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All Journal Jurnal Ilmu Dasar AL KAUNIYAH Indonesian Journal of Biotechnology Jurnal Bioteknologi & Biosains Indonesia (JBBI)
Widhi Dyah Sawitri
Universitas Gadjah Mada
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Control & Systems Engineering Immunology & microbiology Materials Science & Nanotechnology Mathematics

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TRANSFORMASI GENETIK DAN EKSPRESI MUTAN SUCROSE PHOSPHATE SYNTHASE PADA TANAMAN TOMAT Fibriani, Suwinda; Agustien, Inyana Dwi; Sawitri, Widhi Dyah; Sugiharto, Bambang
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 6 No. 1 (2019): June 2019
Publisher : Balai Bioteknologi, Badan Pengkajian dan Penerapan Teknologi (BPPT)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (699.256 KB) | DOI: 10.29122/jbbi.v6i1.3341

Abstract

Genetic Transformation and Expression of Sucrose Phosphate Synthase Mutant in Tomato Plant ABSTRACTSucrose phosphate synthase (SPS) is a key enzyme responsible for sucrose biosynthesis. In its regulation, SPS activity is modulated by an allosteric effector glucose-6-phosphate (G6P) suggested to have an ability to bind SPS N-terminus domain. To understand the role of N-terminus in regulating SPS, the SPS gene was mutated with the deletion of N-terminus domain (∆N-SPS). The ∆N-SPS gen was transformed into tomato plants with 5% transformation efficiency. Three transgenic tomato plant 4.20, 5.5.1, and 5.10 were obtained and confirmed by PCR analysis. Transgenic tomato expression was characterized by enzymatic analysis. Result showed that the G6P allosteric regulation in transgenic ∆N-SPS had lost and the SPS activity increased by 2-fold compared to non-transgenic plant. This showed that N-terminus domain-deleted SPS could be actively expressed in plant. Keywords: enzyme, genetic transformation, N-terminus domain deletion, sucrose phosphate synthase, tomato ABSTRAKSucrose phosphate synthase (SPS) merupakan enzim kunci yang bertanggung jawab dalam sintesis sukrosa. Dalam regulasinya, aktifitas SPS dipengaruhi oleh alosterik efektor glukosa-6-fosfat (G6P) yang diduga dapat berikatan pada domain N-terminus SPS. Untuk mengetahui peran N-terminus pada regulasi SPS, dilakukan mutasi SPS dengan penghilangan domain N-terminus (∆N-SPS). Gen ∆N-SPS diinsersi pada tanaman tomat melalui transformasi genetik dengan efisiensi transformasi 5%. Tiga tanaman transgenik tomat (event4.20; 5.5.1; dan 5.10) didapatkan dan positif terkonfirmasi melalui analisis PCR. Ekspresi mutan dikarakterisasi melalui analisis enzimatik. Hasil menunjukkan bahwa tanaman tomat transgenik ∆N-SPS tidak dipengaruhi regulasi alosterik G6P dan aktifitas SPS 2 kali lipat lebih tinggi daripada tanaman bukan transgenik. Ini menunjukkan bahwa SPS dengan delesi domain N-terminus dapat terekspresi aktif pada tanaman.  Kata Kunci: delesi domain N-terminus, enzim, sucrose phosphate synthase, tomat, transformasi genetik 
Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production Natalia Tri Astuti; Nurmalasari Darsono; Suvia Widyaningrum; Widhi Dyah Sawitri; Sri Puji Astuti; Win Darmanto
Indonesian Journal of Biotechnology Vol 24, No 1 (2019)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (72.263 KB) | DOI: 10.22146/ijbiotech.45551

