The Interference Effects Of Dual ShRNAs Expression Vector On The UL27 And UL54 Genes Of HSV-2

Objective: A kind of plasmid vector for expression dual sh RNAs armed to HSV-2’s UL27 and UL54 was to be constructed, the interference effects on the target genes and the effects on the proliferation of HSV-2 virus were investigated.Methods: The dual sh RNAs(Dual-sh RNA) expression vectorstargeted UL27 and UL54, single sh RNA expression vectors(UL27-sh RNA) targeted UL27 and single sh RNA expression vectors(UL54-sh RNA) targeted UL54, were constructed. And then those vectors were transfected into HEK293 cells with liposomes. Observed the transfect efficiency of them with fluorescence microscopy, and then inoculated with HSV-2. In 24 h later, we detected the survival ratio of cells with Four methyl thiazolyl tetrazolium(MTT) colorimetric method method, the viral titer in supernatant of cell cultrue was determined by end point assay, the m RNA level of HSV-2 UL27 and UL54 were detected with quantitative real-time fluorescent PCR(QPCR). The protein expression of g B and ICP27 were detected with western blotting(WB). Thus we campared the interference effects between Dual-sh RNA and UL27-sh RNA+ UL54-sh RNA(joint interference), then we investigated the effects of double sh RNA expression vectors on the proliferation of HSV-2.Results:(1) MTT assay indicated, the survival ratio of cells in each groups can be improved in varying degree, the survival ratio of UL27-sh RNA and UL54-sh RNA groups are 78±2.65% and 68.67±3.21%; the survival ratio of jointed interference(UL27-sh RNA+UL54-sh RNA) and Dual-sh RNA groups are 91.67±1.53% and 90.33±1.15% respectively, compared with the negative control group, the differences are significant(P<0.05). There is no significant difference between joint interference and Dual-sh RNA groups.(2) The results of end point assay showed, the titers of progeny virus in the supernatant of each interference groups can be reduced in varying degree. UL27-sh RNA and UL54-sh RNA groups are 10-3.2±0.3 and 10-3.4±0.1 respectively; the jointed interference and Dual-sh RNA groups are 10-2.07±0.15 and 10-2.2±0.26, compared with the single interference and negative control groups, the differences are significant(P<0.05).(3) The results of QPCR showed, the m RNA expression level of UL27 and UL54 in each groupscan be differently inhibited, apart from negative control group. In the single gene interference groups, the inhibition rates of UL27 m RNA in UL27-sh RNA are 65.3±2.52%; the inhibition rates of UL54 m RNA in UL54-sh RNA are 71.6±3.51%, compared with the negative control group respectively, the differences are significant(P<0.05). The inhabition ratios of jointed interference group are 85.3±1.53% and 81.3±6.51%; Dual-sh RNA group are 84±1% and 87.7±2.08%, compared with single interference groups, the differences are significant(P<0.05). There is no significant difference between jointed interference and Dual-sh RNA groups.(4) WB method revealed, the jointed interference and Dual-sh RNA groups can lower the contents of g B and ICP27 protein in varying degree, compared with single gene interference and negative control groups the differences are significant(P<0.05). There is no significant difference between joint interference groups and Dual-sh RNA groups.Conclusion: Dual sh RNAs expression plasmid can inhibit the expression of target genes effectively. Dual sh RNAs expression plasmid mediated double genes interference and two kind of single sh RNA expression plasmids mediated double genes interference can inhibit the replication of HSV-2 virus at the same level. Dual genes interference is beter than single gene interference to inhibit the proliferation of HSV-2.