Isolasi Konstruksi dan Rekombinasi Gen Pengkode Reverse Transcriptase asal Simian Retrovirus-2 (SRV-2)

Abstract

The availability of reverse transcriptase enzym as a valuable component for Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique has been providing by foreign companies. Therefore, it needs more cost and time to provide it. This study is an early step to produce in house reverse transcriptase rekombinant enzym from SRV-2. The purpose of this study was isolated, constructed, and rekombined SRV-2 reverse transcriptase gene to entry clone and destination vector. Isolation was done by PCR amplification technique using three pairs of primers, which are RT I (3282-3683), RTII (3261-3900), and RTIII (3169-4133). Construction and recombination in this study used Gateway method from Invitrogen. SRV-2 reverse transcriptase gene was isolated in this study with A-549 cell inoculated SRV-2 as source of virus. This gene amplify by PCR technique and resulted specific band for RTI 401 bp, RTII 639 bp, and RTIII 964 bp. Analysis of pENTR/SD/TOPO and pDEST17 indicated SRV-2 reverse transcriptase gene code has already inserted to this plasmid. This result indicated SRV-2 reverse transcriptase gene have isolated, constructed, and recombined to entry clone and destination vector.