KLONING GEN PENYANDI 10 kDa CULTURE FILTRATE PROTEIN (CFP-10) Mycobacterium tuberculosis

FRANCISCA ROMANA S.S, Prof. Dr. drh. Wayan Tunas Artama

2015 | Tesis | S2 Ilmu Kedokteran Tropis

Pendahuluan: CFP-10 merupakan antigen dominan di wilayah RD1 lokus genom M. tuberculosis dan tidak ditemukan pada M. bovis strain vaksin BCG. CFP-10 disekresikan pada tahap awal infeksi, diduga berhubungan dengan patogenesis dan penentu virulensi. Tujuan: Penelitian ini bertujuan melakukan kloning gen penyandi antigen CFP-10 M. tuberculosis untuk mendapatkan plasmid rekombinan sebagai dasar penelitian selanjutnya guna pengembangan alat uji diagnostik molekuler TB. Metode Penelitian: Isolat M. tuberculosis dipergunakan sebagai template untuk mengamplifikasi gen penyandi CFP-10 dengan PCR. Produk PCR diligasi ke dalam vektor pET SUMO kemudian ditransformasi ke bakteri E. coli BL21 (DE3). Hasil transformasi dikultur pada media Luria Bertani (LB) agar yang mengandung kanamisin 50 ug/ml, pada suhu 37ËšC overnight. Koloni tunggal hasil kultur dipilih kemudian ditumbuhkan dalam media LB cair pada shaker incubator, suhu 37ËšC overnight. Analisis DNA insert dilakukan dengan PCR menggunakan primer spesifik CFP-10 dan sekuensing menggunakan primer SUMO forward dan T7 reverse untuk mengkonfirmasi urutan dan orientasi nukleotida terletak pada frame yang benar. Hasil Penelitian: Hasil PCR menunjukkan single fragmen berukuran 304 bp. Produk PCR berhasil diligasi dan ditransformasi ke bakteri E. coli BL21. Transforman berhasil dikultur dan plasmid rekombinan berhasil diisolasi. Analisis plasmid rekombinan menunjukkan adanya klon positif membawa insert. Hasil sekuensing plasmid dan insert menunjukkan urutan nukleotida 100% homolog sesuai dengan Genbank dan orientasi DNA insert berada pada frame yang benar. Kesimpulan: Berhasil diklon gen penyandi antigen CFP-10 M. tuberculosis, sebagai dasar penelitian selanjutnya untuk pengembangan alat uji diagnostik molekuler Tuberkulosis.

Introduction: The CFP-10 was a dominant antigen in the RD1 locus genome of M. tuberculosis and was not found in M. bovis vaccine strain. The CFP-10 was secreted in early stages of infection, allegedly associated with pathogenesis and virulence determinants. Objective: The purpose of this study was to cloned the gene encoding for CFP-10 antigen of M. tuberculosis to obtain recombinant plasmid for based the further studies to development of molecular diagnostic kit for TB. Methods: M. tuberculosis isolates from TB patients were used as a template. The encoding gene for CFP-10 was amplified by PCR. The PCR product was ligated into the pET SUMO vector and then transformed into E. coli BL21 (DE3). Transformant were cultured on agar plate Luria Bertani (LB) medium containing kanamycin 50 ug/ml, at 37ºC overnight. A single colonies were selected and cultured in liquid LB medium in shaker incubator, at 37ºC overnight. The analysis of insert performed by PCR using CFP-10 specific primers and sequencing using SUMO forward and T7 reverse primer to confirm the order and orientation of the nucleotide located in the correct frame. Result: The PCR product obtained sigle fragment size of 304 bp. The PCR product has ligated and transformed into E. coli BL21. The transformants successfully were cultured and recombinant plasmid was isolated. The analytical of recombinant plasmid showed positive clone insert carrier. The sequencing analyzed showed 100% homologous with nucleotide sequence according to Genbank and orientation of insert is in the correct frame. Conclusion: It has been successfully cloned the gene encoding for CFP-10 antigens of M. tuberculosis, for based the further studies to development of molecular diagnostic kit for Tuberculosis.

Kata Kunci : Cloning, gene encoding for CFP-10 antigen, M. tuberculosis