shRNA表达载体对HSV-2 ICP4基因的干扰效应

Inhibition effect of shRNA expressing vector on herpes simplex virus type 2 ICP4

目的构建针对Ⅱ型单纯疱疹病毒(HSV-2) ICP4基因的短发夹RNA(shRNA)重组表达载体,探究该载体表达的shRNA对HSV-2的抑制作用。方法重组表达载体通过脂质体转染法转染HEK293细胞,并接种HSV-2,48h后,实时荧光定量RT-PCR检测mRNA转录情况,Western blot检测ICP4蛋白的表达情况,终点滴定法测定病毒的滴度。结果与对照组相比,干扰组对mRNA抑制率为71%,使蛋白表达减少71.81%,并且病毒滴度也明显降低(P<0.05)。结论构建的pGPU6/Neo-shRNA ICP4重组质粒表达载体能在体外细胞水平上干扰HSV-2 ICP4的基因表达,抑制HSV-2在细胞中复制。

Objective To construct the expressing vector encording small hairpin RNA （shRNA） a- gainst herpes simplex virus type 2 （ HSV-2 ） ICP4 gene and investigate its efficiency. Methods The ex- pressing vector encording shRNA against ICP4 （termed as pSh -ICP4） was constructed and transiently transfected into HEK293 cell with liposomes. Then, cells were infected with HSV-2. After 48 hours, the viral titer was measured using end point assay Moreover, the transcription level of HSV-2 ICP4 was de- tected using Real time RT - PCR. Finally, the expression of ICP4 protein was determined using Western blot Results Compared with the control groups, the ICP4 mRNA inhibition rate of the interference group was 71%, and the protein expression decreased 71.81% in pSh -ICP4 transfected group. Furthmore, the virus titer also decreased significantly （P 〈 0. 05 ）. Conclusion The expression vector encording shRNA against ICP4 could effectively reduce the expression of ICP4. and the reproduction of HSV-2. virus.