载体法筛选人巨细胞病毒截短UL54基因小干扰RNA靶位体系的建立

RNA screening of efficient small interfering RNA target sites directed against truncated region of UL54 gene in human cytomegalovirus by small interfering RNA expression vectors

目的建立载体法筛选人巨细胞病毒（HCMV）截短UL54基因（UL54S）小干扰RNA（siRNA）靶位的体系。方法利用质粒pAVU6±27设计并构建2个小发夹RNA（shRNA）表达载体（psiUL54-1和psiUL54-2），与融合蛋白表达载体pUL54S-绿色荧光蛋白（EGFP）共转染AD293细胞，荧光显微镜观察融合蛋白荧光表达，RT-PCR法分析UL54SmRNA水平变化，流式细胞仪评价siRNA对融合蛋白的抑制作用，采用方差分析对流式细胞检测结果进行统计分析，筛选出有效的siRNA作用靶位。结果shRNA表达载体psiUL54—2与融合蛋白表达载体pUL54S-EGFP共转染AD293细胞48h后，pUL54S-EGFP、psiUL54-2共转染组EGFP表达量较pUL54S-EGFP、pAVU6±27共转染组下降；凝胶分析显示，psiUL54—2与pUL54S-EGFP共转染48h后，UL54S mRNA相对表达量为19．6，明显低于pUL54S-EGFP、psiUL54—1共转染组的96．6与对照组的100．0；psiUL54—1、pUL54S—EGFP共转染48h后对融合蛋白UL54S-EGFP表达无影响（P〉0．05），但psiUL54—2、pUL54S-EGFP共转染抑制融合蛋白UL54S—EGFP的表达（19．43×10^4±2．29×10^4比27．89×10^4±5．50×10^4，P＜O．01）。结论成功建立载体法筛选HCMV截短UL54S siRNA靶位体系，UL54S序列中1532～1550位点是有效的siRNA靶位点。

Objective To establish a screening system of efficient small interfering RNA （siRNA） target sites directed against truncated region of human cytomegalovirus （HCMV） UL54 gene （UL54S） with siRNA expression vectors. Methods Two small hairpin RNA （shRNA） expression vectors targeting truncated region of HCMV UL54 gene were constructed based on pAVU6 ± 27 vector, and eotransfeeted into AD293 cells with the fusion protein expression vectors pUL54S- enhanced green fluorescent protein （EGFP）. The levels of mRNA and EGFP were evaluated by means of reverse transcription-polymerase chain reaction （RT-PCR）, fluorescence microscopy and flow cytometry so as to assess the inhibitory efficiency of siRNA. Analysis of variance was applied to analyze the variance of total fluorescence intensity to screen out the efficient target sites of siRNA. Results shRNA expression vector psiUL54-2 and fusion protein expression vector pUL54S-EGFP were cotransfected into AD293 cells. The EGFP expression level in pUL54S-EGFP/psiUL54-2 cotransfected group was lower than that in pUL54S-EGFP/pAVU6±27 cotransfected group after 48 h of transfection. Gel analysis showed that the mRNA relative level of UL54S was 19.6 after 48 h of psiUL54-2/pUL54S-EGFP cotransfection, which was significantly lower than those in pUL54S- EGFP/psiUL54-1 group （96.6） and control group （100.0）. Cotransfection of psiUL54-1/pUL54S- EGFP for 48h didn＇t show any effects on the expression of fusion protein UL54S-EGFP （P〉0.05）. While psiUL54-1/pUL54S-EGFP cotransfection inhibited the expression of fusion protein UL54S- EGFP （19.43×10^4±2.29×10^4 vs 27.89×10^4±5.50×10^4, P〈0.01）.Conelusion The screening system of efficient siRNA targeting truncated region of HCMV UL54S is established successfully. The 1532th-1550th nucleotide acids of UL54S coding sequence are efficient siRNA target sites.