【摘要】：目的 研究外部引導(dǎo)序列(External Guide Sequences,EGS)對(duì)人巨細(xì)胞病毒(Human Cytomegalovirus, HCMV)臨床病毒株UL148基因表達(dá)的抑制作用,為探討UL148基因功能奠定基礎(chǔ),為抗HCMV治療尋找新的治療途徑。 方法 細(xì)胞外實(shí)驗(yàn):1.根據(jù)靶基因UL148序列特征設(shè)計(jì)并合成DNA性質(zhì)EGS;2.從HCMV臨床病毒株基因組中擴(kuò)增UL148基因,克隆入pGEM-T-EASY質(zhì)粒, PCR擴(kuò)增UL148小片段,制備底物轉(zhuǎn)錄模板,參入32P-UTP后體外轉(zhuǎn)錄;3.從大腸桿菌DH5α基因組擴(kuò)增M1RNA基因,將其克隆入pUC18質(zhì)粒,線(xiàn)性化后,體外合成M1RNA;4.將底物UL148 RNA、EGS、M1RNA混合于切割反應(yīng)液,37℃反應(yīng)1h,以聚丙烯酰胺凝膠分離產(chǎn)物,應(yīng)用Typhoons掃描儀分析切割結(jié)果。 細(xì)胞內(nèi)實(shí)驗(yàn):將UL148基因克隆入pEGFP-N1質(zhì)粒,重組質(zhì)粒轉(zhuǎn)染HeLa細(xì)胞,同時(shí)以pEGFP-N1質(zhì)粒轉(zhuǎn)染HeLa細(xì)胞作為陰性對(duì)照,均以1mg/mL G418篩選4周。選取細(xì)胞外實(shí)驗(yàn)證實(shí)有切割作用的EGS4,分別以50nM、100nM、200nM、400nM終濃度轉(zhuǎn)染G418篩選后的細(xì)胞。應(yīng)用熒光顯微鏡觀(guān)察上述細(xì)胞熒光量的變化;應(yīng)用熒光定量PCR進(jìn)行絕對(duì)定量,分析EGS4對(duì)UL148表達(dá)量的沉默效果。 結(jié)果 細(xì)胞外實(shí)驗(yàn):1.共設(shè)計(jì)5條EGS;2.成功構(gòu)建pT148重組質(zhì)粒,PCR后擴(kuò)增獲得UL148小片段轉(zhuǎn)錄模板,經(jīng)體外轉(zhuǎn)錄獲得32P標(biāo)記的UL148小片段RNA;3.成功構(gòu)建pUC-M1重組質(zhì)粒,線(xiàn)性化后,體外轉(zhuǎn)錄合成M1RNA;4.在細(xì)胞外進(jìn)行切割實(shí)驗(yàn),篩選出多條具有特異性切割UL148 RNA的EGS。 細(xì)胞內(nèi)實(shí)驗(yàn):成功構(gòu)建p148-EGFP重組質(zhì)粒;重組質(zhì)粒p148-EGFP及pEGFP-N1質(zhì)粒分別轉(zhuǎn)染HeLa細(xì)胞,經(jīng)G418篩選后,成功構(gòu)建穩(wěn)定表達(dá)UL148基因的HeLa細(xì)胞系(UL148-HeLa)和穩(wěn)定表達(dá)EGFP的HeLa細(xì)胞系(EGFP-HeLa)。熒光顯微鏡觀(guān)察50nM、100nM、200nM、400nM的EGS4作用后,UL148-HeLa細(xì)胞的熒光量明顯減少,而EGFP-HeLa細(xì)胞熒光量無(wú)明顯改變。熒光定量PCR分析顯示50nM、100nM、200nM、400nM EGS4對(duì)UL148的抑制效率分別為74.9 %(P0.05)、78.6%(P0.05)、81.1%(P0.05)、87.0%(P0.05)。 結(jié)論 1.經(jīng)細(xì)胞外實(shí)驗(yàn)篩選出2條具有特異性切割UL148 RNA作用的EGS,分別為EGS1和EGS4; 2.在細(xì)胞內(nèi)EGS4能有效沉默HCMV UL148基因的表達(dá),并隨濃度的增加,沉默效果逐漸明顯; 3.細(xì)胞外EGS切割實(shí)驗(yàn)系統(tǒng)可作為細(xì)胞內(nèi)切割實(shí)驗(yàn)前的一種有效篩選途徑; 4.經(jīng)細(xì)胞外和細(xì)胞內(nèi)切割實(shí)驗(yàn)篩選出的有效EGS,可用于研究UL148的基因功能,并且有望發(fā)展成為抗HCMV的小分子核酸藥物。
[Abstract]:Aim to study the inhibitory effect of (External Guide Sequences,EGS on the expression of UL148 gene in human cytomegalovirus (Human Cytomegalovirus, HCMV) clinical strain, to lay a foundation for exploring the function of UL148 gene and to find a new therapeutic way for anti-HCMV therapy. Methods: 1. Design and Synthesis of DNA EGS;2. based on the characteristics of UL148 sequence of Target Gene The UL148 gene was amplified from the genome of HCMV clinical virus strain, cloned into pGEM-T-EASY plasmid, UL148 small fragment was amplified by PCR, substrate transcription template was prepared and transcribed in vitro after incorporation into 32P-UTP. M1RNA gene was amplified from Escherichia coli DH5 偽 genome and cloned into pUC18 plasmid. After linearization, M1RNA4 was synthesized in vitro. The substrate UL148 RNA,EGS,M1RNA was mixed in the cutting solution and reacted at 37 鈩，

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