【作者】 段群军；

【导师】 尚世强； 张泽伟；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 目的人巨细胞病毒(HCMV)可造成人体多系统多方面的严重损害,应用RNA干扰技术抑制HCMV感染具有诱人的前景。HCMV UL122基因和UL54基因是HCMV复制和存活的必需基因,在HCMV与宿主细胞相互作用及抗HCMV药物作用中起不可或缺的作用。但是目前对UL122基因和UL54基因的有效小干扰RNA(siRNA)作用靶位尚缺乏全面的认识。因此有必要进一步研究,以明确UL122基因和UL54基因的有效siRNA作用靶位,开发其潜在应用价值,为应用RNA干扰技术抑制HCMV感染奠定实验基础。方法本研究主要包括以下四大部分：(1)融合蛋白表达载体的构建：设计引物应用PCR法从HCMV AD 169株DNA中扩增出UL122基因的优势表位相应区域以及UL54基因的全长,并克隆入pEGFP-N1质粒载体,构建pUL122-EGFP、pUL54-EGFP融合蛋白表达载体,实现UL122基因和UL54基因的体外表达。(2)短发夹结构RNA (shRNA)表达载体的构建：针对UL122及UL54基因设计siRNA作用靶位点,合成相应序列并克隆入pAVU6+27质粒载体,构建针对UL122和UL54基因的shRNA表达载体。(3)筛选高效作用靶位：将shRNA表达载体和融合蛋白表达载体共转染入AD293细胞,应用实时荧光定量PCR、流式细胞检测技术和倒置荧光显微镜从mRNA和蛋白质水平筛选出理想、高效的siRNA作用靶位。(4)慢病毒载体构建及转染：参照上述筛选出的高效siRNA作用靶位,构建能够表达shRNA的慢病毒载体,并将此慢病毒载体和融合蛋白表达载体转染细胞,应用流式和Western Blot法检测shRNA的慢病毒表达载体对HCMV目标基因体外表达的抑制作用。结果(1)本研究成功构建融合蛋白表达载体pUL122-EGFP和pUL54-EGFP,实现UL122基因和UL54基因的体外表达。该融合蛋白表达载体在转染后24小时和48小时均能表达绿色荧光,但是转染后48小时表达的绿色荧光明显强于转染后24小时表达的绿色荧光。(2)成功将一小段设计合成好的、具有形成shRNA能力的DNA序列插入pAVU6+27质粒载体U6启动子的下游,构建成具有表达shRNA能力的重组质粒载体,分别命名为psiUL122-1、psiUL122-2、psiUL122-3、psiUL54-1、psiUL54-2和psiUL54-3。(3)将融合蛋白表达载体pUL122-EGFP分别与shRNA表达载体psiUL122-1、psiUL122-2和psiUL122-3共转染。倒置荧光显微镜下发现转染后24h重组质粒载体psiUL122-1、psiUL122-2和psiUL122-3对融合蛋白的表达无明显抑制作用,转染后48hpsiUL122-1和psiUL122-2对融合蛋白荧光表达具有明显抑制作用,而psiUL122-3对融合蛋白荧光表达只有轻度抑制作用。流式(FCM)结果显示转染后48hpsiUL122-1、psiUL122-2和psiUL122-3对融合蛋白的抑制率分别为82.0±1.0%、79.5±2.5%和53.5±2.5%,与对照组相比差异有统计学意义(p<0.05),且psiUL122-1和psiUL122-2对pUL122-EGFP的抑制效果较psiUL122-3明显。荧光定量PCR结果显示psiUL122-1、psiUL122-2和psiUL122-3对融合蛋白mRNA的抑制率分别为97.3±0.6%、98.0±0.1%和90.0±3.5%。(4)将融合蛋白表达载体pUL54-EGFP分别与shRNA表达载体psiUL54-1、psiUL54-2和psiUL54-3共转染。倒置荧光显微镜下发现转染后24h重组质粒载体psiUL54-1、psiUL54-2和psiUL54-3对融合蛋白的表达无明显抑制作用,转染后48h psiUL54-1对融合蛋白荧光表达具有明显抑制作用,而psiUL54-2和psiUL54-3对融合蛋白表达只有轻微抑制作用。流式结果显示转染后48hpsiUL54-1对融合蛋白的抑制率为85.4±1.2%,与对照组相比具有显著差异(p<0.05),而psiUL54-2和psiUL54-3对融合蛋白的抑制率分别为14.9±2.9%和20.4±6.2%,与对照组相比没有显著差异(p>0.05)。荧光定量PCR结果显示psiUL54-1对pUL54-EGFP融合蛋白基因mRNA的抑制率为97.4±0.7%,与对照组相比差异有统计学意义(p<0.05),而psiUL54-2和psiUL54-3的抑制率分别为20.3±6.9%和28.2±5.6%，与对照组相比无显著差异(p>0.05)。(5)参照psiUL54-1对UL54基因体外表达的高效抑制作用,进一步成功构建针对UL54基因的慢病毒表达载体。将慢病毒载体与融合蛋白表达载体pUL54-EGFP共转染,倒置荧光显微镜下发现慢病毒载体PscSI-1对融合蛋白荧光表达在转染后24h有轻度抑制作用,在转染后48h和72h具有明显抑制作用,而慢病毒载体PscSI-2、PscSI-3和PscSI-4对融合蛋白荧光表达在各时间点均无明显抑制作用。Western Blot检测显示PscSI-1与融合蛋白表达载体在1：2和1：1比例下均有明显抑制作用,PscSI-3在1：1比例下有轻度抑制作用,而PscSI-2和PscSI-4在两种比例下均无明显抑制作用。结论1.融合蛋白表达载体pUL122-EGFP和pUL54-EGFP能够成功表达融合蛋白UL122-EGFP和UL54-EGFP,实现UL122基因和UL54基因的体外表达,又能通过融合蛋白表达后仍能发出绿色荧光。融合蛋白的表达在转染后48小时较为明显。2.UL122基因的靶位点618-638bp (psiUL122-1和1103-1123bp(psiUL122-2)是高效的siRNA作用靶位点,是应用RNA干扰抗HCMV感染基因治疗的潜在靶位点。UL122基因的靶位点1414-1434bp(psiUL122-3)不是高效的siRNA作用靶位点。3.靶位点1479-1497bp (psiUL54-1和PscSI-1)是一个高效的siRNA作用靶位点,是应用RNA干扰抗HCMV感染基因治疗的潜在靶位点。靶位点2419-2437bp(psiUL54-2)、靶位点2886-2904bp(psiUL54-3和PscSI-2)、靶位点3058-3076bp (PscSI-3)和靶位点419-437bp(PscSI-4)不是高效的siRNA作用靶位点。4.重组干扰质粒转染后48h对融合蛋白的抑制作用较明显,但转染24小时无明显抑制效果。5.与质粒载体相比,慢病毒载体是一种转染率高、起效快、抑制作用持久且高效的基因载体,具有重要的应用价值。6.慢病毒载体与质粒载体在筛选有效siRNA作用靶位方面具有一致性。更多还原

