A simple one-step method for producing dsRNA from E. coli to inhibit shrimp virus replication

作者：Posiri Pratsaneeya****; Ongvarrasopone Chalerm****^*; Panyim Sakol

来源：Journal of Virological Methods, 2013, 188(1-2): 64-69.

DOI：10.1016/j.jviromet.2012.11.033

• Long hairpin dsRNA
• YHV-infected shrimp
• dsRNA production
• RNAi
• PmRab7

While dsRNA has been used experimentally to inhibit replication and thus disease caused by several shrimp viruses, it can be tedious, time consuming and costly to produce. Here we describe a simple method for obtaining long-hairpin dsRNA from RNase III-minus HT115 Escherichia coli cells following its expression from a plasmid containing a RNA promoter. All the method requires is for bacterial cells to be fixed briefly in 75% ethanol in phosphate buffer saline (PBS) before suspension in 150 mM NaCl. When injected into the haemal sinuses of shrimp, hairpin dsRNA specific to the PmRab7 gene prepared by this method was found to inhibit PmRab7 mRNA expression as effectively as hairpin dsRNA purified using TRIzol reagent. Shrimp injection of either dsRNA-PmRab7 or hairpin dsRNA specific to yellow head virus (YHV) prepared similarly also inhibited YHV replication effectively. Based on these findings, this simple and cheap method of producing dsRNA should be adaptable to various commercial-scale applications of RNA interference (RNAi) in shrimp.

• 出版日期2013-3