本文選題：人巨細(xì)胞病毒　切入點(diǎn)：UL122基因　出處：《浙江大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 目的 人巨細(xì)胞病毒(HCMV)可造成人體多系統(tǒng)多方面的嚴(yán)重?fù)p害,應(yīng)用RNA干擾技術(shù)抑制HCMV感染具有誘人的前景。HCMV UL122基因和UL54基因是HCMV復(fù)制和存活的必需基因,在HCMV與宿主細(xì)胞相互作用及抗HCMV藥物作用中起不可或缺的作用。但是目前對UL122基因和UL54基因的有效小干擾RNA(siRNA)作用靶位尚缺乏全面的認(rèn)識。因此有必要進(jìn)一步研究,以明確UL122基因和UL54基因的有效siRNA作用靶位,開發(fā)其潛在應(yīng)用價值,為應(yīng)用RNA干擾技術(shù)抑制HCMV感染奠定實驗基礎(chǔ)。 方法 本研究主要包括以下四大部分： (1)融合蛋白表達(dá)載體的構(gòu)建：設(shè)計引物應(yīng)用PCR法從HCMV AD 169株DNA中擴(kuò)增出UL122基因的優(yōu)勢表位相應(yīng)區(qū)域以及UL54基因的全長,并克隆入pEGFP-N1質(zhì)粒載體,構(gòu)建pUL122-EGFP、pUL54-EGFP融合蛋白表達(dá)載體,實現(xiàn)UL122基因和UL54基因的體外表達(dá)。 (2)短發(fā)夾結(jié)構(gòu)RNA (shRNA)表達(dá)載體的構(gòu)建：針對UL122及UL54基因設(shè)計siRNA作用靶位點(diǎn),合成相應(yīng)序列并克隆入pAVU6+27質(zhì)粒載體,構(gòu)建針對UL122和UL54基因的shRNA表達(dá)載體。 (3)篩選高效作用靶位：將shRNA表達(dá)載體和融合蛋白表達(dá)載體共轉(zhuǎn)染入AD293細(xì)胞,應(yīng)用實時熒光定量PCR、流式細(xì)胞檢測技術(shù)和倒置熒光顯微鏡從mRNA和蛋白質(zhì)水平篩選出理想、高效的siRNA作用靶位。 (4)慢病毒載體構(gòu)建及轉(zhuǎn)染：參照上述篩選出的高效siRNA作用靶位,構(gòu)建能夠表達(dá)shRNA的慢病毒載體,并將此慢病毒載體和融合蛋白表達(dá)載體轉(zhuǎn)染細(xì)胞,應(yīng)用流式和Western Blot法檢測shRNA的慢病毒表達(dá)載體對HCMV目標(biāo)基因體外表達(dá)的抑制作用。 結(jié)果 (1)本研究成功構(gòu)建融合蛋白表達(dá)載體pUL122-EGFP和pUL54-EGFP,實現(xiàn)UL122基因和UL54基因的體外表達(dá)。該融合蛋白表達(dá)載體在轉(zhuǎn)染后24小時和48小時均能表達(dá)綠色熒光,但是轉(zhuǎn)染后48小時表達(dá)的綠色熒光明顯強(qiáng)于轉(zhuǎn)染后24小時表達(dá)的綠色熒光。 (2)成功將一小段設(shè)計合成好的、具有形成shRNA能力的DNA序列插入pAVU6+27質(zhì)粒載體U6啟動子的下游,構(gòu)建成具有表達(dá)shRNA能力的重組質(zhì)粒載體,分別命名為psiUL122-1、psiUL122-2、psiUL122-3、psiUL54-1、psiUL54-2和psiUL54-3。 (3)將融合蛋白表達(dá)載體pUL122-EGFP分別與shRNA表達(dá)載體psiUL122-1、psiUL122-2和psiUL122-3共轉(zhuǎn)染。倒置熒光顯微鏡下發(fā)現(xiàn)轉(zhuǎn)染后24h重組質(zhì)粒載體psiUL122-1、psiUL122-2和psiUL122-3對融合蛋白的表達(dá)無明顯抑制作用,轉(zhuǎn)染后48hpsiUL122-1和psiUL122-2對融合蛋白熒光表達(dá)具有明顯抑制作用,而psiUL122-3對融合蛋白熒光表達(dá)只有輕度抑制作用。流式(FCM)結(jié)果顯示轉(zhuǎn)染后48hpsiUL122-1、psiUL122-2和psiUL122-3對融合蛋白的抑制率分別為82.0±1.0%、79.5±2.5%和53.5±2.5%,與對照組相比差異有統(tǒng)計學(xué)意義(p0.05),且psiUL122-1和psiUL122-2對pUL122-EGFP的抑制效果較psiUL122-3明顯。