本文選題：單純皰疹病毒1型 + 即早蛋白4�。� 參考：《鄭州大學(xué)》2010年碩士論文

【摘要】： 單純皰疹病毒1型(HSV-1)為雙鏈包膜DNA病毒,具有親神經(jīng)的特性,在人群中感染廣泛。HSV-1感染外周神經(jīng)后可逆行跨越神經(jīng)元進(jìn)入中樞神經(jīng)系統(tǒng)并建立潛伏感染,病毒裂解后引起中樞神經(jīng)系統(tǒng)嚴(yán)重?fù)p傷。HSV-1基因組表達(dá)有很高時序性,分即早基因(IE),早期基因(E)和晚期基因(L)。作為IE基因產(chǎn)物之一的即早蛋白ICP4可刺激E基因表達(dá)和DNA合成,誘導(dǎo)L基因的表達(dá),是HSV-1基因表達(dá)從而引發(fā)感染的關(guān)鍵激活因子。因此,有效抑制ICP4的表達(dá)可以通過阻止HSV-1的復(fù)制達(dá)到治療HSV-1感染性疾病的目的。RNA干擾(RNAi)是一種細(xì)胞本身固有的對抗外源基因侵犯的自我保護(hù)現(xiàn)象,雙鏈RNA(dsRNA)分子在mRNA水平關(guān)閉相應(yīng)序列及基因的表達(dá)使其沉默。在此基礎(chǔ)上建立的RNAi技術(shù)不僅僅是一種經(jīng)濟(jì)、快捷、高效的抑制基因表達(dá)的技術(shù)手段,更為研究者在基因功能測定和基因治療等方面開辟了新思路。 本實(shí)驗(yàn)構(gòu)建表達(dá)ICP4 siRNA (小干擾RNA)的重組真核慢病毒質(zhì)粒PLKO.1-puro-ICP4-siRNA,將產(chǎn)生的重組病毒顆粒感染Vero細(xì)胞,建立穩(wěn)定表達(dá)ICP4 siRNA的重組Vero-ICP4-siRNA細(xì)胞系；進(jìn)一步探討干擾HSV-1 ICP4基因后對HSV-1復(fù)制能力的影響,為臨床上運(yùn)用RNAi治療HSV-1感染性疾病提供思路和實(shí)驗(yàn)依據(jù)。 目的： 1建立穩(wěn)定表達(dá)ICP4特異性siRNA的重組細(xì)胞系。 2初步分析靶向ICP4的siRNA對HSV-1在Vero細(xì)胞中復(fù)制能力的影響。 方法： 1基因工程技術(shù)構(gòu)建重組慢病毒質(zhì)粒PLKO.1-puro-ICP4-siRNA,雙酶切及測序鑒定。 2三質(zhì)粒共轉(zhuǎn)染293T細(xì)胞產(chǎn)生重組慢病毒顆粒。 3重組病毒顆粒感染Vero細(xì)胞,嘌呤霉素篩選法建立ICP4-siRNA表達(dá)陽性的重組細(xì)胞系。 4采用Realtime PCR和Western blot鑒定重組Vero-ICP4-siRNA細(xì)胞系。 5通過觀察HSV感染重組Vero細(xì)胞后CPE(細(xì)胞病變效應(yīng))表達(dá)和TCID50(半數(shù)組織培養(yǎng)感染劑量)法測定HSV-1滴度,檢測靶向ICP4的siRNAs對HSV-1病毒復(fù)制能力的影響。 結(jié)果： 1成功構(gòu)建重組真核慢病毒質(zhì)粒PLKO.1-puro-ICP4-siRNA。 2獲得有效濃度的穩(wěn)定表達(dá)ICP4的siRNAs的重組慢病毒顆粒。 3成功建立穩(wěn)定表達(dá)ICP4特異性siRNA的重組細(xì)胞系。 4在mRNA和蛋白水平證實(shí)重組Vero-ICP4-siRNA細(xì)胞中的HSV-1 ICP4表達(dá)被抑制。 5感染野生型HSV-1后,siRNA組Vero細(xì)胞的CPE表達(dá)率與陰性對照組比較有明顯降低。進(jìn)一步采用TCID50法測定病毒滴度并對TCID值進(jìn)行AVONA統(tǒng)計(jì)學(xué)分析發(fā)現(xiàn),siRNA組HSV滴度與對照組存在顯著差異(P0.001), siRNA組低于對照組；且針對兩個靶點(diǎn)的siRNA聯(lián)合組HSV-1病毒滴度低于任何單一抑制組(P0.001)。 結(jié)論： 1成功建立穩(wěn)定表達(dá)抗ICP4 siRNA的重組Vero細(xì)胞系。 2 SiRNA能夠有效地干擾HSV-1 ICP4基因表達(dá),且不同位點(diǎn)聯(lián)合干擾對HSV-1 ICP4基因的表達(dá)有協(xié)同的抑制效果,進(jìn)而對HSV-1病毒復(fù)制有協(xié)同抑制效果。
[Abstract]:Herpes simplex virus type 1 (HSV-1) is a double-strand envelope DNA virus, which has the characteristics of neurobiosis. HSV-1 can retrograde into the central nervous system (CNS) and establish latent infection after infection of peripheral nerves with HSV-1. The genome expression of HSV-1 was highly sequential, I. E. early gene (IE), early gene (E) and late gene (L). ICP4, one of the products of IE gene, can stimulate the expression of E gene and DNA synthesis, induce the expression of L gene, and is the key activator of HSV-1 gene expression. Therefore, the effective inhibition of ICP4 expression can be used to treat HSV-1 infectious diseases by preventing HSV-1 replication. RNA interference (RNAi) is an inherent self-protection phenomenon of cells against foreign gene invasion. Double stranded RNA (dsRNA) silences the sequence and gene expression at the mRNA level. The RNAi technology established on this basis is not only an economical, fast