UL27、UL29基因shRNA表达载体的构建及对HSV-2的干扰效应研究

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

探讨Ⅱ型单纯疱疹病毒(Herpes simplex virus type 2,HSV-2)UL27、UL29基因联合靶向siRNA对HSV-2复制的影响。构建UL27、UL29基因的siRNA的重组表达载体并转染293细胞,通过实时荧光定量PCR技术和蛋白质印迹方法检测UL27、UL29基因的表达,终点滴定法检测细胞上清液中的各组病毒滴度,MTT法检测细胞的存活率。结果显示:(1)成功构建短发夹RNA(shRNA)重组表达载体。(2)转染后48 h,与空白组(空载体)相比,UL27 shRNA75组对UL27基因mRNA的抑制率为75.17%(P<0.05),UL29 shRNA1461组对UL29基因mRNA抑制率为66.08%,具有显著性差异(P<0.05)。UL27 shRNA75联合UL29 shRNA1461联合干扰组对UL27基因抑制率约为91.28%,UL29基因表达抑制率约为80.40%,与空白组比较具有显著性差异(P<0.05)。(3)终点滴定法结果显示单干扰组和联合干扰组可不同程度降低上清液中的病毒感染滴度,与空白组比较差异性显著(P<0.01)。(4)Western blot检测目的基因蛋白,单干扰组与联合干扰组可不同程度降低目的基因蛋白的表达,其中UL27shRNA75+UL29 shRNA1461联合干扰组能显著抑制相应的蛋白表达水平,蛋白表达量明显减少,与单干扰组相比具有显著差异(P<0.05)。(5)经MTT法检测,UL27 shRNA75、UL29 shRNA1461、UL27 shRNA75+UL29 shRNA1461联合干扰组的细胞存活率明显提高,差异有显著意义(P<0.05)。构建的pGPU6/GFP/Neo-UL27、pGPU6/GFP/Neo-UL29重组表达载体,能在体外细胞水平上不同程度的干扰HSV-2 UL27、UL29基因表达,UL27、UL29联合干扰效率更高,抑制HSV-2在HEK293细胞中复制。

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. MTr 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〈O.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〈O.05 ） . （ 5 ） MTF assay showed that, UL27 shRNA75, UL29 shRNA1461, UL27 shRNA75 ＋ UL29 shRNA1461 group cell survival rate was significantly improved, there are significant differences （ P〈O.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.