Abstract

Sugarcane mosaic virus (SCMV, genus Potyvirus, family Potyviridae) is a prominent pathogen of sugarcane (Saccharum sp. hybrids). It can cause losses in susceptible varieties, in crop as well as sugar production, economically. Although it has been studied in major sugar-producing countries, research on the definement of SCMV from Indonesian isolates based on molecular study has been very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate in order to produce polyclonal antibodies that cann be used for immunodiagnosis assays in a subsequent study. A gene-encoding coat protein of SCMV (CP-SCMV) was amplified using RT-PCR and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and over-expressed in Escherichia coli BL21(DE3) to produce a recombinant protein. The highest expression was found in 0.1M IPTG induction media for 5 h at 37oC. SDS-PAGE analysis clarified that the recombinant CP-SCMV remained as an insoluble fraction. Purifications was carried out by the affinity Ni-NTA resin, followed by electroelution to obtain a highly purified protein. To meet the quality requirements of a proper antigen, the highly purified protein was concentrated. A molecular weight of the rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL. 
The efficacy of a chicken antibody for the development of immunoassay‐based rapid detection in sugarcane mosaic virus disease Nurmalasari Darsono; Widhi Dyah Sawitri; Retnosari Apriasti; Agus Heri Setyo Wahyudi; Putri Andreyna Saragi; Victorin Mega Putri; Sugiharto Sugiharto; Win Darmanto
Indonesian Journal of Biotechnology Vol 28, No 1 (2023)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/ijbiotech.74104

Abstract

Sugarcane Mosaic Virus (SCMV) infection is one of the most serious problems that can result in severe yield loss of sugarcane. Since the symptoms of SCMV infection are similar to other biotic and abiotic stress symptoms, the development of a rapid diagnostic with high precision is required. The use of laboratory animals such as rabbits is required for antibody production in immunoassay‐based detection. However, due to its many advantages, specific chicken egg yolk immunoglobulin (IgY) has received considerable attention as an alternative antibody production in immunodiagnostics for infectious diseases. In this study, IgY antibody against SCMV recombinant coat protein (CP) was successfully obtained from chicken blood serum and tested to compare its efficacy against antibody from rabbit (IgG) using immunocapture reverse transcription‐polymerase chain reaction (IC‐RT‐PCR). The result showed that IgY and IgG could detect 0.1 g SCMV infected leaves using 1000‐times‐diluted antibodies. The IgY antibody was also confirmed to be reproducible and potentially applicable in plant disease diagnostics using an antibody‐based detection.
Produksi Antibodi Poliklonal Menggunakan Protein Rekombinan RBD-spike Untuk Deteksi SARS-CoV-2 Iryani Endah Febrianti; Yayuk Fatmawati; Intan Ria Neliana; Widhi Dyah Sawitri; Erlia Narulita; Bambang Sugiharto
Al-Kauniyah: Jurnal Biologi Vol 16, No 2 (2023): AL-KAUNIYAH JURNAL BIOLOGI
Publisher : Department of Biology, Faculty of Science and Technology, Syarif Hidayatullah State Islami

