IIsolation of GAG-CA Subunit of Jembrana Virus from the Viral Genome by RT-PCR and its cloning in pCR2.1-topolasmid using topoisomerase-based system

Jembrana virus is agent of acute Jembrana virus disease (JVD) in Bos javanicus. The genome of Jembrana virus is composed of a single-stranded RNA. We described in this paper the isolation of gag-ca subunit gene from the viral genome by a single-step RT-PCR reaction. The isolated gene was inserted in pCR2.1-TOPO plasmid by TOPO TA cloning kit, based on topoisomerase system. It allowed the cloning procedure using the RT-PCR products without prior purification or restriction enzyme digestion. A high number of positive recombinant bacterial colonies were obtained which were further analysis by BamHI-EcoRI digestion for a definite characterization of positive clones. The cloning procedure we used is highly efficient enabling rapid and easy gene isolation. Keywords: Jembrana virus disease- gag ca gene - RT-PCR-Topoisomerase clonin