Cloning and Expression of Serotype-2 Simian Betaretrovirus Reverse Transcriptase Gene Isolated from Indonesian Cynomolgus Monkey in Escherichia coli

UUS SAEPULOH (PRC IPB)
DIAH ISKANDRIATI
FUNGKEY HOETAMA
SELA SEPTIMA MARIYA
DEDY DURYADI SOLIHIN
JOKO PAMUNGKAS
DONDIN SAJUTHI

Microbiology Indonesia
Vol. 7 No. 2 (2013): June 2013

In this study, we isolated the simian betaretrovirus serotype-2 (SRV-2) reverse transcriptase (RT) gene from infected Indonesian cynomolgus monkey (Macaca fascicularis). The gene was then cloned in Escherichia coli expression system. The SRV-2 RT gene is located between nucleotides 3284-4925 in the polyprotein (Pol) region encodes 547 amino acids. Analysis of expression using SDS-PAGE and western blot techniques showed a specific band of 64.9 kDa, indicating SRV-2 RT recombinant enzyme. Purification of SRV-2 RT recombinant enzyme produced 312 μg mL-1 protein with 7.1 U μL-1 enzyme activities. Application of this recombinant enzyme in reverse transcription-PCR (RT-PCR) of β-globin and β-actin genes produced DNA fragments of 206 and 350 bp, indicating amplification of β-globin and β-actin genes, respectively. Therefore, the expressed SRV-2 RT enzyme was proven to be functional, although the activity was low.