The interference effects on UL29 shRNA expression vectors on HSV-2 / 实用医学杂志

ABSTRACT

Objective To construct short hairpin RNA (shRNA) recombinant expression vector for herpes simplex virus typeⅡ(HSV-2) UL29 gene and observe its inhibitory effect on HSV-2. Methods Four interference target sites of HSV-2UL29 gene were selected to construct 4 groups of small hairpin RNA respectively,named shRNA recombinant expression vector. The expression vectors were transfected into HEK293 cells with liposome. HEK293 cells were infected with HSV-2 after expression vector being transfected. The viral titer was estimated by end-point titration assay. The level of transcription was estimated by Real-Time PCR method. The expressing effect of protein was detected by Western-blot. Results Recombinant expression vector pGPU6/GFP/Neo-shRNA was constructed successfully. The result of end-point titration assay showed that the viral titer was reduced comparing with blank control (P<0.05). The result of RT-PCR showed that inhibition rates were respectively 28.80%, 59.95%, 66.08%and 36.27% comparing with blank control, and there were significant differences (P < 0.05). The effect of UL29shRNA1461 group was the best one. The result of Western-blot showed that the expressing quantity of ICP8 was reduced. Conclusion Recombinant expression vector pGPU6/GFP/Neo-shRNA can interfere HSV-2 UL29 gene expression from different cell level in vitro, which can inhibit the replication of HSV-2 genome in HEK293 cells. Thus, RNA interference (RNAi) is conducive to the further exploration of viral therapy.