应用EGS基因沉默技术研究人巨细胞病毒临床病毒株UL148基因功能
发布时间：2019-03-19 14:37
【摘要】：目的 研究外部引导序列(External Guide Sequences,EGS)对人巨细胞病毒(Human Cytomegalovirus, HCMV)临床病毒株UL148基因表达的抑制作用,为探讨UL148基因功能奠定基础,为抗HCMV治疗寻找新的治疗途径。 方法 细胞外实验:1.根据靶基因UL148序列特征设计并合成DNA性质EGS;2.从HCMV临床病毒株基因组中扩增UL148基因,克隆入pGEM-T-EASY质粒, PCR扩增UL148小片段,制备底物转录模板,参入32P-UTP后体外转录;3.从大肠杆菌DH5α基因组扩增M1RNA基因,将其克隆入pUC18质粒,线性化后,体外合成M1RNA;4.将底物UL148 RNA、EGS、M1RNA混合于切割反应液,37℃反应1h,以聚丙烯酰胺凝胶分离产物,应用Typhoons扫描仪分析切割结果。 细胞内实验:将UL148基因克隆入pEGFP-N1质粒,重组质粒转染HeLa细胞,同时以pEGFP-N1质粒转染HeLa细胞作为阴性对照,均以1mg/mL G418筛选4周。选取细胞外实验证实有切割作用的EGS4,分别以50nM、100nM、200nM、400nM终浓度转染G418筛选后的细胞。应用荧光显微镜观察上述细胞荧光量的变化;应用荧光定量PCR进行绝对定量,分析EGS4对UL148表达量的沉默效果。 结果 细胞外实验:1.共设计5条EGS;2.成功构建pT148重组质粒,PCR后扩增获得UL148小片段转录模板,经体外转录获得32P标记的UL148小片段RNA;3.成功构建pUC-M1重组质粒,线性化后,体外转录合成M1RNA;4.在细胞外进行切割实验,筛选出多条具有特异性切割UL148 RNA的EGS。 细胞内实验:成功构建p148-EGFP重组质粒;重组质粒p148-EGFP及pEGFP-N1质粒分别转染HeLa细胞,经G418筛选后,成功构建稳定表达UL148基因的HeLa细胞系(UL148-HeLa)和稳定表达EGFP的HeLa细胞系(EGFP-HeLa)。荧光显微镜观察50nM、100nM、200nM、400nM的EGS4作用后,UL148-HeLa细胞的荧光量明显减少,而EGFP-HeLa细胞荧光量无明显改变。荧光定量PCR分析显示50nM、100nM、200nM、400nM EGS4对UL148的抑制效率分别为74.9 %(P0.05)、78.6%(P0.05)、81.1%(P0.05)、87.0%(P0.05)。 结论 1.经细胞外实验筛选出2条具有特异性切割UL148 RNA作用的EGS,分别为EGS1和EGS4; 2.在细胞内EGS4能有效沉默HCMV UL148基因的表达,并随浓度的增加,沉默效果逐渐明显; 3.细胞外EGS切割实验系统可作为细胞内切割实验前的一种有效筛选途径; 4.经细胞外和细胞内切割实验筛选出的有效EGS,可用于研究UL148的基因功能,并且有望发展成为抗HCMV的小分子核酸药物。
[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 伪 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，
本文编号：2443617
[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 伪 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，
本文编号：2443617
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