Articles
<![CDATA[Teknik Long Polymerase Chain Reaction (LPCR) Untuk Perbanyakan Kerangka Baca Terbuka Gen Pengkode Polimerase Virus Hepatitis B ]]>
<![CDATA[Hutapea, Hotma]]>;
<![CDATA[Retnoningrum, Debbie]]>;
<![CDATA[Rahman, Ernawati Giri]]>;
<![CDATA[Rostinawati, Tina]]>
<![CDATA[ JURNAL PLASMA Vol 1, No 2 Jun (2015) ]]>
Publisher : <![CDATA[Balai Penelitian dan Pengembangan Biomedis Papua ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
Full PDF (150.775 KB)
<![CDATA[Teknik PCR dapat dikembangkan untuk mengamplifikasi potongan DNA dengan ukuran panjang. Salah satu teknik yang digunakan adalah Long PCR (LPCR). Teknik LPCR sering digunakan untuk mengamplifikasi gen atau potongan DNA yang berukuran lebih panjang ketika PCR standar tidak dapat diaplikasikan untuk amplifikasi tersebut. Tujuan dari penelitian ini adalah untuk memperoleh kerangka baca terbuka gen pengkode polimerase virus hepatitis B (PolHBV) menggunakan metode LPCR, dan menentukan urutan nukleotida potongan DNA tersebut. Amplifikasi dilakukan menggunakan Taq polimerase. Kerangka baca terbuka gen pengkode PolHBV telah berhasil diamplifikasi dengan LPCR yang telah dimodifikasi. Hal tersebut diindikasikan dengan keberadaan pita DNA berukuran  pasangan basa (bp) pada elektroforesis gel agarosa yang mendekati ukuran teoritisnya yaitu 2532 bp. Hasil penentuan urutan nukleotida parsial menunjukkan bahwa potongan DNA yang diperoleh memiliki homologi 98% dengan gen PolHBV yang dideposit di GenBank.Technique of PCR can be improved to permit the amplification of longer DNA fragment. One of this technique is Long PCR (LPCR). LPCR is often used to amplify larger genes or large segment of DNA which standard PCR is not applicable. The objective of this study is to obtain the open reading frame of gene encoding polymerase of HBV (polHBV) using LPCR method, and to determine the nucleotide sequence of DNA fragment encoding polHBV. The amplification was conducted using Taq polymerase. The open reading frame of gene encoding polHBV was successfully amplified using modified LPCR method. It  was  identified by  a  band  between  2000 dan  3000  base  pairs  (bp) DNA marker.  The determination of nucleotide sequence informed that DNA fragment obtained was 98% homologue to the gene encoding PolHBV deposited on GenBank. .]]>
<![CDATA[Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction ]]>
<![CDATA[Patricia Gita Naully]]>;
<![CDATA[Neni Nurainy]]>;
<![CDATA[Elvi Restiawaty]]>;
<![CDATA[Dessy Natalia]]>;
<![CDATA[Debbie Soefie Retnoningrum]]>;
<![CDATA[Wardono Niloperbowo]]>;
<![CDATA[Ernawati Arifin Giri-Rachman]]>
<![CDATA[ HAYATI Journal of Biosciences Vol. 27 No. 4 (2020): October 2020 ]]>
Publisher : <![CDATA[Bogor Agricultural University, Indonesia ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
DOI: 10.4308/hjb.27.4.283
<![CDATA[Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction.]]>
<![CDATA[Sequence Analysis of Putative potB, potC, and potD Genes from Serratia rubidae ]]>
<![CDATA[SONY SUHANDONO]]>;
<![CDATA[RASI FITRIA]]>;
<![CDATA[ERNAWATI ARIFIN GIRI RACHMAN]]>
<![CDATA[ Microbiology Indonesia Vol. 4 No. 2 (2010): August 2010 ]]>
Publisher : <![CDATA[Indonesian Society for microbiology ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
Full PDF (6698.416 KB)
|
DOI: 10.5454/mi.4.2.8
<![CDATA[Amplification of putative potBCD genes from Serratia rubidae was conducted by PCR using a pair of primers FERC and RERC. A fragment with ~1800 bp size was ligated to pGEM-T Easy vector then cloned to competent Escherichia coli DH5α. The recombinant plasmid was sequenced using SP6, T7 and two internal primers, FPC and RPC. A sequence similarity search and analysis was performed with the BLASTN program. The sequence was found to have 83% similarity to potABCD genes from E. coli. Those genes encoded a spermidine-preferential uptake system that consists of four kinds of protein: PotA is a membrane-associated ATPase, PotB and PotC are transmembrane proteins that form channels, and PotD is a periplasmic substrate-binding protein. Alignment analysis showed that the isolated clone consisted of potB (partial), potC (full length) and potD (partial). The sequences for potBCD genes from S. rubidae are not available in the NCBI database. Furthermore, we have submitted this sequence, potBCD from S. rubidae, on GeneBank with Acc. number FJ447342.]]>
