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论文摘要目录： |
| 关键词：医药学论文 学术论文 |
【关键词】 正粘病毒科
RNA interference mediated inhibition of influenza virus NP and PA gene expressions and multiplication in MDCK cells
【Abstract】 AIM: To apply the small interfering RNAs targeting NP or/and PA gene of influenza virus to inhibit the expression of NP or/and PA and multiplication of influenza virus in MadinDarby canine kidney (MDCK) cells. METHODS: The recombinant plasmids, pEGFP/NP, pGenesil/PA and pEGFP/NP+ PA were constructed and transfected into MDCK cells. The transfected MDCK cells were infected with subtype H5N1 strain. The transfection efficiency was determined using fluorescence microscopy. The expression levels of NP gene were determined by Western blot and the transcriptions of NP or/and PA gene were detected by semiquantitative RTPCR. The culture supernatants were assayed for hemagglutination activity (HA) at different time points after infection in order to determine the effect of the siRNA on the multiplication of infectious virus. RESULTS: The results from fluorescence microscopy suggested that the transfection efficiency was about 65.0%. The introductions of plasmids pEGFP/ NP and pEGFP/NP+PA showed efficient in specifically inhibiting the expression of NP according to the results of Western blot, with inhibitory rates of 64.5% and 69.6%. Semiquantitative RTPCR showed that the transcription of NP gene was reduced by nearly 66.0% in infected MDCK cells after transfected by pEGFP/NP, and PA gene was reduced by nearly 63.0% in infected MDCK 无忧论文 【http://www.uklunwen.com】cells after transfected by pGenesil/PA. The transcriptions of NP and PA genes were reduced by nearly 71.4% and 69.3% synchronously in infected MDCK cells transfected by pEGFP/NP+PA. In contrast, the control plasmid did exhibit no inhibitory effect on the protein expressions and the transcription of NP or/and PA gene. The inhibitory rates of influenza virus in MDCK cells transfected with pEGFP/ NP, pGenesil/PA and pEGFP/NP+PA were 87.0%, 75.0% and 96.9% respectively according to the HA results, which demonstrated that the inhibitory ability of pEGFP/NP+PA was the strongest among the various groups. CONCLUSION: The small interfering RNAs targeting NP or/and PA gene shows a dramatic inhibition on protein expression, RNA transcription and multiplication of influenza virus in MDCK cells.
【Keywords】 RNA interferece; orthomyxoviridae; MDCK cell line; NP gene; PA gene
【摘要】 目的： 应用RNA干扰技术（RNAi）研究针对流感病毒NP或/和PA基因的siRNA抑制NP或/和PA基因的表达及抑制流感病毒在MDCK细胞中的增殖. 方法： 分别构建针对NP，PA及同时干扰NP和PA的三种siRNA表达质粒pEGFP/NP，pGenesil/PA和pEGFP/NP+PA，转染MDCK后，H5N1亚型流感病毒感染细胞，荧光显微镜判断转染效率，蛋白质印迹和半定量RTPCR检测NP及PA蛋白表达及基因转录水平的变化，并在不同时间点检测细胞培养上清中的血凝值（HA），观察siRNA抑制流感病毒增殖的能力. 结果： 荧光显微镜结果表明，重组质粒转染效率达65.0%. 蛋白质印迹结果表明，重组质粒pEGFP/NP和pEGFP/NP+PA均能抑制NP蛋白在MDCK细胞内的表达，两者的抑制率分别为64.5%和69.6%. 半定量RTPCR检测到pEGFP/NP质粒在MDCK细胞内对NP基因的转录抑制率为66.0%；pGenesil/PA质粒对PA基因的转录抑制率为63.0%；pEGFP/NP+PA质粒对NP和PA基因的转录同时抑制率，分别为71.4%和69.3%. 而对照质粒pEGFP/HK对NP或/和PA基因的蛋白表达及转录均没有抑制作用. HA结果表明，三种质粒均能抑制流感病毒在MDCK细胞中的增殖，以pEGFP6/NP+PA的作用最为显著，它们抑制流感病毒增殖的能力分别为87.0 |
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