RNA干擾特異抑制單純皰疹病毒1型衣殼蛋白基因及US12基因的研究
發(fā)布時(shí)間：2018-07-29 14:09
【摘要】：目的：篩選高效沉默HSV-1病毒衣殼相關(guān)基因的siRNA;研究siRNA沉默目的基因后對HSV-Ⅰ感染與增殖的影響,為抗單純皰疹病毒藥物開發(fā)提供依據(jù)；探究衣殼蛋白與宿主細(xì)胞,衣殼蛋白與信號(hào)通路的相互關(guān)系；篩選高效沉默HSV-1 US12基因的siRNA,研究siRNA沉默US12基因后對HSV-Ⅰ增殖的影響。 方法：設(shè)計(jì)并化學(xué)合成針對7個(gè)HSV-1衣殼蛋白基因的siRNA,轉(zhuǎn)染Vero細(xì)胞,通過熒光定量PCR檢測相關(guān)siRNA對目的基因mRNA表達(dá)的抑制效果。空斑減數(shù)實(shí)驗(yàn)評價(jià)siRNA抑制目的基因?qū)SV-1感染與增殖的影響。形態(tài)學(xué)觀察siRNA抑制感染細(xì)胞病變的情況。透射電鏡觀察siRNA干擾對核內(nèi)病毒衣殼復(fù)制及出核的影響。構(gòu)建可以用于高效篩選目的基因及蛋白表達(dá)的pEGFP-N1-US12融合表達(dá)質(zhì)粒。采用QUICK技術(shù)研究VP5相互作用蛋白。 結(jié)果：共轉(zhuǎn)染實(shí)驗(yàn)篩選出高效抑制UL18、UL19、UL26、UL26.5、UL35、UL38mRNA表達(dá)的siRNA,但空斑減數(shù)實(shí)驗(yàn)發(fā)現(xiàn)只有針對UL18,UL19基因的siRNA對HSV-1的增殖有顯著抑制效果。siRNA干擾UL19基因能夠顯著抑制細(xì)胞病變情況。透射電鏡觀察發(fā)現(xiàn)干擾UL19基因可以降低核內(nèi)病毒衣殼數(shù)量,抑制病毒衣殼釋放。成功構(gòu)建可以用于高效篩選目的基因及蛋白表達(dá)的pEGFP-N1-US12融合表達(dá)質(zhì)粒。通過QUICK技術(shù)篩選出5個(gè)與VP5相互作用作用蛋白。 結(jié)論：設(shè)計(jì)合成的siRNA可以有效降低HSV-1衣殼蛋白各相關(guān)基因的表達(dá),針對UL18和UL19的siRNA能顯著地抑制病毒的感染與增殖,因此推測UL18基因與UL19基因是開發(fā)HSV-1抗病毒藥物的潛在靶點(diǎn)。RNA干擾US12基因能顯著降低US12的mRNA水平和融合蛋白表達(dá),但是對HSV-1的體外增殖無顯著影響,這表明US12表達(dá)的立即早期蛋白ICP47的功能可能與HSV-1體外增殖無直接相關(guān)。
[Abstract]:Objective: to screen siRNA for highly efficient silencing of HSV-1 virus capsid related genes, to study the effect of siRNA silent target gene on HSV- I infection and proliferation, to provide a basis for the development of anti herpes simplex virus drugs, to explore the relationship between capsid protein and host cells, capsid protein and signal pathway, and to screen the efficient and silent HSV-1 US12 gene. SiRNA, to study the effect of siRNA silencing US12 gene on the proliferation of HSV- I.
Methods: siRNA was designed and synthesized by chemical synthesis of 7 HSV-1 capsid protein genes, Vero cells were transfected and the inhibitory effect of related siRNA on the expression of mRNA was detected by fluorescence quantitative PCR. The effect of siRNA inhibition gene on HSV-1 infection and proliferation was evaluated by space spot subtraction. The effect of siRNA interference on the replication and nucleation of viral capsid in the nucleus was observed by transmission electron microscopy. The pEGFP-N1-US12 fusion expression plasmid which could be used to efficiently screen the expression of the target gene and protein was constructed. The QUICK technique was used to study the VP5 interaction protein.
