HAIRPIN-RNA EXPRESSION CASSETTE BY TWO-STEP CLONING SYSTEM AND ITS POTENTIAL AS AN IMMUNE STIMULANT IN SHRIMP

Vanvimon Saksmerprome*, Patai Charoonart, Boonsirm Withyachumnarnkul

National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani and Centex Shrimp, Mahidol University, Bangkok Thailand
E-mail: vanvimon.sak@biotec.or.th We demonstrate an improved method for delivering RNAi-based immunity to large shrimp populations. Long sequences of double-stranded RNA (dsRNA) have recently been used to enhance viral resistance, through an RNA interference (RNAi) mechanism, in shrimp aquaculture. Since dsRNA-mediated knockdown efficiency of viral genes appears to be dose dependent, a large-scale production of dsRNA is necessary for antiviral/therapeutic applications of RNAi for shrimp farm operations. A new design of hairpin-RNA expression vector, followed by transformation into RNase-deficient E.coli HT115, offers a quick preparation of a large amount of long dsRNA (normally >300 nt). The hairpin RNA consists of a forward strand and a 100-base shortened reverse strand, and the unpaired 100-base region on the forward strand serves as a loop. This strategy reduces 3-step to 2-step cloning of hairpin construct into DNA expression cassette, thus bypassing difficulty in joining a small loop piece into a large carrier vector. A total RNA of ~4 mg is generally obtained from 100 mL bacterial culture.

The bacterially expressed hairpin RNA specific to RNA-dependent RNA polymerase (RdRp) gene of yellowhead virus (YHV) can induce antiviral immunity in Penaeus vannamei shrimp. Viral protection by exogenous dsRNA demonstrates the potential of dsRNA produced by 2-step-cloning hairpin cassette as an immune stimulant in shrimp (Figure 1). We investigate further if the putative viral promoters can be regulated by shrimp transcriptional activators. The hairpin specific to YHV is cloned into the viral promoter-contained plasmids, followed by intramuscular injection of the constructs into shrimp. We examine protection against YHV and level of viral mRNA after plasmid injection. Successful hairpin expression driven by such promoters demonstrates the potential of DNA-based vaccine for effective antiviral immunity in shrimp.