猪Ⅲ型干扰素原核表达及其抗病毒效果研究

doi:10.3969/j.issn.1004-1524.2020.05.04

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (5): 779-788.DOI: 10.3969/j.issn.1004-1524.2020.05.04

猪Ⅲ型干扰素原核表达及其抗病毒效果研究

陈韫陆¹, 单颖¹, 罗浩¹, 徐计东¹, 赵灵燕², 方维焕¹, 李肖梁^1,*

1.浙江大学动物科学学院浙江省动物预防医学重点实验室,浙江杭州 310058;
2.浙江省动物疫病预防控制中心,浙江杭州 310020

收稿日期:2019-11-02 出版日期:2020-05-25 发布日期:2020-05-29
通讯作者: *,李肖梁,E-mail:xlli@zju.edu.cn
作者简介:陈韫陆(1995—),女,江西吉安人,硕士研究生,主要从事动物病原生物学和免疫学研究。E-mail: 511965628@qq.com
基金资助:
浙江省科技重点研发计划(2018C02028); 浙江省“三农六方”科技协作项目(2019SNLF020); 中央财政重大农业技术推广项目(YY2018003)

Prokaryotic expression of porcine type Ⅲ interferon and studying on its antiviral activity

CHEN Yunlu¹, SHAN Ying¹, LUO Hao¹, XU Jidong¹, ZHAO Lingyan², FANG Weihuan¹, LI Xiaoliang^1,*

1. Zhejiang Provincial Key Lab of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China;
2. Zhejiang Provincial Center for Animal Disease control and Prevention, Hangzhou 310020, China

Received:2019-11-02 Online:2020-05-25 Published:2020-05-29

摘要/Abstract

摘要： Ⅲ型干扰素(IFN-λ)具有较好的广谱抗病毒能力和免疫功能调节能力,一般在肠道等黏膜部位呈现高表达。猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)和猪德尔塔冠状病毒(porcine deltacoronavirus,PDCoV)是引起仔猪腹泻的重要肠道病原,严重困扰生猪产业健康发展。研究构建了携带猪PoIFN-λ3基因的重组表达质粒,转化大肠埃希菌Rosetta感受态细胞原核表达后,经SDS-PAGE和Western blotting检测分析,重组PoIFN-λ3蛋白成功表达,大小约为27 ku,且以包涵体形式存在。变性、纯化、复性后的PoIFN-λ3重组蛋白刺激IPEC-J2细胞,ISG-15、OAS1、Mx-1、IFIT1、IFIT3、IFITM1和IFITM3等干扰素刺激基因(ISGs)表达均显著上调,且在12 h达到峰值(P<0.001)。IPEC-J2细胞分别感染PEDV和PDCoV病毒时,用PoIFN-λ3重组蛋白预处理、同时处理和感染后处理这3种方式处理后观察病毒增殖情况,与对照组相比,PEDV和PDCoV病毒拷贝数均显著下降(P<0.05)。上述结果表明,变性、纯化、复性后的PoIFN-λ3重组原核表达蛋白具有较好的生物活性,不仅可以诱导IPEC-J2细胞免疫刺激基因表达上调,在体外也可以抑制PEDV和PDCoV在IPEC-J2细胞中的复制,为防治仔猪病毒性腹泻提供了基础。

关键词: 猪Ⅲ, 型干扰素, 原核表达, 包涵体, PEDV, PDCoV

Abstract: Normally, type III interferon (IFN-λ) was highly expressed in intestinal mucosa and other mucosal systems, with relatively stronger broad-spectrum antiviral ability and immune regulating ability. Porcine epidemic diarrhea virus (PEDV) and porcine deltacorona virus (PDCoV) were two major intestinal pathogens causing diarrhea in piglets, which hindered the swine industry severely. In this study, the recombinant plasmid containing PoIFN-λ3 fragment was constructed. The recombinant plasmid was then transformed into E.coli Rosaetta cells for recombinant expression. Analyzed by SDS-PAGE and western blotting, the results showed that the recombinant PoIFN-λ3 (27 ku) was successfully expressed in the inclusion bodies of E. coli cells. After denaturation, purification and renaturation, the active PoIFN-λ3 recombinant protein was obtained and was used to treat IPEC-J2 cells. Challenged by recombinant PoIFN-λ3, the immune-stimulating genes (ISGs), such as ISG-15, OAS1, Mx-1, IFIT1, IFITM1 and IFITM3 were all up-regulated significantly and reached to the peak (P<0.001) at 12 h post challenge. For PEDV and PDCoV infection in IPEC-J2, the virus replication was detected after pretreatment, simultaneous treatment and post-infection treatment of recombinant PoIFN-λ3. Compared with the control, the viral copy numbers of both PEDV and PDCoV were decreased significantly (P<0.05) after treatment of recombinant PoIFN-λ3. The above results indicated that the purified and renatured PoIFN-λ3 recombinant protein could have good biological activity. The recombinant PoIFN-λ3 can induce high expression of different ISGs in IPEC-J2, and can also inhibit PEDV and PDCoV replication in the host cells. In summary, our study provided a basis for preventing and treating viral diarrhea in piglets.

