中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2008年
10期
585-590
,共6页
胡妙凤%段群军%陶然%尚世强
鬍妙鳳%段群軍%陶然%尚世彊
호묘봉%단군군%도연%상세강
巨细胞病毒%基因,病毒%RNA干扰%RNA,小分子干扰
巨細胞病毒%基因,病毒%RNA榦擾%RNA,小分子榦擾
거세포병독%기인,병독%RNA간우%RNA,소분자간우
Cytomegatovirus%Genes,viral%RNA interference%RNA,small interfering
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UL54S mRNA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48 h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48 h后,UL54SmRNA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48 h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(19.43×104±2.29×104比27.89×104±5.50×104,P<0.01).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.
目的 建立載體法篩選人巨細胞病毒(HCMV)截短UL54基因(UL54S)小榦擾RNA(siRNA)靶位的體繫.方法 利用質粒pAVU6+27設計併構建2箇小髮夾RNA(shRNA)錶達載體(psiUL54-1和psiUL54-2),與融閤蛋白錶達載體pUL54S-綠色熒光蛋白(EGFP)共轉染AD293細胞.熒光顯微鏡觀察融閤蛋白熒光錶達,RT-PCR法分析UL54S mRNA水平變化,流式細胞儀評價siRNA對融閤蛋白的抑製作用,採用方差分析對流式細胞檢測結果進行統計分析,篩選齣有效的siRNA作用靶位.結果 shRNA錶達載體psiUL54-2與融閤蛋白錶達載體pUL54S-EGFP共轉染AD293細胞48 h後,pUL54S-EGFP、psiUL54-2共轉染組EGFP錶達量較pUL54S-EGFP、pAVU6+27共轉染組下降;凝膠分析顯示,psiUL54-2與pUL54S-EGFP共轉染48 h後,UL54SmRNA相對錶達量為19.6,明顯低于pUL54S-EGFP、psiUL54-1共轉染組的96.6與對照組的100.0;psiUL54-1、pUL54S-EGFP共轉染48 h後對融閤蛋白UL54S-EGFP錶達無影響(P>0.05),但psiUL54-2、pUL54S-EGFP共轉染抑製融閤蛋白UL54S-EGFP的錶達(19.43×104±2.29×104比27.89×104±5.50×104,P<0.01).結論 成功建立載體法篩選HCMV截短UL54S siRNA靶位體繫,UL54S序列中1532～1550位點是有效的siRNA靶位點.
목적 건립재체법사선인거세포병독(HCMV)절단UL54기인(UL54S)소간우RNA(siRNA)파위적체계.방법 이용질립pAVU6+27설계병구건2개소발협RNA(shRNA)표체재체(psiUL54-1화psiUL54-2),여융합단백표체재체pUL54S-록색형광단백(EGFP)공전염AD293세포.형광현미경관찰융합단백형광표체,RT-PCR법분석UL54S mRNA수평변화,류식세포의평개siRNA대융합단백적억제작용,채용방차분석대류식세포검측결과진행통계분석,사선출유효적siRNA작용파위.결과 shRNA표체재체psiUL54-2여융합단백표체재체pUL54S-EGFP공전염AD293세포48 h후,pUL54S-EGFP、psiUL54-2공전염조EGFP표체량교pUL54S-EGFP、pAVU6+27공전염조하강;응효분석현시,psiUL54-2여pUL54S-EGFP공전염48 h후,UL54SmRNA상대표체량위19.6,명현저우pUL54S-EGFP、psiUL54-1공전염조적96.6여대조조적100.0;psiUL54-1、pUL54S-EGFP공전염48 h후대융합단백UL54S-EGFP표체무영향(P>0.05),단psiUL54-2、pUL54S-EGFP공전염억제융합단백UL54S-EGFP적표체(19.43×104±2.29×104비27.89×104±5.50×104,P<0.01).결론 성공건립재체법사선HCMV절단UL54S siRNA파위체계,UL54S서렬중1532～1550위점시유효적siRNA파위점.
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 cotransfected 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(19.43×104±2.29×104vs27.89×104±5.50×104, P<0.01).Conclusion Thescreening 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.
