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UL27、UL29基因shRNA表达载体的构建及对HSV-2的干扰效应研究

, PP. 202-209

Keywords: RNA干扰,短发夹RNA,Ⅱ型单纯疱疹病毒,UL27基因,UL29基因

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Abstract:

探讨Ⅱ型单纯疱疹病毒(Herpessimplexvirustype2,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48h,与空白组(空载体)相比,UL27shRNA75组对UL27基因mRNA的抑制率为75.17%(P<0.05),UL29shRNA1461组对UL29基因mRNA抑制率为66.08%,具有显著性差异(P<0.05)。UL27shRNA75联合UL29shRNA1461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)Westernblot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+UL29shRNA1461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,UL27shRNA75、UL29shRNA1461、UL27shRNA75+UL29shRNA1461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-2UL27、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。

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