<![CDATA[Molecular Analysis of Immune-Escape Mutants of Hepatitis B Virus from Local Clinical Samples ]]>
<![CDATA[CHANDRA JINATA]]>;
<![CDATA[ERNAWATI ARIFIN GIRI-RACHMAN]]>;
<![CDATA[DEBBIE SOEFIE RETNONINGRUM]]>
<![CDATA[ Microbiology Indonesia Vol. 6 No. 1 (2012): March 2012 ]]>
Publisher : <![CDATA[Indonesian Society for microbiology ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
Full PDF (379.113 KB)
|
DOI: 10.5454/mi.6.1.2
<![CDATA[Small hepatitis B surface antigen (sHBsAg) is used as a component of hepatitis B vaccine. Even though this vaccine is known to be effective in preventing hepatitis B disease, natural mutation may induce Hepatitis B Virus (HBV) to form immune-escape mutant. This mutant is not only capable of infecting hepatitis B-vaccinated people, but also causing commercial diagnostic assay failure. Immune-escape mutant is generally detected from amino acid change at Major Hydrophilic Region (MHR) of sHBsAg while the change occurred outside the region may also lead to immune-escape mutant formation. This research was aimed to investigate the presence of HBV immune-escape mutants in local clinical samples in Indonesia. sHBsAg gene of seventeen HBV samples from local patients were amplified by polymerase chain reactions then subjected to two-directional sequencing. The DNA sequences later were analyzed by bioinformatics programs. Fifteen out of seventeen samples were genotype B and subtype adw2, while the other two were genotype C and subtype adrq+. Among fifteen genotype B samples, twelve of them were not immune-escape mutants, two were immune-escape mutants that have been previously reported (Gln129Arg and Met133Leu), and one was a mutant outside MHR that has not been previously reported as an immune-escape mutant (Tyr161Ser). Both samples of genotype C group were not immune-escape mutants. As conclusion, by investigating seventeen local clinical HBV samples, it was known that two of seventeen samples were confirmed as immune-escape mutants and one of seventeen samples was a mutant outside MHR.]]>
<![CDATA[Construction of Binary Vector With Wound Inducible Promoter for Hbsag Expression: Development of Plant-Based Edible Hepatitis B Vaccine from Indonesian Isolate ]]>
<![CDATA[Sony Suhandono]]>;
<![CDATA[Ernawati A.Giri Rachman]]>;
<![CDATA[Ima M. Zainuddin]]>;
<![CDATA[Putri Dwi Utari]]>;
<![CDATA[Apsari Supraba]]>;
<![CDATA[Amy Estiati]]>
<![CDATA[ ANNALES BOGORIENSES Vol 11, No 1 (2007): Annales Bogorienses ]]>
Publisher : <![CDATA[Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI) ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
Full PDF (3283.881 KB)
|
DOI: 10.14203/ann.bogor.2007.v11.n1.7-14
<![CDATA[Hepatitis B is a serious infectious disease In the third world countries including Indonesia . Vaccination is the most effective way to prevent the spread of the disease; therefore the demand for HBV vaccine is high. In order to produce more vaccine at lower cost, transgenic plant can be chosen to express the vaccine with the above criteria. Several researches were successfully producing transgenic plants expressing HBsAg that formed virus-like particles and induced immune response in human. However. HBsAg expression in transgenic plant needs to be improved especially on gene expression control system. Here, we describe the construction of HBsAg . structural gene under the control of wound inducible promoter, MeEFl promoter from manihot esculenta Crantz. The HBsAg gene was amplified using PCR from HBV genome isolated from an Indonesian patient. The gene was subsequently fused with VSPaS signal peptide, which targeted the reticulum endoplasm of plant cell . The construct was cloned into binary expression vector for Agrobacterium plant Transformation in near future.Keywords: HBsAg. VSPaS signal peptide and MeEFI promoter]]>
<![CDATA[SARS-CoV-2 Neutralization Assay System using Pseudo-lentivirus ]]>
<![CDATA[Anastasia Armimi]]>;
<![CDATA[Afina Firdaus Syuaib]]>;
<![CDATA[Katherine Vanya]]>;
<![CDATA[Marselina Irasonia Tan]]>;
<![CDATA[Dessy Natalia]]>;
<![CDATA[David Virya Chen]]>;
<![CDATA[Chikako Ono]]>;
<![CDATA[Yoshiharu Matsuura]]>;
<![CDATA[Anita Artarini]]>;
<![CDATA[Ernawati Arifin Giri-Rachman]]>
<![CDATA[ The Indonesian Biomedical Journal Vol 15, No 2 (2023) ]]>
Publisher : <![CDATA[The Prodia Education and Research Institute (PERI) ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
DOI: 10.18585/inabj.v15i2.2212
