Beti Ernawati Dewi
Department of Microbiology, Faculty of Medicine, University of Indonesia, Jakarta10430

Published : 4 Documents Claim Missing Document
Claim Missing Document
Check
Articles

Found 4 Documents
Search

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.
RESPON IMUN SELULER DAN HUMORAL MENCIT YANG DIIMUNISASI KANDIDAT VAKSIN DNA DENGUE BERBASIS GEN preM-E SEROTIPE 4 STRAIN INDONESIA Eleanor Louana Urfa; Beti Ernawati Dewi; T. Mirawati Sudiro
Majalah Kedokteran Andalas Vol 37, No 2 (2014): Published in September 2014
Publisher : Faculty of Medicine, Universitas Andalas

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (623.477 KB) | DOI: 10.22338/mka.v37.i2.p75-85.2014

Abstract

AbstrakInfeksi virus dengue (DENV) terkadang tanpa gejala atau dapat menunjukkan gejala klinis yang luas, berkisar dari sindrom flu ringan (dengue fever/DF), dengue haemorrhagic fever (DHF), hingga syok hipovolemik (dengue shock syndrome/DSS). Hipotesis yang berkaitan dengan tingkat keparahan infeksi DENV meliputi mekanisme antibody-dependent enhancement (ADE) dan keterlibatan sitokin. Hingga kini, belum ada obat antiviral yang efektif untuk mengeradikasi dan mencegah infeksi DENV, sehingga pencegahan berupa vaksin perlu dikembangkan. Kandidat vaksin DNA berbasis gen preM-E serotipe 4 strain Indonesia yang dikembangkan pada penelitian terdahulu disuntikkan ke mencit ddY, kemudian diuji tantang dengan DENV. Pada hari ke-4 dan ke-21 pascauji tantang, keberadaan sitokin IL-2 dalam serum dideteksi dengan metode ELISA. Serum hari ke-21 digunakan dalam uji ADE menggunakan sel K562. Sel limpa diambil pada hari ke-21 pascauji tantang, kemudian keberadaan IL-2 dan antibodi in vitro dideteksi dengan metode ELISA. Tingkat IL-2 tertinggi terdapat pada serum hari ke-4 pada kelompok mencit yang tidak diimunisasi namun diuji tantang, yaitu sebesar 69,83 pg/ml. Konsentrasi IL-2 terendah ditunjukkan oleh kelompok mencit yang diimunisasi namun tidak diuji tantang, yaitu 0 pg/ml. Pengukuran IL-2 pada serum dan supernatan sel limpa hari ke-21 tidak mendapatkan konsentrasi IL-2. Titer antibodi tertinggi terdapat pada kelompok sel limpa mencit yang diimunisasi, diuji tantang, dan diinduksi in vitro dengan DENV. Hasil uji ADE menunjukkan tingkat pengenceran serum berpengaruh terhadap jumlah sel yang terinfeksi oleh DENV, namun tidak ditemukan kondisi netralisasi dan enhancing. Berdasarkan metode yang digunakan, kandidat vaksin DNA tersebut dapat memicu respon imun seluler dan humoral.AbstractDengue virus (DENV) infection can be asymptomatic or cause wide range of clinical symptoms, from mild febrille ilness (dengue fever/DF), dengue haemorrhagic fever (DHF), to hipovolemic shock (dengue shock syndrome/DSS). Hypotheses related to the severity of DENV infection mechanisms including antibody-dependent enhancement (ADE) and cytokines involvement. Until now, there are no effective antiviral drugs can eradicate and prevent DENV infection, therefore the development of vaccines is the alternative. DNA vaccine candidate preM-E serotype 4 strain of Indonesia which was developed in previous studies injected into ddY mice, then challenge with DENV. At day 4 and 21 post-challenge, serum was taken to detect the presence of cytokines IL-2 using ELISA method. Day 21 serum used in the antibody-dependent enhancement (ADE) assay using K562 cell line. Splenocytes were taken at day 21 post-challenge to measure the presence of IL-2 and in vitro antibody using ELISA method. Measurement of IL-2 on day 4 serum produced the highest levels of IL-2 (69.83 pg/ml) in the group of non-immunized, challenged mice, whereas the lowest concentration (0 pg/ml) shown by the group of immunized, non-challenged mice. Measurement of IL-2 in serum and splenocytes day 21 did not get the concentration of IL-2. The highest result of in vitro antibody measurements shown by the group of splenocytes from immunized, challenged mice then in vitro induced with DENV. ADE assay results showed that level of serum dilution has effect on the number of dengue-infected cells, but netralization and enhancing condition were not found in this assay. Based on this methods, the DNA vaccine candidate can trigger cellular and humoral immune responses.
Levels of TNF-α 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

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (709.517 KB) | DOI: 10.5454/mi.13.2.4

Abstract

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 ±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-α 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-α 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

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (606.52 KB) | DOI: 10.5454/mi.13.3.4

Abstract

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 ºC 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 ± 22,779 pg  mL-1 and 576,787 ± 16,901 pg  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.