Karakteristik Molekular dan Imunogenisitas Protein Rekombinan Serta Epitop Imunodominan Polymorphic GC-Rich Repetitive Sequences (PE-PGRS) 24 dan 35 M. tuberculosis Isolat Lokal

ANDANI EKA PUTRA, DR., M.SC, Prof.dr. Marsetyawan HNE Soesatyo, M.Sc, Ph.D; Prof. drh. Widya Asmara, SU., Ph.D; dr. Abu Tholib Aman, M.Sc.,PhD.,Sp.MK (K)

2016 | Disertasi | S3 Ilmu Kedokteran

Mycobacterium tuberculosis merupakan bakteri intraselular patogen yang berdiam di dalam makrofag yang merupakan komponen paling penting dalam sistem imun. M. tuberculosis menghasilkan 2 kelompok protein yang sangat polimorfik, yaitu kelompok Proline-Glutamic acid (PE) dan proline-proline-glutamic acid (PPE). Protein kelompok ini merupakan 10% dari total protein M tuberculosis. Protein ini sangat terkait dengan virulensi dan menjadi target pengembangan diagnosis dan terapi. Penelitian ini bertujuan untuk menganalisis karakteristik molekular dan imunogenisitas protein PE, khususnya PE-PGRS 24 dan 35 dari M. tuberculosis isolat lokal. Tahap awal penelitian adalah genotyping M. tuberculosis isolat lokal, sehingga dapat dideferensiasi Strain Beijing dan bukan. Analisis genomik dilakukan terhadap variasi molekular gen PE-PGRS 24 dan 35 berdasarkan hasil sekuensing. Beberapa analisis in silico dilakukan untuk mengetahui lokalisasi protein dan identifikasi epitop imunogenik. Rekombinan protein diproduksi menggunakan teknik Gateway. Dilakukan uji potensi imunogenik dalam bentuk respon antibodi secara Elisa terhadap epitop peptida dan protein rekombinan PE-PGRS 24 dan 35 pada penderita TB dan non TB. Analisis molekular dilakukan terhadap 10 sampel, yang terdiri dari 5 sampel M. tuberculosis Strain Beijing dan 5 non Beijing serta 10 sekuen pembanding. Analisis terhadap PE-PGRS 24 memperlihatkan adanya 0.83% variasi dibandingkan dengan isolat referen sedangkan pada PE-PGRS 35 ditemukan 1.13% variasi Rv1983. Analisis lokalisasi memperlihatkan bahwa PE-PGRS 24 adalah protein sekresi sedangkan PE-PGRS 35 merupakan protein membran. Respon antibodi terlihat berbeda pada epitop peptida 024*A, 035*A dan 035*C antara penderita TB maupun non TB. Kondisi yang sama juga ditemukan antara epitop peptida dengan protein rekombinan. Berdasarkan hasil penelitian ini dapat disimpulkan terdapat variasi molekuler gen PE-PGRS 24 dan 35, PE-PGRS 24 merupakan protein ekstrasel sedangkan PE-PGRS 35 adalah bagian dari protein membrane. Kedua protein dan epitop linearnya bersifat imunogenik dan mampu membedakan TB dengan non TB serta berpotensi dikembangkan sebagai model diagnostik.

Mycobacterium tuberculosis is an intracellular pathogen and it resides inside the macrophage, which is considered to be the most important component of the immune system. M. tuberculosis possesses two highly polymorphic sets of genes called the Proline-Glutamic acid (PE) dan proline-proline-glutamic acid (PPE) families. These unique families of proteins account for about 10% of the Mycobacterial genome. PE and PPE proteins are closely related to bacterial virulence and and become the target of the development of diagnostics and vaccines. This study aims to assess the characteristics genomic and proteomic of PE family proteins, especially PE- PGRS 24 and 35. At the first stage this research, we have done genotyping analysis from M. tuberculosis, so we can differentiate Beijing and non Beijing strain. Molecular analyzes have performed on molecular variation PE-PGRS 24 and 35 genes based on the sequencing results. Some insilico analysis have conducted to determine protein sublocalization and identification of immunogenic epitopes. Recombinant proteins are produced using Gateway techniques. Immunogenic potency have conducted based on antibody response against recombinant protein epitope peptide and PE-PGRS 24 and 35 in patients with TB and non-TB using Elisa Method. Molecular analysis have performed on 10 samples, consist of 5 Beijing strains and 5 non-Beijing and 10 comparison sequences. Molecular variation on PE-PGRS 24 was 0.83% that was compared with the referent isolates, while on PE-PGRS 35 was 1.13%. Localization analysis have showed that PE-PGRS 24 is a secretion protein while PE-PGRS 35 is a membrane protein. Antibody responses on peptide epitope 024*A, 035*A and 035*C were different between TB with non-TB. The same condition was also found between the peptide epitope with recombinant proteins. Based on these results we can conclude there are molecular variations in PE-PGRS 24 and 35. PE-PGRS 24 is , extracellular protein while PE-PGRS 35 is part of a membrane protein. Both proteins recombinan and immunogenic linear epitopes were immunogenic and able to distinguish TB and non TB an also potent as diagnostic candidate.

Kata Kunci : Tuberkulosis, PGRS protein, Molekular, Imunogenik, epitop