The Construction and Interference Effects to HSV-2 of UL27, UL29 shRNA Expression Vectors

Abstract

Abstract: To investigate the effect of siRNA targeting herpes simplex virus type 2(HSV-2)UL27 and UL29 simultaneously on HSV-2 replication. siRNA recombinant expression vectors targeting UL27 and UL29 simultaneously were constructed and the expression of UL27 mRNA and UL29 mRNA were tested by FQ-PCR 48 hours after transfecting in 293 cells. The viral titer are estimated by using titration end-point assay. The expression and changes(gray scale value)of protein was assessed by western-blot. MTT was used to examine the proliferative activity of cell. The results showed that：(1)The shRNA expression vectors which were constructed by pGPU6/GFP/Neo-UL27、pGPU6/GFP/Neo-UL29.(2)At 48 hours after transfection, the results of FQ-PCR showed that compared to blank group, UL27 shRNA75 the inhibition rate are 75.17%, there are significant differences(P<0.05). UL29 shRNA1461 the inhibition rate are 66.08%, there are significant differences(P<0.05). UL27 shRNA75 +UL29 shRNA1461 joint group of UL27 gene inhibition rate are about 91.28%, UL29 gene inhibition rate are about 80.40%, comparing with blank group, with significant differences(P<0.05).(3)The result of end-point assay showed each interference group could reduce the virus titers of filial generation in the supernatant in different degree, comparing with blank group, with significant differences(P<0.01).(4)Using Western blot to detect the objective gene protein has revealed that single interference group and joint group can reduce the protein expression of target genes in different degree, the joint interference group of UL27 shRNA75 + UL29 shRNA1461 can significantly inhibit the expression level of target genes protein, protein expression of joint interference group is obviously on the decline, compared with the single interference group, there is significant differences(P<0.05).(5)MTT assay showed that, UL27 shRNA75, UL29 shRNA1461, UL27 shRNA75 + UL29 shRNA1461 group cell survival rate was significantly improved, there are significant differences(P<0.05).The construction of pGPU6/GFP/Neo-UL27, pGPU6/GFP/Neo-UL29 recombinant expression vector shRNA can interfere HSV-2 UL27, UL29 gene expression from different cell level in vitro. The joint interference has more effect, which can inhibit the the replication of HSV-2 in HEK293 cells.

Key words: RNA interference, Small hairpin, RNA Herpes, Simplex virus type 2, UL27, UL29

Lü Yancheng, Pan Xiaoyu, Huang Chang, Ding Juan. The Construction and Interference Effects to HSV-2 of UL27, UL29 shRNA Expression Vectors[J]. Biotechnology Bulletin, 2014, 0(5): 202-209.

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