【作者】 王凯；

【导师】 郭抗抗；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2021， 硕士

【摘要】 猪瘟(classical swine fever,CSF)是猪的一种病毒性疾病,具有高传播率和死亡率。猪瘟病毒(classical swine fever virus,CSFV)是CSF的病原体,其自然宿主是猪科动物,主要通过直接或间接接触传播。虽然采用疫苗免疫接种及扑杀措施控制了CSF的大面积暴发及流行,但CSF在一定地区依然存在,甚至存在地方性流行现象。同时,疫苗的免疫压力使得中低毒力毒株引起的发病增多,存在持续性感染现象。对CSFV致病机理的阐明可以为更好的防控CSF提供理论支持。病毒是细胞内专性寄生物,依赖宿主细胞相关蛋白完成复制。Rab25属于小GTPase,亚细胞定位于循环内体,调节顶端囊泡向质膜的运输或循环,支配细胞表面受体的回收和细胞信号通路的激活,调控细胞增殖、细胞运动和细胞凋亡等过程,Rab25的异常表达会引起细胞囊泡运输机制的破坏,导致细胞功能紊乱。我们前期研究发现,CSFV感染引起猪脐静脉血管内皮细胞(SUVEC)中4种mi RNA表达下调,而mi R-140的下调最显著。mi R-140能够抑制感染细胞内CSFV的复制,其胞内靶标为Rab25蛋白。目前,对Rab25蛋白在CSFV感染中的作用还不清楚,研究Rab25蛋白对CSFV复制的作用,鉴定其与CSFV的相互作用,将为CSFV的致病机理研究提供新的科学资料。本研究进行Rab25蛋白对CSFV复制的影响,及其与CSFV NS5A蛋白的相互作用研究。获得了以下结果。(1)CSFV促进宿主细胞Rab25的表达用RT-qPCR、Western blot检测CSFV感染细胞Rab25基因转录和蛋白表达,发现CSFV感染细胞后36 h、48 h和72 h,Rab25 m RNA转录显著上调(P<0.05);感染后12 h、36 h、48 h、72 h,Rab25蛋白表达极显著上调(P<0.001)。(2)Rab25蛋白促进CSFV的复制将pEGFP-Rab25、pEGFP-C1重组质粒分别转染PK-15、ST以及SUVEC细胞,24 h后感染CSFV,病毒感染后12 h、24 h、36 h、48 h和72 h用RT-q PCR检测CSFV,结果显示过表达Rab25蛋白促进CSFV的复制;将Rab25-sh N以及Rab25-sh2转染PK-15、ST以及SUVEC细胞,病毒感染后12 h、24 h、36 h、48 h和72 h用RT-q PCR检测CSFV核酸,结果显示下调Rab25的表达对CSFV的复制有抑制作用。构建了Rab25突变体,转染细胞后感染CSFV,RT-q PCR检测CSFV核酸,CSFV复制受到抑制。以上结果表明Rab25对CSFV在感染细胞中的复制具有促进作用。(3)Rab25蛋白与CSFV NS5A蛋白存在相互作用通过免疫共沉淀(Co-IP)试验发现,Rab25蛋白与CSFV NS5A蛋白在细胞内存在相互作用,GST Pull-down试验发现Rab25蛋白与NS5A蛋白在细胞外存在相互作用,将荧光标记的Rab25蛋白转染宿主细胞,通过激光扫描共聚焦显微镜(CLSM)观察到Rab25蛋白与CSFV NS5A蛋白存在共定位现象。本研究明确了CSFV感染能够促进宿主细胞Rab25蛋白的表达,Rab25蛋白对CSFV在细胞内的复制具有促进作用,Rab25蛋白与CSFV NS5A蛋白存在互作,提示Rab25可能通过与CSFV NS5A相互作用影响病毒的复制。更多还原

