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Konstruksi Plasmid Pengekspresi Antigen Rekombinan HCV Berbasis Multiepitop untuk Deteksi Antibodi Anti-HCV Dian Amirulloh; Silvia Tri Widyaningtyas; Budiman Bela
Media Penelitian dan Pengembangan Kesehatan Vol 28 No 3 (2018)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/mpk.v28i3.39

AbstractHepatitis C virus (HCV) infection can cause chronic liver disease that develops into cirrhosis and liver cancer. It is estimated that are more than 170 million of th world’s population suffering from HCV. Accurate diagnosis is needed to provide appropriate early treatmen, including preventing further transmission of the virus. The purpose of this study was to construct plasmid expression of recombinant antigen for detection of anti-HCV antibodies. The antigen coding gene is designed so that is composed to epitopes that are immunodominant, sustainable and and represent HCV subtypes circulating in Indonesia and globally. Furthermore, the gene was made by synthetic DNA techniques by DNA synthesis service providers and accepted by the researchers in the form of blinding on the PUC57 plasmid to pQE80L plasmid with BamHI and HindIII cloning sites. Subcloned recombinant plasmids were then propagated on Top10 Escherichia coli cells and verified by PCR colony tecnique, restriction, and sequencing analysis. HCV recombinant antigen coding gene is 1200 bp. Cloning of these gene on the PUC57 vector produced a plasmid pUC57-HCV_ME (3910 bp) and subcloned in the pQE80L vector producing pQE80L-HCV_ME plasmid (5909bp). Based on verification results of pQE80L-HCV_ME plasmid the expression of recombinant antigen for detection of anti-HCV antibodies has been successfully constructed. AbstrakInfeksi hepatitis C virus (HCV) dapat menyebabkan penyakit hati kronis yang berkembang menjadi sirosis dan kanker hati. Diperkirakan terdapat lebih dari 170 juta penduduk dunia menderita HCV. Diagnosis yang akurat diperlukan untuk memberikan penanganan tepat secara dini, termasuk mencegah penularan virus tersebut lebih lanjut. Tujuan penelitian ini adalah mengonstruksi plasmid pengekspresi antigen rekombinan untuk deteksi antibodi anti-HCV. Gen pengode antigen tersebut dirancang sedemikian rupa sehingga tersusun atas epitop yang bersifat imunodominan, lestari, serta mewakili subtipe HCV yang bersirkulasi di Indonesia maupun global. Selanjutnya gen tersebut dibuat dengan teknik DNA sintetik oleh perusahaan penyedia jasa sintesis DNA dan diterima oleh peneliti dalam bentuk terklona pada plasmid pUC57. Untuk ekspresi pada sel Escherichia coli, gen penyandi antigen rekombinan disubklona dari plasmid pUC57 ke plasmid pQE80L dengan situs pengklonaan BamHI dan HindIII. Plasmid rekombinan hasil subklona kemudian dipropagasi pada sel Escherichia coli Top10 dan diverifikasi dengan teknik PCR koloni, analisis dengan enzim restriksi dan sekuensing. Gen penyandi antigen rekombinan HCV berbasis epitop multipel (HCV_ME) berukuran 1200 pb. Pengklonaan gen tersebut pada vektor pUC57 menghasilkan plasmid pUC57-HCV_ME (3910 pb) dan subklona pada vektor pQE80L menghasilkan plasmid pQE80L-HCV_ME (5909 pb). Berdasarkan pada hasil verifikasi plasmid pQE80L-HCV_ME pengekspresi antigen rekombinan untuk deteksi antibodi anti-HCV telah berhasil dikonstruksi.

