摘要
为探究Rab18调控禽偏肺病毒C型(aMPV/C)复制的分子机制,将aMPV/C感染A549细胞后进行激光共聚焦显微镜观察并用Image J软件分析共定位系数。结果显示,Rab18与aMPV/C N蛋白均在细胞质中表达并存在共定位,而未接毒组无明显的N蛋白荧光信号,接毒组共定位系数极显著高于未接毒组(P<0.01)。将pCMV-Flag-Rab18、pCMV-Flag、siRab18以及siNC分别转染A549细胞,接种aMPV/C后收集细胞和细胞上清,分别用qPCR、Western-blot以及TCID50检测aMPV/C N基因的转录水平、N蛋白的表达水平以及病毒效价。结果显示,过表达Rab18后aMPV/C N基因的转录水平、N蛋白的表达水平以及病毒效价均极显著升高(P<0.01),而干扰Rab18表达后N基因的转录水平、N蛋白的表达水平以及病毒效价均极显著降低(P<0.01)。将pCMV-mcherry-Rab18分别与可融合表达GFP蛋白的aMPV/C各基因的质粒共转染A549细胞后进行激光共聚焦显微镜观察。结果显示,Rab18均可与aMPV/C各蛋白在细胞质中发生共定位。将pCMV-Flag-Rab18分别与可融合表达GFP蛋白的aMPV/C各基因的质粒共转染HKE293T细胞后进行免疫共沉淀试验。结果显示,Rab18与aMPV/C N、M2-1和L2共表达的IP样品中出现特异性条带,其他蛋白无特异性条带。这些结果表明,Rab18通过与N、M2-1和L2蛋白的互作影响aMPV/C的复制。研究结果为进一步深入解析Rab18调控aMPV/C复制的分子机理奠定了基础。
This work aimed to investigate how Rab18 influences the replication of avian metapneu-movirus type C(aMPV/C)in A549 cells.Confocal images were obtained by laser confocal microscope af-ter being infected with aMPV/C.The colocalization coefficient was analyzed by Image J software.The results showed that Rab18 and aMPV/C N protein were expressed in the cytoplasm and showed obvious co-localization,while no fluorescent signal of N protein was observed in the mock group.The co-local-ization coefficient between Rab18 and aMPV/C N protein was significantly higher than that in the mock group(P<0.01).The plasmids(pCMV-Flag-Rab18 and pCMV-Flag)and nucleic acids(siRab18 and siNC)were transfected into A549 cells before being infected with aMPV/C,respectively.Cell samples and cell su-pernatant were collected for detection of the transcription level of aMPV/C N gene,the expression level of N protein,and viral titers by qPCR,Western-blot,and TCIDso,respectively.The results showed that the transcription level of aMPV/C N gene,the expression level of N protein,and the titer of virus were significantly increased after overexpression of Rab18,respectively(P<0.01).On the contrary,the transcription level of aMPV/C N gene,the expression level of N protein,and the viral titers were significantly decreased after knockdown expression of Rab18,respectively(P<0.01).A549 cells were cotransfected with pCMV-mcherry-Rab18 and GFP-tagged aMPV/C genes before confocal imaging.The results showed that Rab18 could co-localize with aMPV/C proteins in the cytoplasm.HKE293T cells were co-transfected with pCMV-Flag-Rab18 and GFP-tagged aMPV/C genes before conducting the co-immunoprecipitation(co-IP)assay.The results showed that specific N,M2-1,and L2 protein bands were found in IP samples while other viral proteins were not detected.These results suggest that Rab18 influences aMPV/C replication via interaction with N,M2-1,and L2 proteins.The results provided a foundation for further analysis of the molecular mechanism of regulation aMPV/C replication by Rab18.
作者
张正洲
王如嘉
亓宇翔
于瀚哲
孟闯
冯旭飞
ZHANG Zhengzhou;WANG Ruja;QI Yuxiang;YU Hanzhe;MENG Chuang;FENG Xufei(College of Veterinary Medicine(Institute of Comparative Medicine),Yangzhou University,Yangzhou 225009,China;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China;Jiangs u Key Laboratory of Zoonosis,Yangzhou University,Yangzhou 225009,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2024年第4期433-440,共8页
Chinese Veterinary Science
基金
江苏省高等学校基础科研(自然科学)面上项目(21KJB230009)
江苏省人兽共患病学重点实验室开放课题项目(R2106)
高等学校学科创新引智计划资助项目(D18007)
江苏高校优势学科建设工程资助项目(PAPD)。
