Path: TopS2-ThesesChemistry-FMIPA2012

ISOLASI DAN KLONING GEN PENGKODE PENGATUR RESPON TRANSKRIPSI mtrA Mycobacterium tuberculosis

ISOLATION AND CLONING OF Mycobacterium transcriptional RESPONSE REGULATOR-A (MTRA) GENE FROM Mycobacterium tuberculosis

Master Theses from JBPTITBPP / 2017-09-27 15:39:37
Oleh : HENDRIKUS MASANG BAN BOLLY (NIM : 20510002); Pembimbing I : Dessy Natalia, Ph.D ; Pembimbing II : Dr. Ihsanawati, S2 - Chemistry
Dibuat : 2012, dengan 7 file

Keyword : MtrA, Mycobacterium tuberculosis, makrofag dan proteasom.

Tuberkulosis (TBC) masih merupakan masalah kesehatan global yang disebabkan oleh infeksi Mycobacterium tuberculosis. Insidensi, morbiditas dan mortalitas infeksi TBC di seluruh dunia dan di Indonesia masih cukup tinggi. Sepanjang








tahun 2010 tercatat 9 juta kasus baru dan 2 juta kematian karena infeksi TBC. Tingginya kasus infeksi TBC disebabkan oleh infeksi penyerta Human Immunodefiency Virus, resistensi obat, serta karakteristik alamiah M. tuberculosis. Infeksi TBC terjadi setelah bakteri masuk ke dalam tubuh melalui udara pernapasan dan bertahan hidup di dalam makrofag paru-paru sel inang. Makrofag yang teraktivasi akan menghasilkan berbagai senyawa oksidatif (ROS, Reactive Oxygen Species) dan nitrosatif (RNI, Reactive Nitric Intermediate), yang memiliki efek bakterisidal. Perubahan kondisi lingkungan intraseluler makrofag ini menyebabkan M. tuberculosis melakukan pemrograman ekspresi gen agar tetap bertahan hidup. Selain itu, aktivitas proteasom yang dimiliki oleh M. tuberculosis mampu melawan efek bakterisidal ROS dan RNI. MtrA adalah protein yang berperan penting dalam patogenesis infeksi tuberkulosis pada manusia. MtrA berfungsi dalam pengaktifan transkripsi, regulasi proliferasi, multiplikasi sel, penghambat fusi fagosom-lisosom di dalam makrofag sel inang dan sebagai salah satu substrat sistem degradasi proteasom M. tuberculosis. Pengetahuan mengenai peran MtrA dalam sistem degradasi protein oleh proteasom akan memberi pemahaman baru patogenesis TBC pada manusia. Penelitian ini bertujuan untuk mengisolasi dan mengkloning gen pengode MtrA dari M. tuberculosis galur murni H37Rv, isolat klinis Y M. tuberculosis yang diambil dari cairan sum-sum tulang belakang (LCS, liquour cerebrospinalis pasien penderita meningitis tuberkulosis serta isolat klinis R26 dan R27 from M. tuberculosis yang Multi Drug Resistance (MDR). Gen pengode MtrA dari keempat isolat tersebut telah berhasil diisolasi dengan metode Polymerase Chain Reaction (PCR)








menggunakan primer maju HMTAR-F dan primer mundur HMTAR-R, yang dirancang berdasarkan urutan nukleotida mtrA M. tuberculosis H37Rv (Genebank Accession number U01971.1). Fragmen gen mtrA isolat galur murni H37Rv dan isolat klinis Y berukuran 687 pb telah berhasil diklon ke vektor pGEM-T.








Pemotongan gen mtrA pada vektor pGEM-T menggunakan enzim restriksi XbaI dan XhoI tidak berhasil sehingga subkloning ke dalam vektor ekspresi belum dilakukan. Hal ini diduga karena adanya metilasi pada urutan nukleotida GATC, sisi restriksi XbaI. Urutan nukleotida gen mtrA M. tuberculosis galur murni H37Rv dan isolat klinis Y (687 pb) mengode 228 asam amino. Urutan nukleotida isolat R26 dan R27 dari hasil PCR adalah masing-masing 680 dan 682 pb.