【Abstract】 Classical swine fever(CSF)is an economically significant,high mortality,highly contagious viral disease of swine.The only natural reservoirs of the aetiological agent,Classical swine fever virus(CSFV),are members of the Suidae family and the disease affects both domestic and wild pigs,which is spread mainly through direct or indirect contact.Strict control policies,including systematic stamping out of infected herds with and without vaccination,have permitted regional virus eradication.Nevertheless,CSFV persists in certain areas of the world and has re-emerged regularly.Simultaneously,the immune pressure of the vaccine has led to increased morbidity caused by moderate and low virulence strains.In addition,there were congenital persistent CSFV infection and postnatal persistent CSFV infection.The scientific research in the fields of pathogenesis of CSFV and its explanation can provide a fundamental basis for better prevention and control of CSF,which requires extensive and in-depth research on CSFV.Viruses,intracellular parasitism in the host cell,utilize host proteins to replicate themselves.Rab25,a small GTPase belongs to the Rab protein family,which is subcellular localization in the recycling endosome,regulate vesicle at the top to the plasma membrane of transportation and circulation.Rab25 governs cell-surface receptors recycling and cellular signaling pathways activation,allowing it to control a diverse range of cellular functions,including cell proliferation,cell motility and cell death.Aberrant expression of Rab25 may result in disorder of the vesicle transport mechanism,eventually lead to some diseases,including cancer.Preliminary study have shown that CSFV infection could induce down-regulation of the expression levels of four mi RNAs in swine umbilical vein endothelial cells(SUVEC),among which mi R-140 was the most significantly down-regulated.Meanwhile,it is also demonstrated that mi R-140 can inhibit the replication of CSFV and can target Rab25.However,the role of Rab25 in CSFV infection has not been illustrated.Explication of Rab25 protein in the replication process of CSFV and identification with its interaction with CSFV virus protein will provide new scientific materials for the study of pathogenic mechanism of CSFV and find potential target proteins for antiviral therapy.Through this study,we explored the influence of Rab25 protein on CSFV replication and its interaction with CSFV NS5 A protein.The results are as follows.(1)CSFV infection promotes Rab25 expression.Rab25 m RNA and protein levels were detected by RT-q PCR and Western blot after CSFV infection,the results showed that the m RNA level of Rab25 was significantly up-regulated after CSFV infection at 36 h,48 h and72h(P<0.05).Rab25 was significantly up-regulated at 12 h,36h,48 h and 72 h after CSFV infection at protein level(P<0.001).In conclusion,the results showed that CSFV infection could induce up-regulation of Rab25 m RNA and protein levels.(2)Rab25 positively regulates replication of CSFV.Rab25 was overexpressed in PK-15,ST and SUVEC cells.We detected the replication of CSFV at 12 h,24h,36 h,48h and 72 h by RT-q PCR after infection with CSFV.The results showed that overexpression of Rab25 promoted the replication of CSFV.Rab25 was interfered in PK-15,ST and SUVEC cells.The replication of CSFV was detected at 12 h,24h,36 h,48h and 72 h by RT-q PCR The results showed that interference with Rab25 inhibited the replication of CSFV.In addition,the Rab25 mutant was constructed.Cells was infected with CSFV after transfection.RT-q PCR was used to detect CSFV.The results showed that CSFV replication was inhibited.These results indicate that Rab25 positively regulates the replication of CSFV.(3)Rab25 interacts with CSFV NS5 A protein.Rab25 protein interacted with CSFV NS5 A protein in cells was confirmed by Co-Immunoprecipitation.Rab25 protein interacted with CSFV NS5 A extracellular was proved by GST Pull-down Meanwhile,we also observed the co-localization of Rab25 protein and CSFV NS5 A protein by Confocal laser scanning microscope.In conclusion,through this study,it is clear that CSFV infection can promote the expression of Rab25.Overexpression and interference with Rab25 affect the replication of CSFV.Moreover,Rab25 interacts with NS5 A,an important component of the CSFV replication complex,which was suggested that Rab25 might mediate viral replication through interaction with CSFV NS5 A.The results of this study provide new scientific data for elucidating the pathogenesis of CSFV and provide a new direction for antiviral therapy.更多还原