The Construction And Interference Effects To HSV-2of UL27, UL29ShRNA Expression Vectors

Objective:Herpes simplex virus type2(HSV-2)UL27, UL29gene can be used as target gene to construct small hairpin RNA, shRNA recombinant expression vector. On cell level In vitro, observe the inhibition of HSV-2UL27, UL29single target gene shRNA expression vector on virus and the influence on the replication of HSV-2by the jointly interfering two target shRNA.Methods:Four interference target sites of HSV-2UL27, UL29gene are selected to construct respectively4groups of small hairpin RNA, shRNA recombinant expression vector. The negative control expression vector expressing shRNA is also constructed. The expression vector transfect into HEK293cell with liposome. HEK293cell were infected with HSV-2after expression vector transfecting for48hours. shRNA which has interference effect is selected for the joint interference. Filter out interference effects of structure and conduct joint interference. The transcription levels of HSV-2UL27, UL29are estimated by using Real time PCR. The viral titer are estimated by using titration end-point assay. The expressing effect of protein is detected by using Western-blot.Results:(1) The result of DNA sequencing and restrictive enzyme digestion confirms that recombinant plasmid of pGPU6/Neo-UL27, pGPU6/Neo-UL29have been successfully constructed.(2) Comparing with blank control, the inhibition rate are6.05%,75.17%,35.38%,30.74%,60.22%with shRNA NC, UL27shRNA75, UL27shRNA151, UL27shRNA963, UL27shRNA2265, recombinant plasmid respectively. There are significant differences (P<0.05) and shRNA NC no significant differences. Comparing with blank control, the inhibition rate are28.80%,59.95%,66.08%,36.27%,.with UL29shRNA897, UL29shRNA939, UL29shRNA1461, UL29shRNA1653recombinant plasmid respectively. There are significant differences (P<0.05). The UL29shRNA1461group effect is the best one. UL27shRNA75combined with UL29shRNA1461joint group of interference suppression rate91.28%, comparing with blank control, with significant differences (P<0.05).(3) The result of titration end-point assay shows that the virus titers of the supernatant is reduced comparing with blank control by UL27shRNA75、 UL27shRNA151、UL27shRNA963, UL27shRNA2265, UL29shRNA897, UL29shRNA1461, UL27shRNA75+UL29shRNA1461(P<0.05).Conclusion:The successful construction of pGPU6/Neo-UL27, pGPU6/Neo-UL29recombinant expression vector shRNA can interfere HSV-2UL27, UL29gene expression from different cell level In vitro. The joint interference has more effect, which can inhibit the the replication of HSV-2genome in HEK293cells. Thus, the theoretical foundation is provided for the further exploration of ati-HSV-2gene therapy by RNA interference(RNAi).