Konstruksi Plasmid Rekombinan untuk Inisiasi Translasi Enhance Green Fluorescent Protein oleh Internal Ribosomal Entry Site HIV-1 Cintera Rahmagiarti; Silvia Tri Widyaningtyas; Budiman Bela
Media Penelitian dan Pengembangan Kesehatan Vol 28 No 2 (2018)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/mpk.v28i2.181

Human immunodeficiency virus (HIV) is a virus that causes acquired immunodeficiency virus syndrome (AIDS). The HIV genome has a cap structure at 5’ and polyadenylation at 3’ on mRNA resulting in a translation initiation through scanning at 5'untranslated region (UTR). The Vpr protein produced during viral replication causes the 5'cap scanning to be inhibited so HIV-1 can directly recruit the ribosome at the start codon via internal ribosomal entry site (IRES). IRES activity is high at G2/M phase and highest expression in monocyte cell line (THP-1) and lymphocyte (HPB-ALL). The role of HIV IRES however, is not yet known in infection of nondividing cells by HIV-1. HIV-1 IRES and egfp from pcDNA5FRT/TO were amplified with PCR. The insert DNA (HIV-1 IRES_egfp) and pcDNA3.1(+) were digested with EcoRI and ApaI and then ligated. The verification was performed with PCR colonies, restriction verification, and sequencing. The size of insert DNA is 1067 bp while the vector is 5379 bp. E. coli transformed with DNA ligation produces 70 colonies, control of ligation produces 5 colonies, and negative control didn’t grow. 19 colonies contain recombinant DNA, restriction verification was of the appropriate size, and the sequence verification didn’t find any mutation. Therefore, the subcloning process pcDNA3.1_IRES HIV-1_egfp was successfully performed. Abstrak Human immunodeficiency virus (HIV) merupakan virus penyebab acquired immunodeficiency virus syndrome (AIDS). Genom HIV memiliki struktur cap di 5’ dan poliadenilasi di 3’ mRNA sehingga proses inisiasi translasi melalui pemindaian 5’cap pada struktur untranslated region (UTR) di 5’ mRNA HIV. Protein Vpr yang dihasilkan selama replikasi virus menyebabkan pemindaian melalui 5’cap terhambat sehingga HIV-1 dapat langsung merekrut ribosom pada kodon awal translasi melalui struktur internal ribosomal entry site (IRES). Aktivitas IRES tinggi pada fase G2/M dan ekspresi gen tinggi pada sel line monosit (THP-1) dan limfosit (HPB-ALL). Namun, peran IRES HIV-1 belum diketahui pada sel tidak membelah yang merupakan sel target pada infeksi HIV-1. DNA sekuen IRES HIV-1 dan egfp dari pcDNA5FRT/TO diamplifikasi dengan PCR. DNA sisipan (IRES HIV-1_egfp) dan pcDNA3.1(+) dipotong dengan EcoRI dan ApaI lalu DNA sisipan diligasi dengan pcDNA3.1(+). Verifikasi hasil klona dilakukan dengan PCR koloni, verifikasi restriksi, dan sekuensing. Restriksi DNA sisipan menghasilkan pita berukuran 1067 pb. Restriksi vektor plasmid menghasilkan pita berukuran 5379 pb. E.coli yang ditransformasi DNA ligasi menghasilkan 70 koloni, kontrol ligasi 5 koloni, dan kontrol negatif tidak tumbuh. 19 koloni terverifikasi mengandung DNA rekombinan, verifikasi restriksi memiliki ukuran sesuai, dan verifikasi sekuensing tidak terdapat perubahan basa. Oleh karena itu, proses subkloning pcDNA3.1_IRES HIV-1_egfp berhasil dilakukan.

