【作者】 蔡新斌；

【导师】 罗廷荣；

【作者基本信息】 广西大学 ， 预防兽医学， 2011， 硕士

【摘要】 猪瘟病毒是猪烈性传染病猪瘟的病原,属于黄病毒科瘟病毒属,是一种单股正链的RNA病毒,能够在感染的猪体内引起持续性感染。ISG15是一种由Ⅰ型干扰素诱导机体产生的抗病毒蛋白,研究表明ISG15对一些病毒如人免疫缺陷病毒(HIV-1)、B型流感病毒等具有抗病毒作用。本实验室使用基因芯片技术检测猪瘟病毒感染猪只的外周血单个核细胞的基因转录谱的研究中,发现猪瘟病毒感染猪后1、3、6、9天采集的外周血淋巴细胞中ISG15基因mRNA分别呈2.63、4.51、4.43、2.18倍的变化。本研究的目的是为了进一步在蛋白水平上验证感染猪瘟病毒后细胞ISG15的表达变化,并探讨在宿主细胞中ISG15与猪瘟病毒之间的相互关系,为进一步探索猪瘟病毒的持续性感染的分子机制打下基础。本研究通过重组人Ⅰ型干扰素IFNα-2b诱导PK15细胞转录干扰素刺激基因,RT-PCR方法扩增ISG15基因,并克隆至pMD18-T载体上,再将ISG15基因亚克隆至原核表达载体pGEX-4T-1与真核表达载体pCDNA3.0。重组原核表达载体pGEX-ISG15转化BL21菌株,IPTG诱导表达ISG15重组蛋白,经过Ni-NTA树脂纯化重组蛋白,将纯化的ISG15重组蛋白与弗氏佐剂乳化免疫小鼠,制备ISG15多克隆抗体。重组真核表达载体pCDNA-ISG15用脂质体介导的方法转染PK-15细胞,用新霉素类抗生素G418筛选获得稳定表达ISG15蛋白的PK-15细胞系。通过制备的ISG15多克隆抗体,用间接免疫荧光与Western-blot方法检测猪瘟病毒感染PK-15细胞后的0、3、6、12、24、48、60 h的ISG15蛋白表达变化,ISG15蛋白在猪瘟病毒感染的PK-15细胞中表达增加,推测是ISG15蛋白介导的对猪瘟病毒蛋白的泛素化水解途径被猪瘟病毒阻断。更多还原

【Abstract】 Classical swine fever virus (CSFV) is a pathogen for highly infectious diseases as swine fever swine, belonging to Flaviviridae pestivirus genus.It is a positive and single strand of RNA virus and can cause persistent infection in pigs. ISG15 is a kind of antiviral protein induced by type I interferon. Some studies shown that ISG15 possessed antiviral activity to some viruses, such as HIV-1, B-type influenza virus. In the previous study of our laboratory, gene chip was used to detect the gene expression profile changes in peripheral blood lymphocytes of classical swine fever virus infection pigs. The results showed that ISG15 mRNA were 2.63,4.51,4.43,2.18 folds change in peripheral blood lymphocytes of pigs post infection CSFV in 1、3、6、9 days. In this study,we further to verify if the expression of ISG15 protein is increased in CSFV infected PK-15 cells, and to explore the interaction between the ISG15 and CSFV in the host cell, laying the foundation for further discover the molecular mechanism of persistent infection of CSFV.Recombinant human interferon IFN a-2b induced PK-15 cell to transcript mRNA of ISG15 gene, then the ISG15 gene was amplified by RT-PCR, and cloned into pMD18-T vector. The ISG15 gene was subcloned into the prokaryotic expression vector pGEX-4T-1 and eukaryotic expression vector pCDNA 3.0. Prokaryotic expression vector pGEX-ISG15 was transformed into BL21 strain, expression of ISG15 recombinant protein under IPTG induced and purified by Ni-NTA resin. The purified recombinant protein ISG15 was emulsified with Freund’s adjuvant and was immunized to mice for preparing anti-ISG15 polyclonal antibody. Eukaryotic expression vector pCDNA-ISG15 was used to transfect into PK-15 cells mediated liposome. to obtain The PK-15 cell line which the ISG15 protein is stably expressed was obtained with the antibiotic neomycin G418 selection.The ISG15 protein expression in PK-15 cells was dectected with Western-blot and indirect immunofluorescence analysis post CSFV infected 0,3, 6,12,24,48 and 60h. ISG15 protein increased expression in the CSFV infected PK-15 cells. Presumably ISG15 protein mediated of viral protein ubiquitination hydrolysis pathway is blocked by classical swine fever virus.更多还原