Perpustakaan Digital ITB

Infectious Myonecrosis Virus ,or IMNV, is a pathogen that caused white necrosis in skeletal muscle of Penaeid shrimp. This disease could increase shrimp mortality 40% - 70%. To cure this disease, dsRNA (double-stranded RNA) has been known to be used as antivirus. Production of dsRNA can be increased using RdRP (RNA dependent RNA Polymerase) from bacteriophage phi-6. RdRP can synthesize dsRNA using template ssRNA (single-stranded RNA) that have M-segment sequence from bacteriophage phi-6. In this research, production of IMNV dsRNA was done by using RdRP from bacteriophage phi-6 in Escherichia coli BL21 as vector. Construction of RdRP producer was done by inserting P2 Replicase sequence ,from L-segment of bacteriophage phi-6, to pCola-Duet plasmid. This plasmid thus called pP2. Construction of ssRNA template was done by inserting ORF1 of IMNV genome and recognition site from bacteriophage phi-6 M-segment to pET-23a (+) plasmid. This plasmid thus called pIMRI. Both insert is transcribed under T7 promoter. Both plasmid was transformed to E. coli using heat-shock method. The success parameter for transformation was proved by selective medium culture and plasmid isolation. The PCR result of isolated plasmid fit to in silico prediction of amplicon length: 281 bp and 875 bp. The parameter of success for ssRNA transcription was proved by synthetizing cDNA from RNA isolate. The result is E. coli was successfully transformed with pP2 and pIMRI plasmid, but this mutant E. coli could not produce dsRNA as expected in this research. This failure might be caused by excess sequence at 3’ RNA tip from pIMRI plasmid. This excess nucleotide caused RdRP unable to detect RNAi ssRNA, thus synthesize of dsRNA could not happen.