Konstruksi Plasmid Pengekspresi Antigen Gag dan Protein Penghantar VP22 untuk Pengembangan Vaksin HIV-1 Melinda Remelia; Budiman Bela; Silvia Tri Widyaningtyas; Fera Ibrahim
Media Penelitian dan Pengembangan Kesehatan Vol 31 No 2 (2021)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/mpk.v31i2.3046

The endogenous HIV-1 vaccine based on Gag protein is expected to stimulate the immune response of CD8+ T cells (cytotoxic). The Gag protein that has been produced by the E.coli prokaryote system is an exogenous antigen. The fusion of VP22 protein is expected to deliver Gag antigen into the cytoplasm of cell, observed by eGFP markers. Sequences of VP22 (114 pb), GagHIV-1 (1506 pb), and eGFP (733 pb) were inserted into the pQE80L, respectively. The recombinant protein was expressed in the E.coli system and purified by the Ni-NTA method. Antigen delivery fused with VP22 and eGFP was observed with fluorescence and confocal microscopy. The recombinant plasmid constructs of protein expression eGFP, VP22-eGFP, GagHIV-1-eGFP, VP22-GagHIV-1-eGFP were verified by DNA sequencing according to the reference. The recombinant plasmid constructs of Gag HIV-1-eGFP and VP22-GagHIV-1-eGFP still need to be optimized so they can be expressed in the E.coli system. The recombinant protein VP22-eGFP (27.02 kDa) was succesfully obtained and fluorescent green (entered) into the cytoplasm and nucleus of vero cells. In addition to the HIV-1 vaccine, this recombinant plasmids pQE80L-eGFP and pQE80L-VP22-eGFP also have the potential to be used as tools in the development of endogenous vaccines for another viruses/microbes. Abstrak Vaksin endogen HIV-1 berbasis protein Gag diharapkan dapat menstimulus respons imun sel T CD8+ (sitotoksik). Protein Gag yang telah diproduksi dengan sistem prokariota E.coli merupakan antigen yang bersifat eksogen. Fusi protein VP22 diharapkan mampumenghantarkan antigen Gag masuk ke sitoplasma sel, diamati dengan marker eGFP. Sekuens VP22 (114 pb), GagHIV1 (1506 pb), dan eGFP (733 pb) telah diinsersikan pada vektor pQE80L. Protein rekombinan diekspresikan pada sistem E.coli dan dipurifikasi dengan metode Ni-NTA. Penghantaran antigen yang difusikan dengan VP22 dan marker eGFP diamati dengan mikroskop fluoresens dan konfokal. Konstruksi plasmid rekombinan pengekspresi protein eGFP, VP22-eGFP, GagHIV1-eGFP, dan VP22-GagHIV1-eGFP telah diverifikasi dengan sekuensing DNA sesuai dengan sekuen referensi. Plasmid rekombinan pengekspresi GagHIV1-eGFP dan VP22-GagHIV1-eGFP masih perlu dioptimasi agar dapat diekspresikan di sistem E.coli. Protein rekombinan VP22-eGFP (27,02 kDa) telah berhasil diperoleh serta berpendar fluoresens hijau (masuk) ke sitoplasma dan nukleus sel vero. Selain vaksin HIV-1, plasmid rekombinan pQE80L-eGFP dan pQE80L-VP22-eGFP juga berpotensi dapat digunakan sebagai ‘tools’ dalam pengembangan vaksin endogen dari virus atau mikroba lainnya.

Cloning and expression of Human Papilloma virus type 16 L1 capsid protein in bacteria Silvia Tri Widyaningtyas; Sofy Meilany; Budiman Bela
Health Science Journal of Indonesia Vol 10 No 2 (2019)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/hsji.v12i2.2442