【Abstract】 OBJECTIVESHuman cytomegalovirus (HCMV) is a widespread human pathogen that causes serious consequences in different patients. RNA interference (RNAi) is a powerful and potent technique to treat HCMV infection. HCMV UL122 gene and UL54 gene are essential genes for HCMV replication and infection. The sufficient knowledge of effective RNAi target site in UL122 gene and UL54 gene is still in shortage. Therefore, it is of great importance to find the most effective RNAi target sites in HCMV UL122 gene and UL54 gene.MethodsThis study included four parts:(1) Construction of fusion protein expression vectors:A truncated cDNA fragment of UL122 gene and the full-length of cDNA of UL54 gene were amplified from the genomic cDNA of HCMV AD 169 strain and cloned into pEGFP-N1 to construct fusion protein expression recombinant plasmids pUL122-EGFP and pUL54-EGFP. These two vectors can express enhanced green fluorescence protein (EGFP) for detection. (2) Construction of small hairpin RNAs (shRNA) expression vectors:Target sites corresponding to the UL122 gene and UL54 gene were selected and cloned into plasmid pAVU6+27 to construct shRNA expression recombinant vectors.(3) Cotransfection of fusion protein expression vectors and shRNAs expression vectors:Fusion protein expression vectors and shRNAs expression vectors were contransfected into AD293 cells. The inhibition effects of the expression of fusion protein were detected by fluorescence microscope, flow cytometry (FCM) and fluorescence quantitative real-time PCR for the screening of the most effective RNAi target sites.(4) Construction of lentiviral vectors and contransfection:In order to explore the long term knock-down regulation of RNAi, we constructed lentiviral vectors according to the effective RNAi target sites screened out using plasmid vector. The lentiviral vectors were then cotransfected into 293T cells to eveluate its know-down effects by FCM and Western blot.Results(1) The fusion protein expression vectors pUL122-EGFP and pUL54-EGFP were successfully constructed. The fluorescence can be detected under fluorescence microscope 24 h and 48 h after transfection. The green fluorescence signals transfected after 48 h were stronger than the green fluorescence signals transfected after 24 h.(2) Six target sequences corresponding to the open reading frame of HCMV UL122 gene and UL54 gene were selected to construct siRNA expression vectors, which were designated as psiUL122-1, psiUL122-2, psiUL122-3, psiUL54-1, psiUL54-2 and psiUL54-3. (3) The shRNA expression vectors were then cotransfected into AD293 cells with fusion protein expression vectors. The difference of the expression of green fluorescence signals between different groups was not apparent 24 h post-cotransfection. However, the green fluorescence signals were significantly reduced 48 h post-cotransfection in the cells cotransfected with pUL122-EGFP and psiUL122-1, psiUL122-2 separately, whereas green fluorescence signal was slightly reduced in the cells cotransfected with pUL122-EGFP and psiUL122-3. The inhibitory effects of the siRNAs on the expression of EGFP were further quantitatively validated by FCM assay 48 h post-transfection. The EGFP expression of the cells transfected with psiUL122-1 and psiUL122-2 was decreased by 82.0%±1.0% and 79.5%±2.5%, respectively. However, psiUL122-3 resulted in a weak reduction in the EGFP expression with an inhibitory rate of 53.5%±2.5%. We then examined the knock-down effectiveness of siRNAs on mRNA level, which was analyzed by fluorescence quantitative real-time PCR at 48 h post-transfection. A remarkable reduction of the expression of pUL122-EGFP mRNA was produced by psiUL122-1 and psiUL122-2, with an inhibitory rate of 97.3%±0.6% and 98.0%±0.1%, respectively. And the inhibitory efficiency of psiUL122-3 was 90.0%±3.5%.