<![CDATA[BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects humans' lower respiratory tracts and causes coronavirus disease-2019 (COVID-19). Neutralizing antibodies is one of the adaptive immune system responses that can reduce SARS-CoV-2 infection. This study aimed to develop a SARS-CoV-2 neutralization assay system using pseudo-lentivirus.METHODS: The plasmid used for pseudo-lentivirus production was characterized using restriction analysis. The gene encoding for SARS-CoV-2 spike protein was confirmed using sequencing. The transfection pseudo-lentivirus optimal condition was determined by choosing the transfection reagents and adding centrifugation step. Optimal pseudo-lentivirus infection was analysed using fluorescent assay and luciferase assay. The optimal condition of pseudo-lentivirus infection was determined by the target cell type and the number of pseudo-lentiviruses used for neutralization test. SARS-CoV-2 pseudo-lentivirus was used to detect neutralizing antibodies from serum samples.RESULTS: The plasmid used for pseudo-lentivirus production was characterized and confirmed to have no mutations. Lipofectamine 2000 reagent generated pseudo-lentivirus with a higher ability to infect target cells, as indicated by a percentage green fluorescent protein (GFP) of 12.68%. Pseudo-lentivirus centrifuged obtained more stable results in luciferase expression. Optimal pseudo-lentivirus infection conditions were obtained using puromycin-selected HEK 293T-ACE2 cells as target cells. The number of pseudo-lentiviruses used in the neutralization assay system was multiplicity of infection (MOI) 0.075. Serum A samples with a 1:10 dilution had the highest neutralizing antibody activity.CONCLUSION: This study shows that SARS-CoV-2 neutralization assay system using pseudo-lentivirus successfully detected neutralizing antibodies in human serum, which were indicated by a decrease in the percentage of pseudo-lentivirus infections.KEYWORDS: COVID-19, neutralizing antibody, neutralization assay, pseudo-lentivirus, SARS-COV-2]]>
<![CDATA[Cloning, Overexpression, and Purification of PhoR CytoplasmicDomain Protein from Mycobacterium tuberculosis strain H37Rv ]]>
<![CDATA[OKTIRA ROKA AJI]]>;
<![CDATA[DYSHELLY NURKARTIKA PASCAPURNAMA]]>;
<![CDATA[FENRYCO PRATAMA]]>;
<![CDATA[IHSANAWATI IHSANAWATI]]>;
<![CDATA[MAELITA RAMDHANI MOEIS]]>;
<![CDATA[ERNAWATI ARIFIN GIRI-RACHMAN]]>
<![CDATA[ Microbiology Indonesia Vol. 8 No. 4 (2014): December 2014 ]]>
Publisher : <![CDATA[Indonesian Society for microbiology ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
Full PDF (758.166 KB)
|
DOI: 10.5454/mi.8.4.1
<![CDATA[Tuberculosis still becomesa major health problem in the world. This infectious disease is caused by Mycobacterium tuberculosis (Mtb). Novel anti-tubercular drug is urgently needed to counter multidrug resistant cases and Mtb's spread. The cytoplasmic domain of PhoR histidine kinase, a part of the two-component system PhoR-PhoP in Mtb, is one of the potential candidates for anti-tubercular drug target.Three dimensional protein of drug target is needed to screen potential drug candidate using rational drug design approaches. Previous studies have successfully characterized and isolated putative cytoplasmic domain of PhoR (CytoPhoR) from Mtb strain H37Rv. This study aimed to clone, overexpress and purify of CytoPhoR protein. CytoPhoR was fused with thioredoxin protein in expression vector pET32b and overexpressed in Escherichia coli (E.coli)BL21 (DE3) as soluble fraction by induction 1 mM IPTG. Purification of his-tagged CytoPhoR was carried out using IMAC Ni-NTA Agarose his-tag affinity column. SDS-PAGE analysis showed that another protein was co-purified (~35 kDa) along with the CytoPhoR protein. Subsequent protein purification using DEAE-ion exchange column generate a strong single band of 37 kDa on SDS–PAGE which is indicated as CytoPhoR protein. The purified CytoPhoR protein was successfully obtained and can be used for further analysis on determining three dimensional structure of CytoPhoR protein.]]>
<![CDATA[Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli ]]>
<![CDATA[ERNAWATI ARIFIN GIRI-RACHMAN]]>;
<![CDATA[FENRYCO PRATAMA]]>;
<![CDATA[OKTIRA ROKA AJI]]>;
<![CDATA[ARUM PATRIATI]]>;
<![CDATA[IHSANAWATI IHSANAWATI]]>;
<![CDATA[MAELITA RAMDANI MOEIS]]>;
<![CDATA[EDY GIRI-RACHMAN PUTRA]]>