Latar belakang: Secara alamiah protein kapsid L1 Human Papillomavirus (HPV) tipe 16 dapat mengalami auto assembly untuk membentuk Viral like particle (VLP). Terkait dengan penelitian vaksin HPV, VLP dapat digunakan untuk berbagai keperluan seperti vaksin, pseudovirion atau SpyTag-Spycatcher. Penelitian ini ditujukan untuk mendapatkan plasmid rekombinan yang digunakan untuk produksi protein L1 HPV 16. Metode: Gen penyandi protein L1 HPV 16 diklona ke dalam vector pQE80L, suatu plasmid yang mengandung sistem ekspresi untuk prokariota. DNA penyandi HPV 16 L1 disisipkan pada situs restriksi BamHI dan Hind III plasmid pQE80L. Plasmid rekombinan yang mengandung gen L1 HPV 16dikonfirmasi menggunakan PCR dan analisis enzim restriksi. Lebih lanjut untuk memastikan bahwa gen rekombinan L1 HPV 16 dapat diekspresikan dalam prokariota, plasmid rekombinan ditransformasikan ke bakteri Escherichia coli BL21 (DE3). Bakteri diinduksi dengan Isopropyl β-D-1-thiogalactopyranoside (IPTG) dengan berbagai konsentrasi dan berbagai waktu inkubasi. Hasil: protein rekombinan L1, berat 56 kDa, telah berhasil diekspresikan dalam sistem prokariota. Protein rekombinan L1 dapat dimurnikan menggunakan TalonR dalam kondisi denaturasi. Kesimpulan: gen L1 HPV 16 telah dikloning ke dalam pQE80L dan berhasil diekspresikan dalam sistem prokariota. (Health Science Journal of Indonesia 2019;10(2):82-9) Kata kunci: L1, HPV 16, cervical cancer Abstract Background: Naturally Human Papillomavirus (HPV) type 16 L1 capsid protein can auto assemble to form Viral like particles (VLP). Concerning to vaccine development for HPV, VLP can be used for a variety of needs such as a vaccine, pseudovirion or SpyTag-Spycatcher. In this study, to obtain a vector expression that can be used in the production of HPV L1 protein, we cloned gene coding HPV 16 L1 protein into pQE80L a plasmid contains an expression system for prokaryote. Methods: The DNA coding HPV 16 L1 was inserted at BamHI and Hind III restriction sites of pQE80L plasmid. The recombinant plasmid containing the HPV L1 gene was confirmed using PCR colony and enzyme restriction. Further to ensure the recombinant HPV 16 L1 gene could be expressed in a prokaryote, the recombinant plasmid was transformed into bacteria Escherichia coli BL21 (DE3). The bacteria were induced with IPTG with various concentrations and various incubation time. Result: L1 recombinant protein, 56 kDa in weight, has successfully been expressed in prokaryote system. L1 recombinant protein can be purified using TalonR under denaturing conditions. Conclusion: L1 HPV 16 gene has been cloned into pQE80L and successfully expressed in prokaryote system. (Health Science Journal of Indonesia 2019;10(2):82-9) Keywords: L1, HPV 16, cervical cancer

Development of Recombinant Immunoblot Assay Diagnostic Test Based on HIV-1 in Indonesia Jeanne Elvia Christian; Silvia Tri Widyaningtyas; Budiman Bela
Molecular and Cellular Biomedical Sciences Vol 6, No 2 (2022)
Publisher : Cell and BioPharmaceutical Institute

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21705/mcbs.v6i2.251

Background: High mutation rates in HIV-1 could affect the accuracy of diagnostic tests. Therefore, recombinant antigen that has an immunodominant and conserved region from HIV-1 need to be developed to detect HIV-1 infection in Indonesia.Materials and methods: The recombinant antigens comprise of Gag (p24), Pol and Env (gp41). Each antigens was expressed in the Escherichia coli expression system and purified using Ni-NTA chromatography. The reactivity of purified antigen against HIV antibodies was tested against a group of 50 HIV-positive plasma samples and 45 HIV-negative plasma samples in a recombinant immunoblot assay (RIBA) platform test. Moreover, 21 of 50 HIV-positive samples and 3 of 45 HIV-negative samples were also tested using HIV blot 2.2 to compare RIBA with a commercial western blot kit. Ten HBV-positive and 10 HCV-positive plasma samples were used to check cross-reactivity with HIV recombinant proteins in RIBA.Results: All HIV-positive samples (100%) tested with RIBA were reactive towards Gag (p24), Pol, Env (gp41). Otherwise, 3 of 21 HIV-positive samples assayed with HIV blot 2.2 were not reactive to Pol protein. All HIV-negative samples tested with RIBA and 3 HIV-negative samples tested with HIV blot 2.2 did not produce any bands of HIV antigens. Few HBV and HCV samples showed reactivity towards HIV recombinant proteins.Conclusion: Each recombinant protein, Gag (p24), Pol, Env (gp41), could be expressed and purified, as well as had reactivity to HIV-positive samples in RIBA test. Therefore, RIBA can be used as a diagnostic test to detect HIV-1 infection in Indonesia.Keywords: diagnostic, HIV-1, immunodominant, recombinant immunoblot assay (RIBA)

