Andrijono Andrijono
Obstetric and Gynaecology Department, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia

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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
Human Papilloma Virus L1 Gene Methylation as a Potential Biomarker for Precancerous Cervical Lesion: a Preliminary Report Suzanna P Mongan; Andrijono Andrijono; Hartono Tjahadi
Indonesian Journal of Obstetrics and Gynecology Volume. 5, No. 2, April 2017
Publisher : Indonesian Socety of Obstetrics and Gynecology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (115.068 KB) | DOI: 10.32771/inajog.v5i2.532

Abstract

Objective: To determine whether HPV L1 gene methylation can be used in triage of precancerous cervical lesions. The main objective is to determine the genotype of HPV in cervical precancerous lesions and to determine the percentage, the sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio of DNA HPV L1 methylation in precancerous cervical lesions. Methods: A number of 57 samples of paraffin blocks (FFPE) from precancerous lesions and cervical cancer biopsies in the Department of Pathology Faculty of Medicine-Cipto Mangunkusumo General Hospital that had been re-evaluated by the pathologist, underwent extraction of HPV DNA. The genotypes of HPV DNA were examined using primers GP5 / 6 and specific HPV 16, HPV 18 and HPV 52 probes and analyzed by real time PCR. Sequencing was performed on samples with unknown HPV DNA type that were detected using the specific probes to determine the type of HPV. Bisulfite conversion procedure was then performed for the samples that met the inclusion criteria. Results: There were 30 samples (52.6%) with CIN 1, 12 samples (21.1%) CIN 2, 9 samples (15.8%) CIN 3 and 6 samples (10.5%) of cervical cancer. Most of the samples were 36-45 years (35.1%). Of the total 57 samples, 55 samples were successfully extracted and determined the DNA genotyping of HPV (96.5%). HPV 16 infections both in the form of single or multiple was found to be 76.36%. The samples were mostly dominated by co-infection of HPV16 and 18 (49.1%) followed by HPV 16 (24.6%) and HPV 18 (14.0%). Based on the sequencing results there were other types of high risk HPV infection found: HPV 33, HPV 35, HPV 58 and also undeterminate risk HPV 53 and low risk HPV 54. After several procedures of optimization for methylation examination of HPV DNA L1 there was thin band found in electrophoresis procedure in 8 of 42 samples (19%) of HPV 16 after bisulfite conversion but once it was purified there weren’t any band found so we can not proceed to the stage sequencing. Until now we are still in the stage of optimizing the methylation procedure. Conclusion: HPV 16 infection were most commonly found in the form of single or multiple. Co-infection of HPV 16 and 18 were found in the majority of the samples. There were no significant correlation between HPV type and the severity of cervical lesions. Until now, the examination of DNA methylation HPV L1 already obtained eight samples of HPV 16 with a thin band on electrophoresis but the result could not be concluded because it is still in the process of optimization. [Indones J Obstet Gynecol 2017; 5-2: 120-126] Keywords: HPV DNA genotype, L1 gene methylation, precancerous cervical lesions