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All Journal Kimia Padjadjaran Annales Bogorienses
Kusharyoto, Wien
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemistry Environmental Science Materials Science & Nanotechnology

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Generation of mCherryBody: an Anti-Transferrin Receptor Antibody Variable Fragment Linked by The Fluorescent Protein mCherry Kusharyoto, Wien; Andriani, Dian; Handayani, Ira
Annales Bogorienses Vol. 20 No. 2 (2016): Annales Bogorienses
Publisher : BRIN

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Abstract

A facile generation of a recombinant antibody fragment with intrinsic fluorescent properties of the monomeric mCherry fluorescent protein is described. The so-called mCherryBody was designed based on the structure model of the variable fragment of anti-transferrin receptor antibody LUCA31 and the X-ray crystallographic structure of the mCherry protein. mCherryBody was constructed to retain optimal spatial geometry between the C- and N-termini of the antibody light-chain (VL) and heavy-chain (VH) by mimicking the domains interface pairing in antibody Fab fragments and incorporation of the mCherry fluorescent protein as a bridging scaffold. The gene encoding the chimeric protein was cloned into the pJExpress414 expression vector, expressed and secreted into the periplasm of Escherichia coli NiCo21(DE3) for assembly and disulphide bond formation. Based on its amino acid sequence, mCherryBody was predicted to have a molecular weight of 51.46 kDa. The modular assembly used in the generation of mCherryBody may permit the interchange of binding sites and of fluorescent proteins to create robust panels of coloured antibody fragments. Thus, the mCherryBody platform facilitates rapid generation of colored single-chain variable fragment (scFv) chimeras that could be used for screening of antibodies against cell surface markers or receptors.
A Preliminary Report on The Syntheses of Oligonucleotide Primers in The National Research and Innovation Agency (NRIA) Atikana, Akhirta; Prasetyoputri, Anggia; Rubiyana, Yana; Herawati, Neng; Desriani, Desriani; Pratiwi, Riyona Desvy; Wulandari, Dwi; Sukmarini, Linda; Kusharyoto, Wien; Santoso, Adi; Putra, Masteria Yunovilsa; Lisdiyanti, Puspita
Annales Bogorienses Vol. 26 No. 1 (2022): Annales Bogorienses
Publisher : BRIN

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2022.v26.n1.21-27

Abstract

A PolyGen DNA Synthesizer is equipment that is used for synthesizing oligonucleotide primers for any amplification targets. Oligonucleotide primers are indispensable components for any Polymerase Chain Reaction (PCR)-based detections. In the present study a number of oligonucleotide primer sets were synthesized to target (1) the Human Insuline Glargin (HIG) and (2) the Human Erythropoietin (EPO), as well as (3) the RNA-dependent RNA Polymerase (RdRp) and (4) the Nucleocapsid (N) genes of the severe acute respiratory syndrome virus 2 (SARS-CoV-2). A solid-phase oligonucleotide synthesis method was used according to the default protocol of the Polygen’s instrument to synthesize primers at a 40 nmol scale. The synthesized primers in this study were compared to commercially produced primers in their ability to amplify the gene target(s) in PCR and quantitative real-time PCR (qPCR) reactions. The first two sets of primers showed similar results in PCR compared to commercial primers; however, these primers were not tested for qPCR due to sample limitation. In contrast, the primer sets 3 and 4 were not able to produce amplicons in PCR reactions and only the primer set 4 successfully amplified the gene target in qPCR. These results indicate that the crude primers synthesized in this study are promising candidates for molecular detection and diagnostics, but these primers would benefit from further optimization for routine applications.
Co-Authors Adi Santoso Antonius Suwanto Arwansyah Arwansyah Asrul Muhamad Fuad, Asrul Muhamad Atikana, Akhirta Bambang Prasetya Desriani Desriani Dian Andriani, Dian Dwi Wulandari Fifi Afiati Gita Syahputra Handayani, Ira Hariyatun, Hariyatun LINDA SUKMARINI Martha Sari Neng Herawati, Neng Partomuan Simanjuntak Prasetyoputri, Anggia PUSPITA LISDIYANTI Putra, Masteria Yunovilsa Ridwanuloh, Asep Muhamad Riyona Desvy Pratiwi, Riyona Desvy Schmid, Rolf D. Vorlop, Klaus Dieter Willke, Thomas Yana Rubiyana, Yana Yatri Hapsari, Yatri