Construction of Bacteria Producing dsRNA as RNAi- Antiviral Targeting Sequence VP28 White Spot Syndrome Virus (WSSV)

• Main Author: Pranyoto, Slamet
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/43074
• Institution: Institut Teknologi Bandung

White Spot Syndrome Virus (WSSV) is a rod-shaped virus that lives in the marine environment and attack crustaceans, such as shrimp. Studies show that WSSV is one of the biggest causes of economic losses in the aquaculture sector of tiger shrimp in Indonesia and several other countries. WSSV can cause mortality rate up to 100% on 3-10 days post acute infection which is characterized by the appearance of white spots in the inner surface of cuticle. The construction of dsRNA-producing bacteria is used as an effort to deal with WSSV virus infection in shrimp. The aim of this study is to produce double stranded-RNA (dsRNA) with the target structural protein domain VP28 from the WSSV genome to trigger the RNA Interference (RNAi) mechanism. The methods used to construct pWSRI by inserting the VP28 sequence into the pET32 + plasmid along with the 3' end-segment M non-coding sequence of the phi6 bacteriophage as an RdRP-binding site. The formation of dsRNA sequences is assisted by RNA dependent RNA polymerase (RdRP) which work in the initiation and elongation of dsRNA sequence. The pP2RE plasmid construction was carried out to produce P2 protein that can catalyze the activity of RdRP. The P2 Replicase/RdRP sequence is from the L segment sequence of bacteriophage phi6 that inserted into the pCOLA-Duet plasmid. The constructed plasmids were then transformed into Escherichia coli BL21 DE3 using heatshock method with the selection of the antibiotic Ampicilyn (50?g/ml) for pWSRI and Kanamycin (50 ?g/ml) plasmids for pP2RE insert. The transformation result was visualized with 1% agarose gel electrophoresis which showed pWSRI and pP2RE plasmid band sizes are 6200 bp and 5500 bp, respectively. Polymerase Chain Reaction (PCR) results with specific primers showed that were in line with the expected size, pWSRI 178 bp and pP2RE 800 bp. Confirmation of dsRNA isolate shows a single band size of 90 bp. The difference in the size of dsRNA isolate with the size of 200 bp of target dsRNA is caused by P2-Replicase / RdRP used is nonspecific and its effectiveness is not yet known in vivo. The use of 3' end- M segment sequences requires further analysis to be used as an RdRP binding site. The conclusion of this study shows that the E.coli BL21 DE3 has not been able to produce dsRNA as long 200 bp as expected.