<![CDATA[ Microbiology Indonesia Vol. 9 No. 2 (2015): June 2015 ]]>
Publisher : <![CDATA[Indonesian Society for microbiology ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
Full PDF (1382.183 KB)
|
DOI: 10.5454/mi.9.2.1
<![CDATA[Globally, tuberculosis (TB) remains a leading cause of death. The emergence of multidrug-resistant strains (MDR-TB) and extensively drug-resistant strains (XDR-TB) has fuelled the discovery for novel drugs and drug targets for its successful and better treatment. One of the potential candidates for drug target is PhoR sensory protein histidine kinase, a part of the Two Component System (TCS) PhoP/PhoR in Mycobacterium tuberculosis (Mtb). This protein system was known for its role on regulating hundred of Mtb virulence factors, from genes for cell wall and lypid synthesis to genes for adaptation in human leukocyte and hypoxia response. Previous studies have successfully characterized, isolated, and cloned the putative sensory domain of PhoR protein gene into pRSET vector expression system. In this study, Escherichia coli was transformed with pRSET-SensPhoR and cultivated at 37oC under IPTG induction to express PhoR sensor-domain protein. Most of the proteins were overexpressed in the form of inclusion bodies. Â Subsequent protein purification in Ni-NTA system under refolding condition on urea gradient was performed to isolate PhoR sensor-domain protein in soluble form. Arginine was supplemented in purified protein solution to prevent aggregation during long term storage. Â While highly purified protein was acquired, small angle X-ray scattering (SAXS) analysis was conducted to obtain 3-dimensional (3D) protein structures in solution. Â Â Â doi:10.5454/mi.9.2.1Â ]]>
<![CDATA[Design of Adenovirus 5 Vector with Adenovirus 26 Hexon Hypervariable Region Sequence using In Silico Approach ]]>
<![CDATA[Afina Firdaus Syuaib]]>;
<![CDATA[Ernawati Arifin Giri-Rachman]]>;
<![CDATA[Aluicia Anita Artarini]]>
<![CDATA[ Microbiology Indonesia Vol. 16 No. 2 (2022): December ]]>
Publisher : <![CDATA[Indonesian Society for microbiology ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
Full PDF (202.696 KB)
|
DOI: 10.5454/mi.16.2.31-36
<![CDATA[Adenovirus type 5 (Ad5) is one of the vaccine vectors, including the COVID-19 vaccine. Pre-existing immunity to Ad5 may suppress the immunogenicity and efficacy of adenovirus vectored vaccine. The neutralizing antibodies are directed specifically toward seven hypervariable regions (HVR) of hexon proteins located on the outer surface of the capsid. This study aims to design an Ad5 vector that may circumvent anti-Ad5 immunity by designing a chimera Ad5 vector with the sequence of Ad26 HVR (Ad5HVR26) using in silico approach. Substitution of the Ad5 HVR DNA sequence may affect the alternative splicing process of adenovirus mRNA, which then influence the protein product. The splice site prediction of Ad5HVR26 chimera vector was found at HVR5, 6, and 7. The codon change in the splice site was performed to decrease the possibility of incorrect splicing, while retaining the original amino acid sequence. The HVR substitution in chimera vector Ad5HVR26 may also affect the interaction of hexon in the capsid. The HVR2 and HVR4 hexon proteins individually interact with other hexon proteins and IX protein. Thus, two designs of the Ad5HVR26 chimera vector were created in this research. The first design was the Ad5 chimera vector with complete substitution of HVR hexon by Ad26 sequence, with codon modification on the splice site. The second design was Ad5HVR26 chimera vector without the HVR2 and HVR4 substitution to maintain the hexon protein interaction with the capsid proteins. Production of the designed vectors are needed to prove the reduction of vector neutralization by pre-existing immunity.]]>
<![CDATA[Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line ]]>
<![CDATA[Meutia Diva Hakim]]>;
<![CDATA[Noriko Yamano- Adachi]]>;
<![CDATA[Takeshi Omasa]]>;
<![CDATA[Marselina Irasonia Tan]]>;
<![CDATA[Lia Dewi Juliawaty]]>;
<![CDATA[Ernawati Arifin Giri-Rachman]]>
<![CDATA[ Journal of Mathematical and Fundamental Sciences Vol. 54 No. 3 (2023) ]]>
Publisher : <![CDATA[Directorate for Research and Community Services (LPPM) ITB ]]>
Show Abstract
|
Download Original
|
Original Source
|
Check in Google Scholar
|
DOI: 10.5614/j.math.fund.sci.2023.54.3.1
<![CDATA[Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells.]]>
