【作者】 张静；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2019， 硕士

【摘要】 猪瘟病毒(Classical swine fever virus,CSFV)引起的猪瘟是一种高度传染性、通常导致死亡的疾病,会造成养猪业重大经济损失。CSFV与其他真核生物病毒一样,必须借助宿主细胞的成分完成自身的复制增殖。Rab6a是定位于宿主细胞高尔基体的一种小GTP酶,系囊泡转运的重要调节因子。本研究旨在探究Rab6a蛋白对CSFV复制的影响,并了解CSFV E2蛋白在细胞内分布的变化,为阐明Rab6a蛋白对CSFV复制的调控作用提供新的科学资料,获得了以下研究结果。(1)Rab6a对CSFV的复制有正向调控作用。敲低Rab6a表达后,CSFV的复制受到显著抑制(P<0.05);过表达Rab6a后,CSFV的复制受到显著促进(P<0.05)。突变体Rab6a Q72L(GTP)、Rab6a T27N(GDP)对CSFV复制均起显著抑制作用(P<0.05),提示Rab6a蛋白GTP形式和GDP形式的正常转换是CSFV复制的重要因素。(2)Rab6a促进CSFV病毒粒子的产生。干扰Rab6a表达后,细胞上清中病毒粒子产量明显降低;过表达Rab6a后,细胞上清中病毒粒子产量明显增加;突变体Rab6a Q72L(GTP)、Rab6a T27N(GDP)分别处理后的细胞上清中病毒粒子产量均明显降低,说明Rab6a具有促进CSFV病毒粒子产生的作用。(3)Rab6a介导E2蛋白的胞内运输。PK-15细胞共同转染Rab6a和E2蛋白重组质粒,通过激光共聚焦显微镜观察E2蛋白在胞内的分布变化,发现Rab6a介导CSFV E2蛋白由核周围向细胞膜的转运。(4)CSFV感染PK-15细胞后对胞内Rab6a的转录水平和蛋白水平无明显影响。应用real-time PCR及Western blot检测CSFV感染PK-15细胞后对Rab6a转录水平和蛋白表达量的影响,结果显示CSFV感染PK-15细胞后Rab6a转录水平和蛋白表达量无显著变化(P>0.05)。综上所述,本研究发现Rab6a蛋白促进CSFV的复制,对CSFV子代病毒的产生具有重要作用;Rab6a蛋白介导CSFV E2由核周围向细胞膜的转运。研究结果为进一步阐明Rab家族对CSFV复制的影响及了解CSFV在宿主细胞内的转运提供了新的科学资料。更多还原

【Abstract】 Classical swine fever,caused by Classical swine fever virus,is a highly contagious,often fatal porcine disease that causes significant economic losses.Like other eukaryotic viruses,CSFV must complete its own replication and proliferation by means of the components of the host cell.Rab6 a is a small GTPase that localizes to the Golgi of the host cell and is an important regulator of vesicle transport.The aim of this study was to investigate the effect of Rab6 a protein on CSFV replication and to understand the changes in the distribution of CSFV E2 protein in cells.This study provides new scientific information for elucidating the regulation of Rab6 a protein on CSFV replication.The following research results were obtained.(1)Rab6a has a positive regulatory effect on CSFV replication.After knocking down Rab6 a expression,CSFV replication was significantly inhibited(P<0.05);after overexpression of Rab6 a,CSFV replication was significantly promoted(P<0.05).Mutant Rab6 a Q72L(GTP)and Rab6 a T27N(GDP)significantly inhibited CSFV replication(P<0.05),it is suggested that the normal conversion of the GTP form and the GDP form of the Rab6 a protein is an important factor in CSFV replication.(2)Rab6a promotes the production of CSFV infectious virions.After the expression of Rab6 a was disturbed,the yield of infectious virions in the supernatant of the cells was significantly decreased;After overexpression of Rab6 a,the yield of infectious virions in the supernatant of the cells was significantly promoted;The yield of infectious virions in the supernatants of the mutants treated with Rab6 a Q72L(GTP)and Rab6 a T27N(GDP)were significantly decreased,indicating that Rab6 a has promotes the production of CSFV infectious virions.(3)Rab6a mediates intracellular transport of E2 protein.PK-15 cells were co-transfected with Rab6 a and E2 protein recombinant plasmids.The distribution of E2 protein in the cells was observed by laser confocal microscopy.It was found that Rab6 a mediates the transport of CSFV E2 protein from the periphery of the nucleus to the cell membrane.(4)There was no significant effect on the transcriptional level and protein level of intracellular Rab6 a in PK-15 cells infected with classical swine fever virus.The effect of CSFV infection with PK-15 cells on the transcription level and protein expression of Rab6 awas detected by real-time PCR and Western blot,the results showed that the transcription level and protein expression of Rab6 a did not change significantly after CSFV infected PK-15 cells(P>0.05).In summary,this study found that Rab6 a protein promotes the replication of CSFV and plays an important role in the production of CSFV progeny virus;Rab6a protein mediates the transport of CSFV E2 from the periphery of the nucleus to the cell membrane.The results provide new scientific information to further elucidate the effect of the Rab family on CSFV replication and to understand the transport of CSFV in host cells.更多还原