Results: the siRNA of UL18, UL19, UL26, UL26.5, UL35, UL38mRNA expressed effectively was screened by CO transfection experiment, but only UL18, siRNA of UL19 gene had significant inhibitory effect on HSV-1 proliferation. In order to reduce the number of viral capsid and inhibit the release of viral capsid, the pEGFP-N1-US12 fusion expression plasmid, which can be used to efficiently screen the target gene and protein expression, was successfully constructed. 5 proteins interacting with VP5 were screened by QUICK technology.
Conclusion: the synthesized siRNA can effectively reduce the expression of HSV-1 capsid protein related genes, and the siRNA of UL18 and UL19 can significantly inhibit virus infection and proliferation. Therefore, it is speculated that the UL18 gene and UL19 gene are potential targets for developing HSV-1 antiviral drugs,.RNA interference US12 gene can significantly reduce US12 mRNA level and fusion. The protein expression has no significant effect on the proliferation of HSV-1 in vitro, which indicates that the function of the immediate early protein ICP47 expressed by US12 may not be directly related to the proliferation of HSV-1 in vitro.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373
本文編號(hào)：2152924
[Abstract]:Objective: to screen siRNA for highly efficient silencing of HSV-1 virus capsid related genes, to study the effect of siRNA silent target gene on HSV- I infection and proliferation, to provide a basis for the development of anti herpes simplex virus drugs, to explore the relationship between capsid protein and host cells, capsid protein and signal pathway, and to screen the efficient and silent HSV-1 US12 gene. SiRNA, to study the effect of siRNA silencing US12 gene on the proliferation of HSV- I.
Methods: siRNA was designed and synthesized by chemical synthesis of 7 HSV-1 capsid protein genes, Vero cells were transfected and the inhibitory effect of related siRNA on the expression of mRNA was detected by fluorescence quantitative PCR. The effect of siRNA inhibition gene on HSV-1 infection and proliferation was evaluated by space spot subtraction. The effect of siRNA interference on the replication and nucleation of viral capsid in the nucleus was observed by transmission electron microscopy. The pEGFP-N1-US12 fusion expression plasmid which could be used to efficiently screen the expression of the target gene and protein was constructed. The QUICK technique was used to study the VP5 interaction protein.
Results: the siRNA of UL18, UL19, UL26, UL26.5, UL35, UL38mRNA expressed effectively was screened by CO transfection experiment, but only UL18, siRNA of UL19 gene had significant inhibitory effect on HSV-1 proliferation. In order to reduce the number of viral capsid and inhibit the release of viral capsid, the pEGFP-N1-US12 fusion expression plasmid, which can be used to efficiently screen the target gene and protein expression, was successfully constructed. 5 proteins interacting with VP5 were screened by QUICK technology.
Conclusion: the synthesized siRNA can effectively reduce the expression of HSV-1 capsid protein related genes, and the siRNA of UL18 and UL19 can significantly inhibit virus infection and proliferation. Therefore, it is speculated that the UL18 gene and UL19 gene are potential targets for developing HSV-1 antiviral drugs,.RNA interference US12 gene can significantly reduce US12 mRNA level and fusion. The protein expression has no significant effect on the proliferation of HSV-1 in vitro, which indicates that the function of the immediate early protein ICP47 expressed by US12 may not be directly related to the proliferation of HSV-1 in vitro.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 朱欽昌;任哲;張春龍;張美英;廖紅娟;劉秋英;張佩琢;李久香;胡巢鳳;王華東;王一飛;;siRNA干擾HSV1 gD糖蛋白基因的研究[J];病毒學(xué)報(bào);2007年01期
2 洪敏;李衛(wèi)中;李琦涵;;單純皰疹病毒Ⅰ型免疫逃避機(jī)制——ICP47抑制抗原加工相關(guān)轉(zhuǎn)運(yùn)蛋白[J];病毒學(xué)報(bào);2007年01期
3 任哲;張美英;王一飛;朱欽昌;張春龍;陸大祥;胡巢鳳;李久香;張佩琢;楊志榮;;人HSV-1 UL40基因重組質(zhì)粒的構(gòu)建及其在siRNA篩選中的應(yīng)用[J];中國病理生理雜志;2007年12期
4 ;siRNA-mediated inhibition of HBV replication and expression[J];World Journal of Gastroenterology;2004年20期
，本文編號(hào)：2152924
本文鏈接：http://sikaile.net/xiyixuelunwen/2152924.html
最近更新
教材專著
