应用RNA干扰技术抑制人巨细胞病毒UL122、UL54基因体外表达的研究

发布时间：2018-03-20 23:07

本文选题：人巨细胞病毒　切入点：UL122基因　出处：《浙江大学》2010年博士论文　论文类型：学位论文

【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(p0.05),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(p0.05),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(p0.05)。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(p0.05),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(p0.05)。 (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。
[Abstract]:objective
Human cytomegalovirus (HCMV) may cause serious damage to human multi system in many aspects, the inhibition of HCMV infection has a prospect of.HCMV UL122 gene and UL54 gene is attractive HCMV replication and survival of essential genes using RNA interference technology plays an indispensable role in the interaction between HCMV and host cells and anti HCMV drugs. But at present the UL122 gene and UL54 gene of effective small interfering RNA (siRNA) target is still a lack of understanding. Therefore, further studies are necessary to effectively target specific UL122 siRNA gene and UL54 gene, its potential application value for the application of RNA interference technology to provide the experimental basis for inhibition of HCMV infection.
Method
This study mainly includes the following four parts:
(1) fusion protein expression vector construction: primers were designed using PCR method to amplify full-length UL122 gene epitope and the corresponding region of the UL54 gene from HCMV AD in 169 strains of DNA, and cloned into pEGFP-N1 plasmid, to construct pUL122-EGFP, pUL54-EGFP fusion protein expression vector, UL122 gene and UL54 gene expression in vitro.
(2) construction of short hairpin RNA (shRNA) expression vector: targeting UL122 and UL54 gene, designing siRNA target site, synthesizing corresponding sequence and cloning into pAVU6+27 plasmid vector, constructing shRNA expression vector targeting UL122 and UL54 gene.
(3) screening effective target: transfection of shRNA expression vector and fusion protein expression vector into AD293 cells. Real-time and real-time PCR, flow cytometry and inverted fluorescence microscope were used to screen ideal and efficient siRNA target from mRNA and protein level.
(4) to construct a lentiviral vector and transfection: efficient siRNA target with reference to the selected position, to construct the Lentivirus Expression Vector shRNA, and the lentivirus vector and fusion protein expression vector transfected cells, detection of shRNA by flow cytometry and Western Blot lentiviral expression vector on the expression of HCMV gene in vitro the inhibitory effect.
Result
(1) this study successfully constructed the fusion protein expression vector pUL122-EGFP and pUL54-EGFP, UL122 gene and UL54 gene expression in vitro. The expression of fusion protein expression of green fluorescence in 24 hours after transfection and 48 hours are but the expression of green fluorescence carrier, 48 hours after transfection the expression of green fluorescence was stronger than the 24 hours after transfection..
(2) successfully designed and synthesized a short sequence of DNA shRNA, has formed the ability of pAVU6+27 plasmid U6 promoter, to construct recombinant plasmid vector with shRNA expression ability, named psiUL122-1, psiUL122-2, psiUL122-3, psiUL54-1, psiUL54-2 and psiUL54-3.
(3) the fusion protein expression vector pUL122-EGFP respectively with shRNA expression vector psiUL122-1, psiUL122-2 and psiUL122-3 were co transfected. After transfection of 24h recombinant plasmid psiUL122-1 under inverted fluorescence microscope, psiUL122-2 and psiUL122-3 had no obvious inhibitory effect on the expression of the fusion protein after transfection, 48hpsiUL122-1 and psiUL122-2 on the expression of fusion protein fluorescence has obvious inhibitory effect. While the psiUL122-3 of fusion protein expression inhibition. Only mild fluorescence flow cytometry (FCM) results showed that after transfection of 48hpsiUL122-1, psiUL122-2 and psiUL122-3 on the inhibition of fusion protein rate were 82 + 1%, 79.5 + 2.5% and 53.5 + 2.5%, compared with the control group the difference was statistically significant (P0.05), and the inhibitory effects of psiUL122-1 and psiUL122-2 the pUL122-EGFP is more obvious than psiUL122-3. Fluorescence quantitative PCR results showed that psiUL122-1, psiUL122-2 and psiUL122-3 of mRNA fusion protein The inhibition rates were 97.3 + 0.6%, 98 + 0.1% and 90 + 3.5%., respectively.