(4) The fusion protein expression vector pUL54-EGFP was cotransfected with psiUL54-1, psiUL54-2 and psiUL54-3, respectively. The difference of the expression of green fluorescence signals between different groups was not apparent 24 h post-cotransfection. The green fluorescence signals were significantly reduced 48 h post-cotransfection in the cells cotransfected with pUL54-EGFP and psiUL54-1, whereas fluorescence signal was slightly reduced in the cells cotransfected with pUL54-EGFP and psiUL54-2, psiUL54-3 separately. When evaluated with FCM, the EGFP expression of the cells transfected with psiUL54-1 was decreased by 85.4%±1.2%. However, psiUL54-2 and psiUL54-3 resulted in a weak reduction in the EGFP expression with an inhibitory rate of 14.9%±2.9%和20.4%±6.2%, respectively. A significant reduction of pUL54-EGFP mRNA was produced by psiUL54-1 with an inhibitory rate of 97.4%±0.7%. And the inhibitory efficiency of psiUL54-2 and psiUL54-3 was 20.3%±6.9%and 28.2%±5.6%, respectively.(5) Lentiviral vectors were successfully constructed and contransfected into 293T cells with pUL54-EGFP. Lentiviral vector PscSI-1 was found to have a mild inhibition of pUL54-EGFP fluorescent signals 24 h after contransfection and the inhibition effects become remarkable 48 h and 72 h after contransfection under fluorescence microscope. However, PscSI-2、PscSI-3 and PscSI-4 showed no inhibition of pUL54-EGFP fluorescent signals. When PscSI-1 and pUL54-EGFP were cotransfected with 1:2 ratio and 1:1 ratio, Western blot analysis displayed that PscSI-1 have significant knock-down of pUL54-EGFP both at these two ratios. PscSI-3 had mild inhibition of pUL54-EGFP at the ratio of 1:1. PscSI-2 and PscSI-4 showed no inhibition of pUL54-EGFP at these two ratios.Conclusion1. Recombinant plasmids pUL122-EGFP and pUL54-EGFP can express fusion protein UL122-EGFP and UL54-EGFP. And the green fluorescence signals can be detected from these two fusion protein. The fusion protein expression is remarkable 48 h after transfection.2. The target sites 618-638bp (psiUL122-1) and 1103-1123bp (psiUL122-2) of UL122 gene are effective RNAi target sites. The target site 1414-1434bp (psiUL122-3) is not an effective RNAi target site.3. The target sites 1479-1497bp (psiUL54-1 and PscSI-1) of UL54 gene is an effective RNAi target site. The target sites 2419-2437bp (psiUL54-2), 2886-2904bp (psiUL54-3 and PscSI-2),3058-3076bp (PscSI-3) and 419-437bp (PscSI-4) are not effective RNAi target sites.4. Recombinant shRNA expression plasmid vectors have the most effective inhibition of fusion protein at the time point of 48 h after cotransfection and have no significant inhibition at the time point of 24 h after cotransfection.5. Lentiviral vector is a kind of vector that shows inhibition effect quickly and has a higher transfection rate, more efficacious and stable knock-down effect, compared with plasmid vector.6. Lentiviral vector shows good accordance with plasmid vector for the the screening of effective RNAi target site.更多还原