利用杆状病毒表达系统表达猪IFN-L3及其抗PEDV活性的研究
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摘要
利用Bac-to-Bac杆状病毒表达系统在昆虫细胞上表达重组猪干扰素L3(rPoIFN-L3)进行抗猪流行性腹泻病毒(PEDV)的研究。首先通过间接免疫荧光和Western-blot验证rPoIFN-L3表达,并用Protein A亲和层析柱进行蛋白纯化;采用表达绿色荧光蛋白的水疱性口炎病毒(VSV~*GFP)检测rPoIFN-L3的抗病毒活性,含rPoIFN-L3的细胞上清的抗病毒活性为1×10~3AU/mL,纯化的rPoIFN-L3抗病毒活性为5×10~5AU/mg。进一步对纯化的rPoIFN-L3蛋白在IPEC-J2细胞上进行抗PEDV活性探究,结果显示,rPoIFN-L3可呈剂量依赖性抑制PEDV复制,并且上调相关干扰素-刺激基因(ISGs)的表达。由此证明,本研究表达的rPoIFN-L3具有抗病毒活性,并可有效抑制PEDV在IPEC-J2上的复制,对预防和治疗PEDV感染有重要意义。
In this paper,the recombinant porcine interferon L3(rPoIFN-L3) was expressed in insect cells by using the Bac-to-Bac baculovirus expression system for anti-porcine epidemic diarrhea virus(PEDV) investigation.First,the expression of rPoIFN-L3 was verified by indirect immunofluorescence and Western-blot,and then we purified rPoIFN-L3 by protein A affinity chromatography column. The antiviral activity of rPoIFN-L3 was detected by vesicular stomatitis virus expressing green fluorescent protein(VSV~*GFP).The antiviral activity of the supernatant containing rPoIFN-L3 was 1×10~3 AU/m L,and the purified rPoIFN-L3 antiviral activity was 5×10~5 AU/mg. Furthermore,we verified that r Po IFN-L3 had a strong antiviral activity by inhibiting the replication of PEDV and up-regulated the expression of interferon-stimulated genes(ISGs) in a dose-dependent manner in IPEC-J2 cells.These results showed that rPoIFN-L3 expressed in this study has antiviral activity and can effectively inhibit the replication of PEDV in IPEC-J2.It is important to the prevention and treatment of PEDV infection.
In this paper,the recombinant porcine interferon L3(rPoIFN-L3) was expressed in insect cells by using the Bac-to-Bac baculovirus expression system for anti-porcine epidemic diarrhea virus(PEDV) investigation.First,the expression of rPoIFN-L3 was verified by indirect immunofluorescence and Western-blot,and then we purified rPoIFN-L3 by protein A affinity chromatography column. The antiviral activity of rPoIFN-L3 was detected by vesicular stomatitis virus expressing green fluorescent protein(VSV~*GFP).The antiviral activity of the supernatant containing rPoIFN-L3 was 1×10~3 AU/m L,and the purified rPoIFN-L3 antiviral activity was 5×10~5 AU/mg. Furthermore,we verified that r Po IFN-L3 had a strong antiviral activity by inhibiting the replication of PEDV and up-regulated the expression of interferon-stimulated genes(ISGs) in a dose-dependent manner in IPEC-J2 cells.These results showed that rPoIFN-L3 expressed in this study has antiviral activity and can effectively inhibit the replication of PEDV in IPEC-J2.It is important to the prevention and treatment of PEDV infection.
引文
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[15]CHEN X,YANG J,YU F,et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus(PEDV)samples from field cases in Fujian,China[J].Virus Genes,2012,45(3):499-507.
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[18]LI L,FU F,XUE M,et al.IFN-lambda preferably inhibits PEDV infection of porcine intestinal epithelial cells compared with IFN-alpha[J].Antiviral Research,2017,140:76-82.
[19]MILLER L K.Baculoviruses as gene expression vectors[J].Annual Review of Microbiology,1988,42:177-199.
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[22]李悦,陈伟贞,陈建国,等.小鼠抑制素α亚基在杆状病毒表达系统中的表达和纯化[J].中国兽医科学,2015,45(12):1295-1299.LI Yue,CHEN Wei-zhen,CHEN Jian-guo,et al.Expression and purification of mouse inhibin-αin baculovirus expression system[J].Chinese Veterinary Science,2015,45(12):1295-1299.(in Chinese)
[23]DELLGREN C,GAD H H,HAMMING O J,et al.Human interferon-lambda3 is a potent member of the typeⅢinterferon family[J].Genes and Immunity,2009,10(2):125-131.
[24]BALDRIDGE M T,NICE T J,MCCUNE B T,et al.Commensal microbes and interferon-lambda determine persistence of enteric murine norovirus infection[J].Science,2015,347(6219):266-269.
[25]MAHLAKOIV T,HERNANDEZ P,GRONKE K,et al.Leuko cyte-derived IFN-alpha/beta and epithelial IFN-lambda constitute a compartmentalized mucosal defense system that restricts enteric virus infections[J].PLoS Pathogens,2015,11(4):e1004782.
[26]LIN J D,FENG N,SEN A,et al.Distinct roles of typeⅠand typeⅢinterferons in intestinal immunity to homologous and heterologous rotavirus infections[J].PLoS Pathogens,2016,12(4):e1005600.
[27]HERNANDEZ P P,MAHLAKOIV T,YANG I,et al.Interferon-lambda and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection[J].Nature Immunology,2015,16(7):698-707.
[28]ZHOU Z,HAMMING O J,ANK N,et al.TypeⅢinterferon(IFN)induces a typeⅠIFN-like response in a restricted subset of cells through signaling pathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases[J].Journal of Virology,2007,81(14):7749-7758.