Computational Analysis of Chromophore Tripeptides FollowingFusion of Enhanced Green Fluorescent Protein and Cell-penetrating Peptides Widyaningtyas, Silvia Tri; Pratiwi, Ekawati Betty; Bela, Budiman
Makara Journal of Science Vol. 24, No. 4
Publisher : UI Scholars Hub

Show Abstract | Download Original | Original Source | Check in Google Scholar

Cell-penetrating peptides (CPPs) are small peptides that can transfer other materials into a cellular compartment. In this research, we studied the effect of fusion of new CPPs to the N-terminal of enhanced Green Fluorescent Protein eGFP on the ability of the latter to fluoresce. Results showed that the recombinant protein CPPs-eGFP could be successfully expressed in Escherichia coli. In contrast to E. coli expressing wild-type eGFP, which could fluoresce under ultraviolet (UV) or visible light, E. coli expressing CPPs-eGFP lost their ability to fluoresce. PyMol, a molecular visualization system, revealed that fusion of the new CPPs to the N-terminal of eGFP alters interactions between chromophore-forming tripeptides and the adjacent amino acids of other tripeptides. Disrupting peptide interactions induced structural changes in eGFP that caused it to lose its fluorescence ability. We suggest performing computational analyses to predict the biological function of new fusion proteins prior to starting laboratory work.

The Use of Cell-penetrating Peptide for Delivery of Recombinant Transcription Factor DNA into Primary Human Fibroblast Melinda Remelia; Budiman Bela; Silvia Tri Widyaningtyas; Radiana Dhewayani Antarianto; Nuzli Fahdia Mazfufah; Jeanne Adiwinata Pawitan
Molecular and Cellular Biomedical Sciences Vol 7, No 1 (2023)
Publisher : Cell and BioPharmaceutical Institute

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21705/mcbs.v7i1.279

Background: Reprogrammed cell therapy has not been applied for clinical purposes due to the malignancy issue. The aim of this study was to design the recombinant vector of the transcription factors and analyze the effectiveness of cell-penetrating peptide delivering system for human primary fibroblast transfection to avoid the malignancy issue.Materials and methods: The constructions of CCAT/enhancer binding protein alpha (CEBPA), hepatocyte nuclear factor 4 alpha (HNF4A), nuclear receptor subfamily 1 group I member 2 (NR1I2) were confirmed with DNA digestion and sequencing. Breast reduction (BRED) and palate (PAL) tissue were used as human primary fibroblast sources. The transcription factors were delivered into BRED and PAL with recombination of avian leukosis sarcoma virus (ALSV), human immunodeficiency virus (HIV) matrix, and regulator of expression of virion proteins (Rev) (ALMR), tagged with enhanced green fluorescence protein (eGFP). Post-transfection cells were then cultivated with optimized medium. Gene expression was measured with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results: Gene expression levels of CEBPA, HNF4A, NR1I2, glutamate-ammonia ligase (GLUL), albumin (ALB), and cytochrome P450 (CYP) were increased. Transfection with ALMR, which were more efficient in BRED than PAL fibroblasts may have the advantage in autologous cell therapy for elderly patients.Conclusion: Transfection of transcription factors to human primary fibroblast may be performed by using constructions of plasmid as designed in this study.Keywords: recombinant plasmid, hepatocyte-like cells, primary fibroblasts, recombinant peptide, cell reprogramming, autologous cells therapy

