【摘要】 A型流感病毒(IAV)在宿主细胞内复制过程中需要许多宿主因子的参与，同时IAV也能调控多种宿主因子的表达。为寻找调控IAV复制的宿主因子，本研究通过SDS-PAGE电泳检测发现H1N1亚型流感病毒A/WSN/1933株(简写为WSN)感染A549细胞会上调表达55 ku左右的特异蛋白条带(感染后9 h)，将该55 ku左右的蛋白条带切胶回收后进行质谱分析，根据质谱分析的筛选标准选择蛋白得分(Mass score)较高、谱图数(Matches)和序列数(Sequences)较为可信的宿主因子DBNL作为调控IAV复制的潜在研究对象进一步研究。本研究针对DBNL设计特异的siRNA和对照Scrambled si RNA，将其分别转染A549细胞，通过荧光定量PCR确定DBNL si RNA在转录水平的干扰效果显著；利用细胞活力测定试验确定DBNL si RNA的干扰不影响细胞活力。利用DBNL si RNA下调表达A549细胞中的DBNL后，以WSN感染该细胞，在感染后24 h、48 h分别收集细胞上清，利用MDCK细胞进行噬斑滴定试验。试验结果显示，与对照组相比，下调表达DBNL后IAV的滴度极显著降低(P<0.01)，表明下调表达DBNL能够抑制A549细胞中的IAV复制。利用si RNA下调表达A549细胞中的DBNL后，以WSN感染A549细胞，采用激光共聚焦试验鉴定IAV NP蛋白的表达与定位情况。结果显示，在病毒感染后6 h，对照组中红色荧光集中分布在细胞质中，而DBNL siRNA组中红色荧光在细胞核与细胞质中均有分布，表明下调表达DBNL能够抑制细胞中IAV NP蛋白的出核。反之，通过转染过表达DBNL的重组质粒，以WSN感染A549细胞，采用激光共聚焦试验鉴定IAV NP蛋白的表达与定位情况。结果显示，在病毒感染后4 h，转染p CAGGS的对照组中红色荧光主要分布在细胞核中，而过表达DBNL组中的红色荧光在细胞核与细胞质中均有分布，表明过表达DBNL能够促进细胞中IAV NP蛋白的出核。以上研究结果证实DBNL正调控IAV的复制与其NP蛋白的出核。本研究丰富了DBNL功能的多样性，为宿主因子调控IAV复制的研究提供参考数据。更多还原

【Abstract】 Replication of influenza A virus(IAV) in host cells requires the involvement of numerous host infectors whose expression is in turn regulated by virus infection. To search for host factors that regulate influenza virus replication, we found that influenza virus infection upregulated the expression of specific proteins with a band of around 55ku in A549 cells 9 hours after infection. After SDS-PAGE, the protein band of approximately 55ku was extracted for mass spectrometry analysis. According to the screening criteria for mass spectrometry analysis, the host factor DBNL with a high mass score, relatively reliable Matches and Sequences was selected as a potential research object of regulating IAV replication for further research. In this study, the specific si RNA and control Scrambled si RNA for DBNL were designed, and transfected into A549 cells, respectively. The transcription level of DBNL was detected by relative fluorescent quantitative PCR. The results showed that compared with the control group,DBNL si RNA significantly decreased the transcription level of DBNL in A549 cells, and DBNL si RNA interference had no significant impact on cell viability. The results indicated that DBNL si RNA could downregulate the expression of DBNL without impairing cell viability. After knocking down the expression of DBNL by si RNA, A549 cells were infected with H1N1 influenza virus, and the cell supernatant was collected at 24 hours and 48 hours post infection and subjected to plaque titration using MDCK cells. Plaque titration test showed that the titer of IAV significantly decreased after down-regulation of DBNL compared to the control group(P<0.01). The results indicated that downregulating the expression of DBNL inhibited IAV replication. After knocking down the expression of DBNL using si RNA, we use indirect immunofluorescence assay(IFA) to observe the expression and localization of IAV NP protein. The results showed that the red fluorescence was concentrated in the cytoplasm in the control group, while distributed in both the nucleus and cytoplasm in the DBNL interference group at 6 hours post virus infection. The results indicated that downregulation of DBNL expression inhibited NP protein export from the nucleus. Similarly, A549 cells were transfected with the DBNL recombinant plasmid, and then infected with IAV, and the expression and localization of IAV NP protein were observed using IFA. The results showed that at 4 hours after virus infection, red fluorescence was mainly distributed in the nucleus in the control group, while in the DBNL overexpression group, red fluorescence was distributed in both the nucleus and cytoplasm. The results indicated that overexpression of DBNL could promote the export of IAV NP protein from nucleus.Together, the above results demonstrated that DBNL promotes the nuclear export of IAV NP, thereby positively regulates the replication of IAV. This study enriched the functional diversity of DBNL and could provide reference for the study of host factors regulating IAV replication.更多还原