本文選題：單純皰疹病毒1型 + 即早蛋白4��； 參考：《鄭州大學》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,具有親神經的特性,在人群中感染廣泛。HSV-1感染外周神經后可逆行跨越神經元進入中樞神經系統(tǒng)并建立潛伏感染,病毒裂解后引起中樞神經系統(tǒng)嚴重損傷。HSV-1基因組表達有很高時序性,分即早基因(IE),早期基因(E)和晚期基因(L)。作為IE基因產物之一的即早蛋白ICP4可刺激E基因表達和DNA合成,誘導L基因的表達,是HSV-1基因表達從而引發(fā)感染的關鍵激活因子。因此,有效抑制ICP4的表達可以通過阻止HSV-1的復制達到治療HSV-1感染性疾病的目的。RNA干擾(RNAi)是一種細胞本身固有的對抗外源基因侵犯的自我保護現(xiàn)象,雙鏈RNA(dsRNA)分子在mRNA水平關閉相應序列及基因的表達使其沉默。在此基礎上建立的RNAi技術不僅僅是一種經濟、快捷、高效的抑制基因表達的技術手段,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。 本實驗構建表達ICP4 siRNA (小干擾RNA)的重組真核慢病毒質粒PLKO.1-puro-ICP4-siRNA,將產生的重組病毒顆粒感染Vero細胞,建立穩(wěn)定表達ICP4 siRNA的重組Vero-ICP4-siRNA細胞系；進一步探討干擾HSV-1 ICP4基因后對HSV-1復制能力的影響,為臨床上運用RNAi治療HSV-1感染性疾病提供思路和實驗依據。 目的： 1建立穩(wěn)定表達ICP4特異性siRNA的重組細胞系。 2初步分析靶向ICP4的siRNA對HSV-1在Vero細胞中復制能力的影響。 方法： 1基因工程技術構建重組慢病毒質粒PLKO.1-puro-ICP4-siRNA,雙酶切及測序鑒定。 2三質粒共轉染293T細胞產生重組慢病毒顆粒。 3重組病毒顆粒感染Vero細胞,嘌呤霉素篩選法建立ICP4-siRNA表達陽性的重組細胞系。 4采用Realtime PCR和Western blot鑒定重組Vero-ICP4-siRNA細胞系。 5通過觀察HSV感染重組Vero細胞后CPE(細胞病變效應)表達和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病毒復制能力的影響。 結果： 1成功構建重組真核慢病毒質粒PLKO.1-puro-ICP4-siRNA。 2獲得有效濃度的穩(wěn)定表達ICP4的siRNAs的重組慢病毒顆粒。 3成功建立穩(wěn)定表達ICP4特異性siRNA的重組細胞系。 4在mRNA和蛋白水平證實重組Vero-ICP4-siRNA細胞中的HSV-1 ICP4表達被抑制。 5感染野生型HSV-1后,siRNA組Vero細胞的CPE表達率與陰性對照組比較有明顯降低。進一步采用TCID50法測定病毒滴度并對TCID值進行AVONA統(tǒng)計學分析發(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；且針對兩個靶點的siRNA聯(lián)合組HSV-1病毒滴度低于任何單一抑制組(P0.001)。 結論： 1成功建立穩(wěn)定表達抗ICP4 siRNA的重組Vero細胞系。 2 SiRNA能夠有效地干擾HSV-1 ICP4基因表達,且不同位點聯(lián)合干擾對HSV-1 ICP4基因的表達有協(xié)同的抑制效果,進而對HSV-1病毒復制有協(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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R346

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