Key words: type Ⅲ, interferon, prokaryotic expression, inclusion bodies, PEDV, PDCoV

中图分类号:

S855.3

陈韫陆, 单颖, 罗浩, 徐计东, 赵灵燕, 方维焕, 李肖梁. 猪Ⅲ型干扰素原核表达及其抗病毒效果研究[J]. 浙江农业学报, 2020, 32(5): 779-788.

CHEN Yunlu, SHAN Ying, LUO Hao, XU Jidong, ZHAO Lingyan, FANG Weihuan, LI Xiaoliang. Prokaryotic expression of porcine type Ⅲ interferon and studying on its antiviral activity[J]. , 2020, 32(5): 779-788.

参考文献

[1] KOONPAEW S, TEERAVECHYAN S, FRANTZ P N, et al.PEDV and PDCoV pathogenesis: the interplay between host innate immune responses and porcine enteric coronaviruses[J]. Frontiers in Veterinary Science, 2019, 6: 34.
[2] BRIAN D A, BARIC R S.Coronavirus genome structure and replication[J]. Current Topics in Microbiology and Immunology, 2005, 287: 1-30.
[3] CHEN X L, ZHANG X X, LI C X, et al.Epidemiology of porcine epidemic diarrhea virus among Chinese pig populations: a meta-analysis[J]. Microbial Pathogenesis, 2019, 129: 43-49.
[4] CHEN X, YANG J X, YU F S, 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.
[5] WANG D, FANG L R, XIAO S B.Porcine epidemic diarrhea in China[J]. Virus Research, 2016, 226: 7-13.
[6] ANK, N, WEST H, BARTHOLDY C, et al.Lambda interferon (IFN-λ), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo[J]. Journal of Virology, 2006, 80(9):4501-4509.
[7] STETSON D B, MEDZHITOV R.Type I interferons in host defense[J]. Immunity, 2006, 25(3): 373-381.
[8] SPIEGEL M, PICHLMAIR A, MÜHLBERGER E, et al. The antiviral effect of interferon-beta against SARS-Coronavirus is not mediated by MxA protein[J]. Journal of Clinical Virology, 2004, 30(3): 211-213.
[9] CHARLEY B, LAUDE H.Induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein E1[J]. Journal of Virology, 1988, 62(1): 8-11.
[10] ROTH-CROSS J K, BENDER S J, WEISS S R. Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia[J]. Journal of Virology, 2008, 82(20): 9829-9838.
[11] WELLS A I, COYNE C B.Type III interferons in antiviral defenses at barrier surfaces[J]. Trends in Immunology, 2018, 39(10): 848-858.
[12] LAZEAR H M, SCHOGGINS J W, DIAMOND M S.Shared and distinct functions of type I and type III interferons[J]. Immunity, 2019, 50(4):907-923.
[13] SANG Y M, ROWLAND R R R, BLECHA F. Molecular characterization and antiviral analyses of porcine type III interferons[J]. Journal of Interferon & Cytokine Research, 2010, 30(11): 801-807.
[14] ZHANG Q Z, KE H Z, BLIKSLAGER A, et al. Type III interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in IRF1 signaling[J]. Journal of Virology, 2017: JVI.01677-17.
[15] SYEDBASHA M, EGLI A.Interferon lambda: modulating immunity in infectious diseases[J]. Frontiers in Immunology, 2017, 8: 119.
[16] 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.
[17] 单颖. 单核细胞增生李斯特菌:无菌斑马鱼感染模型及Mmp-9在抗细菌感染中的作用机制[D]. 杭州: 浙江大学, 2016.
SHAN Y.Listeria monocytogenes: germ-free zebrafish infection model and the antibacterial role of mmp-9 during infection[D]. Hangzhou: Zhejiang University, 2016.(in Chinese with English abstract)
[18] 单颖, 刘子琦, 施杏芬, 等. 浙江及周边地区猪流行性腹泻病毒S基因分子特征分析[J]. 浙江大学学报(农业与生命科学版), 2018, 44(5): 610-618.