and efficient technique to inhibit gene expression, but also opens up new ideas for gene function determination and gene therapy. In this experiment, the recombinant eukaryotic lentivirus plasmid PLKO.1-puro-ICP4-siRNAs expressing ICP4 siRNA was constructed. The recombinant virus particles were infected into Vero cells, and a recombinant Vero-ICP4-siRNA cell line was established which expressed ICP4 siRNA stably. To further investigate the effect of interference with HSV-1 ICP4 gene on HSV-1 replication ability, and to provide an experimental basis for the clinical treatment of HSV-1 infectious diseases with RNAi. Aim: 1 to establish a recombinant cell line stably expressing ICP4 specific siRNA. 2 to analyze the effect of ICP4 targeting siRNA on HSV-1 replication in Vero cells. Methods: 1Recombinant lentivirus plasmids PLKO.1-puro-ICP4-siRNAs were constructed by genetic engineering technique and identified by double enzyme digestion and sequencing. 2Recombinant lentivirus particles were produced by co-transfection of three plasmids into 293T cells. A recombinant cell line with positive expression of ICP4-siRNA was established by purine mycin screening. 4 Recombinant Vero-ICP4-siRNA cell line was identified by Realtime PCR and Western blot. 5 CPE (fine) after HSV infection of recombinant Vero cells was observed. The expression of cytopathic effect and the titer of HSV-1 were measured by TCID50 method. The effect of siRNAs targeting ICP4 on the replication ability of HSV-1 virus was detected. Results: 1Recombinant lentivirus particles were successfully constructed into recombinant eukaryotic lentivirus plasmid PLKO.1-puro-ICP4-siRNA.2 with stable concentration of siRNAs expressing ICP4. 3 To establish a recombinant cell line stably expressing ICP4-specific siRNA. 4 the expression of HSV-1 ICP4 in recombinant Vero-ICP4-siRNA cells was confirmed at mRNA and protein levels. 5 Vero of siRNA group after infection with wild-type HSV-1 was confirmed by inhibition of HSV-1 ICP4 expression in recombinant Vero-ICP4-siRNA cells. The expression rate of CPE in the cells was significantly lower than that in the negative control group. The titer of HSV in siRNA group was significantly higher than that in control group (P0.001), and the titer of HSV in siRNA group was lower than that in control group (P0.001). The titer of HSV-1 virus in siRNA combination group was lower than that in any single inhibition group (P0.001). Conclusion: 1Recombinant Vero cell line with stable expression of anti-ICP4 siRNA was successfully established. 2SiRNA could effectively interfere with the expression of HSV-1 ICP4 gene. The co-interference of different sites had synergistic inhibitory effect on HSV-1 ICP4 gene expression and then on HSV-1 virus replication.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

【共引文獻(xiàn)】

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