Pemilihan Peptida Immunogenik E6 Human Papiloma Virus 16 sebagai Kandidat Vaksin kuratif dengan menggunakan Next-Generation IEDB Tools Widyaningtyas, Silvia Tri; Pratiwi, Ekawati Betty; Bela, Budiman
InaBHS (Indonesian Journal of Biomedicine and Health Science) Vol 3 No 2 (2024): Indonesian Journal of Biomedicine and Health Science
Publisher : Fakultas Kedokteran UGJ Cirebon

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.33603/inabhs.v3i2.10519

ABSTRAK Kanker serviks atau kanker mulut rahim merupakan salah satu penyebab kematian tinggi sampai saat ini. Kanker ini dikaitkan dengan infeksi Human Pappiloma virus (HPV). Vaksinasi merupakan salah satu metode pencegahan yang cukup efektif untuk mencegah kanker serviks. Vaksin yang beredar saat ini efektif untuk mencegah infeksi HPV, namun kurang efektif jika diberikan pada orang yang telah terinfeksi. Hal tersebut dikaitkan dengan jalur kekebalan tubuh yang diinduksi oleh vaksin tersebut. Dalam penelitian ini dirancang antigen HPV 16 yang dapat digunakan dalam pengembangan vaksin terapetik yang ditujukan untuk meningkatkan respon kekebalan seluler yand secara spesifik dapat menghancurkan sel kanker servik. Pemilihan dan karakterisasi antigen dilakukan dengan menerapkan Bioinformatika. Parameter yang dinilai antara lain kemampuan antigen diproses dan dipresentasikan oleh sel tubuh pada sel kekebalan tubuh yang bertanggung jawab pada eliminasi sel kanker, yaitu sel CD8. Hasil studi menunjukkan diperoleh epitop CD8 yang dikenali oleh beberapa jenis HLA. Bioinformatika dapat digunakan untuk mencari epitop.

Mengatasi Tantangan Skrining HPV pada Perempuan: Sebuah Kajian Umum Budiman Bela; Soni Hartono; Ekawati Betty Pratiwi; Hesti Pramesti Cahyani; Silvia Tri Widyaningtyas
Majalah Kedokteran Indonesia Vol 75 No 2 (2025): Journal of The Indonesian Medical Association - Majalah Kedokteran Indonesia, Vo
Publisher : PENGURUS BESAR IKATAN DOKTER INDONESIA (PB IDI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.47830/jinma-vol.75.2-2025-1898

Introduction: A major challenge in the success of HPV screening programs is achieving 70% coverage of women aged 30-69. This study conducted a survey to examine the factors influencing the willingness of woman in that age group to actively engage HPV screening program.Method: Socio-demographic data, knowledge levels, cervical cancer screening uptake, and willingness to participate in the government’s routine screening program were collected digitally using cross-sectional study. Factors associated with knowledge, participation, and willingness to engage program measured using logistic regression analysis.Result: Among the 87 eligible respondents, 96.6% had heard about cervical cancer mostly via internet, and 51% categorized as having low level knowledge. The percentage of participants willingness to participate in program was 67.82% below the percentage government target coverage. Factors correlated with willingness included: age group 36-40 (aOR = 1.865, 95% CI = 0.567-6.129) and 41-45 (aOR = 2.93, 95% CI = 0.762-11.272), monthly household expense (aOR = 1.682, 95% CI = 0.684-4.138), having relatives suffer cervical cancer (aOR = 2.764, 95% CI = 0.790-9.672), history of HPV vaccination (aOR = 1.187, 95% CI = 0.593-7.539).Conclusion: Respondent willingness to participate in HPV screening program remains to be improved. Various ways to increase participation in screening programs need to be developed to reach various populations of women.

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