SHAN Y, LIU Z Q, SHI X F, et al.Molecular characteristic analysis of porcine epidemic diarrhea virus S gene in Zhejiang and surrounding areas[J]. Journal of Zhejiang University(Agriculture and Life Sciences), 2018, 44(5): 610-618.(in Chinese with English abstract)
[19] 单颖, 许伟成, 施杏芬, 等. 猪代尔塔冠状病毒荧光定量PCR检测方法的建立及其S基因分子特征分析[J]. 浙江农业学报, 2018, 30(2): 220-227.
SHAN Y, XU W C, SHI X F, et al.Establishment of one-step Taqman quantitative PCR detection method and molecular S gene characterization analysis of porcine Deltacoronavirus[J]. Acta Agriculturae Zhejiangensis, 2018, 30(2): 220-227.(in Chinese with English abstract)
[20] SHAN Y, LIU Y J, LIU Z Q, et al.Development and application of an indirect enzyme-linked immunosorbent assay using recombinant S1 for serological testing of porcine epidemic diarrhea virus[J]. Canadian Journal of Microbiology, 2019, 65(5): 343-352.
[21] MAHLAKÕIV T, HERNANDEZ P, GRONKE K, et al. Leukocyte-derived IFN-α/β and epithelial IFN-λ constitute a compartmentalized mucosal defense system that restricts enteric virus infections[J]. PLoS Pathogens, 2015, 11(4): e1004782.
[22] LI M L, XU W W, GAO Y D, et al.Interferon-Lambda3 (IFN-λ3) and its cognate receptor subunits in tree shrews (Tupaia belangeri): genomic sequence retrieval, molecular identification and expression analysis[J]. PLoS One, 2013, 8(3): e60048.
[23] 范向东, 孙在柏. L-精氨酸助溶菌酶复性过程动力学研究[J]. 化学与生物工程, 2010, 27(10): 50-54.
FAN X D, SUN Z B.Study on kinetics of lysozyme refolding facilitated by L-arginine[J]. Chemistry & Bioengineering, 2010, 27(10): 50-54.(in Chinese with English abstract)
[24] BATAS B, CHAUDHURI J B.Protein refolding at high concentration using size-exclusion chromatography[J]. Biotechnology and Bioengineering, 1996, 50(1): 16-23.
[25] POTT J, MAHLAKOIV T, MORDSTEIN M, et al.IFN-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.
[26] SOMMEREYNS C, PAUL S, STAEHELI P, et al.IFN-lambda (IFN-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo[J]. PLoS Pathogens, 2008, 4(3): e1000017.
[27] 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.
[28] LAZEAR H M, NICE T J, DIAMOND M S.Interferon-λ: immune functions at barrier surfaces and beyond[J]. Immunity, 2015, 43(1): 15-28.
[29] HORISBERGER M A, WATHELET M, SZPIRER J, et al.cDNA cloning and assignment to chromosome 21 of IFI-78K gene, the human equivalent of murine Mx gene[J]. Somatic Cell and Molecular Genetics, 1988, 14(2): 123-131.
[30] HALLER O, STAEHELI P, SCHWEMMLE M, et al.Mx GTPases: dynamin-like antiviral machines of innate immunity[J]. Trends in Microbiology, 2015, 23(3): 154-163.
[31] CHAKRABARTI A, JHA B K, SILVERMAN R H.New insights into the role of RNase L in innate immunity[J]. Journal of Interferon & Cytokine Research, 2011, 31(1): 49-57.
[32] KOMANDER D, RAPE M.The ubiquitin code[J]. Annual review of Biochemistry, 2012, 81(1): 203-229.
[33] BRASS A L, HUANG I C, BENITA Y, et al.The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, west Nile virus, and dengue virus[J]. Cell, 2009, 139(7): 1243-1254.

猪Ⅲ型干扰素原核表达及其抗病毒效果研究

Prokaryotic expression of porcine type Ⅲ interferon and studying on its antiviral activity

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