siRNA Interfering HSV-1 UL30 DNA Polymerase Gene in vitro

Objective:To screen siRNA silencing HSV-1 UL30 gene effectively and to explore the influence of siRNA on the reproduction of HSV-1.Method:12 siRNAs targeting UL30 gene were designed,chemosynthesized and were cotransfected into VERO cells with the fusion expression plasmid pEGFP-N1-Fi,fluorescence-activated cell sorter was used to screen siRNAs effectively inhibiting the fusion protein Fi-EGFP,the inhibitive effect on the expression of UL30 mRNA in infected cells was detected via FQ-real time PCR,and the inhibitive effect on the reproduction of HSV-1 was assayed via cytopathic effect and Plaque Reduction Experiment.Result:siRNA4,siRNA10 and siRNA8 which could effectively inhibit the fusion protein Fi-EGFP were screened via cotransfection experiment and could significantly reduce the expression level of intracellular UL30 mRNA and the titer of progeny virus released into supernatant,both siRNA4 and siRNA10 showed significant inhibitive effect on the reproduction of HSV-1 at 36h post infection,the plaque numbers decreased separately by 61.17%,51.46%(P0.05),the diameter of plaques in siRNA4,siRNA10 and siRNA8 groups decreased separately by 29.94%,23.49%,21.69%(P0.01).Conclusion:3 siRNAs effectively inhibiting UL30 were screened,both siRNA4 and siRNA10 showed significant inhibitive effect on the reproduction of HSV-1 in early stage of plaque formation,siRNA4,siRNA10 and siRNA8 could delay the spread and increase of plaques and showed definite inhibitive effect on the reproduction of HSV-1.