ISG15的原核表达和多克隆抗体的制备及其与猪瘟病毒相互作用的初步研究
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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。
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.
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。
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.
引文
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[2]Bazzigher L, Pavlovic J, Haller O, et al. Mx genes show weaker primary response to virus than other interferon-regulated genes. Virology,1992,186(1):154-60.
[3]Staehelia P, Hallera O, Bollb W, et al. Mx protein:Constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell, 1986,44(1):147-158.
[4]Der SD, Zhou A, Williams BR, et al. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc Natl Acad Sci U S A,1998,95(26):15623-8.
[5]Viralzone. http://expasy.org/viralzone/all_by_protein/723.html.
[6]Haas AL, Ahrens P, Bright PM, et al. Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin. J Biol Chem,1987,262(23):11315-11323.
[7]Skaug B and Chen ZJ. Emerging role of ISG15 in antiviral immunity. Cell,2010, 143(2):187-90.
[8]Durfee LA, Lyon N, Seo K, et al. The ISG15 conjugation system broadly targets newly synthesized proteins:implications for the antiviral function of ISG15. Mol Cell,2010, 38(5):722-32.
[9]Malakhov MP, Malakhova OA, Kim KI, et al. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J Biol Chem,2002,277(12):9976-9981.
[10]Zhao C, Denison C, Huibregtse JM, et al. Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc Natl Acad Sci U S A,2005,102(29):10200-5.
[11]Nyman TA, Matikainen S, Sareneva T, et al. Proteome analysis reveals ubiquitin-conjugating enzymes to be a new family of interferon-alpha-regulated genes. Eur J Biochem,2000,267(13):4011-9.
[12]Yuan W and Krug RM. Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. Embo J,2001,20(3):362-71.
[13]Yuan W, Aramini JM, Montelione GT, et al. Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus:a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus. Virology,2002,304(2):291-301.
[14]Chang YG, Yan XZ, Xie YY, et al. Different roles for two ubiquitin-like domains of ISG15 in protein modification. J Biol Chem,2008,283(19):13370-7.
[15]Sridharan H, Zhao C and Krug RM. Species specificity of the NS1 protein of influenza B virus:NS1 binds only human and non-human primate ubiquitin-like ISG15 proteins. J Biol Chem,2010,285(11):7852-6.
[16]Versteeg GA, Hale BG, van Boheemen S, et al. Species-specific antagonism of host ISGylation by the influenza B virus NS1 protein. J Virol,2010,84(10):5423-30.
[17]Zhao C, Hsiang TY, Kuo RL, et al. ISG15 conjugation system targets the viral NS1 protein in influenza A virus-infected cells. Proc Natl Acad Sci U S A,2009,107(5): 2253-2258.
[18]Tang Y, Zhong G, Zhu L, et al. Herc5 attenuates influenza A virus by catalyzing ISGylation of viral NS1 protein. J Immunol,2010,184(10):5777-90.
[19]Lenschow DJ, Lai C, Frias-Staheli N, et al. IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses. Proc Natl Acad Sci U S A,2007,104(4):1371-6.
[20]Osiak A, Utermohlen O, Niendorf S, et al. ISG15, an Interferon-Stimulated Ubiquitin-Like Protein, Is Not Essential for STAT1 Signaling and Responses against Vesicular Stomatitis and Lymphocytic Choriomeningitis Virus. Mol Cell Biol,2005, 25(15):6338-6345.
[21]Lai C, Struckhoff JJ, Schneider J, et al. Mice lacking the ISG15 El enzyme UbE1L demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza B virus infection. J Virol,2009,83(2):1147-51.
[22]Hsiang TY, Zhao C and Krug RM. Interferon-induced ISG15 conjugation inhibits influenza A virus gene expression and replication in human cells. J Virol,2009,83(12): 5971-7.
[23]Lenschow DJ, Giannakopoulos NV, Gunn LJ, et al. Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo. J Virol,2005,79(22):13974-83.
[24]Giannakopoulos NV, Arutyunova E, Lai C, et al. ISG15 Argl51 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of Sindbis virus. J Virol,2009,83(4):1602-10.
[25]Nicholl MJ, Robinson LH and Preston CM. Activation of cellular interferon-responsive genes after infection of human cells with herpes simplex virus type 1. J Gen Virol,2000,81(Pt 9):2215-8.
[26]Johnson KE and Knipe DM. Herpes simplex virus-1 infection causes the secretion of a type I interferon-antagonizing protein and inhibits signaling at or before Jak-1 activation. Virology,2010,396(1):21-9.
[27]Malakhova OA and Zhang DE. ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response. J Biol Chem,2008,283(14):8783-7.
[28]Okumura A, Pitha PM and Harty RN. ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc Natl Acad Sci U S A,2008,105(10):3974-9.
[29]Kim JH, Luo JK and Zhang DE. The level of hepatitis B virus replication is not affected by protein ISG15 modification but is reduced by inhibition of UBP43 (USP18) expression. J Immunol,2008,181(9):6467-72.
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