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15408/kauniyah.v16i2.25664

Abstract

 AbstrakSARS-CoV-2 merupakan virus yang menyebabkan Coronavirus Disease 2019 (COVID-19) di seluruh dunia dan sampai saat ini kasus terbaru masih terus dilaporkan. Diagnostic test merupakan hal yang krusial untuk dikembangkan. Prinsip diagnostic test COVID-19 berbasis antigen, yaitu mendeteksi virus SARS-CoV-2 melalui respon antibodi dari penderita. Penelitian ini bertujuan untuk memproduksi antibodi poliklonal menggunakan protein rekombinan RBD-Spike untuk mendeteksi virus SARS-CoV-2 berbasis antibodi. Penelitian dimulai dengan penentuan domain RBD-Spike menggunakan pensejajaran asam amino, dan konstruksi DNA untuk RBD-Spike pada vektor ekspresi pET28a menggunakan sintetik nukleotida. Produksi protein rekombinan RBD-Spike diekspresikan pada sel bakteri Escherichia coli. Purifikasi dilakukan untuk memperoleh protein RBD-Spike dan selanjutnya digunakan sebagai antigen untuk induksi antibodi poliklonal pada kelinci. Hasil penelitian menunjukkan bahwa ekspresi protein rekombinan RBD-Spike SARS-CoV-2 memerlukan induksi IPTG 0,1 mM dan terekspresi dalam bentuk inclusion bodies dengan ukuran 39 kDa. Purifikasi protein RBD-Spike dilakukan menggunaan resin afinitas NiNTA, elektroelusi, dan dialisis. Total protein RBD-Spike yang diperoleh sebanyak 4 mL dengan konsentrasi 10 mg/mL. Analisa Ouchterlony menunjukkan bahwa antibodi poliklonal terdeteksi pada minggu kedua setelah injeksi booster dan analisa spesifitas antibodi terhadap antigen menunjukkan bahwa antibodi poliklonal dapat mendeteksi protein RBD-Spike pada konsentrasi 0,1 µg. Selanjutnya diharapkan antibodi poliklonal dapat digunakan untuk deteksi keberadaan virus SARS-CoV-2 dan dapat dikembangkan untuk kit deteksi berbasis antibodi.AbstractSARS-CoV-2 is the virus that causes Coronavirus Disease 2019 (COVID-19) worldwide and the latest cases are still being reported until now. The diagnostic test is a crucial to be developed. The principle of the antigen-based COVID-19 diagnostic test is to detect the SARS-CoV-2 virus through antibody response from the patients. This study was conducted to produce polyclonal antibodies using recombinant protein RBD-Spike. The research was carried out by determining the RBD-Spike domain using amino acid alignment and constructing the DNA of RBD-Spike to the expression vector of pET28a using nucleotide synthesis. Production of RBD-Spike recombinant protein was expressed in Escherichia coli. Purification was carried out to obtain RBD-Spike protein and used to induce polyclonal antibody ina rabbit. The results showed that the expression of RBD-Spike recombinant protein required induction of IPTG 0.1 mM and was expressed in inclusion bodies with molecular size of 39 kDa. The purification of RBD-Spike protein was carried out using resin affinity, electroelution, and dialysis. The total protein of RBD-Spike obtained was 4 mL with a concentration of 10 mg/mL. Ouchterlony analysis revealed that polyclonal antibody was detected in the second week after booster injection and analysis of antibody specificity showed that polyclonal antibodies detected RBD-Spike protein at the concentration of 0.1µg of RBD-Spike protein. Moreover, it is expected that our polyclonal antibody detect the SARS-CoV-2 virus and can be developed for antibody-based detection kits.
The Expression of The PfEMP1-DBL2β Recombinant Protein of Plasmodium falciparum Isolated From Indonesia Hasanah, Fathul Hidayatul; Sulistyaningsih, Erma; Sawitri, Widhi Dyah
Jurnal ILMU DASAR Vol 21 No 1 (2020)
Publisher : Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Jember

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (123.49 KB) | DOI: 10.19184/jid.v21i1.10494

Abstract

The binding of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) to Intracellular Adhesion Molecule-1 (ICAM-1) is a major pathological mechanism in severe malaria including cerebral malaria. The binding is mediated by PfEMP1-DBL2β domain. The study aimed to explore there combinant protein of PfEMP1-DBL2β domain of P. falciparum isolated from Indonesia. DNA was isolated from a severe malaria patient. The DBL2β domain was amplified using Polymerase Chain Reaction (PCR) with specific primer and cloned into the pJET1 cloning vector. The DBL2β recombinant protein was constructed from DBL2β-pJET1 clone using pET-30a expression vector and expressed in Escherichia coli BL21-DE3. PCR colony and digestion of plasmid clones using restriction enzymes were conducted to confirm cloning result, and the expression of recombinant protein was analyzed using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The expression of DBL2β-PfEMP1 domain is higher in pellet than in supernatant fraction. In conclusion, the DBL2β-PfEMP1 domain recombinant protein of P. falciparum isolated from Indonesia expressed as a ~66 kDa protein in full length. Keywords: DBL2β domain, Indonesia, PfEMP1, Plasmodium falciparum, recombinant protein.
Co-Authors Agus Heri Setyo Wahyudi Agustien, Inyana Dwi Bambang Sugiharto Erlia Narulita Erma Sulistyaningsih Fathul Hidayatul Hasanah, Fathul Hidayatul Fibriani, Suwinda Intan Ria Neliana Iryani Endah Febrianti Natalia Tri Astuti Nurmalasari Darsono Nurmalasari Darsono Putri Andreyna Saragi Retnosari Apriasti Sri Puji Astuti Sugiharto Suvia Widyaningrum Victorin Mega Putri Win Darmanto Yayuk Fatmawati