Biosynthesis and function verification of short hairpin RNA targeting to Influenza A virus / 第三军医大学学报

ABSTRACT

Objective To investigate the inhibitive effects of biosynthesized short hairpin RNA(shRNA)on the duplication of Influenza A virus(IAV)in human bronchial epithelium(HBE)cells.Methods NP-shRNA and PA-shRNA targeting to the highly conservative sequences of IAV nucleoprotein(NP)and acidic RNA polymerase(PA)were designed and biosynthesized by in vitro transcription,and then were inserted into the plasmid pGenSil-1.After sequencing analysis,the recombinant plasmids NP-shRNA and/or PA-shRNA were transduced into HBE cells followed by infected with IAV A/PR/8/34 H1N1(A/PR8)virus.The cytopathogenic effect(CPE)of the HBE cells,hemagglutination assay(HA)and 50% tissue culture infective dose(TCID50)titer of A/PR8 in the culture supernatants were determined respectively.The mRNA and protein expressions of NP and PA were detected by RT-PCR and Western blotting respectively,and the results were compared in order to evaluate the inhibitive efficiency of NP-shRNA and/or PA-shRNA to A/PR8 duplication in HBE cells.Results The content of NP-shRNA and PA-shRNA biosynthesized by in vitro transcription were respectively 59.4 nmol and 50.6 nmol in each 100 ?l.The purity was more than 2.0 and the sequences were verified to be correct after sequencing analysis.The CPE of HBE cells and the virus titer in the culture supernatants of cells transfected with NP-shRNA,PA-shRNA or NP-shRNA+PA-shRNA were markedly lower than those of the control groups.In 3 h after 1 TCID50 A/PR8 infection,the mRNA level of NP in NP-shRNA group,the mRNA level of PA in PA-shRNA group and those in NP-shRNA+PA-shRNA group were 41.7%,43.4%,68.5% and 73.7% respectively,lower than those of the control group.At 48 h after 1 TCID50 A/PR8 infection,NP synthesis were 92.3%,84.0% and 91.7% respectively of those of the control group.Conclusion Anti-IAV NP-shRNA and PA-shRNA are successfully prepared by in vitro transcription.Both of those markedly inhibit the reproduction of IAV A/PR8 and produce a favorable protective effect on HBE cells.