Analisis urutan nukleotida gen mtrA keempat sampel tersebut menunjukkan tingkat kemiripan 100% dengan gen mtrA M. tuberculosis galur H37Rv pada Genebank. Pada gen mtrA isolat Y ditemukan mutasi bisu C261T. Mutasi ini tidak








menyebabkan perubahan asam amino Lys-85. Adapun urutan nukelotida gen mtrA isolat R26 dan R27 sebagian besar tidak menunjukkan adanya perubahan nukelotida. Hasil ini berimplikasi pada protein MtrA yang tetap aktif meregulasi








ekspresi gen-gen M. tuberculosis yang MDR maupun M. tuberculosis pada infeksi meningitis TBC.

Deskripsi Alternatif :

Tuberculosis (TB) remains a global health problem caused by Mycobacterium tuberculosis infection. Incidence, morbidity and mortality of TB infection in the world and in Indonesia are still high. There are 9 million of new cases with








almost 2 million mortality of TB around the world. Human Immunodefiency Virus infection, drug resistance, late diagnosis, and the natural characteristics of bacteria increase the number of M. tuberculosis cases. The infection occurs when some bacil are inhaled through aerosol and survive in the host alveolar macrophages. Activated alveolar macrophage will release a variety of oxidative








compounds, ROS (Reactive Oxygen Species) and nitrosative compounds (RNI, Reactive Nitric Intermediate), which have bactericidal effects against intracellular M. tuberculosis. In order to survive in the host macrophages, Mtb is able to








reprogram its gene expression. Moreover, M. tuberculosis has a proteasome activity, which is resistable to bactericidal effects of ROS and RNI. MtrA is a protein that plays an important role in the pathogenesis of tuberculosis in humans. MtrA has major roles in transcription activation, proliferation regulation, cell multiplication, inhibiting phagosome-lysosome fusion and as a substrate of M. tuberculosis proteasome. Understanding the role of MtrA in the protein degradation system of M. tuberculosis proteasome will provide clear understanding of tuberculosis pathogenesis in human. This study was aimed to isolate and clone the gene encoding MtrA from M. tuberculosis strains H37Rv and clinical isolates, Y, which was obtained from liquour cerebrospinalis of patients with meningitis tuberculosis,; and two isolates, R26 and R27, which were








MDR. Genes encoding MtrA of all isolates were amplified successfully by the Polymerase Chain Reaction (PCR) method using a forward primer HMTAR-F and a reverse primer HMTAR-R. Both primers were designed based on the nucleotide








sequence of M. tuberculosis mtrA (Genebank Accession number U01971.1). Gene fragments of mtrA (0,687 kb) from H37Rv strain and Y clinical isolate had been cloned into the vector pGEM-T and digested by XhoI and XbaI. However, we








could not obtain the fragment of mtrA gene from the vector due to DNA methylation of GATC sequences on XbaI restriction site. The sequencing results of the mtrA gene from H37Rv and Y clinical isolate showed 687 bp encoding 228 amino acids. Furthermore, the mtrA gene sequence from R26 and R27 isolate showed 680 bp and 682 bp, respectively. Sequence homology analysis from all samples showed 100% homology with that of mtrA in the Genebank database.








There was a silent mutation C261T, which was not change Lys-85 from the Y clinical isolate and there was no mutation on the mtrA gene from the R26 and R27 clinical isolates. These results implied that the MtrA protein is still active in regulating genes expression of MDR M. tuberculosis and meningococal tuberculosis.

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  • Pembimbing I : Dessy Natalia, Ph.D ; Pembimbing II : Dr. Ihsanawati, Editor: Alice Diniarti

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