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All Journal Microbiology Indonesia Media Penelitian dan Pengembangan Kesehatan Health Science Journal of Indonesia
Budiman Bela
Department of Microbiology, Medicine Faculty, University of Indonesia, Jakarta 10430
Biochemistry, Genetics & Molecular Biology Decision Sciences, Operations Research & Management Dentistry Health Professions Immunology & microbiology Medicine & Pharmacology Nursing Public Health

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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

Abstract

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.
Construction of plasmids expressing recombinant B cell epitopes of PD1 Sofy Meilany; Andrijono Andrijono; Pauline Phoebe Halim; Budiman Bela
Health Science Journal of Indonesia Vol 10 No 1 (2019)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/hsji.v10i1.1848

Abstract

Latar Belakang: Pengobatan kanker di Indonesia umumnya menggunakan pengobatan dengan kemoterapi atau dengan operasi. Efek samping dari pengobatan ini antara lain adalah kerontokan rambut, mual dan penurunan berat badan. Saat ini sedang berkembang alternatif terapi kanker dengan menggunakan immunoterapi. Kemampuan sel kanker untuk menghindar dari sistem imun disebabkan adanya protein PD-1 pada sel T yang berikatan dengan ligannya PD-L1. Metode: Penelitian ini merupakan penelitian awal yaitu pembuatan rekombinan PQE PD-1 dan menggunakan bagian soluble dari PD-1 yang disebut dengan EP2PD1 yang akan digunakan untuk pembuatan antibodi monoklonal dan sistem pendeteksi antibodi monoklonal. Metode pembuatan rekombinan PD-1 dan EP2PD1 dengan cara penentuan sekuens epitop sel B yang paling imunogenik dilanjutkan dengan amplifikasi sekuen tersebut dengan PCR dan diligasi ke vektor pengekspresi PQE80. Hasil: Telah terbentuk konstruksi rekombinan PQE80 PD-1 dan PQEEP2PD1 yang diverifikasi menggunakan PCR koloni, pemotongan enzimatik dan sekuensing. Hasil penelitian menunjukkan bahwa epitop PD1 telah terklona ke PQE 80 dan tidak ditemukan mutasi dalam urutan asam amino. Kesimpulan: Konstruksi yang dibuat tidak mempunya mutasi dan dapat dilanjutkan untuk pembuatan antibodi monoklonal. Kata Kunci: PD1, Epitop, Kanker, Immunotherapy Abstract Background: Medications on cancer to date in Indonesia is mostly by surgical or chemotherapy, this type of medications is not always curing the patients. The side effect of the chemotherapy drugs sometimes more challenging such as hair loss, nausea and lost weight. One of the promising targets for cancer is using immune therapy. Cancer cells can avoid immune response by surprising immunity through activation of specific inhibitory signalling pathways, referred to as immune checkpoints. Immune check points like PD-1, PD-L1 are breakthrough therapies in oncology and this monoclonal antibody have been approved by the FDA for treatment. In this research we develop full PD-1 and part of PD1 sequence as an insert then we construct with plasmid PQE80L. This recombinant called PQE PD-1 and PQEEP2PD1. The aim of this study is to make recombinant which would be used to detect PD1 full clone monoclonal antibodies. Methods: In this study, we designed our recombinants using Indonesian HLA and others using in silico models, this prototype will not only cover Indonesian patients but also other country. Results: The result showed that the epitope sequence of PD1 has been clone to PQE 80 wt and verified using colony PCR, Enzyme Digestion and Sanger Sequencing. The Clone than will be expressed and injected to animal model to produce antibody. Conclusion: Construction of recombinant PQE PD-1 and PQE EP2PD1 are constructed without any mutation in the sequence, this recombinant can be used in the next study for protein expression of PQE PD-1 and PQE EP2PD1. Keywords: PD1, Epitope , Cancer, Immunotherapy
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

Abstract

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
Cloning and Expression of HA1 Gene of H1N1 Influenza Virus 2009 Pandemic (H1n1pdm09) Indonesia Strain in the Pichia Pastoris Expression System for the Development of Influenza Vaccine ASRI SULFIANTI; ANDI YASMON; BUDIMAN BELA; FERA IBIRAHIM
Microbiology Indonesia Vol. 9 No. 2 (2015): June 2015
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1209.728 KB) | DOI: 10.5454/mi.9.2.7

Abstract

Among influenza viral proteins, hemagglutinin 1 (HA1) is the target for neutralizing antibodies which inhibit virus binding to receptor of target cells. This protein is widely developed as subunit recombinant vaccine. In this research, we expressed HA1 protein recombinant from DKI271/2011 Indonesian strain in Pichia pastoris. The identity of this protein was confirmed by western blotting using anti-His T ag and mouse specific antibody HA H1N1pdm09. The use of yeast P . pastoris as an alternative strategy to solve the problems which commonly found in influenza vaccine productions. Expression protein in E. coli has been known to have many problems, while mammalian and insect cells requires special skills and relatively high cost. The analysis of HA1 gene sequences showed no mutation in epitope region which recognized by T dan B cells. Further, this recombinant protein can be used as vaccine candidate in influenza vaccine development.Keywords: Hemagglutinin; Pichia pastoris; vaccine; Influenza Virus; H1N1pdm09.
Co-Authors Andi Yasmon Andrijono Andrijono ASRI SULFIANTI FERA IBIRAHIM Fera Ibrahim Melinda Remelia Pauline Phoebe Halim Silvia Tri Widyaningtyas Sofy Meilany