(4) the fusion protein expression vector pUL54-EGFP respectively with shRNA expression vector psiUL54-1, psiUL54-2 and psiUL54-3 were co transfected. After transfection of 24h recombinant plasmid psiUL54-1 under inverted fluorescence microscope, psiUL54-2 and psiUL54-3 had no obvious inhibitory effect on the expression of the fusion protein after transfection of 48h psiUL54-1 on the expression of fusion protein fluorescence has obvious inhibitory effect, and psiUL54-2 and psiUL54-3 on the expression of fusion protein is only slightly inhibited. Flow cytometry showed that after transfection of 48hpsiUL54-1 on inhibition of fusion protein was 85.4 + 1.2%, compared with the control group with significant difference (P0.05), psiUL54-2 and psiUL54-3 on the inhibition of fusion protein rate were 14.9 + 2.9% and 20.4 + 6.2%, there was no significant difference compared with the control group (P0.05). Fluorescence quantitative PCR results showed that psiUL54-1 inhibition of pUL54-EGFP fusion protein gene mRNA was 97.4 + 0.7%, and the control group The difference was statistically significant (P0.05), while the inhibition rates of psiUL54-2 and psiUL54-3 were 20.3 + 6.9% and 28.2 + 5.6%, respectively, and there was no significant difference compared with the control group (P0.05).
(5) according to the high inhibitory effect of psiUL54-1 on the expression of UL54 gene in vitro, and further to UL54 gene lentiviral expression vector was successfully constructed. The lentivirus vector and fusion protein expression vector pUL54-EGFP co transfection, lentiviral vector of PscSI-1 fusion protein fluorescence expression in transfected 24h had slight inhibition was found in the inverted fluorescence microscope. After transfection of 48h and 72h has obvious inhibitory effect, and lentiviral vector PscSI-2, PscSI-3 and PscSI-4 on the expression of fusion protein fluorescence at each time point had no obvious inhibitory effect of.Western Blot showed that PscSI-1 and fusion protein expression vector in both 1:2 and 1:1 ratio significantly inhibited PscSI-3 with slight inhibitory effect on the 1:1 ratio. While PscSI-2 and PscSI-4 in two the proportion had no obvious inhibitory effect.
conclusion
1. fusion protein expression vector pUL122-EGFP and pUL54-EGFP can successfully express the fusion protein of UL122-EGFP and UL54-EGFP, UL122 gene and UL54 gene expression in vitro, and the expression of fusion protein could emit green fluorescence. The fusion protein expression in 48 hours after transfection was very significant.
The target site of 2.UL122 gene, 618-638bp (psiUL122-1 and 1103-1123bp (psiUL122-2)), is an efficient target site for siRNA action. It is a potential target site for RNA interference and anti HCMV infection gene therapy. The target site of.UL122 gene 1414-1434bp (psiUL122-3) is not an efficient target site for the treatment of siRNA.
3. target sites of 1479-1497bp (psiUL54-1 and PscSI-1) is an efficient siRNA target sites, is the application of RNA interference potential target sites for gene therapy against HCMV infection. Target sites of 2419-2437bp (psiUL54-2), 2886-2904bp (psiUL54-3 and PscSI-2 target sites), target sites of 3058-3076bp (PscSI-3) and the target sites of 419-437bp (PscSI-4) is not high the siRNA target sites.
4. the inhibitory effect of 48h on the fusion protein was obvious after the transfection of the recombinant plasmid, but there was no obvious inhibitory effect on the transfection for 24 hours.
5. compared with plasmid vector, lentivirus vector is a kind of gene vector with high transfection rate, fast effect, persistent inhibition and high efficiency. It has important application value.
The 6. lentivirus vector and plasmid carrier are consistent in the screening of effective siRNA target.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

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