Construction Of PRNA Chimera Harboring SiRNA Targeting The NS1 Gene Of Avain Influenza Virus

Continuing outbreaks of highly pathogenic Avian Influenza lead to the great economic loss and death in humans. The high risk of recombination of avian influenza virus is raising concerns that AIV might develop into highly pathogenic human infectious strains with high transmissibility in the human population. How to control this disease is still open. RNA interference is a process by which double-stranded RNA referred to siRNA(small interfering RNA)directs sequence-specific degradation of virus RNA, findings have raised the possibility that siRNA could inhibit viral gene expression and protect cells from viral infection. NS1 protein, a non-structural protein of AIV, could have an antagonistic effect on the production of IFN-α/βand contribute to virus infection, so NS1 gene may be used as major candidate for designing specific siRNA. The successful application of siRNA for the control of viral infection requires overcoming the obstacles such as siRNA degradation by exonucleases within the cell. A bacteriophage encoded small RNA named pRNA(packaging RNA) has been shown the potential as a carrier. In this study, siRNA was screened out for silencing NS1 gene and pRNA chimera harboring siRNA(pRNA-siRNA) was constructed.To construct the chimera of pRNA-siRNA,Firstly, the target sequences in the coding region of NS1 gene were determined for the design of specific siRNA. Secondly, the template, 120bp DNA oligos according to the sequence of pRNA, was synthesized. Primers containing T7 promoter and target sequences of siRNA were designed for PCR. The PCR product was named as dpRNA-siRNA(t), which were used as the templates in vitro transcription to prepare all the pRNA-siRNA(t) with T7 RNA polymerase. siRNA has been connected to the 5'and 3'ends of pRNA to form pRNA-siRNA. The pRNA-siRNA(c) is prepared as the negative control harboring a siRNA whose sequence will not find in the genome of mouse, human,Canine, avain or AIV .To determine the inhibition efficiency of the NS1 expression by pRNA-siRNA, enhanced green fluorescent protein (EGFP) expression plasmid pcEGFP and NS1-EGFP fusion expression plasmid pcNS1-EGFP were constructed using pcDNA3.1(+) vector as backbone. NS1 expression in MDCK cells was evaluated by the strength of fluorescence according to the expression of green fluorescent protein (GFP). The pRNA-siRNA(287) targeting the nts 287～307 of NS1 gene was cotransfected with the plasmid pcNS1-EGFP to determine its effect of inhibition on the expression of NS1. Cotransfections of pRNA-siRNA(287) with plasmid pcEGFP or pRNA-siRNA(c) with plasmid pcNS1-EGFP was carried out respectively as negative controls. The results showed that pRNA-siRNA(287) could specifically inhibit NS1 expression in MDCK cells.