[29]DOYLE S E,SCHRECKHISE H,KHUU-DUONG K,et al.Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes[J].Hepatology,2006,44(4):896-906.
[2]MARCELLO T,GRAKOUI A,BARBA-SPAETH G,et al.Interferons alpha and lambda inhibit hepatitis C virus replication with distinct signal transduction and gene regulation kinetics[J].Gastroenterology,2006,131(6):1887-1898.
[3]ROBEK M D,BOYD B S,CHISARI F V.Lambda interferon inhibits hepatitis B and C virus replication[J].Journal of Virology,2005,79(6):3851-3854.
[4]MUIR A J,SHIFFMAN M L,ZAMAN A,et al.Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis C virus infection[J].Hepatology,2010,52(3):822-832.
[5]SOMMEREYNS C,PAUL S,STAEHELI P,et al.IFN-lambda is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo[J].PLoS Pathogens,2008,4(3):e1000017.
[6]MORDSTEIN M,NEUGEBAUER E,DITT V,et al.Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections[J].Journal of Virology,2010,84(11):5670-5677.
[7]DAVIDSON S,MCCABE T M,CROTTA S,et al.IFN-lambda is a potent anti-influenza therapeutic without the inflammatory side effects of IFN-alpha treatment[J].EMBO Molecular Medicine,2016,8(9):1099-1112.
[8]LAZEAR H M,NICE T J,DIAMOND M S.Interferon-lambda:immune functions at barrier surfaces and beyond[J].Immunity,2015,43(1):15-28.
[9]ZANONI I,GRANUCCI F,BROGGI A.Interferon(IFN)-lambda takes the helm:immunomodulatory roles of typeⅢIFNs[J].Frontiers in Immunology,2017(8):1661.
[10]SANG Y,ROWLAND R R,BLECHA F.Molecular characterization and antiviral analyses of porcine typeⅢinterferons[J].Journal of Interferon and Cytokine Research,2010,30(11):801-807.
[11]ZHANG Q,KE H,BLIKSLAGER A,et al.TypeⅢinterferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in IRF1signaling[J].Journal of Virology,2018,92(4):e01677-17.
[12]WANG D,FANG L,XIAO S.Porcine epidemic diarrhea in China[J].Virus Research,2016,226:7-13.
[13]DEBOUCK P,PENSAERT M.Experimental infection of pigs with a new porcine enteric coronavirus,CV777[J].American Journal of Veterinary Research,1980,41(2):219-223.
[14]SONG D,PARK B.Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology,diagnosis,and vaccines[J].Virus Genes,2012,44(2):167-175.
[15]CHEN X,YANG J,YU F,et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus(PEDV)samples from field cases in Fujian,China[J].Virus Genes,2012,45(3):499-507.
[16]LI W,LI H,LIU Y,et al.New variants of porcine epidemic diarrhea virus,China,2011[J].Emerging Infectious Diseases,2012,18(8):1350-1353.
[17]POTT J,MAHLAKOIV T,MORDSTEIN M,et al.IFN-lambda determines the intestinal epithelial antiviral host defense[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(19):7944-7949.
[18]LI L,FU F,XUE M,et al.IFN-lambda preferably inhibits PEDV infection of porcine intestinal epithelial cells compared with IFN-alpha[J].Antiviral Research,2017,140:76-82.
[19]MILLER L K.Baculoviruses as gene expression vectors[J].Annual Review of Microbiology,1988,42:177-199.
[20]LUCKOW V A,SUMMERS M D.Signals important for high-level expression of foreign genes in autographa californica nuclear polyhedrosis virus expression vectors[J].Virology,1988,167(1):56-71.
[21]KOST T A,CONDREAY J P,JARVIS D L.Baculovirus as versatile vectors for protein expression in insect and mammalian cells[J].Nature Biotechnology,2005,23(5):567-575.
[22]李悦,陈伟贞,陈建国,等.小鼠抑制素α亚基在杆状病毒表达系统中的表达和纯化[J].中国兽医科学,2015,45(12):1295-1299.LI Yue,CHEN Wei-zhen,CHEN Jian-guo,et al.Expression and purification of mouse inhibin-αin baculovirus expression system[J].Chinese Veterinary Science,2015,45(12):1295-1299.(in Chinese)
[23]DELLGREN C,GAD H H,HAMMING O J,et al.Human interferon-lambda3 is a potent member of the typeⅢinterferon family[J].Genes and Immunity,2009,10(2):125-131.
[24]BALDRIDGE M T,NICE T J,MCCUNE B T,et al.Commensal microbes and interferon-lambda determine persistence of enteric murine norovirus infection[J].Science,2015,347(6219):266-269.
[25]MAHLAKOIV T,HERNANDEZ P,GRONKE K,et al.Leuko cyte-derived IFN-alpha/beta and epithelial IFN-lambda constitute a compartmentalized mucosal defense system that restricts enteric virus infections[J].PLoS Pathogens,2015,11(4):e1004782.
[26]LIN J D,FENG N,SEN A,et al.Distinct roles of typeⅠand typeⅢinterferons in intestinal immunity to homologous and heterologous rotavirus infections[J].PLoS Pathogens,2016,12(4):e1005600.
[27]HERNANDEZ P P,MAHLAKOIV T,YANG I,et al.Interferon-lambda and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection[J].Nature Immunology,2015,16(7):698-707.
[28]ZHOU Z,HAMMING O J,ANK N,et al.TypeⅢinterferon(IFN)induces a typeⅠIFN-like response in a restricted subset of cells through signaling pathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases[J].Journal of Virology,2007,81(14):7749-7758.
[29]DOYLE S E,SCHRECKHISE H,KHUU-DUONG K,et al.Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes[J].Hepatology,2006,44(4):896-906.