tumor formation. Therefore, we conducted an in vitro study using Huh 7it-1 cells co-cultured with peripheral blood mononuclear cells (PBMCs) to investigate CXCL10 chemokine induction during DENV infection. Huh 7it-1 cells were grown on 96 micro well plate until a monolayer was formed. The cells were infected with DENV-2 at an MOI of 0.5 FFU/cell and 1 FFU/cell in the presence of PBMCs. Heat inactivated DENV-2 and Huh7 cell medium were used as control. After 2 hours of infection, cells were co-cultured with PBMCs and incubated at 37 ÂºC with 5% CO 2 for 48 h. Cell supernatant was collected and CXCL10 chemokine levels were measured using CXCL10 Quantikine ELISA Kit. Statistical analysis was performed by SPSS 23. In the presence of PBMCs, CXCL10 levels from DENV infected Huh 7it-1 at an MOI of 0,5 FFU/cell and MOI of 1 FFU/cell were 552,653 Â± 22,779 pg Â mL-1 and 576,787 Â± 16,901 pg Â mL-1 . Those levels were higher when compared with supernatan from heat inactivated DENV-2 and control cells. Without PBMCs, all of treatments showed lower level of CXCL10. DENV-2 infection in Huh 7it-1 cells co-cultured with PBMCs was able to induce CXCL10 secretion. Furthermore, heat inactivated DENV-2 also still capable to inducen the secretion of CXCL10 chemokine in Huh 7it-1 cells.

QUALITY OF AIR BACTERIA IN OPERATING THEATER IN SOME HOSPITALS IN JAKARTA AND SURROUNDINGS AREAS IN 2018-2019 CONNY RIANA TJAMPAKASARI; NABILA NAURA; TJAHJANI MIRAWATI SUDIRO
Microbiology Indonesia Vol. 14 No. 4 (2020): December 2020
Publisher : Indonesian Society for microbiology

Nosocomial infection is an infection obtained by a patient or hospital staff while in hospital. This infectionplays a role in causing morbidity and mortality in hospitals and can occur in various hospitals rooms, includingoperating theaters. Nosocomial infections can occur due to various factors, one of which is contamination fromairborne bacteria. In some countries, regulations are set to limit the concentration of airborne bacteria, both in theoperating theaters and other rooms in hospitals, hence the need for monitoring and supervision of air quality asareflection on the cleanliness conditions in hospitals. Based on this, it is necessary to know the bacteriological airquality in the operating theaters in several hospitals in Jakarta and surrounding areas as one of the steps to preventnosocomial infections. The method uses an air sampler with the principle of impaction. Air sampler works byseparating the particles from the air by utilizing the inertia of the particles to force the bacteria to settle to thesurface of the medium. A total of 217 examinations in the operating theaters were carried out in 17 hospitals inJakarta and surrounding areas during January 2018 to June 2019. The majority of the operating theaters inhospitals in Jakarta and surrounding areas have air quality that met appropriate quality standards. In 2018, 120 of137 (87.59%) examination in the operating theaters met the quality standar. Meanwhile in 2019, 70 of 80(87.50%) operating theaters met the standard determined by the Ministry of Health of the Republic of Indonesia. Key words: air sampler method, operating theater, quality of air bacteria

Co-Authors . Andriansjah Agus Syahrurachman Angky Budianti Beti Ernawati Dewi DEKA LARASATI EVA DAMAYANTI FITRIYAH SJATHA Heni Pujiastuti NABILA NAURA OCTAVIA CHANDRA MUSTIKA OKTIVIA CHANDRA MUSTIKA PAISAL PAISAL SJATHA, FITHRIYAH Tjampakasari, Conny Riana

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