熒光定量PCR結(jié)果顯示psiUL122-1、psiUL122-2和psiUL122-3對融合蛋白mRNA的抑制率分別為97.3±0.6%、98.0±0.1%和90.0±3.5%。 (4)將融合蛋白表達(dá)載體pUL54-EGFP分別與shRNA表達(dá)載體psiUL54-1、psiUL54-2和psiUL54-3共轉(zhuǎn)染。倒置熒光顯微鏡下發(fā)現(xiàn)轉(zhuǎn)染后24h重組質(zhì)粒載體psiUL54-1、psiUL54-2和psiUL54-3對融合蛋白的表達(dá)無明顯抑制作用,轉(zhuǎn)染后48h psiUL54-1對融合蛋白熒光表達(dá)具有明顯抑制作用,而psiUL54-2和psiUL54-3對融合蛋白表達(dá)只有輕微抑制作用。流式結(jié)果顯示轉(zhuǎn)染后48hpsiUL54-1對融合蛋白的抑制率為85.4±1.2%,與對照組相比具有顯著差異(p0.05),而psiUL54-2和psiUL54-3對融合蛋白的抑制率分別為14.9±2.9%和20.4±6.2%,與對照組相比沒有顯著差異(p0.05)。熒光定量PCR結(jié)果顯示psiUL54-1對pUL54-EGFP融合蛋白基因mRNA的抑制率為97.4±0.7%,與對照組相比差異有統(tǒng)計學(xué)意義(p0.05),而psiUL54-2和psiUL54-3的抑制率分別為20.3±6.9%和28.2±5.6%，，與對照組相比無顯著差異(p0.05)。 (5)參照psiUL54-1對UL54基因體外表達(dá)的高效抑制作用,進(jìn)一步成功構(gòu)建針對UL54基因的慢病毒表達(dá)載體。將慢病毒載體與融合蛋白表達(dá)載體pUL54-EGFP共轉(zhuǎn)染,倒置熒光顯微鏡下發(fā)現(xiàn)慢病毒載體PscSI-1對融合蛋白熒光表達(dá)在轉(zhuǎn)染后24h有輕度抑制作用,在轉(zhuǎn)染后48h和72h具有明顯抑制作用,而慢病毒載體PscSI-2、PscSI-3和PscSI-4對融合蛋白熒光表達(dá)在各時間點(diǎn)均無明顯抑制作用。Western Blot檢測顯示PscSI-1與融合蛋白表達(dá)載體在1：2和1：1比例下均有明顯抑制作用,PscSI-3在1：1比例下有輕度抑制作用,而PscSI-2和PscSI-4在兩種比例下均無明顯抑制作用。 結(jié)論 1.融合蛋白表達(dá)載體pUL122-EGFP和pUL54-EGFP能夠成功表達(dá)融合蛋白UL122-EGFP和UL54-EGFP,實現(xiàn)UL122基因和UL54基因的體外表達(dá),又能通過融合蛋白表達(dá)后仍能發(fā)出綠色熒光。融合蛋白的表達(dá)在轉(zhuǎn)染后48小時較為明顯。 2.UL122基因的靶位點(diǎn)618-638bp (psiUL122-1和1103-1123bp(psiUL122-2)是高效的siRNA作用靶位點(diǎn),是應(yīng)用RNA干擾抗HCMV感染基因治療的潛在靶位點(diǎn)。UL122基因的靶位點(diǎn)1414-1434bp(psiUL122-3)不是高效的siRNA作用靶位點(diǎn)。 3.靶位點(diǎn)1479-1497bp (psiUL54-1和PscSI-1)是一個高效的siRNA作用靶位點(diǎn),是應(yīng)用RNA干擾抗HCMV感染基因治療的潛在靶位點(diǎn)。靶位點(diǎn)2419-2437bp(psiUL54-2)、靶位點(diǎn)2886-2904bp(psiUL54-3和PscSI-2)、靶位點(diǎn)3058-3076bp (PscSI-3)和靶位點(diǎn)419-437bp(PscSI-4)不是高效的siRNA作用靶位點(diǎn)。 4.重組干擾質(zhì)粒轉(zhuǎn)染后48h對融合蛋白的抑制作用較明顯,但轉(zhuǎn)染24小時無明顯抑制效果。 5.與質(zhì)粒載體相比,慢病毒載體是一種轉(zhuǎn)染率高、起效快、抑制作用持久且高效的基因載體,具有重要的應(yīng)用價值。 6.慢病毒載體與質(zhì)粒載體在篩選有效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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346

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