Ekspresi enzim rekombinan reverse transcriptase (rtrnase h) simian betaretrovirus serotipe-2 asal macaca fascicularis indonesia Dalam sistem ekspresi eschericia coli

Abstract

Reverse transcriptase (RT) enzyme is a key tool in molecular biology for the synthesis of complementary DNA (cDNA) from messenger RNA (mRNA). Combining RT activity with PCR amplification has been a gold standard as the first step in cloning the coding region of any gene of interest. Evidently, RTs have been critical in advancing molecular biology, genetics and medicine to their current stage. In this study, we were developing the RTRNase H recombinant enzyme isolated from serotype-2 simian betaretrovirus-2 (SRV-2) infected Indonesian Macaca fascicularis using Escherichia coli expression system. The study was conducted using RT SRV-2 gene expression using E. coli expression system, proteins purification, and application to RT PCR technique. The SDS PAGE expression analysis showed a specific band size of 32.7 kDa assumed as RT protein enzyme. Application of RT SRV-2 enzyme generated to the RT PCR technique of β-globin CDV and SRV-2 env gene target showed that the RT SRV-2 enzyme was capable to reverse transcribed mRNA into cDNA as indicated by the presence of specific DNA band compared with commercial RT enzymes. This RT SRV-2 enzyme showed its activity similar to that of commercial one, although the activity was lower.

Enzim reverse transcriptase (RT) merupakan salah satu reagen yang sangat penting dalam bidang biologi molekuler, yaitu untuk mensintesis DNA komplementer (cDNA) dari messenger RNA (mRNA). Kombinasi antara aktivitas RT dan amplifikasi PCR telah menjadi suatu gold standard dalam proses pengklonan daerah pengkode gen dari berbagai target yang diinginkan. Dengan demikian, enzim RT telah menjadi bagian penting dalam perkembangan biologi molekuler, genetik, dan biomedis. Tujuan dari penelitian ini ialah menghasilkan enzim rekombinan RTRNase H simian betaretrovirus serotype-2 (SRV-2) yang diisolasi dari monyet ekor panjang (Macaca fascicularis) asal Indonesia. Tahapan penelitian yang telah dilakukan ialah ekspresi gen penyandi RT SRV-2 menggunakan sistem ekspresi Escherichia coli, pemurnian, analisis hasil ekspresi, dan aplikasi pada teknik RT PCR. Hasil analisis ekspresi protein dengan teknik SDS PAGE menunjukkan ada pita protein spesifik berukuran sekitar 32,7 kDa yang kemungkinan merupakan pita RT SRV-2. Aplikasi enzim RT SRV-2 dalam teknik RT PCR berhasil mentranskripsi balik mRNA pada beberapa target gen, yaitu β-globin, virus CDV, dan env virus SRV-2. Hal ini menunjukkan bahwa enzim rekombinan RT SRV-2 berhasil dikembangkan dan menunjukkan aktivitas walaupun masih rendah dibandingkan enzim komersial.